• A Kinetic Analysis of Calcium-Triggered Exocytosis

      Blank, Paul S.; Vogel, Steven S.; Malley, James D.; Zimmerberg, Joshua; Institute of Molecular Medicine and Genetics (2001-08-1)
      Although the relationship between exocytosis and calcium is fundamental both to synaptic and nonneuronal secretory function, analysis is problematic because of the temporal and spatial properties of calcium, and the fact that vesicle transport, priming, retrieval, and recycling are coupled. By analyzing the kinetics of sea urchin egg secretory vesicle exocytosis in vitro , the final steps of exocytosis are resolved. These steps are modeled as a three-state system: activated, committed, and fused, where interstate transitions are given by the probabilities that an active fusion complex commits (a ) and that a committed fusion complex results in fusion, p . The number of committed complexes per vesicle docking site is Poisson distributed with mean n. Experimentally, p and n increase with increasing calcium, whereas a and the p/n ratio remain constant, reducing the kinetic description to only one calcium-dependent, controlling variable, . On average, the calcium dependence of the maximum rate (R max ) and the time to reach R max (T peak ) are described by the calcium dependence of n . Thus, the nonlinear relationship between the free calcium concentration and the rate of exocytosis can be explained solely by the calcium dependence of the distribution of fusion complexes at vesicle docking sites.
    • Kynurenine is a novel endothelium-derived vascular relaxing factor produced during inflammation

      Wang, Yutang; Liu, Hanzhong; McKenzie, Gavin; Witting, Paul K; Stasch, Johannes-Peter; Hahn, Michael; Jackman, Katherine A; Changsirivathanathamrong, Dechaboon; Wu, Ben J; Ball, Helen J; et al. (2009-08-11)
    • The Lack of AC1 Impairs the Inhibitory Effects of cAMP on Cell Mitigation and Proliferation in Pancreatic Cancer

      Quinn, Sierra; Sabbatini, Maria; College of Science and Mathematics (2015-02-06)
      Introduction: Adenylyl cyclase (AC) is an important enzyme in signal transduction processes. This protein catalyzes the conversion of ATP to cAMP. We found five isoforms of AC in human pancreatic carcinoma HPAC cell line: AC1, AC3, AC6, AC7 and AC9. Both AC1 and AC3 are very important because they are up-regulated in HPAC cells. We also found that forskolin (FSK}, inhibits cell proliferation and cell migration but not cell invasion in HPAC cells. Objective: To silence AC1 and AC3 and determine their participation in the inhibitory effect of cAMP on cell migration and proliferation of HPAC cells. Results: To study the functional roles of AC1 and AC3, the expression of both isoforms were knocked-out using siRNA (human). The lack of either AC1 or AC3 proteins was assessed using Western-blotting. When testing for cell proliferation and cell migration, we found that the inhibitory effect of FSK was impaired in the presence of siRNA AC1, but not in the presence of siRNA AC3. Discussion: We conclude that AC1 mediates the inhibitory effect of cAMP in cell proliferation and migration. Begin Time: 26:09 End Time: 44:52
    • Lack of an association of miR-938 SNP in IDDM10 with human type 1 diabetes

      Mi, Xiaofan; He, Hongzhi; Deng, Yangxin; Levin, Abert M; She, Jin-Xiong; Mi, Qing-Sheng; Zhou, Li; Center for Biotechnology and Genomic Medicine (2011-10-20)
      MicroRNAs (miRNAs) are a newly discovered type of small non-protein coding RNA that function in the inhibition of effective mRNA translation, and may serve as susceptibility genes for various disease developments. The SNP rs12416605, located in human type 1 diabetes IDDM10 locus, changes the seeding sequence (UGU[G/A]CCC) of miRNA miR-938 and potentially alters miR-938 targets, including IL-16 and IL-17A. In an attempt to test whether miR-938 may be a susceptibility gene for IDDM10, we assessed the possible association of the miR-938 SNP with T1D in an American Caucasian cohort of 622 patients and 723 healthy controls by TaqMan assay. Our current data do not support the association between the SNP in miR-938 and type 1 diabetes.
    • Lack of correlation between the levels of soluble cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and the CT-60 genotypes.

      Purohit, Sharad; Podolsky, Robert H.; Collins, Christin; Zheng, Weipeng; Schatz, Desmond; Muir, Andy; Hopkins, Diane; Huang, Yi-Hua; She, Jin-Xiong; Center for Biotechnology and Genomic Medicine (2005-11-24)
      BACKGROUND: Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) plays a critical role in downregulation of antigen-activated immune response and polymorphisms at the CTLA-4 gene have been shown to be associated with several autoimmune diseases including type-1 diabetes (T1D). The etiological mutation was mapped to the CT60-A/G single nucleotide polymorphism (SNP) that is believed to control the processing and production of soluble CTLA-4 (sCTLA-4). METHODS: We therefore determined sCTLA-4 protein levels in the sera from 82 T1D patients and 19 autoantibody positive (AbP) subjects and 117 autoantibody negative (AbN) controls using ELISA. The CT-60 SNP was genotyped for these samples by using PCR and restriction enzyme digestion of a 268 bp DNA segment containing the SNP. Genotyping of CT-60 SNP was confirmed by dye terminating sequencing reaction. RESULTS: Higher levels of sCTLA-4 were observed in T1D (2.24 ng/ml) and AbP (mean = 2.17 ng/ml) subjects compared to AbN controls (mean = 1.69 ng/ml) with the differences between these subjects becoming significant with age (p = 0.02). However, we found no correlation between sCTLA-4 levels and the CTLA-4 CT-60 SNP genotypes. CONCLUSION: Consistent with the higher serum sCTLA-4 levels observed in other autoimmune diseases, our results suggest that sCTLA-4 may be a risk factor for T1D. However, our results do not support the conclusion that the CT-60 SNP controls the expression of sCTLA-4.
    • Large Scale Gene Expression Analysis Reveals Insight into Pathways Related to Type 1 Diabetes and Associated Complications

      Carey, Colleen M.; Center for Biotechnology and Genomic Medicine (2013-08)
      Type 1 Diabetes (T1D) is a chronic inflammatory disease resulting from complex interactions between susceptibility genes, the environment, and the immune system, ultimately leading to the destruction o f pancreatic islet cells and insulin deficiency. Previous studies have examined the series o f molecular, cellular, and protein changes occurring within subsets of individuals and how these are associated with particular disease states. Genome wide association studies have revealed a large number o f genetic susceptibility intervals including those implicated in disease pathogenesis, the identification o f various markers for risk assessment, the classification o f disease or complications, and finally markers for monitoring therapies for disease. However, none of these studies to date is without seriously limitations. First, although microarray based gene expression profiling is a powerful tool in discovery; results must be validated by alternate techniques. Second, due to the inherent heterogeneity of the human population large sample sizes in each group must be used in order to handle the expected large expression variations among individual subject. Third, for accurate normalization of Real-Time PCR expression data appropriate reference genes must be selected. We proposed a large scale gene expression validation study to address the limitations of previous studies. Validation studies were performed using high throughput Real-Time RT-PCR on peripheral blood mononuclear cells (PBMCs) o f 928 individuals with T1D and 922 individuals as antibody negative (AbN) controls, recruited through the Prospective Assessment in Newborns of Diabetes Autoimmunity (PANDA) study. This dissertation work validated the gene expression changes among 28 genes shown to have differential expression in T1D patients as compared to controls. These genes were selected based on their function, role in inflammatory or the immune response, and any previously documented reference to a role in T1D. Our aims were to 1) identify gene expression changes which may be occurring specifically in diabetic complications, and 2) identify gene expression changes which may result in an increased state o f oxidative stress in the diabetic state. For validation studies, we divided the 28 genes into two subsets based on related function to ask whether any gene expression signatures could be associated with diabetes, diabetic complications, or oxidative stress in the diabetic state. Our studies revealed genes that are involved in inflammation, immune regulation, and antigen processing and presentation are significantly altered in the PBMCs o f T1D patients. Eight genes (S100A8, S100A9, MNDA, SELL, TGFB1, PSMB3, CD74, and IL12A) were shown to have higher expression, with three genes (GNLY, PSMA4, and SMAD7) having lower expression, in T1D when compared to controls. The data also suggested that inflammatory mediators secreted mainly by myeloid cells are implicated in T1D and its complications (Odds ratios OR = 1.3-2.6, adjusted P value= 0.005- 1.08 x 10 8), and particularly in those patients with nephropathy (OR=4.8-7.9, adjusted P value < 0.005). Validation studies also revealed nine genes (LAT2, MAPK1, APOBEC3B, SOD2, NDUFB3, STK40, PRKD2, ITGB2, and COX7B) with higher expression in T1D. These genes are involved in general pathways of inflammation and immune response; however SOD2, NDUFB3, and COX7B (OR=l.l-1.27, adjusted P value= 0.007-0.47) are functionally involved in the mechanisms o f the mitochondria and may play a role in the increased state of oxidative stress seen in T1D. In these studies we have validated and confirmed the gene expression differences between T1D and control subjects initially suggested by microarray. Our experimental design has addressed each of the limitations posed by earlier studies in the largest scale study to date on gene expression profiles in human T1D. We have demonstrated that gene expression is significantly different between autoantibody negative (AbN) controls and T1D patients without any complications. Genes implicated in immune function (S100A8, S100A9, MNDA, IL12A), immune regulation and promotion (TGFB1, SELL), antigen processing and presentation (CD74, PSMB3), and mitochondrial function (SOD2, NDUFB3, COX7B) have higher expression in T1D and support the notion that chronic inflammation and cellular oxidative stress contribute to the development of T1D and associated complications. The understanding gained from our results implies a translational potential for the use o f gene expression profiles in the classification o f at risk individuals for both T1D and complication. Further, our understanding into the role that the immune system plays in cellular oxidative stress leading to the diabetic state may serve to provide prevention therapies however there remains much to be learned before this is attainable.
    • Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes.

      Merkley, Mark A.; Hildebrandt, Ellen; Podolsky, Robert H.; Arnouk, Hilal; Ferris, Daron G.; Dynan, William S.; Stöppler, Hubert; Institute of Molecular Medicine and Genetics; Center for Biotechnology and Genomic Medicine; Department of Obstetrics and Gynecology; et al. (2009-09-16)
      BACKGROUND: Infection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features. RESULTS: Six replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE). The median within-group coefficient of variation was 19-21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23%) of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2%) were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1); and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27). CONCLUSION: This large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.
    • Lectin-based food poisoning: a new mechanism of protein toxicity.

      Miyake, Katsuya; Tanaka, Toru; McNeil, Paul L.; Institute of Molecular Medicine and Genetics; Department of Cellular Biology and Anatomy (2007-08-01)
      BACKGROUND: Ingestion of the lectins present in certain improperly cooked vegetables can result in acute GI tract distress, but the mechanism of toxicity is unknown. In vivo, gut epithelial cells are constantly exposed to mechanical and other stresses and consequently individual cells frequently experience plasma membrane disruptions. Repair of these cell surface disruptions allows the wounded cell to survive: failure results in necrotic cell death. Plasma membrane repair is mediated, in part, by an exocytotic event that adds a patch of internal membrane to the defect site. Lectins are known to inhibit exocytosis. We therefore tested the novel hypothesis that lectin toxicity is due to an inhibitory effect on plasma membrane repair. METHODS AND FINDINGS: Repair of plasma membrane disruptions and exocytosis of mucus was assessed after treatment of cultured cell models and excised segments of the GI tract with lectins. Plasma membrane disruptions were produced by focal irradiation of individual cells, using a microscope-based laser, or by mechanical abrasion of multiple cells, using a syringe needle. Repair was then assessed by monitoring the cytosolic penetration of dyes incapable of crossing the intact plasma membrane. We found that cell surface-bound lectins potently inhibited plasma membrane repair, and the exocytosis of mucus that normally accompanies the repair response. CONCLUSIONS: Lectins potently inhibit plasma membrane repair, and hence are toxic to wounded cells. This represents a novel form of protein-based toxicity, one that, we propose, is the basis of plant lectin food poisoning.
    • Let's talk about sex: understanding psychosocial and high risk sexual behaviors among detained juveniles

      Swartz, Sarah; Willcox, Lyndsey; Gates, Madison L.; Augusta University (2016-07)
    • LGN regulates mitotic spindle orientation during epithelial morphogenesis

      Zheng, Zhen; Zhu, Huabin; Wan, Qingwen; Liu, Jing; Xiao, Zhuoni; Siderovski, David P.; Du, Quansheng; Institute of Molecular Medicine and Genetics; Department of Neurology (2010-04-19)
      Coordinated cell polarization and mitotic spindle orientation are thought to be important for epithelial morphogenesis. Whether spindle orientation is indeed linked to epithelial morphogenesis and how it is controlled at the molecular level is still unknown. Here, we show that the NuMA- and Ga-binding protein LGN is required for directing spindle orientation during cystogenesis of MDCK cells. LGN localizes to the lateral cell cortex, and is excluded from the apical cell cortex of dividing cells. Depleting LGN, preventing its cortical localization, or disrupting its interaction with endogenous NuMA or Ga proteins all lead to spindle misorientation and abnormal cystogenesis. Moreover, artificial mistargeting of endogenous LGN to the apical membrane results in a near 90° rotation of the spindle axis and profound cystogenesis defects that are dependent on cell division. The normal apical exclusion of LGN during mitosis appears to be mediated by atypical PKC. Thus, cell polarizationâ mediated spatial restriction of spindle orientation determinants is critical for epithelial morphogenesis.
    • LIM and SH3 protein-1 modulates CXCR2-mediated cell migration.

      Raman, Dayanidhi; Sai, Jiqing; Neel, Nicole F; Chew, Catherine S; Richmond, Ann; Institute of Molecular Medicine and Genetics (2010-04-26)
      BACKGROUND: The chemokine receptor CXCR2 plays a pivotal role in migration of neutrophils, macrophages and endothelial cells, modulating several biological responses such as angiogenesis, wound healing and acute inflammation. CXCR2 is also involved in pathogenesis of chronic inflammation, sepsis and atherosclerosis. The ability of CXCR2 to associate with a variety of proteins dynamically is responsible for its effects on directed cell migration or chemotaxis. The dynamic network of such CXCR2 binding proteins is termed as "CXCR2 chemosynapse". Proteomic analysis of proteins that co-immunoprecipitated with CXCR2 in neutrophil-like dHL-60 cells revealed a novel protein, LIM and SH3 protein 1 (LASP-1), binds CXCR2 under both basal and ligand activated conditions. LASP-1 is an actin binding cytoskeletal protein, involved in the cell migration. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that CXCR2 and LASP-1 co-immunoprecipitate and co-localize at the leading edge of migrating cells. The LIM domain of LASP-1 directly binds to the carboxy-terminal domain (CTD) of CXCR2. Moreover, LASP-1 also directly binds the CTD of CXCR1, CXCR3 and CXCR4. Using a site-directed and deletion mutagenesis approach, Iso323-Leu324 of the conserved LKIL motif on CXCR2-CTD was identified as the binding site for LASP-1. Interruption of the interaction between CXCR2-CTD and LIM domain of LASP-1 by dominant negative and knock down approaches inhibited CXCR2-mediated chemotaxis. Analysis for the mechanism for inhibition of CXCR2-mediated chemotaxis indicated that LASP-1/CXCR2 interaction is essential for cell motility and focal adhesion turnover involving activation of Src, paxillin, PAK1, p130CAS and ERK1/2. CONCLUSIONS/SIGNIFICANCE: We demonstrate here for the first time that LASP-1 is a key component of the "CXCR2 chemosynapse" and LASP-1 interaction with CXCR2 is critical for CXCR2-mediated chemotaxis. Furthermore, LASP-1 also directly binds the CTD of CXCR1, CXCR3 and CXCR4, suggesting that LASP-1 is a general mediator of CXC chemokine mediated chemotaxis. Thus, LASP-1 may serve as a new link coordinating the flow of information between chemokine receptors and nascent focal adhesions, especially at the leading edge. Thus the association between the chemokine receptors and LASP-1 suggests to the presence of a CXC chemokine receptor-LASP-1 pro-migratory module in cells governing the cell migration.
    • Linear Approaches to Intramolecular Forster Resonance Energy Transfer Probe Measurements for Quantitative Modeling

      Birtwistle, Marc R.; von Kriegsheim, Alexander; Kida, Katarzyna; Schwarz, Juliane P.; Anderson, Kurt I.; Kolch, Walter; GHSU Cancer Center (2011-11-16)
      Numerous unimolecular, genetically-encoded Forster Resonance Energy Transfer (FRET) probes for monitoring biochemical activities in live cells have been developed over the past decade. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells and tissues, they are an attractive technology for obtaining data to develop quantitative, mathematical models of spatiotemporal signaling dynamics. However, to be useful for such purposes the observed FRET from such probes should be related to a biological quantity of interest through a defined mathematical relationship, which is straightforward when this relationship is linear, and can be difficult otherwise. First, we show that only in rare circumstances is the observed FRET linearly proportional to a biochemical activity. Therefore in most cases FRET measurements should only be compared either to explicitly modeled probes or to concentrations of products of the biochemical activity, but not to activities themselves. Importantly, we find that FRET measured by standard intensity-based, ratiometric methods is inherently non-linear with respect to the fraction of probes undergoing FRET. Alternatively, we find that quantifying FRET either via (1) fluorescence lifetime imaging (FLIM) or (2) ratiometric methods where the donor emission intensity is divided by the directly-excited acceptor emission intensity (denoted Ralt) is linear with respect to the fraction of probes undergoing FRET. This linearity property allows one to calculate the fraction of active probes based on the FRET measurement. Thus, our results suggest that either FLIM or ratiometric methods based on Ralt are the preferred techniques for obtaining quantitative data from FRET probe experiments for mathematical modeling purposes.
    • Loss of Jak2 selectively suppresses DC-mediated innate immune response and protects mice from lethal dose of LPS-induced septic shock.

      Zhong, Jixin; Yang, Ping; Muta, Kenjiro; Dong, Robert; Marrero, Mario; Gong, Feili; Wang, Cong-Yi; Center for Biotechnology and Genomic Medicine; Vascular Biology Center (2010-03-16)
      Given the importance of Jak2 in cell signaling, a critical role for Jak2 in immune cells especially dendritic cells (DCs) has long been proposed. The exact function for Jak2 in DCs, however, remained poorly understood as Jak2 deficiency leads to embryonic lethality. Here we established Jak2 deficiency in adult Cre(+/+)Jak2(fl/fl) mice by tamoxifen induction. Loss of Jak2 significantly impaired DC development as manifested by reduced BMDC yield, smaller spleen size and reduced percentage of DCs in total splenocytes. Jak2 was also crucial for the capacity of DCs to mediate innate immune response. Jak2(-/-) DCs were less potent in response to inflammatory stimuli and showed reduced capacity to secrete proinflammatory cytokines such as TNFalpha and IL-12. As a result, Jak2(-/-) mice were defective for the early clearance of Listeria after infection. However, their potency to mediate adaptive immune response was not affected. Unlike DCs, Jak2(-/-) macrophages showed similar capacity secretion of proinflammatory cytokines, suggesting that Jak2 selectively modulates innate immune response in a DC-dependent manner. Consistent with these results, Jak2(-/-) mice were remarkably resistant to lethal dose of LPS-induced septic shock, a deadly sepsis characterized by the excessive innate immune response, and adoptive transfer of normal DCs restored their susceptibility to LPS-induced septic shock. Mechanistic studies revealed that Jak2/SATA5 signaling is pivotal for DC development and maturation, while the capacity for DCs secretion of proinflammatory cytokines is regulated by both Jak2/STAT5 and Jak2/STAT6 signaling.
    • Loss of Zebrafish lgi1b Leads to Hydrocephalus and Sensitization to Pentylenetetrazol Induced Seizure-Like Behavior

      Teng, Yong; Xie, Xiayang; Walker, Steven L.; Saxena, Meera T.; Kozlowski, David J.; Mumm, Jeff S.; Cowell, John K.; GHSU Cancer Center; Department of Cellular Biology and Anatomy; Vision Discovery Institute; et al. (2011-09-16)
      Mutations in the LGI1 gene predispose to a hereditary epilepsy syndrome and is the first gene associated with this disease which does not encode an ion channel protein. In zebrafish, there are two paralogs of the LGI1 gene, lgi1a and lgi1b. Knockdown of lgi1a results in a seizure-like hyperactivity phenotype with associated developmental abnormalities characterized by cellular loss in the eyes and brain. We have now generated knockdown morphants for the lgi1b gene which also show developmental abnormalities but do not show a seizure-like behavior. Instead, the most striking phenotype involves significant enlargement of the ventricles (hydrocephalus). As shown for the lgi1a morphants, however, lgi1b morphants are also sensitized to PTZ-induced hyperactivity. The different phenotypes between the two lgi1 morphants support a subfunctionalization model for the two paralogs.
    • Measuring Surface Tension Using the Pendant Drop Method

      Jaleel Bolden; Zane Corder; Charlene Higdon; Miller, Camille; College of Science and Mathematics; Millan, Josefa Guerrero (2015-04-17)
      Measuring surface tension between fluids is of a great practical importance in the oil, food, chemical, cosmetic, etc. industries. Also, the measurement of the contact angle tl1at is formed between the fluid interface and a restricting wall are of prime importance in surface science. Instruments that employ the DuNouy ring and Wilhelmy plate methods are in common use at industry and research laboratories but they are very labor intensive. Th ere is a need for a rapid, easy and low cost technique with satisfactory accuracy and reproducibility. Reliable measurement of these parameters require significant computer programming and image analysis. The goal of the project has been the development of a computer program written in Matlab which use the pendant drop method to measure t he surface tension between two fluids. In addition, an experimental setup has been developed to test the accuracy of this method. In the framework of the soft matter, where tl1e surface tension of tl1e fluids plays a key role, the use of more complex and biological fluids make harder to find data a bout how interact these fluids. This is the reason that codes like these are inclispensable tools in these laboratories.
    • Mechanisms of Neuroprotection by Estrogen and Selective Estrogen Receptor Modulators

      Dhandapani, Krishnan M.; Institute of Molecular Medicine and Genetics (2003-03)
      Specific Aim #1: To determine whether 17b-E2 and SERMs directly influence neuronal survival. Specific Aim #2: To determine whether astrocyte-derived TGF-b protects neurons from cell death. Specific Aim #3: To determine whether 17b-E2 or SERMs regulate the release of TGF-b from astrocytes. Specific Aim #4:To elucidate the mechanism of TGF-b mediated neuroprotection in GT1-7 Neurons. Specific Aim #5: To identify genes potentially mediating the neuroprotective effects of 17b-E2 and/or tamoxifen through the use of high density gene chip arrays.
    • Mechanisms of Vessel Obliteration in Oxygen-Induced Retinopathy

      Gu, Xiaolin; Vascular Biology Center (2001-11)
      The overall goal of this study was to explore the possible molecular mediators of vaso-obliteration in retinopathy o f prematurity. Vaso-obliteration is the early hyperoxiainduced pathology. It leads to the later relative hypoxia in the retina tissue, because the insufficient blood supply cannot meet the increasing demands o f oxygen from the developing retina. Such retinal hypoxia then causes the blinding outcome through the formation o f neovessels and subsequent vitreous bleeding and fibrotic change in both retina and vitreous. Therefore, identification o f the possible mediators o f hyperoxiainduced vaso-obliteration will help us to understand more about the pathogenesis o f ROP and provide new and better strategies of treating and preventing this disease. Previous studies have shown that administration o f exogenous antioxidants can attenuate retinopathy in certain animal models and that hyperoxia is able to upregulate the expression and activity o f eNOS in vascular endothelial cells (Liao et al., 1995; North et al., 1996; Phelan and Faller, 1996). Hyperoxia also increases formation o f O2 ' which rapidly combines with NO to form the highly reactive oxidant ONOO*. Therefore, it is hypothesized that the NO and O2 'derived oxidant, ONOO', play an important role in the initial vascular injury leading to obliteration of the developing retinal capillaries in oxygen-induced retinopathy (OIR). It is further proposed that ONOO' causes vascular injury by modifying the critical intracellular signaling pathway that controls endothelial cell survival (Fig 5). This hypothesis has been tested by accomplishing the following specific aims: 1. Establish the OIR mouse model for ROP. Analyze NOS expression and assay the formation of NO and ONOO' in the vaso-obliteration phase o f OIR. 2. Determine whether deletion o f the eNOS or iNOS gene alters the vaso-obliteration phase o f OIR. If so, determine whether the gene deletion also reduces ONOO' formation in the vaso-obliteration phase o f OIR. 3. Test whether or not pharmacological inhibition o f NOS reduces vascular obliteration in wild-type mice with OIR. 4. Establish a tissue culture model for oxygen-induced endothelial cell injury. Determine the effect o f hyperoxia on endothelial cell survival and test whether the effects are mediated by NO, O2 ', and /or ONOO'. 5. Test whether ONOO' alters the signal transduction pathway for endothelial cell survival by altering the activity o f PI3K/AKT.
    • Mechanosensory Hair Cell Precursors in the Zebrafish Lateral Line

      Floyd, Tiffany L.; Institute of Molecular Medicine and Genetics (2009-07)
      The vertebrate inner ear mediates the senses of hearing and balance. Contained within both the auditory and vestibular compartments of the inner ear are specialized mechanosensory hair cells that function as receptors and transducers of environmental stimuli. In all vertebrates, these sensory hair cells are particularly susceptible to ototoxic insults resulting in cell death and, in mammals, the irreversible loss of hair cells underlies deafness and balance disorders. In contrast to mammals, several non-mammalian vertebrates (including zebrafish) possess the innate capacity to produce new hair cells throughout life as well as regenerate hair cells that have been lethally damaged. A long-term strategy of the hearing research field is to determine the molecular mechanisms of hair cell regeneration using regenerating model systems such as zebrafish, then to apply this information to mammalian models where sensory hair cell regeneration is limited or nonexistent. During embryogenesis, sensory hair cell fates are specified through a mechanism of Notch-Delta-mediated lateral inhibition. The gamma secretase complex is an upstream regulator of Notch signaling, responsible for proteolytic\ cleavage and activation of the Notch receptor. Recent evidence suggests that Notch signaling may also play a role during the process of hair cell regeneration in zebrafish (Ma et al., 2008). I used a chemical inhibitor of the gamma secretase complex to examine the role of Notch signaling in the regulation of hair cell number maintenance in larval zebrafish. Results presented in this thesis provide novel insight into the mechanisms regulating the maintenance of resident hair cell precursors within the sensory epithelium. Moreover, this new information is directly relevant to research efforts in mammalian models by providing the molecular framework for therapeutic strategies designed to replace or regenerate lethally damaged hair cells in the mammalian cochlea by reactivating resident precursors to differentiate into hair cells.
    • MedEdPORTAL Module Guides Evaluation of Faculty Fellowship

      Richardson, D; Villarosa, M; Palladino, Christie; Thomas, Andria M.; Education Discovery Institute; Medical College of Georiga; Georgia Health Sciences University (Georgia Health Sciences University, 2011)
      Professional development of faculty is critical to the future of health sciences education. The Education Discovery Institute (EDI) at Georgia Health Sciences University (GHSU) developed a year-long Educational Research Fellowship designed to nurture faculty career progression by providing training in health sciences education research and fostering career development. As we developed the program, we realized the need for a careful evaluation plan.