2024-03-29T08:42:48Zhttps://thescholarship.ecu.edu/oai/requestoai:TheScholarship.intra.ecu.edu:10342/45632021-03-03T20:57:57Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Intratracheal instillation of silver nanoparticles exacerbates cardiac ischemia/reperfusion injury in male sprague-dawley rats
Holland, Nathan A.
Wingard, Christopher J.
The uses of engineered nanomaterials have expanded in biomedical technology and consumer manufacturing. Exposure to particulate matter has been demonstrated to negatively influence cardiovascular health and expand myocardial infarction. Furthermore, pulmonary exposure to various engineered nanomaterials has, likewise, demonstrated the ability to exacerbate cardiac ischemia reperfusion (I/R) injury. We hypothesized that pulmonary exposure to AgNP induces cardiovascular toxicity in the form of expanded I/R injury, electrical dysfunction and inducing a persistent increase in circulating proinflammatory cytokines. To test this hypothesis, we exposed male SD rats to an intratracheal (IT) instillation of 200 µg of 20 or 110 nm polyvinylprryolidone (PVP) or citrate capped AgNP, in 200 ul of the respective PVP or citrate vehicle. Serum samples were collected prior to instillation and 1, 3, 6, 24, 48, 72, and 168 hours following instillation. Serum samples were analyzed by multiplex assay for concentrations of: G-CSF, GM-CSF, MIP-1a, IL -1b, IL-2, IL-5, IL-6, IL-10, IL-13, IL-17a, IL-18, MCP-1, IFNy, RANTES, and TNFa. Twenty four and 168 hours after IT exposure, cardiac ischemia was induced by left anterior descending coronary artery ligation for 20 minutes followed by 2 hours of reperfusion. Intraoperative ECG was monitored throughout cardiac I/R surgery for heart rate (HR), PR interval, and QT interval. To test the impact of silver ion exposure on cardiac I/R injury we administered 200 ul of 0.01 mg/mL, 0.1 mg/mL, or 1 mg/mL silver acetate (AgAc) and induced cardiac I/R 24 hours later. Intratracheal instillation of AgNP resulted in expansion of I/R injury for both sizes of citrate and PVP capped AgNP at both 24 hours and 168 following instillation; exposure to 0.1 and 1 mg/mL AgAc also resulted in expansion of I/R injury. Intratracheal instillation of AgNP did not result in increased serum concentrations of selected proinflammatory cytokines, however post I/R serum levels of IL-2, IL-6, and IL-18 were significantly elevated in rats exposed to 20 nm PVP capped AgNP compared to vehicle controls at 24 hours post instillation. Instillation of AgNP had no impact on HR or QT interval. However, exposure to 20 nm AgNP resulted in a differential prolongation or shortening of PR interval during reperfusion based on capping agent. In conclusion IT instillation of AgNP exacerbates cardiac I/R injury 24 and 168 hours following instillation, without inducing a strong systemic inflammatory response or electrical dysfunction. Exposure to AgNP may result in a sensitization of the immune system in response to a secondary insult (e.g., cardiac I/R) which are largely correlated with capping agents and particle size and may drive expansion of I/R injury at 24 and 168 hours following IT instillation of AgNP. Â
East Carolina University
2014
Master's Thesis
http://hdl.handle.net/10342/4563
https://thescholarship.ecu.edu/bitstream/10342/4563/1/Holland_ecu_0600O_11215.pdf
291c44e02f16e1c97ded2e2d5ae741d6
https://thescholarship.ecu.edu/bitstream/10342/4563/2/Holland_ecu_0600O_11215.pdf.txt
459d5150cb02fddcfa55a3e9a62dc30c
https://thescholarship.ecu.edu/bitstream/10342/4563/3/NEDL_Holland.pdf
894fcb5eefe752fd531ee4ea8ff806f4
Physiology
Toxicology
Cardiac
Inflammation
Ischemia
Nanomaterial
Reperfusion
Silver
Biology, Physiology
Myocardial Infarction
Reperfusion Injury
Nanoparticles--adverse effects
oai:TheScholarship.intra.ecu.edu:10342/64002021-03-03T21:15:21Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Targeting Kremen1 Downregulation with RVG-9R/siRNA Complexes in the Triple-Transgenic Mouse Model of Alzheimer’s Disease
Baker, Kelly E.
Murashov, Alexander K.
Alzheimer's disease (AD) is a progressive disease characterized by cognitive decline and memory loss. Memory loss observed in AD results from the loss of neurons and synapses which may be caused by the disruption of the canonical Wnt signaling pathway by Dickkopf-1 (Dkk-1). Under normal conditions, the canonical Wnt signaling pathway is responsible for normal neuronal development, synaptic plasticity, and overall normal brain function. Amyloid-[beta] (A[beta]) plaques and neurofibrillary tangles are two characteristic morphological changes observed in AD. An increased level of A[beta] has been associated with increased expression of Dkk-1, which may be linked to synaptic loss seen in AD. Kremen1 (Krm1) is a receptor for Dkk-1. Published and unpublished observations from our laboratory showed that silencing Krm1 with miR-431 can promote regenerative axon growth and prevent synaptic loss in a cell culture model. This study focused on downregulating Krm1 the triple-transgenic mouse model of AD (3xTg-AD). It was hypothesized that application of siRNA-431 in vivo would downregulate Krm1 thereby preventing synaptic loss and memory deficits in the 3xTg-AD mouse model of AD. Tail vein injections of RVG-9R/siRNA complexes and control injections were administered to 3xTg-AD mice and wild-type (WT) mice at 4, 6, or 12 months of age. Within each age cohort there were three different groups: 3xTg-AD mice injected with RVG/siRNA, 3xTg-AD mice injected with control peptide/siRNA, and WT mice injected with saline. Each group of mice was approximately half male and half female. Following the injections, the Barnes Maze was administered to each mouse in order to assess memory function. Data gathered from the Barnes Maze shows 3xTg-AD mice have a longer primary latency in the probe phase compared to WT mice. Of mice tested, fewer 3xTg-AD mice have been successful in finding the target hole during probe phase compared to WT mice. After completion of the Barnes Maze, mice were sacrificed and brains were collected for analysis. The brains were analyzed for Krm1 downregulation at the protein and mRNA levels via Western blot and qPCR, respectively. In 4 month old mice, WT mice showed the lowest levels of Krm1 protein and mRNA expression levels and 3xTg-AD CPep/siRNA treated mice showed the highest. The 4 month old 3xTg-AD RVG-9R/siRNA treated mice had Krm1 protein and mRNA expression levels that fell between the other two groups. Immunofluorescence was performed on coronal brain sections to analyze number of synapses. Six month old 3xTg-AD CPep/siRNA treated mice had significantly fewer synapses than both the WT and 3xTg-AD RVG-9R/siRNA treated groups. In conclusion, IF, qPCR, and Western blot data reveal the potential for RVG-9R/siRNA treatment to target and downregulate Kremen1 in vivo and provide protection from synaptic loss. However, further studies are need to confirm the ability of RVG-9R/siRNA treatment to downregulate Kremen1.
East Carolina University
2017-07-19
Master's Thesis
en
http://hdl.handle.net/10342/6400
https://thescholarship.ecu.edu/bitstream/10342/6400/1/BAKER-MASTERSTHESIS-2017.pdf
0a2fc27f0becfc4ea2cd52b98d5bf18a
https://thescholarship.ecu.edu/bitstream/10342/6400/2/LICENSE.txt
171663e6cdb606402e2fda76765fee74
https://thescholarship.ecu.edu/bitstream/10342/6400/3/PROQUEST_LICENSE.txt
b857219098972b01777f2c826aadfdfd
https://thescholarship.ecu.edu/bitstream/10342/6400/4/signature%20page.pdf
d82fe217e9e6885737f175bb18bb03eb
https://thescholarship.ecu.edu/bitstream/10342/6400/5/NEDL.KEB%20copy.pdf
1e12f0f9a9f543cad8085b0b8a784f90
https://thescholarship.ecu.edu/bitstream/10342/6400/7/BAKER-MASTERSTHESIS-2017.pdf.txt
542fd764e33112b2876db5ee08832ea6
Kremen1
Wnt Signaling
3xTg-AD Mouse Model
Alzheimer Disease
Down-Regulation
RNA, Small Interfering
Mice, Ttransgenic
oai:TheScholarship.intra.ecu.edu:10342/46622021-03-03T20:56:29Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
TMEFF2 is an epigenetic modulator that promotes androgen independent growth in castration-resistant prostate cancer cells
Corbin, Joshua Moses
Ruiz-Echevarria, Maria
While the ability to detect PCa has improved significantly due to PSA screenings, the survival rate for men diagnosed with PCa has remained stagnant, and the disease remains the second leading cause of cancer related deaths in men. Most patients initially respond to androgen deprivation treatment; however, a significant percentage of patients relapse with currently untreatable castration resistant prostate cancer (CRPC), during which the PCa cells develop the ability to grow in androgen depleted conditions. The androgen receptor (AR) plays a vital role in prostate development and homeostasis, and the deregulation of AR drives PCa tumorigenesis and progression to CRPC. Delineating molecular mechanisms that contribute to AR activity and/or PCa cell growth in androgen-depleted conditions may aid in the development of future CRPC therapies Epigenetic alterations play a critical role in differentiation during development, and aberrations in epigenetic regulation are associated with tumorigenesis and cancer progression. Two types of epigenetic modifications, DNA methylation and the methylation of multiple histone lysines, play significant roles in prostate cancer (PCa). Many histone methyltransferases (HMT) and demethylases (HDM), including the JMJD2 family of histone demethyalses, act as coregulators of AR, and many of these enzymes are implicated in CRPC. Because of this, HDMSs and HMTs have proven as attractive targets for therapeutic intervention. We have been studying TMEFF2, a protein that is regulated transcriptionally and translationally by the AR, and is overexpressed in PCa and CRPC suggesting a role in this disease. Data presented here demonstrate that TMEFF2 modulates JMJD2 controlled methyl histone marks and increases growth in androgen depleted conditions in CRPC cells. In correlation with its effect on histone methylation and growth, TMEFF2 overexpression increases resistance to the anti-growth effects of the pan-jumonji demethylase inhibitor, JIB-04, suggesting that TMEFF2 modulates growth, at least in part, by increasing jumonji demethylase activity. Additionally, TMEFF2 positively regulates PSA expression without altering AR levels in CRPC cells, indicating that TMEFF2 is a novel activator of AR. All together this data suggests a model in which TMEFF2, by modulating the activity of AR and JMJD2 enzymes, increases CRPC cell growth. Because CRPC remains to be a significant obstacle in the successful treatment of metastatic PCa, the results presented have the potential to be of therapeutic value.
East Carolina University
2014
Master's Thesis
http://hdl.handle.net/10342/4662
https://thescholarship.ecu.edu/bitstream/10342/4662/1/Corbin_ecu_0600O_11285.pdf
a5c9a6b3510e8ec034a0f5866eb4c92e
https://thescholarship.ecu.edu/bitstream/10342/4662/2/CorbinNEDL.PDF
3ce8069d8bdd8bf992e81a832c3e896a
https://thescholarship.ecu.edu/bitstream/10342/4662/3/Corbin_ecu_0600O_11285.pdf.txt
456e742de0615419340064447e6fa4b8
Oncology
Prostatic Neoplasms--pathology
Prostatic Neoplasms--genetics
Prostatic Neoplasms, Castration-Resistant--pathology
Prostate--metabolism
oai:TheScholarship.intra.ecu.edu:10342/50192021-03-03T20:58:25Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Genetic control of cell fate specification in Caenorhabditis elegans germline.
Mamillapalli, Srivalli Swathi
Lee, Myon-Hee
The precise regulation of germ cell fates (sperm or oocyte) lies at the heart of reproduction and fertility. The nematode Caenorhabditis elegans hermaphrodites produce a discrete number of sperm during larval development and then switch to produce oocyte during adulthood. A number of positive (e.g., fbf genes) and negative (e.g., gld-3) regulators are important for this switch. Here, we found that aberrant activation of MPK-1 (an ERK homolog) by removal of both fbf-1 and lip-1 partially inhibits sperm-oocyte switch, resulting in Mog (masculinization of germline) sterility. The fbf-1 gene encodes a conserved PUF (Pumilio and FBF) RNA-binding protein and the lip-1 gene encodes an MPK-1/ERK phosphatase. Notably, inhibition of MPK-1/ERK signaling by either genetic mutation or chemical inhibition reprograms the germ cell fate and thus helps in regaining the fertility. We also found that fbf-1; lip-1 Mog sterility was enhanced by the depletion of G2/M cell cycle regulators, including CYB-3/Cyclin B, CDK-1/CDK1, and CDC-25.1/CDC25. Markedly, cdc-25.1 mRNA is a direct target of FBF-1. These results suggest that FBF-1 and LIP-1 may promote sperm-oocyte switch by activating MPK-1/ERK signaling and G2/M cell cycle progression.
East Carolina University
2015
Master's Thesis
http://hdl.handle.net/10342/5019
https://thescholarship.ecu.edu/bitstream/10342/5019/1/MAMILLAPALLI_ecu_0600O_11516.pdf
4c7f9de0d3f0b26814c68efec2c7a9a3
https://thescholarship.ecu.edu/bitstream/10342/5019/2/MAMILLAPALLI_ecu_0600O_11516.pdf.txt
facfb804b5ec0a62ed9135611edc5fb0
https://thescholarship.ecu.edu/bitstream/10342/5019/4/MAMILLAPALLI_NEDL.pdf
dda0c66e2cdac942019b6419df1391c9
Developmental biology
Genetics
Medicine
C. elegans
Cell cycle regulation
Cell fate specification
Genetic controls
Germlines
MPK-1/ERK signaling
Caenorhabditis elegans
Spermatogenesis--physiology
Disorders of Sex Development-- metabolism.
Disorders of Sex Development-- genetics
Caenorhabditis elegans--growth & development
Caenorhabditis elegans--metabolism
RNA-Binding Proteins--metabolism
oai:TheScholarship.intra.ecu.edu:10342/91242022-12-14T17:29:51Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
PUF-8 and MPK-1: Genetic and Chemical Control of Spermatocyte Dedifferentiation in Caenorhabditis elegans
Gaddy, Matthew A.
Lee, Myon-Hee
Stem cells face a number of major fate decision during their development: the decision to self-renew or differentiate, and then whether to remain differentiated or dedifferentiate, as occurs in some oncogenesis. A regulatory network controlling these decisions is vital to the development of all multicellular organisms, including humans. Aberrant regulation can result in either loss of specific cell type or uncontrolled cell proliferation, leading to tumors. However, our understanding of how differentiated cell can be reverted to an undifferentiated state remains far more limited.Using the nematode C. elegans germline as a model system, we previously reported that PUF-8 (a PUF RNA-binding protein) and LIP-1 (a dual-specificity phosphatase) inhibit the formation of germline tumors via repressing the dedifferentiation of spermatocytes into mitotic cells (termed "spermatocyte dedifferentiation") at least in part by inhibiting MPK-1 (an ERK MAPK homolog) activation. To gain insight into the molecular competence for spermatocyte dedifferentiation, we compared the germline phenotypes between two competent mutants -- puf-8(q725); lip-1(zh15) with a high MPK-1 activity and puf-8(q725); fem-3(q20gf) with a low MPK-1 activity. puf-8(q725); lip-1(zh15) mutants developed germline tumors more aggressively than puf-8(q725); fem-3(q20gf) mutants at 25°C with aging. This result suggests that MPK-1 activation is critical to induce the formation of germline tumors via spermatocyte dedifferentiation. This idea was confirmed by treatment of puf-8(q725); fem-3(q20gf) mutant worms with Resveratrol, which stimulates MPK-1 activation. Our results show that 100 mM RSV significantly induced the formation of germline tumors via spermatocyte dedifferentiation at 25°C with aging. Therefore, we conclude that MPK-1 activation is required to promote the formation of germline tumors via spermatocyte dedifferentiation in the absence of PUF-8. Since PUF-8 and MPK-1 are broadly conserved, we therefore suggest that similar molecular mechanisms may control dedifferentiation-mediated tumorigenesis in other organisms, including humans.
East Carolina University
2021-05-03
Master's Thesis
en
http://hdl.handle.net/10342/9124
https://thescholarship.ecu.edu/bitstream/10342/9124/1/GADDY-MASTERSTHESIS-2021.pdf
37a1ef4e92a1c4f57d6c4c10934ab272
https://thescholarship.ecu.edu/bitstream/10342/9124/2/LICENSE.txt
1120b03448b5757a40f3f22b5f5e7cba
https://thescholarship.ecu.edu/bitstream/10342/9124/3/PROQUEST_LICENSE.txt
45577290cf638eca545618af68341b1d
https://thescholarship.ecu.edu/bitstream/10342/9124/4/ECUThesisSignaturePage.pdf
c6259a6b1f6f13a66162849b0599c276
https://thescholarship.ecu.edu/bitstream/10342/9124/5/Vireo-NonExclusive-Distribution_License_Edited-Verio-Version_L0615-2.pdf
67ecc0b8fab6c2953d9c894c77929d3a
https://thescholarship.ecu.edu/bitstream/10342/9124/7/GADDY-MASTERSTHESIS-2021.pdf.txt
59b7420af2bbb4b22a66acc6e2a55f7d
Differentiation
Dedifferentiation
Tumorigenesis
MPK-1
PUF-8
Germline
Resveratrol
Spermatocytes
Caenorhabditis elegans
Animals
Caenorhabditis elegans Proteins
oai:TheScholarship.intra.ecu.edu:10342/44072022-11-28T15:38:28Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Use of monoamine oxidase and redox enzymes in atrial tissue as novel predictors of postoperative atrial fibrillation
Darden, Timothy M.
Anderson, Ethan
Postoperative atrial fibrillation (POAF) occurs in approximately 30% of cardiac surgery patients. The complication occurs despite advances in surgical procedures. It is associated with increased mortality, morbidity, and healthcare cost. The exact pathogenesis of this complication is unknown, but oxidative stress and inflammation are considered to be significant factors.   A precisely controlled balance between reactive oxygen species (ROS) generation and ROS scavenging influence the oxidative environment within cells and tissues. Increased activity of monoamine oxidase (MAO) activity, a ROS generating enzyme, can produce a more oxidative environment. A decrease in glutathione (GSH), a ROS scavenging molecule, can also yield an oxidative environment. The enzymes GSH-peroxidase (GPx) and GSH-reductase (GR) are responsible for maintaining the cellular redox environment within a range that is compatible with favorable homeostasis. Despite the well-characterized role that these enzymes play in maintaining redox environment, a comprehensive evaluation of these enzymes in human myocardium has never been attempted.  Furthermore, since reports have recently documented the association between oxidative stress in atrial tissue and the incidence of POAF, we tested the hypothesis that these enzymes are associated with POAF, in a cohort of patients undergoing cardiac surgery at Vidant Medical Center, East Carolina Heart Institute (ECHI).  Human right atrial appendage tissue were obtained from 244 patients undergoing elective coronary artery bypass graft surgery at ECHI between January, 2010, and December, 2012. The generation of ROS were determined using assays on MAO, NADPH oxidase (NOX), along with the activity of glutathione reductase (GR) and glutathione peroxidase (GPx). Patient outcomes, including POAF development, were analyzed in relation to the assays performed. A statistical model was then created to measure the association for risk of POAF development.   This was the first study to determine that MAO activity is a major source of ROS in human atrial tissue. MAO activity is significantly associated with POAF. Total glutathione (GSHt) activity is inversely related to POAF development. GPx is also significantly associated with POAF as well, but the trend is not a linear. GR activity is not correlated with POAF.   MAO, GSHt and GPx are enzymes that contribute to the atrial oxidative environment and increased risk of POAF development. Because this is a pilot study, further exploration is needed to validate our study and to determine if and where these enzymes fit in the etiology of POAF. Â
East Carolina University
2014
Master's Thesis
http://hdl.handle.net/10342/4407
https://thescholarship.ecu.edu/bitstream/10342/4407/3/Darden.pdf
dbb780ef5fb2e4c7c3436dbdd616d404
https://thescholarship.ecu.edu/bitstream/10342/4407/1/Darden_ecu_0600O_11170.pdf
648fbf6bfc49b84a803f3a1cff4e313e
https://thescholarship.ecu.edu/bitstream/10342/4407/2/Darden_ecu_0600O_11170.pdf.txt
73b6d305af669a13d6d6e7b62dcaccc4
Health sciences
Pharmacology
Surgery
Cardiopulmonary bypass
Catecholamines
Monoamine oxidase
Oxadative stress
Postoperative atrial fibrillation
Redox
Atrial Fibrillation--metabolism
Atrial Fibrillation--prevention & control
Postoperative Complications--prevention & control
Monoamine Oxidase--metabolism
oai:TheScholarship.intra.ecu.edu:10342/91352022-12-13T18:17:23Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
The Circadian Rhythm and its Role in the Dynamic Dopamine Neuron Phenotype
Barker, Samantha
Eells, Jeffrey B.
The circadian rhythm is strongly implicated in many neuropsychiatric and neurodegenerative disorders, all of which are associated with altered dopamine (DA) neurotransmission in the substantia nigra and ventral tegmental area. Progress has been made in elucidating the circadian rhythm-dopaminergic network and its role in the onset of neuropsychiatric and neurodegenerative disorders. Previous research suggests that circadian rhythm transcription factors are responsible for directly regulating the DA phenotype; however, it is currently unknown what the relationship between the circadian rhythm and dopaminergic genes looks like with respect to age and time of day. Using a transgenic mouse model with Cre recombinase expression under control of dopamine transporter (DATCre) and yellow fluorescent protein (YFP) Cre reporter and immunohistochemistry techniques, we are able to characterize sub-populations of DA neurons in the ventral midbrain (VMB) that are responsive to the circadian rhythm. Here, we demonstrate a dynamic DA neuron phenotype, where classic dopaminergic markers, such as dopamine transporter (DAT) and tyrosine hydroxylase (TH) are not detected in dopaminergic neurons, due to regulation by the circadian rhythm. In this study, mice transgenic for DATCre/YFP were analyzed at postnatal day 0 (P0), P21, P35 and adulthood (>P60). Each time point included mice taken at subjective dawn (circadian time 0) and subjective dusk (CT12), excluding P0 mice. Results revealed that between P21 and P35, there was a significant loss of the DA neuron phenotype at CT12, as compared to CT0. There was no statistical difference between P35 and adults at CT0 or CT12. This suggests that between P21 and P35, DA neurons begin to transition to a 'former' phenotype throughout the circadian rhythm. Additionally, qRT-PCR data revealed abnormal dopaminergic gene mRNA levels at P21. Elucidating the molecular characteristics of these DA neurons is crucial to understanding the biological mechanisms behind the dopaminergic-circadian rhythm network, which will have future implications in understanding neuropsychiatric and neurodegenerative disorders.
East Carolina University
2021-05-04
Master's Thesis
en
http://hdl.handle.net/10342/9135
https://thescholarship.ecu.edu/bitstream/10342/9135/1/BARKER-MASTERSTHESIS-2021.pdf
54278c4d5bdb037db0848aa336594343
https://thescholarship.ecu.edu/bitstream/10342/9135/2/LICENSE.txt
85fc3b2cd2d595d32892ed112bc5a60f
https://thescholarship.ecu.edu/bitstream/10342/9135/3/PROQUEST_LICENSE.txt
2f0179ce9fd4c0b521f26124654f09a2
https://thescholarship.ecu.edu/bitstream/10342/9135/4/SBarker_Thesis%20Signiture%20Page.pdf
f8b038f509889a08dbfe3ea9840e63b0
https://thescholarship.ecu.edu/bitstream/10342/9135/5/SBarker_Thesis%20NEDL.pdf
16502429b92be1a9926952d0dbc3d73b
https://thescholarship.ecu.edu/bitstream/10342/9135/7/BARKER-MASTERSTHESIS-2021.pdf.txt
c6572d7def3a9a135c667a8185887e63
Neurobiology
Circadian Rhythm
Dopaminergic Neurons
Phenotype
Retina
oai:TheScholarship.intra.ecu.edu:10342/42472021-03-03T20:56:06Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Using TIRF microscopy to analyze stimulated and basal state B-cell MHC II clustering in response to ageing and dietary fish oil
Melton, Mark T.
Shaikh, Saame Raza
This research focused on developing an efficient TIRF microscopy approach to evaluate membrane protein organization. More specifically, the data demonstrate that TIRF microscopy can detect changes in ex vivo B-cell MHC II lateral organization using a monoclonal antibody under differing conditions. MHC II clustering is dependent on the underlying lipid environment and upon LPS stimulation MHC II expression is dramatically increased. Using mice fed a fish oil or control diet for three weeks, or using mice aged for nine months, we imaged splenic B-cell MHC II clustering using TIRF microscopy. We also used LPS to stimulate B-cells from both experimental conditions to determine if either ageing the animals or feeding them fish oil could affect MHC II clustering. We then determined cluster quantity, size, and intensity using the NIH ImageJ software. The data showed that neither a relevant dose of fish oil, nor aging the mice approximately nine months, had an affect on MHC II clustering in the absence of LPS stimulation. However upon LPS stimulation, MHC II clustering dramatically changed in aged mice as well as fish oil fed mice compared to control animals. Taken together, the data establish the TIRF microscopy protocol as a relevant alternative to more costly and time consuming approaches to address membrane protein clustering. Moreover, either ageing the animals or feeding them fish oil significantly affects MHC II clustering upon stimulation with LPS. Â
East Carolina University
2013
Master's Thesis
http://hdl.handle.net/10342/4247
https://thescholarship.ecu.edu/bitstream/10342/4247/2/melton.pdf
08e18500464278dacfe879da19948497
https://thescholarship.ecu.edu/bitstream/10342/4247/3/Melton_ecu_0600M_11039.pdf.txt
86d3418edcb737c7124828145befcf56
https://thescholarship.ecu.edu/bitstream/10342/4247/1/Melton_ecu_0600M_11039.pdf
491493ec120e67d569ff137b26e6014c
Chemistry, Biochemistry
Immunology
B cells
Lipid rafts
MHC class II
Plasma membrane
Protein clustering
TIRF
Biochemistry
Microscopy--methods
Membrane Microdomains
Cell Membrane Structures
Biochemical Phenomena
oai:TheScholarship.intra.ecu.edu:10342/74732022-12-12T18:51:05Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
30-Day Immunotoxicity Study of PFMOAA in C57BL/6 Mice
Vance, Samuel
DeWitt, Jamie C.
Within the past five years, two classes of per- and polyfluoroalkyl substances (PFAS) were phased out of production in the U.S., which led to the development and production of PFAS to replace these two major classes. One family of these PFAS are perfluoro-ether carboxylic acids (PFECA), which have emerged in the public and scientific arenas due to their presence in drinking water systems across the U.S., including Wilmington, NC. Although manufacturers have touted them as having more favorable environmental and toxicological properties very little is known about the toxicity and environmental fate these emerging PFECA. One compound, perfluoro-2-methoxyacetic acid (PFMOAA), was identified as the dominant PFECA in the Cape Fear River, in concentrations as high as 35,000 ng/L. There is very little mention of PFMOAA in the publicly available scientific literature and to our knowledge, we are the first to investigate its potential for toxic effects. In this 30-day study, we orally administered 25,000, 2,500,000, or 250,000,000 ng/L of PFMOAA in water to male and female C57BL/6 mice and investigated immune and liver alterations following exposure. Mice given PFMOAA showed no signs of overt toxicity during the study and no evident changes were observed in liver mass or peroxisomal enzyme activity. While mild alterations in splenic and thymic lymphocyte sub-populations were observed in males, these results do not point to any definitive alterations in immune function. Ultimately, we concluded that the doses administered were too low to achieve an internal dose sufficient to induce changes to immune endpoints, likely due to rapid excretion of PFMOAA in mice. Further investigation into serum and organ concentrations of PFMOAA as well its effects on antibody production will be more conclusive of immunotoxic effects.
East Carolina University
2019-07-18
Master's Thesis
en
http://hdl.handle.net/10342/7473
https://thescholarship.ecu.edu/bitstream/10342/7473/1/VANCE-MASTERSTHESIS-2019.pdf
48265cfcd282c16dbf4896f0f97d173d
https://thescholarship.ecu.edu/bitstream/10342/7473/2/LICENSE.txt
9a65d290b845fc4c88d46d008f169922
https://thescholarship.ecu.edu/bitstream/10342/7473/3/PROQUEST_LICENSE.txt
4fd0c626e20c595311bb19755219619a
https://thescholarship.ecu.edu/bitstream/10342/7473/4/SUNCW20519071110490.pdf
e8b9348dd7c1724d36e1a1cfa56c7cb4
https://thescholarship.ecu.edu/bitstream/10342/7473/5/liscensepfmoaa.pdf
5b83118da5ec66d3b84e7e64469b0f67
https://thescholarship.ecu.edu/bitstream/10342/7473/7/VANCE-MASTERSTHESIS-2019.pdf.txt
1359fb786cf580a7a7b8763a88aa3131
PFAS
PFMOAA
PFOA
Environmental Pollutants
Mice
Mice, Inbred C57BL
oai:TheScholarship.intra.ecu.edu:10342/63932021-03-03T21:15:19Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Structural and functional analysis of the Vaccinia virus O1 virulence protein
Weeks, Anastasia C.
Mannie, Mark D.
Poxviruses are double-stranded DNA viruses capable of causing disfiguring and deadly disease in a wide range of hosts, from insects to mammals. Orthopoxviruses (OPXV) encode many proteins that are not essential for viral replication, but are responsible for vast differences in pathogenesis. Of the>200 proteins in the prototypical OPXV vaccinia virus (VACV), many remain functionally cryptic. The objective of these studies was to understand how the VACV O1 protein functions by investigating cell-specific effects that may contribute to virulence. The O1L gene is expressed early as the O1 protein, a 78 kDa protein that lacked N-linked glycosylation. These data are the first to demonstrate the reduced ability of an O1 deletion mutant ([delta]O1) to induce cell migration compared to the parental VACV Western Reserve strain (VACV-WR). [delta]O1-infected cell monolayers also exhibited reduced plaque diameter and clearance in plaque foci. These observations indicated that O1 is a significant contributor to VACV cytopathic effects (CPE) in vitro, in agreement with published reports. The results reported herein are the first to describe an altered immunological response with [delta]O1, as levels of anti-VACV immunoglobulin significantly increased with [delta]O1 infection at a time point (seven days post-infection) when VACV-WR induced VACV-specific antibody levels were comparable to sera from mock-infected mice. [delta]O1 was more immunogenic in an ex vivo antigen presentation assay, although mitogen-induced CD4+ T cell activation during [delta]O1 infection was equivalent to VACV-WR infection. Surprisingly, of all the immune cell types tested, [delta]O1 significantly differed from VACV-WR infection in the metabolic readout of only one cell type - RAW 264.7 macrophages. VACV-WR infected RAW 264.7 macrophages were more metabolically active than [delta]O1-infected cells at higher infectious doses, which may be indicative of a specialized niche for O1 function. Taken together, these data may provide clues into the mechanism of O1 virulence.
East Carolina University
2017-07-26
Master's Thesis
en
http://hdl.handle.net/10342/6393
https://thescholarship.ecu.edu/bitstream/10342/6393/1/WEEKS-MASTERSTHESIS-2017.pdf
2294fd7086527ad59f04b30f612a354a
https://thescholarship.ecu.edu/bitstream/10342/6393/2/LICENSE.txt
2abd76b8122cb1b45f21ad6c1221026a
https://thescholarship.ecu.edu/bitstream/10342/6393/3/PROQUEST_LICENSE.txt
8f0723c351fa5676b1af12c6910dc586
https://thescholarship.ecu.edu/bitstream/10342/6393/4/Anastasia%20Weeks066.pdf
43655ae444b84468f91828352a494720
https://thescholarship.ecu.edu/bitstream/10342/6393/5/AWeeks%20Nonexclusive%20License.pdf
0e7e1cac495e24708cf42bf6e4429203
https://thescholarship.ecu.edu/bitstream/10342/6393/6/AWeeks%20Nonexclusive%20License_title%20edit.pdf
3b8ab517f5f21616ac465fcd6e745f1b
https://thescholarship.ecu.edu/bitstream/10342/6393/7/AWeeks%20Nonexclusive%20License_title%20edit_1.pdf
3b8ab517f5f21616ac465fcd6e745f1b
https://thescholarship.ecu.edu/bitstream/10342/6393/9/WEEKS-MASTERSTHESIS-2017.pdf.txt
e74b7313d6e20e1258993ce78de49419
poxvirus
O1L
mutant
immune
attenuated
plaque
morphology
cell
migration
Virulence
Viral Proteins
Vaccinia virus
oai:TheScholarship.intra.ecu.edu:10342/43282023-05-31T13:03:32Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
The contribution of motility and chemotaxis in the Borrelia burgdorferi infectious life cycle
Yerke, Aaron
Motaleb, M. D.
Lyme disease has emerged as an increasing problem for people in the east and northeastern part of the United States. It can cause a chronic debilitating infection if left untreated and is difficult to diagnose. The illness is caused by an infection with the spirochete known as Borrelia burgdorferi. B. burgdorferi is a Gram-negative bacterium that is transmitted by ticks of the Ixodes genus. The primary carriers in regions of high Lyme disease incidence are Ixodes scapularis vector and white-footed Mus musculus rodents. B. burgdorferi is not known to produce common virulence factors such as toxins or capsules. Chemotaxis and motility are important for B. burgdorferi to cause infection and are considered as invasive attributes of this organism. Only a handful of studies have reported that non-chemotactic and non-motile B. burgdorferi mutants are unable to disseminate in hosts, and are, therefore, non-infectious in mice. Although motility and chemotaxis has been shown to be crucial for the infectious life cycle of B. burgdorferi, little is known about the mechanism of motility or assembly of periplasmic flagella. It is not known how incremental reductions of motility affect B. burgdorferi's virulence. Recent cryo-electron microscopy tomography revealed that spirochetes possess a unique flagellar structure called the collar. However, the gene or genes that encode for the B. burgdorferi collar proteins are unknown. Because of its location in the periplasmic flagellar motor, we hypothesize that this organelle is important for flagellar assembly as well as motility. We also hypothesize that less motile or less chemotactic mutants will exhibit a reduced invasive phenotype in vivo. Accordingly, using various comprehensive approaches, we have identified several putative genes in the B. burgdorferi genome with no significant similarity to other bacterial species. In vitro gene mutant analyses indicate that the cells are motility deficient. Additionally, fliZ putatively encodes a regulator of the flagella assembly complex. In other bacteria, inactivation of fliZ creates a reduced motile phenotype. To demonstrate if reduced motility is important for survivability or transmission between hosts, we plan to assay these mutants in mouse-tick-mouse infection cycle experiments. Importantly, mutants deficient in chemotaxis response regulator cheY2 exhibit normal motility and chemotaxis in vitro but exhibit reduced virulence in mice. Specifically, the cheY2 mutants were significantly attenuated in mouse infection and dissemination to distant tissues after needle inoculation. Additionally, while ΔcheY2 mutant cells can survive normally in the Ixodes ticks, mice fed upon by the ΔcheY2-infected ticks failed to establish persistent infection. These data suggest that CheY2, despite resembling a typical chemotaxis response regulator, functions distinctively from most other CheY proteins. These data lead us to propose that CheY2 serves as a regulator for a virulence determinant that is required for productive infection within murine, but not Ixodes tick hosts.
East Carolina University
2013
Master's Thesis
http://hdl.handle.net/10342/4328
https://thescholarship.ecu.edu/bitstream/10342/4328/3/Yerke_ecu_0600M_11053.pdf.txt
51056399e9bd13af5607a69a7431c938
https://thescholarship.ecu.edu/bitstream/10342/4328/1/Yerke_ecu_0600M_11053.pdf
27a9c98cda552c81aca9e161590f8217
https://thescholarship.ecu.edu/bitstream/10342/4328/5/Yerke%2c%20Aaron%20Matthew.pdf
e712259a829aeebf4a41ca087a29e17c
https://thescholarship.ecu.edu/bitstream/10342/4328/7/Re_%20Available%20Thesis.pdf
cf74516983c5f96130be469309eab3c1
2024-05-16
Biology, Molecular
Borrelia
Burgdorferi
Chemotaxis
Collar
Flagella
Motility
Molecular biology
Lyme Disease
Ticks
Ixodes
Tick-Borne Diseases
Erythema Chronicum Migrans
Borrelia burgdorferi--pathogenicity
Chemotaxis
Mice
Treponema pallidum
Gram-Negative Anaerobic Bacteria
Gram-Negative Bacterial Infections
Borrelia Infections
oai:TheScholarship.intra.ecu.edu:10342/46632022-12-09T16:06:56Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Fesselin, an intrinsically disordered smooth muscle protein, organizes and stabilizes actin-myosin and myosin
Kingsbury, Nathaniel
Chalovich, Joseph
Fesselin is an intrinsically disordered protein that is known to bind a large variety of cytoskeletal proteins. The proteins fesselin is known to bind include: actin (Leinweber et al. 1999), [alpha]-actinin (Pham et al. 2006), calmodulin (Schroeter et al. 2004), filamin (Weins et al. 2001), and smooth myosin (Schroeter et al. 2005). The binding of fessilin to smooth myosin is of particular interest because unphosphorylated smooth muscle myosin filaments are unstable in the presence of ATP (Trybus et al. 1982, Ikebe et al. 1983, Suzuki et al. 1978). However, in smooth muscle cells unphosphorylated myosin filaments are maintained (Milton et al. 2011). Several proteins have been identified that stabilize myosin filaments and actin-myosin filament interactions. Our experiments show that fesselin may be one such protein. The organization of F-actin and myosin filaments by fesselin was observed by monitoring the rate of dissociation of actin-myosin by ATP in a stopped-flow device. Actin-myosin dissociation was measured by light scattering (a measure of particle size) and by pyrene-actin or acrylodan-tropomyosin fluorescence (a measure of myosin-actin bond breaking). The stopped-flow studies were further supported with electron microscopy analysis. These experiments showed that fesselin was able to tether actin and myosin filaments together without significantly impacting the rate of the actin-myosin bond breaking. The stabilization of myosin filaments by fesselin was tested using a similar method. First, stopped-flow rapid kinetics were used to measure the rate of ATP induced myosin filament break down. Next, electron microscopy was used to support the stopped-flow data and observe the effects of fesselin on myosin filament organization. Through the stopped-flow and electron microscopy experiments it was found that fesselin stabilizes myosin filaments. The electron microscopy experiments further revealed that fesselin enhanced myosin filament size and organized them into bundles. Previously published results showed that the interaction between fesselin and actin is regulated by calmodulin (Schroeter et al. 2004). The final set of experiments presented here examined the possibility that calmodulin regulates the interactions between fessilin and myosin. The calmodulin regulation of fesselin myosin interactions would greatly expand the role that fesselin has within smooth muscle by placing it within the calcium signaling pathway. The regulation of fesselin-myosin interactions by calmodulin was tested using pyrene labeled actin as well as N,N'-Dimethyl-N-(Iodoacetyl)-N'-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Ethylenediamine labeled fesselin (IANBD-fesselin). These experiments showed that the interactions between fesselin and myosin are regulated by calmodulin. Overall, our results support that fesselin plays a critical role within smooth muscle to organize contractile elements.
East Carolina University
2014
Master's Thesis
http://hdl.handle.net/10342/4663
https://thescholarship.ecu.edu/bitstream/10342/4663/1/Kingsbury_ecu_0600O_11348.pdf
032b696c8fe3c15eafe5113f6b36293f
https://thescholarship.ecu.edu/bitstream/10342/4663/2/Kingsbury_NEDL.pdf
ac73b3640db19090d83618c88c720b99
https://thescholarship.ecu.edu/bitstream/10342/4663/3/Kingsbury_ecu_0600O_11348.pdf.txt
5819b2e16ebd472bdc5b14cba48bae83
Biochemistry
Molecular biology
Actin
Fesselin
Myosin
Rapid kinetics
Synaptopodin-2
Smooth Muscle Myosins--metabolism
Cytoskeletal Proteins--metabolism
Actins--metabolism
Adenosine Triphosphatases--metabolism
Membrane Proteins--physiology
Myosins--metabolism
oai:TheScholarship.intra.ecu.edu:10342/70162021-03-03T21:19:48Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Identification of Biomarkers in Non-Small Cell Lung Cancer Patients Treated with PD-1 Monoclonal Antibody Immunotherapy
Atwell, Druid Carlisle
Yang, Li V
Cancer immunotherapy works by taking a patient's existing immune system and priming it to recognize cancer cells in order for immune cells to mount an effective response to the disease. This is a less invasive means of treating cancer for the patient. However current immunotherapy does come with its own unique side effects such as auto immune disorders that manifest in the patients' treatment due to the blocking of essential immune regulatory checkpoints. In this study, patients are treated with drugs nivolumab and pembrolizumab, both of which are PD-1 (Programmed Death Receptor 1) monoclonal antibodies. These antibodies bind to PD-1 and prevent ligand interaction with PD-L1. PD-1 is a receptor expressed on the surface of activated B-cells, macrophages and T-cells. When PD-1 is activated by PD-L1 a signal propagates from the receptor to inside the cell that results in the apoptosis of the cell that expresses PD-1. The activation of PD-1 on activated T-cells ultimately results in a reduction of T-cell proliferation and IFN-[gamma] secretion. An apoptotic signal occurs through the inhibition of the cell survival signal that is propagated through the PI3K pathway. While there is knowledge on how the expression and activation of PD-1 on immune cells regulates the progression of cancer, there is a lack of evidence to suggest biomarkers in non-small cell lung cancer patients for optimizing immunotherapy. This study serves to identify biomarkers in non-small cell lung cancer patients undergoing PD-1 monoclonal antibody immunotherapy. To accomplish this, blood samples were collected from non-small cell lung cancer patients undergoing the immunotherapy treatment and the cell counts were taken. Cell types of interest include cytotoxic T-cells, helper T-cells, B-cells, and granulocytes. Cytotoxic T-cells were identified by CD8 expression, a known marker of cytotoxic T-cells. Helper T-cells were identified by CD4 expression and B-cells were identified by CD19 expression, both of which are known markers of helper T-cells and B-cells, respectively. Secondly, this study investigated the expression levels of known immune regulatory genes and how these changed over the course of the immunotherapy treatment. Known immune regulatory genes included PD-L1, PD-1, CTLA4, CD28, A2A, CD80 and CD86. The expression levels of the proton sensing family of G-protein coupled receptors (G2A, GPR4, OGR1 and TDAG8) were also investigated. Thirdly, we investigated how tumor cell expression of PD-1 and PD-L1 was altered when introduced into an acidic environment. Due to the tumor microenvironment being characteristically acidic this would provide insight on how anti PD-1 and anti PD-L1 immunotherapies could potentially be used in various cancers and may also lead to the development of potential future combination therapies. Our study shows that approximately 90% of patients exhibited an increase in cytotoxic T-cell counts with 50% of patients achieving healthy donor cytotoxic T-cell counts after receiving immunotherapy. Additionally 2 patients out of the total 16 patients achieved and sustained cytotoxic T-cell counts above that of healthy donors. There was an observable trend that indicated a possible correlation that PD-1 levels at baseline could predict patient response to the PD-1 monoclonal antibody immunotherapy. In addition to our research into the clinical aspects of PD-1 monoclonal antibody immunotherapy, we also investigated the change in expression of PD-1 and PD-L1 mRNA in several cancer cell lines. We observed that there was a variation in how cancer cells responded to acidosis. PD-1 and PD-L1 mRNA expression was shown to be regulated through several variables such as the acidity of the media, duration of exposure to acidic conditions and cancer cell type. It was also observed that there was PD-1 and PD-L1 expression in these cancer cell lines, at 5 hour and 24 hour treatment times, with a prominent level of PD-L1 mRNA expression in most of these cancer cells.
East Carolina University
2018-08-22
Master's Thesis
en
http://hdl.handle.net/10342/7016
https://thescholarship.ecu.edu/bitstream/10342/7016/1/ATWELL-MASTERSTHESIS-2018.pdf
8df50c2bfdb03ff828617a66f11f7484
https://thescholarship.ecu.edu/bitstream/10342/7016/2/PROQUEST_LICENSE.txt
dcd77021d7d25508ea4bf44e085f1b60
https://thescholarship.ecu.edu/bitstream/10342/7016/3/LICENSE.txt
4fa84c5a807ba44258fd43ef51faaf50
https://thescholarship.ecu.edu/bitstream/10342/7016/4/thesis%20signature%20page%208.9.18_1.jpg
5d58c826b6a5dba7c1e1bd8a96738a1a
https://thescholarship.ecu.edu/bitstream/10342/7016/5/thesis%20signature%20page%208.9.18.jpg
5d58c826b6a5dba7c1e1bd8a96738a1a
https://thescholarship.ecu.edu/bitstream/10342/7016/6/Thesis%20Defense%20Signature%20page%20CCI07302018.jpg
e1de49e9de53523150be21ae9255a6de
https://thescholarship.ecu.edu/bitstream/10342/7016/7/ECU%20Non-Exclusive%20Distribution%20License.pdf
cde49298c080b772d83efaad866412ce
https://thescholarship.ecu.edu/bitstream/10342/7016/9/ATWELL-MASTERSTHESIS-2018.pdf.txt
a84c27df15c882b7a35529b2bea42b0c
PD-1
PD-L1
Antibodies, Monoclonal
Carcinoma, Non-Small-Cell Lung
Lung Neoplasms
Immunotherapy
Humans
oai:TheScholarship.intra.ecu.edu:10342/76252022-12-05T17:48:02Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Matrix Metalloproteinase 12 is Critical for Granuloma Formation in the Murine Model of Granulomatous disease
Neequaye, Nicole N
Mohan, Arjun
Matrix Metalloproteinase 12 (MMP12) is a protein produced primarily by alveolar macrophages that degrades elastin in the extracellular matrix (ECM) and enables infiltration of immune cells that participate in the inflammatory response. To our knowledge, few studies have been conducted to clarify the role of MMP12 in granulomatous diseases such as sarcoidosis, a chronic inflammatory disease characterized by granuloma formation primarily in the lungs. Previous studies have shown an increase in gene and protein expression of MMP12 in lung tissue and bronchoalveolar lavage (BAL) of patients with sarcoidosis as well as a correlation between MMP12 elevation and disease severity. Our murine model uses multiwall carbon nanotubes (MWCNT) to mimic the characteristics (gene, protein expression, and granuloma formation) observed in sarcoidosis patients. Based on these observations, we hypothesized that MMP12 plays a role in the acute and late inflammatory response in pulmonary sarcoidosis. MMP12KO mice were used to address this hypothesis. Analysis of gene expression of BAL cells in C57BL/6 (wildtype) mice shows a significant elevation in MMP12 after oropharyngeal instillation of MWCNT at all time points (3, 10, 20, 60, 90 days). We observed similar trends in proinflammatory genes chemokine (C-C motif) ligand 2 (CCL2), matrix metalloproteinase 14 (MMP14), and interferon-gamma (IFNIÌ‚Ä‘) at all time points and osteopontin (OPN) at 20, 60, and 90 days. MMP12 protein levels increased in BAL cells at all time points. Evaluation of BAL cells from MMP12KO mice shows a similarity in the expression of all proinflammatory genes explored with wildtype at 10 days. CCL2 and MMP14, identified through gene expression profiling of the wildtype to be directly regulated by MMP12, is significantly reduced at 60 days in MMP12KO MWCNT instilled mice compared to wildtype. Histological analyses at 3, 10, 20, and 60 days shows increasing exacerbation in wildtype and continuous attenuation of granuloma formation in MMP12KO mice after exposure to MWCNT. A proposed mechanism for the reduction of granulomas at 60 days in MMP12KO, lead to an investigation into the relationship between MMP12, a pro-inflammatory mediator and PPAR[gamma], an anti-inflammatory modulator. Gene analysis showed a significant increase in MMP12 in PPAR[gamma]KO mice compared to wildtype and a substantial rise in PPARIÌ‚Ä‘ in MMP12KO mice compared to wildtype. Interestingly, MMP12 significantly increased, and PPAR[gamma] decreased dramatically in African American sarcoid patient's vs. controls when adjusted for race. MMP12 is seemingly instrumental in driving granuloma pathogenesis during inflammation. Evaluation of genes in MMP12KO mice suggests that the macrophage-secreted cytokines and matrix genes explored are necessary for granuloma formation. The significant increase in PPAR[gamma] intrinsically and after instillation with MWCNT in MMP12KO and its' decrease in wildtype mice after MWCNT instillation at 60 days suggests an inverse relationship between MMP12 and PPAR[gamma]. A reduction in granuloma formation in MMP12KO mice compared to wildtype supports a critical role for MMP12 in granuloma formation.
East Carolina University
2019-08-16
Master's Thesis
en
http://hdl.handle.net/10342/7625
https://thescholarship.ecu.edu/bitstream/10342/7625/1/NEEQUAYE-MASTERSTHESIS-2019.pdf
1e4c1a0f4cec43af1b9f4d9a566ed093
https://thescholarship.ecu.edu/bitstream/10342/7625/2/LICENSE.txt
c0184cee0c2d81b46385323a935dac4e
https://thescholarship.ecu.edu/bitstream/10342/7625/3/PROQUEST_LICENSE.txt
9e022dbe6b35e8be9c8bd9c51d15d555
https://thescholarship.ecu.edu/bitstream/10342/7625/4/scanned%20and%20signed%20signature%20page.pdf
7f2cced7e9ed3ffc04cb0fae52eeb307
https://thescholarship.ecu.edu/bitstream/10342/7625/5/corrected%20signature%20page.pdf
ba5482002a1f591cc8a20f9680095214
https://thescholarship.ecu.edu/bitstream/10342/7625/6/corrected%20signature%20page_1.pdf
ba5482002a1f591cc8a20f9680095214
https://thescholarship.ecu.edu/bitstream/10342/7625/7/Distribution%20License.pdf
6cbaeaf7b982959a4c3029d2a14d17c1
https://thescholarship.ecu.edu/bitstream/10342/7625/9/NEEQUAYE-MASTERSTHESIS-2019.pdf.txt
50a0c4f76716fda1005f29895bfe9924
sarcoidosis
MMP12
granulomas
inflammation
osteopontin
PPARg
Granuloma
Matrix Metalloproteinase 12
Mice
Disease Models, Animal
oai:TheScholarship.intra.ecu.edu:10342/75902022-12-14T17:29:05Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
DEVELOPMENT OF SMALL-MODELCULE INHIGITORS OF THE INTIATING PROTEASES, C1r AND C1s, OF THE CLASSICAL COMPLEMENT PATHWAY
Rohlik, Denise
Garcia, Brandon L.
Complement is a proteolytic cascade that upon activation plays a key effector role in the innate immune system and acts to prime the adaptive immune response. During normal homeostatic events, complement is tightly regulated for its roles in immune complex clearance, lysis of target cells, opsonization, and recruitment of leukocytes and monocytes to target areas. Several endogenous regulators are responsible for the control of complement activation, however when dysregulation occurs, aberrant complement activation has been linked to autoimmune, proinflammatory, and neurodegenerative diseases, including Alzheimer's disease. Inhibition of the classical complement component C1 may ameliorate hallmarks of autoimmune and inflammatory disease. The serine proteases within the C1 complex, C1r and C1s, are promising therapeutic targets for structure-based small-molecule drug development. We investigated the activity of a series of small-molecule compounds identified in a large-scale fragment library screen and those from a cheminformatics computational docking screen in which hit compounds were predicted to bind the C1r or C1s proteases. Using surface plasmon resonance and ELISA-based assays for hit validation, we analyzed the binding affinities and the inhibitory IC50's of several compounds predicted to bind and inhibit the activation of C1r or C1s in a dose-dependent manner. In this study, we have identified four lead compounds (cmp-1611, cmp-1663, cmp-1696, cmp-1827) and their 10 active structural analogues that target and inhibit C1r activation. Given their abilities to bind and inhibit C1r and favorable physicochemical properties, our lead compounds may provide a starting point for optimizing affinity and specificity necessary for developing novel routes of therapeutic upstream complement inhibition.
2019-12
Thesis
en_US
http://hdl.handle.net/10342/7590
https://thescholarship.ecu.edu/bitstream/10342/7590/3/ROHLIK-MASTERSTHESIS-2019.pdf
b88b910900c48c6aadda6e488b0136e6
https://thescholarship.ecu.edu/bitstream/10342/7590/2/license.txt
8a4605be74aa9ea9d79846c1fba20a33
https://thescholarship.ecu.edu/bitstream/10342/7590/4/Rohlik_Non-exclusive%20dist%20form_1.pdf
9d8aeed32be1642048a311865ec442c2
https://thescholarship.ecu.edu/bitstream/10342/7590/5/Rohlik_Thesis%20Committee%20Approval%20Form_2.pdf
658c223a586cc8ae4d1974ccd7ccacfe
https://thescholarship.ecu.edu/bitstream/10342/7590/6/ROHLIK-MASTERSTHESIS-2019.pdf.txt
bfe7a7a6b5e8c3ed34d4549fabed5b34
small-molecule inhibitor
C1s
Complement Pathway, Classical
Peptide Hydrolases
Endopeptidases
Complement C1r
oai:TheScholarship.intra.ecu.edu:10342/64642021-03-03T21:15:59Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
TGF-β Signaling, Via Smad3, Mediates Notch Pathway-Induced Stemness and Epithelial to Mesenchymal Transition in Colon Cancer Cells
Clark, Alexander G.
Sigounas, George
Late stage colorectal cancer (CRC) remains a challenging disease to treat due to several factors including stemness and epithelial to mesenchymal transition (EMT). Dysfunctional signaling pathways in CRC contribute to these phenomena, including the Notch and Transforming Growth Factor-Beta (TGF-[beta]) pathways. These pathways integrate external signals by cross-talking with one another to fine-tune cellular responses. We previously found that cells expressing constitutively active Notch1 also had increased expression of Smad3, an important member of the TGF-[beta] signaling pathway. Therefore, we hypothesized that the TGF-[beta] pathway, via Smad3, mediates the Notch-induced stemness and EMT observed in these cells. To test our hypothesis, we used the human colorectal carcinoma cell line HCT-116 and derivative cell lines GFP-v (a control cell line transfected with a retrovirus containing the green fluorescent protein), and ICN1 (a cell line transfected with a retrovirus containing both the green fluorescent protein and constitutively active intracytoplasmic Notch1). These cells were treated with different combinations of TGF-[beta]1 (a TGF-[beta] receptor ligand), DAPT (a [gamma]-secretase inhibitor), or SIS3 (a novel Smad3 inhibitor). Western blot procedures and statistical analysis were performed to determine the cross-talk between the Notch and TGF-[beta] pathways and to assess the effects of Smad3 stimulation and inhibition on Notch and its downstream targets. The role of Smad3 on colosphere formation was also determined using the aforementioned cell lines. Smad3 inhibition induced a decrease in Notch1 and Notch3 receptor expression. Additionally, SIS3 effectively inhibited key stemness and EMT markers such as CD44, Slug, Snail, and Hes1. Colosphere forming ability was also considerably reduced in cells treated with SIS3. These results indicate a key role of TGF-[beta] signaling in Notch1-induced tumorigenesis, and they also suggest a potential use for Smad3 inhibitors in combination with Notch1 inhibitors that are already in use for CRC treatments.
East Carolina University
2017-08-21
Master's Thesis
en
http://hdl.handle.net/10342/6464
https://thescholarship.ecu.edu/bitstream/10342/6464/1/CLARK-MASTERSTHESIS-2017.pdf
9711cf56d08d4702a029aa359becdb33
https://thescholarship.ecu.edu/bitstream/10342/6464/2/LICENSE.txt
d294917a595a7dedbff90a4bc37bf10c
https://thescholarship.ecu.edu/bitstream/10342/6464/3/PROQUEST_LICENSE.txt
df8be1449157f616703349a82a87a637
https://thescholarship.ecu.edu/bitstream/10342/6464/4/Alex%20Clark%20signature%20page.pdf
a996d612a664e25d25cb311c291d0636
https://thescholarship.ecu.edu/bitstream/10342/6464/5/Alex%20Clark%20NEDL%20-%20Sigounas%20permission.pdf
ef8caa26df3f6b7838fc1faebd76c151
https://thescholarship.ecu.edu/bitstream/10342/6464/7/CLARK-MASTERSTHESIS-2017.pdf.txt
91f36dcba9cd7fffbbb31dab9b63f5ee
Notch signaling
Colonic Neoplasms
Epithelial-Mesenchymal Transition
Smad3 Protein
Signal Transduction
Cell Count
oai:TheScholarship.intra.ecu.edu:10342/43272021-03-03T20:52:53Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Genetic and hypoxic effects on germline tumor development in caenorhabditis elegans
Datla, Udaya Sree
Lee, Myon-Hee
The process of differentiation of stem cells to committed, progenitor specific cell types is well studied but the reverse process of the dedifferentiation of these committed cells back to the undifferentiated state still remains a major challenge in stem cell biology. In my project, we studied some of the major regulators of the dedifferentiation process taking C. elegans germline model system. In C. elegans, the germline stem cells at the distal end that initially divide mitotically then enter meiosis and differentiate into gametes as they progress towards the proximal end. In the first part of my project, we proposed that the Ras-ERK MAPK signaling activated by the removal of two negative regulators, PUF-8 RNA-binding protein and LIP-1 dual specificity phosphatase, plays an important role in controlling the dedifferentiation of secondary spermatocytes at the proximal end to a more undifferentiated, multipotent state forming proximal germline tumor. Further, by RNAi screening, the RSKN-1/P90RSK, a downstream effector of MPK-1/ERK was identified to be critical for this (already published).   As a continuation of my project, next, the HIF-1 (Hypoxia inducible factor-1) transcription factor that plays a key role in oxygen homeostasis during hypoxic conditions for the survival of the organism has been studied for its pronounced effect on this dedifferentiation-mediated tumorigenesis process. We found that under hypoxic conditions or under the conditions that mimic hypoxia (by exposing the worms to cobalt chloride, a chemical inducer of hypoxia), the activation of HIF-1 inhibits dedifferentiation-mediated tumorigenesis in puf-8; lip-1 mutant germline, probably through Ras-ERK MAPK signaling. Besides focusing on the effect of hypoxia on dedifferentiation-mediated tumorigenesis, we also studied the effect of hypoxia on glp-1(ar202), also called glp-1(gf) gain-of-function mutant - used to study GLP-1/Notch-mediated tumorigenesis where the tumor development occurs via a different pathway where the meiotic entry of germline stem cells are inhibited and instead self-renews and in the process of self-renewal forms the tumor cell-like cells. Interestingly, the activation of HIF-1 by either genetic manipulation or cobalt chloride treatment also inhibited GLP-1/Notch-mediated tumorigenesis. It suggests that there might be a common mechanism underlying the action of HIF-1 in both dedifferentiation-mediated and GLP-1/Notch-mediated germline tumors. Â
East Carolina University
2013
Master's Thesis
http://hdl.handle.net/10342/4327
https://thescholarship.ecu.edu/bitstream/10342/4327/3/Datla_ecu_0600M_11056.pdf.txt
c072abcffba4858834bc802a5c1c9e11
https://thescholarship.ecu.edu/bitstream/10342/4327/1/Datla_ecu_0600M_11056.pdf
36e8a9dd3c8002336f9b4eb31b8ac100
https://thescholarship.ecu.edu/bitstream/10342/4327/5/Datla%2c%20Udaya%20Sree.pdf
215555200dae8a56fe669c9ea3469e13
Oncology
Biology, Cellular
Caenorhabditis elegans
Dedifferentiation
Hypoxia
MAPK signaling
PUF RNA binding protein
Tumorigenesis
Cellular biology
C. elegans
Caenorhabditis elegans--genetics
Caenorhabditis elegans--metabolism
Stem Cells
Germ Cells--cytology
Germ Cells--metabolism
Neoplasms--genetics
Neoplasms--metabolism
RNA-Binding Proteins--genetics
RNA-Binding Proteins--metabolism
Tumor Suppressor Proteins--genetics
Tumor Suppressor Proteins--metabolism
Hypoxia-Inducible Factor 1
HIF-1 protein, C elegans
Glucagon-Like Peptide 1
Cobalt
Carcinogenesis
Cell Dedifferentiation
Models, Animal
oai:TheScholarship.intra.ecu.edu:10342/50802021-03-03T20:58:52Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Pulmonary Pathologies Following Inhalation of Multi-Walled Carbon Nanotubes at Occupational Levels
Phipps, Marshal Anthony, Jr.
Walters, Dianne M.
Multi-walled carbon nanotubes (MWCNT) are a nanomaterial that is growing in use and popularity. The health effects of occupational pulmonary exposure to MWCNT are currently unknown. The goals of this study were to build a dust generator capable of producing occupational levels of MWNCT and to examine pulmonary effects of occupational levels of inhaled dry MWCNT. We designed, built, and tested a dust generator capable of producing MWCNT concentrations in the occupational exposure range (25,000 to 50,000 particles/cm3). In a time course study, C57BL/6J male mice were exposed to either a dust of MWCNT at a daily average of approximately 37,000 particles/cm3 and a daily peak of about 50,000 particles/cm3 or to air alone. Six mice per group were exposed for 4 hours per day for 5 days a week for 2 weeks and sacrificed 1, 3, 7, 10, 14, 28, and 84 days post-exposure. In a strain comparison study DBA/2J and A/J mice underwent the same exposure and were sacrificed at 14 days post-exposure. For both studies bronchoalverlavage (BAL) fluid was collected to assess cellular profile and protein content. The right lung was used for collagen measurement. The left lung was used for histological evaluation. Total protein, a measure of lung permeability, did not change significantly after MWCNT exposure compared to air controls at any time point. Likewise, eosinophil and neutrophil cell counts were not significantly different between air and MWCNT-exposed mice. However, compared to air controls, MWCNT-exposed mice had increased numbers of total cells and macrophages at 28 days post exposure and increased monocytes from 10 to 14 days exposure. The presence of MWCNT was visually noted in BAL cell pellets, histology slides, and lung homogenate membrane pellets at all of the time points. Dark field microscopy showed MWCNT in BAL cells at all of the time points in MWCNT exposed mice. Collagen levels were not different between exposure groups at any time point. The strain comparison found that C57BL/6J mice had increases in monocytes an lymphocytes at 14 days post exposure and A/J mice showed a trend towards an increase in lung protein levels in MWCNT exposed mice at 14 days post exposure. MWCNT were visually detected in lung homogenate membrane pellets and in the histology slides from all strains. Although short-term inhalational exposure to occupationally relevant levels of dry dusts of MWCNTs did not elicit significant increases in measures of lung injury or fibrogenesis, increases in mononuclear cells and lack of particle clearance may indicate an altered host-defense capacity which could lead to disease with further particle accumulation or subsequent pathogen exposure.
East Carolina University
1/13/16
Master's Thesis
http://hdl.handle.net/10342/5080
https://thescholarship.ecu.edu/bitstream/10342/5080/1/Phipps_ecu_0600O_11571.pdf
39577a18eba1756d15f776def6a0e1f9
https://thescholarship.ecu.edu/bitstream/10342/5080/2/Phipps_NEDL.pdf
74c8a130892955f9277c8448c55d984e
https://thescholarship.ecu.edu/bitstream/10342/5080/3/Phipps_ecu_0600O_11571.pdf.txt
d550d0d3dd6aa3787bee759f6b5e9141
Toxicology
Occupational safety
Physiology
MWCNT
Administration, Inhalation
Lung
Nanotubes, Carbon
oai:TheScholarship.intra.ecu.edu:10342/45652021-03-03T21:10:44Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Developmental lead exposure and the exacerbation of Alzheimer's
pathology: an immunological analysis
Vonderembse, Annalise Noelle
DeWitt, Jamie C.
Early neuroimmune dysfunction may play a driving role in the etiopathology of Alzheimer's disease (AD), stemming from the hypothesis that many late-stage adult diseases have an early-life basis. Here we explore whether exposure to a known neuroimmunotoxicant during a period of developmental susceptibility in the central nervous system (CNS) parenchyma exacerbates the pathologies in an AD prone triple transgenic mouse model (3xTgAD). This "double-hit" research design is optimal for modeling the high variability in AD due to detrimental exogenous influences, rather than the minority of AD cases that have a well-defined genetic origin and regular neurodegenerative progression. We gavaged triple transgenic mice with lead acetate from postnatal day 5-15, a critical developmental window for microglia, immune cells of the CNS that are thought to play a major role in shaping the CNS. We then analyzed microglial phenotype and colocalization with amyloid-beta, the protein associated with AD senile plaques, to appropriately detect the change in pathological severity due to the intimate correlation of microglia with amyloid-beta plaques. Our data indicate early exposure to a neurotoxicant increases the number of activated microglia with age, which we hypothesize is due to either degradation of homeostatic inhibitory signaling pathways associated with early onset synaptic degeneration or over-burdened microglial phagocytic load. Furthmore, microglial activation states differed between genders and fluctuated with age, suggesting a sex-specific component to AD pathology and potential correlation of neurodegenerative diseases with hormone receptors in the sexual differentiation of the developing brain. Â
East Carolina University
2014
Master's Thesis
http://hdl.handle.net/10342/4565
https://thescholarship.ecu.edu/bitstream/10342/4565/1/Vonderembse_ecu_0600O_11257.pdf
5682f79b25ffe6b950a26a0f1caceacf
https://thescholarship.ecu.edu/bitstream/10342/4565/2/Vonderembse_ecu_0600O_11257.pdf.txt
51c4975f0a9e74331d5fcacf549dc306
https://thescholarship.ecu.edu/bitstream/10342/4565/3/NEDL_Vonderembse.pdf
7970b7dc0b77d76c8aac39ebb2aaeecc
Toxicology
Neuroscience
Developmental biology
Alzheimer's disease
Developmental immunotoxicology
Microglia
Neurotoxicology
Biology, Neuroscience
Alzheimer Disease--pathology
Disease Progression
Amyloid beta-Protein Precursor--metabolism
Plaque, Amyloid--pathology
Lead--toxicity
oai:TheScholarship.intra.ecu.edu:10342/63982021-03-03T21:15:20Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Notch 3 Affects Chemoresistance in Colorectal Cancer via DNA Base Excision Repair Enzymes
Khan, Azeem
Sigounas, George
Approximately 1.2 million cases of colorectal cancer (CRC) arise each year, and 40-50% of CRC patients will reach metastasis. The Notch pathway is known to be dysregulated in CRC, and its relationship with DNA repair mechanisms, which contribute to drug resistance, is currently being established. Previously, we observed a decrease in DNA base excision repair (BER) enzymes and drug resistance upon Notch 1 targeting. We have also observed that Notch 1 signaling is associated with promoting cancer stemness and epithelial to mesenchymal transition in CRC via upregulation of the Notch 3 receptor. Thus, we hypothesized that targeting Notch 3 will increase drug sensitivity in CRC via signaling effects on proteins associated with the DNA BER mechanism. Methods: In order to assess our hypothesis, the colon cancer cell line HCT 116 was transduced with a small hairpin messenger RNA construct that effectively knocked down the Notch 3 receptor, creating the Sh-N3 cell line. Culturing and Western blot analyses were conducted using standard methodology. Drug resistance was analyzed by treating cells with cisplatin or cytarabine, potent DNA damaging agents, and cytotoxicity was assessed. Microscopy was used to confirm effects of the DNA damaging agents. Proteins from the Notch 3 receptor targeted cell line treated with the DNA damaging agents were also studied. Results: Notch 3 targeted (Sh-N3) cells resulted in 1.5-fold lower plating efficiency compared to their counterpart controls (p<0.01). Western blot analysis showed that Notch 3 targeting led to a decrease in poly (ADP-ribose) polymerase (PARP1) expression by 34% in comparison to the parental cell line control (p<0.05), while apurinic/apyrmidinic endonuclease (APE1) expression was decreased by 47% (p<0.05). Sh-N3 cells treated with 20 [micro]g/mL of cisplatin for 48 hours showed a 2-fold increase in cell death compared to the controls (p<0.001). Additionally, cytotoxicity in Notch 3 null cells treated with 0.64 [micro]g/mL of cytarabine at 48 hours displayed a 1.7-fold increase compared to the controls (p<0.001). Microscopic observations confirmed these cytotoxicity results. Conclusions: This study further reinforces the importance of Notch signaling in drug resistance, and highlights the potential use of Notch 3 inhibition in conjunction with DNA BER protein inhibitors to effectively target chemoresistant CRC cells.
East Carolina University
2017-07-28
Master's Thesis
en
http://hdl.handle.net/10342/6398
https://thescholarship.ecu.edu/bitstream/10342/6398/1/KHAN-MASTERSTHESIS-2017.pdf
ead467d9005041e87b44719219a2e4b3
https://thescholarship.ecu.edu/bitstream/10342/6398/2/LICENSE.txt
02430d7f90ab39a2b0e744ef2806a435
https://thescholarship.ecu.edu/bitstream/10342/6398/3/PROQUEST_LICENSE.txt
69059b45a19cbcd23f49977d617297ca
https://thescholarship.ecu.edu/bitstream/10342/6398/4/Committee%20Signature%20Form.docx
100bc333e1254b7d3a797059020d36dd
https://thescholarship.ecu.edu/bitstream/10342/6398/5/NEDL.docx
e7483e300cea586d3d8430145ab6ba00
https://thescholarship.ecu.edu/bitstream/10342/6398/7/KHAN-MASTERSTHESIS-2017.pdf.txt
86d345117c0c5a61575e16e1905c7357
Notch 3
PARP1
APE1
Notch Pathway
Colorectal Neoplasms
DNA Repair
oai:TheScholarship.intra.ecu.edu:10342/91122023-05-01T08:01:56Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Borrelia burgdorferi ErpB and ErpQ inhibit C1 complex of the classical pathway of complement through a novel mechanism
Garrigues, Ryan
Garcia, Brandon L.
The complement system is an organized proteolytic cascade of dozens of proteins that functions in the recognition, opsonization, and lysis of pathogenic and altered-host cells. Bloodborne pathogens like the etiologic agent of Lyme disease, Borrelia burgdorferi, encounter complement during their bloodmeal and in their dissemination through the body. Therefore, to avoid complement mediated destruction, these pathogens have developed mechanisms that aid in complement evasion and defense. The spirochete B. burgdorferi, has nearly a dozen known complement recruiting or inhibiting surface exposed lipoproteins. Here, we uncover a novel inhibitory mechanism for two surface exposed lipoproteins, ErpB and ErpQ, that were recently identified using a lipoprotein gain of function library. Using surface plasmon resonance, ErpB and ErpQ were found to bind C1 complex proteases C1r and C1s with high affinity. Gel-based biochemical assays showed that ErpB and ErpQ specifically inhibit C1s-mediated cleavage of both C2 and C4 making them the only known bacterial inhibitors of C1s. Furthermore, they were shown to block C1s outside of the active site indicating that they function by a novel mechanism. Additional site-directed mutagenesis of C1s exosites revealed determinants for high affinity inhibitor interactions that have been shown to be important for C1s recognition of C4 outside of the active site. The discovery of a novel mechanism of complement inhibition by a medically-relevant human pathogen expands our knowledge of host pathogens interactions and contributes to previously unknown pathophysiological immune evasion by B. burgdorferi. Mechanistic studies on ErpB and ErpQ also support further understanding of molecular interactions between complement proteases and their substrates, which provides alternative means for the development of specific complement therapeutics toward complement-mediated diseases.
East Carolina University
2021-05-03
Master's Thesis
en
http://hdl.handle.net/10342/9112
https://thescholarship.ecu.edu/bitstream/10342/9112/1/GARRIGUES-MASTERSTHESIS-2021.pdf
13d9e656b54d287337168064a6ba8e5d
https://thescholarship.ecu.edu/bitstream/10342/9112/2/LICENSE.txt
5e57835aaaa9e6540b9181895a181721
https://thescholarship.ecu.edu/bitstream/10342/9112/3/PROQUEST_LICENSE.txt
2b5c223ca76cb543db84b6735ccd83d8
https://thescholarship.ecu.edu/bitstream/10342/9112/4/ECUThesisSignaturePage.pdf
2ccef6552ad0314962feb051a9ce7612
https://thescholarship.ecu.edu/bitstream/10342/9112/5/Vireo-NonExclusive-Distribution_License_Edited-Verio-Version_L0615-2.pdf
255ef31393488646ee17d756da0f4999
https://thescholarship.ecu.edu/bitstream/10342/9112/7/GARRIGUES-MASTERSTHESIS-2021.pdf.txt
6c2c952639ab1d24607f0b29c18552a8
Complement
Protease inhibitors
Immune evasion
Borrelia burgdorferi
Lyme Disease
oai:TheScholarship.intra.ecu.edu:10342/49482021-03-03T20:57:55Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Role of Notch signaling in tumorigenesis, stemness, and epithelial to mesenchymal transtion in colorectal cancer.
Fender, Alexander W.
Sigounas, George
Colorectal cancer is the third most commonly diagnosed cancer in both men and women in the United States. Surgical resection and combination chemotherapy are often used for treatment, but in later stages of the disease, these therapies are often unsuccessful. Previous studies have revealed that circulating cancer cells with stem-cell like properties are associated with disease progression and metastatic potential. The Notch signaling pathway has been found to be critical for proliferation and proper functioning in the stem cell compartment of the colon. Preliminary studies from our lab have shown a marked increase in Notch-1 levels from colon tumor tissue as compared to normal colon tissue. This study hypothesizes that overexpression of Notch-1 signaling in colon cancer results in enhanced Epithelial to Mesenchymal Transition (EMT) and stemness mediated by other Notch family members via the Jagged-1 ligand. Overexpression of Notch-1 resulted in a cell phenotype which resembles that of a cancer stem cell, with a slower dividing time, and enhanced aggressiveness. Furthermore, cells with constitutively active Notch-1 overexpressed proteins associated with stemness and EMT such as CD44 and Slug. The results which we obtained provide an indication that Notch signaling plays a significant role in the upregulation of EMT and stemness in colon cancer. Â
East Carolina University
2015
Master's Thesis
http://hdl.handle.net/10342/4948
https://thescholarship.ecu.edu/bitstream/10342/4948/1/Fender_ecu_0600O_11469.pdf
c4eef937ba3c12cd4e54462f036a4871
https://thescholarship.ecu.edu/bitstream/10342/4948/2/FenderNEDL.docx
8a86a921070498d38c9246573f4bfe80
https://thescholarship.ecu.edu/bitstream/10342/4948/3/Fender_ecu_0600O_11469.pdf.txt
de17ed8e76896064d3ba3720e84a0a8e
Biology
Biochemistry
Molecular biology
Colorectal Neoplasms--metabolism
Receptors, Notch--metabolism
Signal Transduction
Transforming Growth Factor beta--metabolism
oai:TheScholarship.intra.ecu.edu:10342/53642021-03-03T21:02:33Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
The Role of the Notch-3 Receptor in Stemness, EMT, and Tumorigenesis in Colorectal Cancer
Vinson, Kaitlyn E.
Sigounas, George
As the second leading cause of cancer death worldwide, colorectal cancer (CRC) poses a significant threat to both men and women alike. Although a small percentage of CRC cases are inherited, the majority of cases arise from environmental causes and are widely attributed to dysfunctional cellular pathways. Currently, the CRC progression model maintains that cancer growth, relapse and metastasis are due primarily to cancer stem cells (CSCs), a cell subpopulation that shares many properties with stem cells through epithelial to mesenchymal transition (EMT). We have recently reported that Notch-1 signaling is highly associated with promoting cancer stemness and EMT in CRC. Furthermore, we observed that expression of the Notch-1 receptor also upregulates Jagged-1, a ligand of the Notch receptors, which may affect stemness and EMT. However, this effect is reversed by treatment with DAPT, a [gamma]-secretase and Notch signaling inhibitor. Thus, we hypothesized that the effect of Notch-1 on stemness and EMT in CRC is mediated by Jagged-1 via another member of the Notch pathway, the Notch-3 receptor. To assess our hypothesis, we utilized the colon cancer cell line HCT-116. The parental HCT-116 cells were transduced with an IRES (internal ribosome entry site)-GFP retrovirus that expressed the human intracytoplasmic domain of Notch-1, thus producing the ICN1 cell line. The ICN1 cells were then transduced with a small hairpin RNA (shRNA) construct that effectively knocked down Notch-3, which created the ICN1-shN3 cell line. Growth ability, plating efficiency and colosphere formation were assessed in each cell line. In these experiments, cell culture and Western blot analysis were performed using standard methodology. We found that targeting Notch-3 via shRNA transduction in the colon tumor cell line ICN1-shN3 resulted in a significant decrease of Notch-3 expression. Further Western blot analysis indicated reduced expression of CD44 and Slug, markers for stemness and EMT, respectively. Further analyses showed that in the absence of functioning Notch-3, plating efficiency decreased by 60% and migration decreased by 22% when compared to the ICN1 cell line. These results were accompanied by significant changes in colosphere formation when the ICN1-shN3 cells were compared to ICN1 cells. ICN1-shN3 cells showed a 35% reduction in colosphere formation compared to the ICN cells. This difference was observed in colospheres of both high (33%) and medium/low (37%) proliferative capacity. These data indicate a key role for Notch-3 signaling in Notch-1-induced tumorigenesis mediated by Jagged-1. They also highlight the potential use of Notch-3 inhibitors to effectively target aggressive CRC tumors.
East Carolina University
2016-05-05
Master's Thesis
en
http://hdl.handle.net/10342/5364
https://thescholarship.ecu.edu/bitstream/10342/5364/1/VINSON-MASTERSTHESIS-2016.pdf
5c51a13966a8b9a1feebe879ea890325
https://thescholarship.ecu.edu/bitstream/10342/5364/2/LICENSE.txt
22e54f6bea62bf7d3c7fb6f8c71f0928
https://thescholarship.ecu.edu/bitstream/10342/5364/3/PROQUEST_LICENSE.txt
b3f6fc4af6137da3cd8c6dc7a525f86f
https://thescholarship.ecu.edu/bitstream/10342/5364/4/kaitlyn.vinson_april2016_thesis_signature.page.pdf
150537a710cce116883a69f3c594cdec
https://thescholarship.ecu.edu/bitstream/10342/5364/5/publication.agreement.pdf
dc1030a1253a1e88fa7c397196d07535
https://thescholarship.ecu.edu/bitstream/10342/5364/7/VINSON-MASTERSTHESIS-2016.pdf.txt
8676c4e57bfd848032ff366ded9e87df
Colorectal Cancer
Notch 3
Notch Signaling
Colorectal Neoplasms
Receptors, Notch
Carcinogenesis
Cell Transformation, Neoplastic
Epithelial-Mesenchymal Transition
oai:TheScholarship.intra.ecu.edu:10342/50142021-03-03T20:58:26Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Proposed regulatory role of noncatalytic Adams in ectodomain shedding
Hoggard, Jason Andrew
Bridges, Lance
Members of the ADAM (A Disintegrin And Metalloprotease) protein family uniquely exhibit both proteolytic and adhesive properties. Specifically, ADAMs catalyze the conversion of cell-surface proteins to soluble, biologically active derivatives through a process known as ectodomain shedding. Ectodomain shedding coordinates normal physiological processes. Aberrant ADAM activity contributes to pathological states, such as chronic inflammation. Understanding how ADAM ectodomain shedding activity is governed may provide new avenues for therapeutic intervention of ADAM-mediated shedding pathologies.
While ectodomain shedding is the hallmark feature of the ADAMs, thirteen of the forty ADAMs identified among various species are catalytically inactive. Noncatalytic ADAMs lack one or more consensus elements (HExxHxxGxxH) within the active site of the metalloprotease domain. Despite lacking the hallmark catalytic activity, noncatalytic ADAMs exhibit function(s) associated with other nonenzymatic domains (e.g. integrin recognition of the disintegrin domain). Disruption/mutation of noncatalytic ADAMs has been associated with perturbation of biological events.
My overall hypothesis is that noncatalytic ADAMs regulate the activity of catalytically active ADAMs by competing for substrates and/or receptors when expressed within the same cellular niche. To begin testing this proposed competitive binding regulatory mechanism, I used noncatalytic human ADAM7 and catalytically active human ADAM28 as a model ADAM pair. Preliminary, unpublished data from our lab demonstrated expression of ADAM7 mRNA in multiple immune cell lines established to express ADAM28 at the protein level. For determination of ADAM7 expression patterns, monoclonal antibodies against ADAM7 were produced by our lab. However, the antibodies failed to exhibit reactivity against exogenous, full-length ADAM7.
Based upon preliminary phylogenetic analysis and genomic location, it is likely that ADAM7 arose from gene duplication of ADAM28, which would allow a genetic copy of the molecular specificity required for regulation (e.g. integrin binding) with eventual silencing of catalytic activity. We predicted that the gross structural integrity of the metalloprotease may be uniquely conserved between ADAM7 and ADAM28. Restoration of the active site glutamate residue of the ADAM7 metalloprotease domain bestowed catalytic activity to ADAM7 in a manner that reflected specificity of ADAM28-mediated catalysis. This is the first demonstration, to our knowledge, of 'awakening' a noncatalytic enzyme through a single point mutation. This discovery provides an initial functional link between ADAM7 and ADAM28 and lends credence to the hypothesis that ADAM7 may regulate ADAM28 through competitive binding. These findings have a broader impact, as 92 of the 570 collective human proteases are noncatalytic.
East Carolina University
2015
Master's Thesis
http://hdl.handle.net/10342/5014
https://thescholarship.ecu.edu/bitstream/10342/5014/1/Hoggard_ecu_0600O_11565.pdf
6f5193bb749976cee0903c6d932301b3
https://thescholarship.ecu.edu/bitstream/10342/5014/2/Hoggard_ecu_0600O_11565.pdf.txt
368eb7541cf522b28df573d0b5ed9cd3
https://thescholarship.ecu.edu/bitstream/10342/5014/4/Hoggard_NEDL.pdf
3e780f3f45d9c439f3ff1f14ba9f3d0d
Biochemistry
Molecular biology
ADAM
Disintegrin
Ectodomain
Metalloprotease
Shedding
MDC
Inflammation--metabolism
ADAM Proteins--metabolism
oai:TheScholarship.intra.ecu.edu:10342/40152021-03-03T20:55:41Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Dopaminergic modulation of the autonomic nervous system: in vitro and in vivo evidence from the mouse
Johnson, Tracy L.
Clemens, Stefan
Central nervous system (CNS) function depends on both the connections between the underlying neurons and neural circuits and their activity. Neuronal activity in turn can be classified as neurotransmission and neuromodulation, where neuromodulation serves as a means to adapt neurotransmission to the different behavioral needs of the animal. Here, using the well-described monosynaptic stretch reflex (MSR) spinal circuit as a tool, we compared in the in vitro mouse spinal cord preparation the modulatory actions of DA in spinal lumbar segments that mediate somatosensory input and locomotor output with segments that additionally house the final common output of the autonomic nervous system (ANS). As the ANS contributes to the activation of the cardiac system and as a dysfunction of the DA system and the DA D3 receptor is associated with an increased prevalence of hypertension, we next addressed the potential role of DA modulation on the ANS in vivo, and in particular its role in hypertension and with age.   In the first part of this thesis, we provide evidence that DA exerts opposing modulatory effects in ANS-containing segments when compared to segments void of ANS innervation and that this switch in DA modulation in the thoracic spinal cord is reversed by gap junction blockers.  In the second part of the study, we show that aging-related increases in blood pressure and cardiac function in control wild-type (WT) animals were accompanied by bradycardia in the oldest animals. Interestingly, young D3 receptor knockout (D3KO) mice displayed blood pressure and heart rate values that were significantly increased over their age-matched WT controls but similar to those of the old WT group. Ultrasound echocardiography revealed aging-related increases in heart ventricle size in WT animals, but no similar changes in D3KO. In contrast, functional analyses revealed that ejection fraction and fractional shortening were compromised in old WT animals and similar to young D3KO mice. Subsequent histological assays demonstrated an aging-related interstitial fibrosis that peaked in old WT and that was similar to old WT in young D3KO mice.  Taken together, our data suggest that DA-mediated neuromodulatory actions of spinal cord circuits are dependent on the underlying spinal circuitry, and they suggest that a dysfunction of the D3 receptor pathway is sufficient to mimic the increased hypertension and cardiac remodeling observed in the aging heart. Â
East Carolina University
2012
Master's Thesis
http://hdl.handle.net/10342/4015
https://thescholarship.ecu.edu/bitstream/10342/4015/1/Johnson_ecu_0600M_10755.pdf.txt
88ed56e0db7a0e82cd5fa3ad9ce3f7ea
https://thescholarship.ecu.edu/bitstream/10342/4015/2/Johnson_ecu_0600M_10755.pdf
a171d042dca777b34d41abe506fdfcce
https://thescholarship.ecu.edu/bitstream/10342/4015/3/license.txt
48d772d9ef478e9dd063ea202fa5e0d9
https://thescholarship.ecu.edu/bitstream/10342/4015/4/Johnson_001.pdf
43ca0132876440d98a6a139a562f966f
Neuroscience
Physiology
Autonomic nervous system
Dopamine
Hypertension
Spinal cord
Spinal reflex
Biology, Neuroscience
Biology, Physiology
Central Nervous System
Dopamine--physiology
Mice
Receptors, Dopamine D2
oai:TheScholarship.intra.ecu.edu:10342/74812022-12-05T17:48:35Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Peroxisome Proliferator-Activated Receptor Gamma Deficiency Promotes a T Lymphocyte Response in a Murine Model of Chronic Pulmonary Sarcoidosis
Sanderford, Victoria L
Mohan, Arjun
Sarcoidosis is a chronic granulomatous disease of unknown etiology. Previous studies from our lab have shown a deficiency of the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPAR[gamma]) in the alveolar macrophages of sarcoidosis patients. We established a murine model of granuloma formation by instillation with multiwall carbon nanotubes (MWCNT), and the expression of PPAR[gamma] was also decreased in MWCNT instilled mice. There is evidence linking lymphocyte reactivity to mycobacterial antigens in sarcoidosis, so we hypothesized that addition of mycobacterial peptide early secretory antigenic target 6 (ESAT-6) to MWCNT might exacerbate the murine T effector cell response. MWCNT with or without ESAT-6 peptide 14 were instilled into macrophage-specific PPAR[gamma]-KO or wild-type mice. Controls received vehicle or ESAT-6 alone. Bronchoalveolar lavage (BAL) fluid was collected for analysis via qPCR, and lymph nodes were collected for histology, qPCR, in vitro studies, or flow cytometric analysis. PPAR[gamma]-KO mice receiving MWCNT+ESAT-6 displayed markedly increased granuloma formation and exhibited mediastinal lymphadenopathy. Additionally, PPAR[gamma]-KO mice treated with MWCNT+ESAT-6 exhibited exacerbated fibrotic severity at 20- and 60- days post-instillation. Similarly, lymphocyte frequency was elevated in the BAL of PPAR[gamma]-KO mice compared to controls at 20- and 60-days post-instillation with MWCNT+ESAT-6. Further, TH17 associated transcription factor ROR[gamma]t and immune regulatory marker PD1 were elevated in PPAR[gamma]-KO mice treated with MWCNT+ESAT-6 compared to sham mice at both time points, with elevation of multiple other TH17 associated genes at 60 days post instillation. Carbon nanotubes were observed in the mediastinal lymph nodes of PPAR[gamma]-KO and wild-type mice post-instillation with MWCNT and MWCNT+ESAT-6, and flow cytometric analyses revealed that CD4+ T cells were significantly elevated in PPAR[gamma]KO mice receiving MWCNT+ESAT-6. These findings suggest that instillation of PPAR[gamma]-KO mice with MWCNT+ESAT-6 elicits an exacerbated granulomatous and T lymphocyte mediated response.
East Carolina University
2019-07-03
Master's Thesis
en
http://hdl.handle.net/10342/7481
https://thescholarship.ecu.edu/bitstream/10342/7481/1/SANDERFORD-MASTERSTHESIS-2019.pdf
329c86f2f0a79af4fa57c8414bb84d56
https://thescholarship.ecu.edu/bitstream/10342/7481/2/LICENSE.txt
f8db45c48462cf31cf211a017c28654d
https://thescholarship.ecu.edu/bitstream/10342/7481/3/PROQUEST_LICENSE.txt
d20fd2b01ffe0019ac44519be8e49807
https://thescholarship.ecu.edu/bitstream/10342/7481/4/Final%20Signature%20Page_6-27-19.pdf
94cf7f6c058893281d5fdf293a7c54fa
https://thescholarship.ecu.edu/bitstream/10342/7481/5/Distribution%20License%20Combined.pdf
872e62a2cf48d1b36acb9aef41a15c00
https://thescholarship.ecu.edu/bitstream/10342/7481/7/SANDERFORD-MASTERSTHESIS-2019.pdf.txt
d4550e957a6363c3c64eebd6d2a7fe4b
Granuloma
Mycobacterial antigens
Carbon nanotubes
Sarcoidosis, Pulmonary
T-Lymphocytes
PPAR gamma
Disease Models, Animal
Lymphocytes
Mice
Transcription Factors
oai:TheScholarship.intra.ecu.edu:10342/47342021-03-03T20:57:03Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
THE EFFECTS OF ACIDIC TUMOR MICROENVIRONMENT ON LYMPHOMA CELL RESPONSES TO CHEMOTHERAPEUTICS
Yu, Zhou
Yang, Li
Acidic tumor microenvironment exists in many types of cancer. Altered glycolytic metabolism of tumor cells and deficient blood supply in tissues are major causes for this phenomenon. Lymphoma cells may have different responses to chemotherapeutics when they are exposed to acidic tumor microenvironment. Bortezomib (BTZ) is a chemotherapeutic drug already approved by FDA for treating multiple myeloma and mantle cell lymphoma, which can inhibit the 26S proteasome and block the protein degradation process. Ramos parental cells (Human Burkitt's lymphoma cell line) and BTZ-resistant Ramos cells (obtained after chronic selection using low concentration of BTZ) were used in the study. Cells were treated from 2h to 24h with RPMI culture media buffered to pH 7.4 and pH 6.4, with or without chemical therapeutics. Cancer therapeutics tested in this study were BTZ, Cyclophosphamide, Doxorubicin, and ABT-737. MTT assay was utilized to detect cell viability under each treatment condition. Flow cytometry was used to examine changes on apoptosis and cell cycle. Western blots were performed to measure the changes of cellular protein levels. The MTT assays showed the acidic microenvironment could decrease cell proliferation rate, and if combined with BTZ treatment it could notably increase the cytotoxic efficacy of BTZ. After chronic selection under low concentration of BTZ, BTZ-resistant Ramos cells were obtained and were able to survive and grow in the presence of BTZ. However, acidic pH with BTZ could sensitize the BTZ-Resistant Ramos cells to BTZ again. Apoptosis assays demonstrated that acidic pH itself did not significantly induce cell apoptosis within 24h. But the combination of acidic pH with BTZ treatment caused much more apoptosis than BTZ alone. Cell cycle analysis showed that acidic pH could block the cell cycle, which led to cell cycle arrest and reduced percentage of cells in the G2/M phase. Western blots illustrated that acidic pH alone could upregulate the level of p53, Phospho-p53 at Ser15, Phospho-CHK1, Phospho-CHK2, pro-apoptotic proteins of the mitochondrial Bcl-2 family, and the pro-apoptotic Caspase family. In addition, if combined with BTZ treatment, acidosis could further increase phosphorylation of p53 at Ser15, expression of the Bcl-2 family proteins, and level of cleaved caspases. In conclusion, the combination of acidosis and BTZ treatment induce higher level of apoptosis and decrease Ramos cell proliferation. Our study demonstrates that arrested cell proliferation and increased apoptosis occur by the inhibition of cell cycle regulators and the activation of p53-mitochondria-caspase apoptosis pathways respectively. Â
East Carolina University
2014
Master's Thesis
http://hdl.handle.net/10342/4734
https://thescholarship.ecu.edu/bitstream/10342/4734/1/Yu_ecu_0600O_11294.pdf
9120d2771a2b35c89e9e59e5352f2fa6
https://thescholarship.ecu.edu/bitstream/10342/4734/2/NEDL%20ZU.pdf
78a10e8523f0f7a31490ff4180f354eb
https://thescholarship.ecu.edu/bitstream/10342/4734/3/Yu_ecu_0600O_11294.pdf.txt
d5ca22a1389b581ff526fe6e5d13c776
Biology
Oncology
Acidosis
Chemotherapeutics
Lymphoma
Tumor microenvironment
Neoplasms--physiopathology
Lymphoma--pathology
oai:TheScholarship.intra.ecu.edu:10342/128722023-06-05T14:05:49Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Kinin B1 Receptor Mediates Bidirectional Interaction between Neuroinflammation and Oxidative Stress
Theobald, Drew
Hypertension is the leading risk factor for cardiovascular disease and affects nearly half the adults in the United States. It is well established that hypertension is a low-grade inflammatory condition and is associated with increased release of proinflammatory cytokines, elevated oxidative stress levels, and increased activity of the kallikrein-kinin system (KKS). The KKS is a family of vasoactive proinflammatory peptides that play a vital role in regulating blood pressure. Activation of kinin B1 receptor (B1R) results in increased inflammation and vasoconstrictive effects which can ultimately lead to hypertension. Previously, we showed that angiotensin II (Ang II) can upregulate B1R expression and can induce oxidative stress and neuroinflammation in primary neurons. However, the order at which this occurs has not yet been investigated. In this study, we aim to determine the relationships between neuroinflammation, oxidative stress, and activation of B1R in both primary hypothalamic neurons and primary hypothalamic microglia. Following stimulation with tumor necrosis factor (TNF), lipopolysaccharide (LPS), or hydrogen peroxide (H2O2), we were able to identify a significant increase in reactive oxygen species production, inflammation, and B1R expression. Furthermore, we showed that B1R blockade using a B1R specific antagonist can attenuate these effects in both neurons and microglia. Together, these data provide novel evidence that the interaction between neuroinflammation, oxidative stress, and B1R activation in the brain is bidirectional and that blocking B1R may serve as a potential therapeutic target for neuroinflammation and oxidative stress in various disease pathologies.
East Carolina University
2023-05-02
Master's Thesis
en
http://hdl.handle.net/10342/12872
https://thescholarship.ecu.edu/bitstream/10342/12872/1/THEOBALD-MASTERSTHESIS-2023.pdf
8af65fb32d2a6f4dd0fc02ccbddac3ce
https://thescholarship.ecu.edu/bitstream/10342/12872/2/LICENSE.txt
3ddfc5f7247848cb8ad4a644cbadfc6e
https://thescholarship.ecu.edu/bitstream/10342/12872/3/PROQUEST_LICENSE.txt
307d83267eb7eda84988db7bf904c733
https://thescholarship.ecu.edu/bitstream/10342/12872/4/Signature%20page%20thesis%20pdf.pdf
290d48986dc55092ec2b0020b3ad91fb
https://thescholarship.ecu.edu/bitstream/10342/12872/5/NEDL%20Confirmation.pdf
7fffeb160fbcfe0a687ebc1e1184ceb6
2025-05-01
Hypertension
Neuroinflammation
Oxidative Stress
Kinin B1 Receptor
Neurons
Microglia
oai:TheScholarship.intra.ecu.edu:10342/58802021-03-03T21:09:45Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Notch Signaling Mediates Drug Resistance in Colorectal Cancer Cells via Effects on DNA Repair Proteins
George, Dennis
Sigounas, George
Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States and the third most commonly diagnosed cancer in men and women. Despite tremendous progress in diagnosis, prevention, and treatment efforts, tumor recurrence and chemoresistance remain as considerable challenges. Treatment options are limited if surgery and chemotherapy are unsuccessful. Several studies have implicated the Notch signaling pathway in conferring drug resistance to tumor cells, in addition to increased CRC aggressiveness and potential for metastatic spread. Our group previously showed that Notch-1 signaling is highly associated with promoting cancer stem cell properties in CRC cells. Furthermore, we have also confirmed that human colon tissue samples isolated from CRC patients indicate higher expression of DNA repair proteins associated with Base Excision Repair (BER). Herein, we hypothesized that Notch signaling confers drug resistance to tumor cells via signaling effects on critical proteins associated with DNA repair. Methods: The experiments conducted in this study utilized the colon cancer cell line HCT-116. These cells were transduced with an IRES-GFP retrovirus expressing human intracytoplasmic domain of Notch-1 (ICN1). Another group of HCT-116 cells was transduced with a small hairpin mRNA construct (sh59) that effectively knocked out Notch-1 action. Cell lines were exposed to varying concentrations of cytarabine and cisplatin, potent DNA damaging agents, and observed for chemosensitivity. Western blot analysis was performed using standard methodology. Results: Small hairpin mRNA (sh59) transduction into the colon tumor cell line HCT-116 resulted in a significantly decreased expression of critical BER DNA repair enzymes, Poly (ADP-Ribose) Polymerase 1 (PARP1) and 8-Oxoguanine glycosylase (OGG1). These changes were accompanied by significantly higher chemosensitivity of these cells to cytarabine and cisplatin treatment as opposed to cells expressing constitutively active Notch-1 signaling. Furthermore, cells with intact Notch signaling expressed upto two-fold increase in expression of APE1 following treatment with cytarabine compared to cells with no Notch-1 expression. Conclusions: These data indicate a key role for Notch signaling in conferring drug resistance to CRC cells via effects on critical proteins of DNA repair. This finding highlights the potential use of Notch-1 inhibitors in combination with PARP1 inhibitors to effectively target highly drug resistant CRC cells.
East Carolina University
2016-07-25
Master's Thesis
en
http://hdl.handle.net/10342/5880
https://thescholarship.ecu.edu/bitstream/10342/5880/1/GEORGE-MASTERSTHESIS-2016.pdf
e0d7bc816caf9ad2da53d7ad1a33b4ec
https://thescholarship.ecu.edu/bitstream/10342/5880/2/LICENSE.txt
f4d1d1b63efcab60305be53c3b09048a
https://thescholarship.ecu.edu/bitstream/10342/5880/3/PROQUEST_LICENSE.txt
b4a69d245454151d07ad73bb83ed7ede
https://thescholarship.ecu.edu/bitstream/10342/5880/4/SignedSignatures.jpeg
a28c3a4b06a4100e1a68d307d791d64a
https://thescholarship.ecu.edu/bitstream/10342/5880/5/SignedLicensePage1.jpeg
1330578153db1d4a477ff606f2f2c711
https://thescholarship.ecu.edu/bitstream/10342/5880/6/SignedLicensePage2.jpeg
896b1a82efd6ebcd34f4581e2453f982
https://thescholarship.ecu.edu/bitstream/10342/5880/8/GEORGE-MASTERSTHESIS-2016.pdf.txt
a203d2c56fac55c476f6f503b09084e1
Colorectal Neoplasms
DNA Repair
Drug Resistance
Signal Transduction
Notch Signaling
oai:TheScholarship.intra.ecu.edu:10342/42462021-03-03T20:55:56Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Activation of the proton sensing G-protein coupled receptor, GPR4, regulates focal adhesion dynamics and delays cell spreading due to increased cytoskeletal tension
Justus, Calvin Richard
Yang, Li
The tumor microenvironment is characteristically acidic due to insufficient blood perfusion, chronic inflammation, hypoxia, and altered cell metabolism. The low pH found in the tumor microenvironment may facilitate the dissemination of cancer cells into the blood stream or lymph system by breaking down extracellular matrix components and degrading the basement membrane. In the murine B16F10 melanoma model, low pH has also been reported to activate the proton sensing G-protein coupled receptor, GPR4, and decrease cell migration in vitro as well as reduce pulmonary metastasis subsequent to tail vein injections. In this study we investigated delayed cell spreading found in B16F10 melanoma cells genetically modified to express GPR4 at a high level, namely B16/GPR4 cells. Attachment assays to tissue culture plates, fibronectin, glass coverslips, and matrigel established that B16/GPR4 cell spreading was significantly delayed when plated in media buffered to pH 6.4. Next, we investigated the specific heterotrimeric G-protein responsible for delayed B16/GPR4 cell spreading by using several chemical activators and inhibitors as well as numerous genetically engineered cell lines. Treatment with either C3-transferase (CT04), a direct Rho inhibitor, or a G12/13 inhibitory construct restored B16/GPR4 cell spreading significantly to a level similar to the vector control group and the physiological pH treatment group. We also evaluated the Gs and Gq G-protein pathways by using 2', 5'-dideoxyadenosine (DDA) and 8-bromo-cAMP or thapsigargin and a Gq inhibitory construct but found little modifications in B16/GPR4 cell spreading indicating the G12/13 G-protein pathway as the responsible variant. The downstream signaling mechanisms for delayed B16/GPR4 cell spreading were investigated. Both the ROCK inhibitor, Y27632, and the MLCK inhibitor staurosporine restored cell spreading. The same chemical inhibitors and activators as well as inhibitory constructs that restored B16/GPR4 cell spreading also reduced the formation of actin stress fibers indicating a proton-induced signaling cascade, which leads to increased cytoskeletal tension and delayed cell spreading. Due to the importance of focal adhesion dynamics in cell spreading and migration we next investigated two focal adhesion proteins that are vital for this process, phospho-paxillin (Y118) and phospho-focal adhesion kinase (Y397). The spatial localization of phospho-paxillin (Y118) and phospho-FAK (Y397) in B16/GPR4 cells after one-hour attachment assays is altered when treated with acidic media, displaying localization to the cell body instead of the cell periphery where dynamic focal adhesions are located. These results indicate that a proton-induced GPR4/G12/13/Rho/Rock/MLCK signaling pathway is responsible for delayed melanoma cell spreading and altered focal adhesion dynamics. Â
East Carolina University
2013
Master's Thesis
http://hdl.handle.net/10342/4246
https://thescholarship.ecu.edu/bitstream/10342/4246/2/justus.pdf
cbda91ac37c897f1aaba7f0b2c880905
https://thescholarship.ecu.edu/bitstream/10342/4246/3/Justus_ecu_0600M_11027.pdf.txt
baf467625f7972907f5fd784951ec8fc
https://thescholarship.ecu.edu/bitstream/10342/4246/1/Justus_ecu_0600M_11027.pdf
793b965d9d7f021e408e2945bd9d731f
Oncology
Biology, Cellular
Cellular biology
Rho-Associated Kinases
Melanoma
Receptors, G-Protein-Coupled
Models, Animal
Myosin-Light-Chain Kinase
Y 27632
TNF-Related Apoptosis-Inducing Ligand
GTP-Binding Proteins
oai:TheScholarship.intra.ecu.edu:10342/91452023-05-01T08:01:59Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
SPINAL CORD REFLEXES IN A BRAIN-IRON DEFICIENT MODEL OF RESTLESS LEGS SYNDROME: ROLE OF DOPAMINE AND ADENOSINE RECEPTORS
Woods, Sydney
Clemens, Stefan
Restless Legs Syndrome (RLS) is a sensorimotor disorder that severely disrupts sleep. RLS patients regularly present with a condition known as Brain Iron Deficiency (BID), and BID is commonly associated with altered dopamine and adenosine neurotransmission in the striatum. Dopamine and adenosine can form receptor heteromers in the striatum, where the adenosine A1 receptor (A1R) modulates dopamine D1 receptor (D1R) function. However, there are no data on the impact of BID in the spinal cord, the ultimate sensorimotor circuitry involved in RLS. We here tested if a diet-induced brain iron-deficient animal model affects spinal cord excitability as reported in other RLS animal models, and we tested the responsiveness of this model to treatment with dopamine and adenosine receptor modulators. Following previously established protocols, C57Bl/6 mice were separated upon weaning into male and female cohorts fed either control iron or iron-reduced diets. The BID diet did not induce an anemic phenotype. To assess spinal cord excitability, we measured thermal pain reflex withdrawal latencies (RWLs) using the Hargreaves system, starting at one-week post-diet exposure. The BID cohorts showed significantly lower RWLs than their respective CTRL cohorts, and these differences remained stable over time. We then tested the responsiveness of this model to dopamine receptor modulators Pramipexole (PPX, D3 receptor agonist, 0.5 mg/kg, i.p.) and SCH 39166 (Ecopipam, D1 receptor antagonist, 0.5 mg/kg + 1.0 mg/kg, i.p.), and adenosine receptor modulators caffeine (A1R/A2R, 50 mg/kg, i.p.) and N6-cyclpentyladenosine (CPA, A1R agonist, 1.0 mg/kg, i.p.). These data indicate that PPX did have significant effects on increasing RWLs but with strong locomotor side effects, SCH 39166 showed significant effects in increasing RWLs in the male BID cohort, but not female BID cohort, and while caffeine did not have significant effects on RWLs in CTRL cohorts, use of CPA led to a significant increase in RWLs in both male and female BID cohorts. Western blot analysis of D1R and A1R expression in the mouse spinal cord revealed an increase in D1R expression and opposing decrease in A1R expression. Finally, the use of proximity ligation assays (PLAs) revealed the presence of A1R-D1R heteromers within the mouse spinal cord, where motoneurons reside, and that these heteromers decrease in number or are no longer functional under BID conditions. Together our data show that diet-induced iron-deficiency leads to a decrease in RWLs, and that use of dopamine and adenosine receptor modulators show significant effects in this model. RLS patients present with BID and are initially highly responsive to dopamine D3 receptor (D3R)-based treatment in the clinic. However, long-term treatment with these compounds can lead to unwanted long-term side effects. We here propose a hypothetical model of RLS and how BID conditions might affect dopamine and adenosine neurotransmission via A1R-D1R heteromers.
East Carolina University
2021-05-04
Master's Thesis
en
http://hdl.handle.net/10342/9145
https://thescholarship.ecu.edu/bitstream/10342/9145/1/WOODS-MASTERSTHESIS-2021.pdf
a0364716655a6c61a00dbfdbbc28a22a
https://thescholarship.ecu.edu/bitstream/10342/9145/2/LICENSE.txt
c1e58c5c0efbfb8fd541dac1e34f48b9
https://thescholarship.ecu.edu/bitstream/10342/9145/3/PROQUEST_LICENSE.txt
1f851c61ff53eca40706cc292fde02fe
https://thescholarship.ecu.edu/bitstream/10342/9145/4/Thesis%20Signature%20Page%20FINAL%20final.pdf
839eb2b154ace7153006c5d2dbfcf5ec
https://thescholarship.ecu.edu/bitstream/10342/9145/5/Vireo-NonExclusive-Distribution_License_Edited-Verio-Version_L0615-2.pdf
ea065c33be5a474dfda7d968b01395de
https://thescholarship.ecu.edu/bitstream/10342/9145/7/WOODS-MASTERSTHESIS-2021.pdf.txt
3a90adb3b774ca2107f82d5802a84422
Adenosine
Reflexes
Restless Legs Syndrome
Dopamine
Iron
Spinal Cord
Brain
Receptors, Purinergic P1
oai:TheScholarship.intra.ecu.edu:10342/46642021-03-03T21:10:49Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Expression of microRNA in Alveolar Macrophages Deficient in PPARy
McPeek, Matthew
Thomassen, Mary Jane
The nuclear transcription factor Peroxisome proliferator-activated receptor gamma (PPARgamma) is a negative regulator of macrophage activation and inflammatory mediators. Alveolar macrophages of healthy individuals constitutively express PPARgamma Decreased activity and expression of PPARgamma are observed in the alveolar macrophages from patients suffering from inflammatory conditions such as pulmonary alveolar proteinosis (PAP) and sarcoidosis. These finding suggest that PPAR<em>f</em>× activity may have an integral role in maintaining lung homeostasis. This study tested the hypothesis that microRNA expression would be dysregulated in murine alveolar macrophages deficient in PPARgamma. microRNA (miR) are small non-coding RNA molecules that post-transcriptionally regulate the expression of messenger RNA.   Evaluation of microRNA in the murine model of PAP, the GM-CSF-KO mouse, demonstrates the elevation of miR-27a and miR-27b which target PPARgamma. The deficiency of PPARgamma and the lipid transporters ABCA1 and ABCG1 have been shown to contribute to the pathology of PAP. The microRNA miR-33-3p and miR-33-5p, which target these lipid transporters, were also elevated in GM-CSF-KO mice. Pulmonary granulomas comparable to those observed in pulmonary sarcoidosis are induced by instillation of multiwall carbon nanotubes (MWCNT) in C57Bl/6 mice. These animals have decreased PPARgamma activity and show elevated expression of miR-27a and miR-27b. It was also observed that the expression of the transporters ABCA1 and ABCG1 were decreased in MWCNT instilled mice. Expression of miR-33-3p and miR-33-5p was elevated in MWCNT instilled animals. The expression of microRNA that affects the activity of NF-£eB is also elevated in both murine models.  We next investigated the use of PPARgamma agonist rosiglitazone on the expression of microRNA and messenger RNA. The use of rosiglitazone altered the expression of microRNA in both GM-CSF-KO and C57Bl/6+MWCNT mice. Rosiglitazone treatment altered the expression of the lipid transporter ABCA1and ABCG1 in C57Bl/6+MWCNT mice. The elevation of proinflammatory cytokines was also observed.   Taken together, these observations support the hypothesis that PPARgamma activity effects the microRNA and gene expression in alveolar macrophages which is critical to overall lung homeostasis. Understanding the relationship between PPARgamma and microRNA in alveolar macrophage biology will provide insight into the regulation of the lung environment and possible therapeutic targets. Â
East Carolina University
2014
Master's Thesis
http://hdl.handle.net/10342/4664
https://thescholarship.ecu.edu/bitstream/10342/4664/1/McPeek_ecu_0600O_11379.pdf
92fb8b6cb801cec81f7bcebb07aa2381
https://thescholarship.ecu.edu/bitstream/10342/4664/2/McPeek_NEDL.pdf
544ec50631826b46f938b5fe26879474
https://thescholarship.ecu.edu/bitstream/10342/4664/3/McPeek_ecu_0600O_11379.pdf.txt
73a0daec4c65279fe34bbba675bc0d2d
Biology
Alveolar proteinosis
Granulomatous lung disease
PPAR gamma
Sarcoidosis
oai:TheScholarship.intra.ecu.edu:10342/74552022-12-05T17:47:46Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Investigating the Role of Poxvirus Virulence Genes A35 and O1L in the Virus Life Cycle
Hayes, Alexandra G
Roper, Rachel L
Poxviruses, some of the largest viruses in existence, have a great impact on the human and animal world due to their ability to infect a broad assortment of organisms and cause significant disease. Today, poxvirus infections remain a danger to human health, as natural and potential bioterrorism threats. Vaccinia virus (VACV), the species of poxvirus used in smallpox vaccines, is the most studied poxvirus, but there is still much to learn in regards to its virulence factors and their role in the virus life cycle. Our laboratory has identified two VACV genes/proteins that play an important role in virulence, A35 and O1L, which we hypothesized were immunoregulatory, so their effects on host immune responses were assessed. We found that the A35 protein inhibits anti-viral antibody production and cytokine responses by T lymphocytes in vivo. However, there was no evidence to suggest that A35 inhibits recall antigen presentation by infected BMDC in vitro. There were also no A35 effects observed on VACV cell killing, replication, or integrin expression for bone marrow dendritic cells (BMDC), which were used as antigen presenting cells (APC). When looking at the function of O1L, we did not find an effect of O1L on anti-viral antibody production or T cell response, so the O1L effects on replication and spread, cell killing, integrin expression, cytokine production, and innate immunity were also measured. In each of these cases, the O1L deletion mutant (O1LDel) had a similar phenotype to the wild type virus. We did observe that plaques formed by the O1LDel virus appeared smaller in some cases compared to wild type plaques, which was due to reduced cell clearance in the center of the O1LDel plaques. However, the biological relevance of this finding is unclear at this time. The fact that the VACV O1L gene encodes a large protein that is conserved in mammalian tropic poxviruses with 92-100% homology supports that the gene performs an important function in the poxvirus life cycle. Our laboratory has shown that both A35 and O1L deletion viral mutants make safer vaccine alternatives against poxviruses. Understanding how poxviruses turn off immune responses will aid in our understanding of viral pathogenesis and support anti-viral drug design, improve vaccines, and may allow us to mimic poxvirus immunosuppression to control autoimmune diseases.
East Carolina University
2019-07-22
Master's Thesis
en
http://hdl.handle.net/10342/7455
https://thescholarship.ecu.edu/bitstream/10342/7455/1/HAYES-MASTERSTHESIS-2019.pdf
f47696d71cd9116d3a45143d1e1edb1a
https://thescholarship.ecu.edu/bitstream/10342/7455/2/LICENSE.txt
2737389d8ef18f80618f0a75c26e1301
https://thescholarship.ecu.edu/bitstream/10342/7455/3/PROQUEST_LICENSE.txt
89c130735991ef804744a47d74cb393f
https://thescholarship.ecu.edu/bitstream/10342/7455/4/AHsignaturepage2019.pdf
8dbb6b533905bcaa795a0f9580406038
https://thescholarship.ecu.edu/bitstream/10342/7455/5/AHNEDL2019.pdf
cff6b5b91ed9613036b9b12ef0536293
https://thescholarship.ecu.edu/bitstream/10342/7455/7/HAYES-MASTERSTHESIS-2019.pdf.txt
1d2c7600c406e4f796aadb5228c8c169
poxvirus
vaccinia virus
A35
O1L
immunoregulatory
virulence
Virulence
Chordopoxvirinae
Poxviridae
oai:TheScholarship.intra.ecu.edu:10342/45642021-03-03T21:10:43Zcom_10342_74com_10342_73com_10342_122col_10342_3934col_10342_124
Interrelated role of Notch signaling and mTORC pathways in prostate cancer cell survival and growth
Nutter, Jennifer Makenzie
Bertrand, Fred E.
Prostate cancer is currently the second highest leading cause of
cancer death in men. Notch is a transmembrane receptor protein that
is part of a signaling pathway necessary in the normal development of
the prostate. Notch1 signaling has been shown to be lost in prostate
adenocarcinoma. One of prostate cancer's biggest risk factors is age
and mTOR has been shown to be linked to longevity and age related
diseases. mTOR exists as two complexes, mTORC 1/2, whose key
functions are to control cell survival, metabolism, and growth.
mTORC1/2 are often overexpressed in cancer. It was also reported that the mTORC1 pathway became inactivated when Notch1 signaling was inhibited in prostate cancer cell line PC-3. Herein, we suggest a link between Notch1 signaling and mTOR pathway activity which led to experiments with DU145 cells manipulated to have decreased Notch1 expression. The data shows that loss of Notch1 signaling causes decreased expression of the mTORC1 component Raptor as well as decreased phosphorylation of mTORC1 downstream target 4E-BP1 in conditions of cell stress. The mTORC2 pathway exhibited decreased phospho-mTOR (Ser2481) in normal conditions and decreased Rictor signaling in both normal and serum starved conditions when Notch1 expression was lost. The data also suggests there is less G[bata]L in conditions of stress in Notch1 knockdown cells. We hypothesize that downregulation of Notch1 signaling leads to the dysfunction of both mTOR pathways.
East Carolina University
2014
Master's Thesis
http://hdl.handle.net/10342/4564
https://thescholarship.ecu.edu/bitstream/10342/4564/1/Nutter_ecu_0600O_11282.pdf
47afd3cc60cd9db554fa6a455d3cba42
https://thescholarship.ecu.edu/bitstream/10342/4564/2/Nutter_ecu_0600O_11282.pdf.txt
20f3c559f300d843b639a2bae0379822
https://thescholarship.ecu.edu/bitstream/10342/4564/3/NEDL_Nutter.pdf
5ed409664ab1ffce91b585ffe71e944e
Oncology
Prostatic Neoplasms--chemistry
Prostatic Neoplasms--pathology
Adenocarcinoma
TOR Serine-Threonine Kinases
Receptor, Notch1