Medical College of Georgia: Theses and Dissertations
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Item Open Access Activation of a Molecular Chaperone (Sigma 1 Receptor) in a Murine Model of Autosomal Dominant Retinitis Pigmentosa(Augusta University, 2022-05) Barwick, Shannon; Biomedical SciencesRetinitis pigmentosa (RP) is a devastating group of inherited retinal diseases that leads to visual impairment and eventually complete blindness. Currently no cure or treatment exists for RP patients, thus research into prolonging the vision in these patients is imperative. Sigma 1 receptor (Sig1R) is a promising small molecule target that appears to have neuroprotective benefits in the retina of fast degenerating mouse models. However, it is not clear whether Sig1R activation can provide similar neuroprotective benefits in more slowly progressing RP models, which are more similar to human patients. In this study, we examined whether Sig1R activation can be neuroprotective (i.e. prolong vision) in a more slowly progressing mouse model of autosomal dominant retinitis pigmentosa, RhoP23H/+. Current studies in the field give a brief overview of the RhoP23H/+ degeneration, but do not give a complete characterization of disease progression. Aim 1 of this study sought to further characterize the degeneration of the RhoP23H/+ mouse using 3 in vivo methods, Optomotor Response (OMR), Electrophysiology (ERG), and Spectral Domain-Optical Coherence Tomography (SD-OCT). A slow retinal degeneration was observed in both male and female RhoP23H/+ mice when compared to WT. Visual acuity showed a gradual decline through 10 months. Interestingly, visual acuity was still detectible, albeit significantly reduced through 10 months in both male and female mutant mice. Females appeared to have significantly lower visual acuity than males. These RhoP23H/+ mice showed a gradual decline in scotopic and photopic responses. Aims 2 and 3 sought to investigate the neuroprotective benefits of Sig1R activation in the RhoP23H/+ mouse model. Mutant mice were treated with a high specificity Sig1R ligand (+)-pentazocine ((+)-PTZ) 3x/week and examined using OMR, ERG, SD-OCT. A significant retention of visual function was observed in both males and females at 10 months of age, with treated females retaining ~50% greater visual acuity than non-treated mutant females. Using ERG, significant retention of scotopic and photopic b-wave amplitudes were observed at 6 months in both male and female mice treated with (+)-PTZ. Further, in vivo analysis of ONL thickness revealed a significant retention in both male and female treated mice. Histological studies using retinal cryosections showed significant retention of IS/OS length (~50%), ONL thickness, and number of rows of PRC nuclei at 6 months in both male and female (+)-PTZ-treated mice. Interestingly, electron microscopy revealed preservation of OS discs in (+)-PTZ treated mutant mice. Taken collectively, the in vivo and in vitro data represent the first report of Sig1R activation rescuing visual function and structure in the RhoP23H/+ mouse model. These results are promising and lay the framework for future studies to investigate Sig1R as a potential therapeutic target in retinal degenerative disease.Item Embargo Characterization of an Inducible p65 Knockout Mouse Model for Studying Glioblastoma(Augusta University, 2022-05) Trang, Amy; Biomedical SciencesGlioblastoma (GBM) is a malignant primary brain tumor that results in patient death within two years following diagnosis. The GBM tumor microenvironment has an important impact on the formation, progression, and drug resistance of this lethal disease. The GBM tumor microenvironment is composed of a variety of cell types that can support tumor growth like microglia. Microglia are immune cells that can switch from a classical, tumoricidal M1 phenotype to an alternative, tumor-promoting M2 phenotype. However, there are limited studies about the specific effect(s) that microglia have on GBM tumors. Given the importance of the tumor microenvironment to GBM tumor progression, targeting key signaling pathways in tumor-associated cells like microglia could be an effective way to reduce tumor progression. One signaling pathway involved in causing different cancers is the nuclear factor-kappa B (NF-κB) pathway, and it has been implicated in M1 to M2 phenotype polarization. Therefore, this study focuses on the characterization of the p65fl/fl/CX3CR1creER/+ mouse model, which is an inducible p65 knockout mouse model for studying how inhibition of canonical NF-κB signaling in microglia affects GBM tumors. After tamoxifen is administered to the mouse, p65, a transcription factor of the canonical NF-κB pathway, should be deleted in microglia due to the activation of a Cre recombinase (CreER) under the control of the CX3CR1 promoter. Characterization of this mouse model is necessary to determine if the p65 gene is effectively deleted in microglia from these mice. Liquid chromatography-mass spectrometry analysis has shown that the orally administered tamoxifen was present and quantifiable in the brains of tamoxifen-treated mice to induce recombination. Based on data from PCR and western blots, tamoxifen given to the p65 knockout mice induced partial deletion of the p65 gene in isolated microglia when compared to control groups. Preliminary flow cytometry data suggested that p65 deletion in microglia occurred in p65fl/fl/CX3CR1creER/+ mice that received GBM implantations and tamoxifen treatment compared to a vehicle control group. Overall, the data from this study suggests that the p65 gene is partially deleted in microglia from the tamoxifen-treated p65 knockout mice compared to the vehicle control mice.Item Open Access An Inactive Receptor-G Protein Complex Maintains the Dynamic Range of Agonist-induced Signaling(Augusta University, 2022-05-19) Jang, Wonjo; Biomedical SciencesG protein-coupled receptors (GPCRs) are 7-transmembrane (TM) proteins that are targets of one-third of approved drugs. In response to agonist binding, GPCRs adopt active-state conformations that promote their association with G protein heterotrimers. The resulting active-state ternary complex (i.e., agonist-GPCR-G protein complex) is the basis for conventional stimulus-response coupling. However multiple studies have also described GPCR-G protein complexes that form prior to agonist binding. While others have previously proposed that this interaction is thought to promote rapid or specific signaling, the role of such “preassociated” complexes is not well understood, and inactive-state receptors are generally considered unable to interact with heterotrimeric G proteins. Here, we show that preassociation of 5-HT7 serotonin receptors with Gs heterotrimers is necessary for agonist-induced signaling. Because inverse agonists and receptor mutations that favor the inactive state of 5-HT7 receptors promote the formation of preassociated complexes, 5-HT7 receptors in their inactive state preassociate with Gs. Upon agonist binding, 5-HT7 receptors adopt conformations that disfavor the formation of inactive-state 5-HT7–Gs complexes, thus permitting the formation of conventional agonist–5-HT7–Gs ternary complexes. Because Gs variants that cannot form inactive-state 5-HT7-Gs complexes are constitutively activated by 5-HT7 receptors, we conclude that this unconventional inactive-state 5-HT7-Gs complex is critical for the dynamic range of agonist-induced signaling. Thus, our findings provide evidence that agonists can initiate signaling via two distinct mechanisms, by promoting the association of conventional ternary complexes and by disrupting inverse-coupled binary complexes.Item Open Access Blood Pressure Impacts the Renal T Cell Profile of Male and Female Spontaneously Hypertensive Rats(Augusta University, 2014-03) Tipton, Ashlee Joy; Medical College of GeorgiaItem Open Access Activation of Arginase and the Endothelin System in Models of Ischemic Retinopathy(Augusta University, 2014-07) Chintan, Patel; Medical College of GeorgiaItem Open Access Angiotensin II-Induced Protein Kinase D Activation and Regulation of Aldosterone Production(Augusta University, 2013-02) Olala, Lawrence O.; Medical College of GeorgiaDysregulated aldosterone production leading to hypertension and its associated complications, such as congestive heart failure, cardiac fibrosis and renal failure, arc important public health concerns with a huge impact on the economy and patient quality of life. Thus, there is a high level of interest in the development of medical interventions and lifestyle changes to reduce the incidence of hypertension. Stimulation of the adrenal zona glomcrulosa with angiotensin II (Angll), potassium (K ) or adrenocorticotropic hormone (ACT! I), increases aldosterone production, to result in increased sodium and \\ater retention. We ha\e recently shown a role for the serine/threonine protein kinase D (PKD) in the regulation of acute aldosterone synthesis upon Ang[( stimulation. In this study, using both molecular and pharmacological approaches, we demonstrate that Src family kinases and protein kinase C (PKC) activate PKD to increase aldosterone production in bovine adrenal glomerulosa cells. We have also shown that PKD positively regu lates expression of steroidogenic acute regulatory (StAR) protein, a protein required for cholesterol transport into the mitochondria, and aldosterone synthesis. PKD plays this role, in part, through activating members of the activating transcription factor (A TF)/cAMP response clement (CRE-) binding protein (CREB) family of leucine tipper transcription factors. Therefore, we hypothesite that PKC and Src family kinase-mediated PKD activation in response to Angll increases the phosphorylation and activation of ATF-2 and CREB, '' hich bind the StAR proximal promoter thereby resulting in induction of StAR expression and stimulation of steroidogenesis.Item Open Access Characterization of the Thioredoxin System in the Diabetic Retina(Augusta University, 2013-07) Lamoke, Folami; Medical College of GeorgiaItem Open Access Association of Risk Factors with School Attendance Among Overweight Children(Augusta University, 2014-03) Hyer, J. Madison; Medical College of GeorgiaItem Open Access Cellular and Molecular Players in Neuromuscular Junction (NMJ) Formation and Function(Augusta University, 2014-04) Barik, Arnab; Medical College of GeorgiaItem Open Access Bioactivity and Mechanism of Action of Resveratrol, a Polyhenolic Phytoalexin, in Sickle Cell Disease(Augusta University, 2013-07) Agyekum, Davies G.; Medical College of GeorgiaItem Open Access Angiogenesis-Associated Gene Expression Changes in Surgical Skin Flaps of Diabetic Rats(Augusta University, 2010-07) Zhou, Miao Xian (Cindy); Medical College of GeorgiaItem Open Access Aquaporin 3 in Keratinocyte Differentiation(Augusta University, 2003-08) Zhan, Xiangjian; Medical College of GeorgiaAquaporin 3 (AQP3) is a channel that transports both water and glycerol. AQP3- null mutant mice exhibit skin defects, including impairment of water holding capacity, barrier recovery and wound healing and decreased glycerol content. We hypothesized that AQP3 is involved in the regulation ofkeratinocyte proliferation and differentiation and this regulation is mediated, at least in part, by the functional interaction between AQP3 and phospholipase D (PLD). Here we demonstrate that AQP3 expression was down-regulated at the transcriptional level and glycerol uptake was reduced when primary mouse keratinocytes were induced to differentiate. In co-transfection experiments, we found that AQP3 decreased the promoter activity of keratin 5, a keratinocyte proliferation marker, but increased the promoter activity of keratin 10 and involucrin, an early and intermediate keratinocyte differentiation marker respectively. These results suggest that AQP3 is a regulator of early keratinocyte differentiation. In further investigatjons to determine the sigualing function of AQP3 in regulating keratinocyte differentiation, we found using sucrose gradient centrifugation, irnmunoprecipitation analysis, confocal microscopy that AQP3 and PLD2 were colocalized in lipid rafts. In addition, we demonstrated that AQP3 could contribute to the synthesis of phosphatidylglycerol (PG) and that PLD2 was able to utilize glycerol as a substrate to synthesize PG. These data suggest that AQP3 transports glycerol for use as a physiological primary alcohol substrate for-adjacent PLD2 to generate PG. Our results, together with the reports that PG is an activator of protein kinases (PKqm and PKCe) and also contributes to protein-protein interactions in membranes, suggest that glycerol AQP3-PLD2-PG is a potential signaling pathway in regulating keratinocyte differentiation.Item Open Access Characterization of Leydig Cell Development in the Rat Testis(Augusta University, 1996-05) Zhai, Juan; Medical College of GeorgiaItem Open Access Androgen Regulation of the Expression of OCTN2, a High Affinity Carnitine Transporter, in the Epididymis and Other Tissues(Augusta University, 1995) Tolson, George E.; Medical College of GeorgiaItem Open Access Androgen Regulation of the Expression of OCTN2, a High Affinity Carnitine Transporter, in the Epididymis and Other Tissues(Augusta University, 2001) Timm, Russell S.; Medical College of GeorgiaOCTNZ is a transport protein and a member of a superfamily of organic cation transporters and has been shown to transport a variety of substrates, including camitine. The transport of the substrate is unique among this family of transport proteins as it is sodium-dependant, and it has been shown that this transporter is a physiologically relevant camitine transporter, as naturally occurring mutations in the gene encoding this transporter result in the clinical manifestation of the syndrome known as primary camitine deficiency. With the acceptance of OCTNZ's physiological role in camitine homeostasis, further scientific inquiries need to be made to definitively show that OCTN2 is a molecular mechanism that underlies many of the observations made in the field of camitine research in past century. Multiple investigators have reported the effect and relationship between androgen levels and carnitine transport, but until the discovery of OCTN2 and its role in camitine homeostasis, the effect of androgens on the molecular mechanism responsible for these observations could not be proven. The results of the research described here should provide conclusive evidence that androgen levels regulate levels of OCTNZ expr!lSsion in both tissues of the male rat and in a human cell line, through either a direct or indirect mechanism, and that the effect on the regulation of expression is consistent with transport data :from previous researchers.Item Open Access Biosynthesis and Modification of Helicobacter Pylori Lipid A(Augusta University, 2010) Stead, Christopher Michael; Medical College of GeorgiaThe secondary acylation steps of Helicobacter pylori lipid A biosynthesis are poorly understood because H. pylori only has one homolog (Jhp0265) to the Escherichia coli secondary acyl transferases Lpxl and LpxM. Jhp0265 was shown to be responsible for the transfer of a secondary C18 acyl chain to the 2' -linked acyl chain of lipid A, making Jhp0265 homologous to Lpxl. An activity was also demonstrated for the addition of a secondary acyl chain to the 3'-linked acyl chain of H. pylori lipid A, although the enzyme responsible for the transfer remains unknown. After synthesis, H. pylori lipid A is modified by the action of five enzymes. Mutation of the candidate modification enzyme Jhp0634 demonstrated that the enzyme catalyzes the removal of the 3'-linked acyl chains of H. pylori lipid A, producing a tetra-acylated lipid A species. Continuing with the characterization of H. pylori lipid A modification enzymes, we were also able to demonstrate an activity for a Kdo trimming enzyme in vitro. Requirement for a Kdo hydrolase in vivo was confirmed after the Kdo transferase of H. pylori was shown to be bifunctional despite the presence of only one Kdo sugar in H. pylori lipopolysaccharide. Attempted identification of the Kdo hydrolase revealed that both Hp0579 and Hp0580 were required for the removal of the Kdo sugar, which occurred in the periplasm. A Kdo hydrolase mutant revealed two unexpected phenotypes related to interaction with the innate immune system. The first was an increased sensitivity to cationic antimicrobial peptides, which was explained by a downstream effect on modification to the 4'- phosphate group of lipid A. The second phenotype related to the expression of 0- antigen on the bacterial cell surface. The Kdo hydrolase mutants produced a reduced amount of fully extended lipopolysaccharide and conversely, an increased amount of core-lipid A. The type of 0-antigen epitope displayed was also affected by a Kdo hydrolase mutation, in a strain specific manner.Item Open Access Caveolin-1 in the Cerebral Microvasculature: Identification and Characterization Within Junctional Domains and Implications for a Role in Blood-Brain Barrier Permeability(Augusta University, 2008) Socha, Matthew John; Medical College of GeorgiaItem Open Access Caveolin-1 in the Cerebral Microvasculature: Identification and Characterization Within Junctional Domains and Implications for a Role in Blood-Brain Barrier Permeability(Augusta University, 2002) Smart, Dee Dee Kay; Medical College of GeorgiaThe restricted permeability properties of cerebral microvascular endothelium are considered to reflect the pal!city, or near complete absence, of transcytotic· vesicles (caveolae) as well as the presence of complex tight junctions. Although cerebral microvasculature lacks caveolae, caveolin-1, the principle structural and functional component of caveolae, is detected predominantly in association with endothelia throughout the adult brain. In this study, I have investigated the expression of caveolin-1 within the microvasculature of the rat neocortex using imrnunolocalization, imrnunoblot, and subcellular fractionation methodologies. Although caveolin-1 immunoreactivity was displayed principally by differentiating astroglial and neuronal cells at early developmental timepoints, caveolin- I immunoreactivity in the adult neocortex distributed predominantly to the microvascular endothelium. The change in the cellular distribution of caveolin-1 immunoreactivity was accompanied by a dramatic change in the detergentsolubility characte~ of the caveolin-·1·, protein. Because these morphological and biochemical changes in caveolin-1 expression and character paralleled the developmental maturation of interendothelial junctions, I pursued studies to determine if caveolin-1 associated with the junctional domain in forming and adult microvascular endothelia. The detergent-i11solubility character of caveoiin-1 in adult cerebral microvasculature was not altered by increased detergent solubilization, depolymerization of the actin or the microtubule cytoskeleton, or disruption of intercellular adhesions. Depletion of membrane cholesterol with saponin, however, increased the solubility of caveolin suggesting that caveolin-1 was localized within cholesterol-enriched microdomains. Both caveolin-1 and occludin revealed an enrichment in isolated junctional domain fractions. Caveolin-1 within the junctional domain fraction resolved as a 150-200 kDa protein complex, although a direct interaction with occludin was not apparent. Taken together, these data suggest that caveolin-1 expression in cerebral microvascular endothelium is associated with the formation and function of the junctional domain in cerebral microvasculatureItem Embargo Cell Death Contributes to Sex Differences in the Control of Blood Pressure in Spontaneously Hypertensive Rats (SHR)(Augusta University, 2021-05) Abdelbary, Mahmoud; Biomedical SciencesCell death is a hallmark of hypertension; however, little is known regarding sex differences in cell death in hypertension. The goal of Aim 1 was to determine if there are sex differences in cell death in young adult spontaneously hypertensive rats (SHR). Cell death was measured by flow cytometric analysis in the kidney, spleen and thoracic aorta. We found greater renal necrosis in male SHR compared to females. Whereas female SHR had greater renal and aortic apoptosis compared to males. Necrosis is a pathologic form of cell death, whereas apoptosis is a physiologic form of cell death. The contribution of cell death to the reported sex differences in renal T cell profile or the control of blood pressure (BP) in either sex is unknown. Therefore, studies in Aims 2 and 3 were designed to determine the contributions of necrosis (Aim 2) and apoptosis (Aim 3) to the development of hypertension, control of BP in established hypertension, and the renal T cell profile in both male and female SHR. We found that necrosis, but not apoptosis contributes to the control of BP and the development of hypertension in male, but not female SHR. In addition, necrosis, but not apoptosis contributed to the pro-inflammatory renal T cell profile in SHR, although to a comparable degree in both sexes. The goal of Aim 4 was to gain additional mechanistic insight into why males had greater necrosis vs. females. It is well-established that the male sex hormone testosterone contributes to the sex differences in BP in SHR. Therefore, Aim 4 was designed to determine the relative contributions of testosterone vs. high BP on renal necrosis in male SHR. To achieve this, male SHR were subjected to surgical castration prior to sexual maturation or treated with BP-lowering drugs prior to the development of hypertension. We found that testosterone, but not high BP contributes to renal necrosis when compared to sham male SHR. The massive workload imposed on renal tubular epithelial cells (TECs) requires them to consume high amounts of energy and studies showed that TECs from male SHR are more prone to stress-induced cell death compared to TECs from male Wistar Kyoto rats (WKY). Additional studies in Aim 4 were further designed to determine if TECs isolated from male SHR are more prone to oxidative stress-induced necrotic cell death when compared to cells from females. TECs from males showed greater necrotic cell death compared to cells from age-matched female SHR. From these studies, we conclude that male SHR exhibit a testosterone-mediated increase in renal necrotic cell death which contributes to the sex differences in BP in SHR by exacerbating increases in BP in male, but not female SHR. Targeting necrotic cell death in males could allow for better control of BP to reduce overall cardiovascular morbidity and mortality.Item Open Access AKAP350 Targets to the Golgi Apparatus Where it Interacts With clic5b and cip4/5(Augusta University, 2002-03) Shanks, Ryan Ayer; Medical College of GeorgiaA-kinase anchoring proteins (AKAPs) are defined by their ability to scaffold PKA, but their function depends upon their targeting of PKA and other scaffolded signaling proteins to specific subcellular compartments. We have investigated one AKAP, AKAP350, which can scaffold a number of protein kinases and phosphatases at the centrosome and the Golgi apparatus. The AKAP350 gene is multiply spliced to create three carboxyl terminal splice variants which we have designated AKAP350A, AKAP350B and AKAP350C. Immunocytochemistry in HCA-7 cells demonstrated that AKAP350A was localized specifically to the Golgi apparatus. GFP-fusion proteins representing the carboxyl terminus of AKAP350A identified a carboxyl terminal region responsible for the Golgi apparatus targeting of AKAP350A. Yeast two-hybrid analysis was utilized to screen a rabbit gastric parietal cell library with a 3.2kb segment of AKAP350 (nucleotides 3611-6813) which is weakly homologous to pericentrin. This screen yielded two positive clones representing rabbit chloride intracellular channel 1 (CLICl) and rabbit Cdc42 interacting protein 5 (CIPS). Further yeast-two hybrid binary analysis determined that CLICl and CIPS bound to AKAP350 through adjacent domains located with in the PHR. CLICl belongs to a family of proteins which all contain a high degree of homology in their carboxyl termini, and this conserved domain is responsible for several CLIC family member's ability to bind AKAP350. We isolated the human homologue of bovine p64, CLIC5B, from an HCA-7 colonic adenocarcinoma ceil eDNA. A splice variant of CLIC5, the predicted molecular weight of CLIC5B corresponds to the molecular weight of a major CLIC immunoreactive protein in HCA-7 cells. Immunocytochemistry determined that CLIC5B colocalized with AKAP350 at the Golgi apparatus. Yeast-two hybrid binary analysis determined that the final 120 amino acids of CLIC5B interacted with AKAP350. Furthermore, expression of a GFP-fusion protein containing the final 120 amino acids targeted to the Golgi apparatus in HCA-7 cells. CIP5 contains high homology to the human protein Cdc42 interacting protein 4 (CIP4). Yeast-two hybrid binary analysis determined that the first 117 amino acids of both human CIP4 and CIP5 interacted with AKAP350. Immunocytochemistry in HCA-7 ceiis determined with an antibody recognizing CIP4 and CIP5 localized to the Golgi apparatus. These results suggest that AKAP350 associates with CLIC proteins and CIP4/5, and · these proteins interact with the AKAP35A splice variant at the Golgi apparatus.