• Diabetic Membrane Repair Deficiency and Repair Promotion By Vitamin E

      Howard, Amber Cyran; Department of Cellular Biology and Anatomy (5/30/2014)
      Myopathy, characterized by muscle necrosis and atrophy, is a diabetic complication. The myopathy of at least one muscular dystrophy is linked to defective membrane repair. We hypothesized that defective membrane repair is also associated with diabetic myopathy. To test this hypothesis, we monitored repair in intact muscle from diabetic type 1 (INS2Akita+/-) and type 2 (db/db) mouse models. Myocytes were laser injured in the presence of a membrane impermeant dye, and cellular dye uptake through the disruption site was monitored. Dye influx of diabetic myocytes was significantly increased, compared to controls, indicating repair deficiency. This defect was mimicked in cultured cell models by high (30 mM) glucose exposure. Inhibiting the high glucose formation of advanced glycation endproducts (AGE) prevented this repair defect, but was induced in the absence of high glucose exposure by enhanced AGE receptor (RAGE) binding. We conclude that high glucose exposure leads to defective membrane repair in skeletal muscle, and that AGE/RAGE interactions underlie this defect. AGE/RAGE binding also induces generation of reactive oxygen species (ROS), which is increased in diabetes. ROS are also produced in skeletal muscle during eccentric contracts, an act that creates muscle membrane disruptions. Using a potent antioxidant, vitamin E (α-tocopherol), we were able to reverse the high glucose exposure repair defect. Interestingly, diets deficient in vitamin E results in a lethal muscular dystrophy. α-Tocopherol partitions into membrane bilayers where it is thought to act as a membrane stabilizer and/or as an antioxidant. We hypothesize that one important biological role of vitamin E is to promote muscle membrane repair. To test this hypothesis, cultured muscle cells were loaded with α-tocopherol and repair assessed with the laser assay. α-Tocopherol loading significantly decreased cellular dye influx, indicating that repair had been promoted. Strikingly, the HeLa cell, a non-muscle cell that normally displays unrestricted dye influx after laser disruption, e.g. not capable of repair via this form of injury, became repair competent after loading with α-tocopherol. Vitamin C, another antioxidant that can be loaded into cells, also significantly decreased dye influx after laser injury. However, horseradish peroxidase, an antioxidant that lacks transport across the plasma membrane was found to be ineffective in promoting repair. Cells injured in the presence of H2O2, displayed significantly more dye influx than controls injured in physiological saline lacking this oxidant. If however cells were loaded with vitamin E the H2O2 did not affect repair. We further tested H2O2 exposure in intact mouse skeletal muscle, and found repair to be significantly impaired. However, comparable to vitamin E loading in the cell model, Trolox (a water soluble analog of vitamin E) pretreatment prevented the H2O2 muscle membrane repair defect. We conclude that vitamin E promotes plasma membrane repair, and that its capacity as an anti-oxidant is crucial in this role.
    • IN VITRO AND IN VIVO STUDIES DEMONSTRATE A ROLE FOR SH3PX1 IN LAMELLIPODIA FORMATION.

      Hicks, Lawrence Joseph; Department of Cellular Biology and Anatomy (5/22/2018)
      Actin remodeling and endocytosis are essential functions for most cells. Defects in these processes present in a variety of diseases. Sorting nexins are known to contribute to endocytic uptake, cytokinesis, the retromer complex, and autophagy. Sorting nexin 9 (Snx9) interacts with major endocytic factors and proteins involved in regulation of actin cytoskeleton dynamics. Nonetheless, Snx9’s exact in vivo roles in these basic cellular processes and disease mechanisms are not known. By examining the roles of Sh3px1, we can better understand the mechanism by which this protein contributes to endocytosis and actin remodeling in vivo. Two additional paralogs, Snx18 and Snx33, complicate studies in mammalian models due to potential redundant mechanisms. Utilizing the single ortholog in Drosophila, sh3px1, this report describes the function of Sh3px1 in membrane organization and actin dynamics. Drosophila S2 cells that are depleted of Sh3px1 fail to form lamellipodia, a process that is also dependent on the actin nucleation factor, Scar. In addition, over-expression of Sh3px1 in S2 cells results in the formation of tubules and also long membrane protrusions, atypical of a classical BAR domain protein. An intact PX-BAR domain is required for these overexpression phenotypes. sh3px1 null flies are viable; however, mutant females have significantly compromised fertility. Female sh3px1 null egg chambers show many morphological defects. The age-dependent degeneration of the null egg chamber is not likely due to compromised endocytosis. Additionally, collective border cell migration is attenuated in the absence of Sh3px1. These cells are known for their reliance on endocytosis and modulation of actin dynamics for migration. We have found that Sh3px1 is essential in efficient lamellipodia production at the start of border cell migration. Our findings also suggest that Scar directly interacts with Sh3px1 and is upregulated in sh3px1 nulls. Mutation of Scar enhances many reproductive defects in sh3px1 nulls. Thus, our work reveals a main in vivo function of Sh3px1 in actin regulation for the production of structures such as lamellipodia.
    • DNA METHYLATION REGULATION IN ACUTE KIDNEY INJURY

      Guo, Chunyuan; Department of Cellular Biology and Anatomy (4/26/2018)
      DNA methylation is a critical epigenetic mechanism, which is heritable during cell division, but does not involve the change of DNA sequence. It plays an essential role in regulating gene transcription in physiological and disease conditions. However, little is known about DNA methylation in renal diseases, especially in acute kidney injury (AKI). In this study, the role of DNA methylation in AKI was determined in both cell culture and mouse models. In cell culture, 5-aza-2’-deoxycytidine (5-aza), a pharmacological DNA methylation inhibitor, was used to inhibit DNA methylation. Interestingly, 5-aza increased both cisplatin- and hypoxia-induced apoptosis. These results suggest pharmacologic blockade of DNA methylation by 5-aza sensitizes renal tubular cells to apoptosis, supporting a cytoprotective role of DNA methylation in AKI. To determine the role of DNA methylation in vivo, we first successfully established conditional knockout mice that were deficient in DNMT1, DNMT3a or both exclusively in proximal tubules. In cisplatin-induced AKI, consistent with the effects of 5-aza in the cell culture, ablation of DNMT1 from proximal tubules exacerbated cisplatin-induced AKI in mice, and primary proximal tubular cells from PT-DNMT1-KO mice were more sensitive to cisplatin-induced apoptosis than wild-type cells. In sharp contrast, PT-DNMT1/3a-DK mice attenuated cisplatin-induced AKI, and primary proximal tubular cells from PT-DNMT1/3a-DK mice were more resistant to cisplatin-induced apoptosis. However, PT-DNMT3a-KO mice and PT-DNMT3a-WT mice showed similar AKI following cisplatin treatment. These results suggest different DNMTs play different roles in cisplatin-induced AKI. In ischemic AKI, none of the conditional knockout models showed differences in response to ischemia-reperfusion injury. Nevertheless, although ablation of both DNMT1 and DNMT3a in proximal tubular cells did not affect ischemia-reperfusion injury, it, indeed, suppressed renal fibroblast activation and ameliorated renal fibrosis. Furthermore, we found that Irf8 was regulated by DNA methylation during cisplatin treatment and knockdown of Irf8 in RPTC cells inhibited cisplatin-induced apoptosis, supporting a pro-death role of Irf8 in renal tubular cells. In ischemic AKI, although Bcl6 is hypermethylated and repressed in mice, overexpression of Bcl6 in RPTC cells had no impact on hypoxia-induced apoptosis. Collectively, these results suggest an important role of DNA methylation in AKI by regulating specific genes expression.
    • In search of genetic mutations for familial keratoconus

      Khaled, Mariam Lotfy; Department of Cellular Biology and Anatomy (Augusta University, 2019-05)
      Keratoconus (KC) is the most common corneal degenerative disorder and a leading cause of corneal transplantation in developed countries. KC is a multi-factorial disease with involvement of genetic, environmental, and hormonal factors. Although KC has been widely studied, the main cause of the disease and the molecular mechanism remain unknown. We aimed to study the molecular genetics of KC via utilizing next-generation sequencing technology including RNA-Seq, whole exome sequencing, and whole genome sequencing. We used RNA-Seq to study the KC-affected corneal transcriptome. We identified 436 coding RNAs and 584 lncRNAs with differential expression in the KC-affected corneas with a |fold change| ≥ 2 and a false discovery rate ≤ 0.05. Pathway analysis, using WebGestalt, indicated the enrichment of the genes involved in the extracellular matrix, protein binding, glycosaminoglycan binding, and cell migration. Co-expression analysis revealed 296 pairs of genes with significant KC-specific correlations. The RNA-Seq data analysis highlighted the potential roles of several genes (CTGF, SFRP1, AQP5, lnc-WNT4-2:1, and lnc-ALDH3A2-2:1) and pathways (TGF-β, WNT signaling, and PI3K/AKT pathways) in KC pathogenesis. Next, we used whole genome and exome sequencing to figure out the causal mutation(s) in a four-generation KC family with a linkage locus on Chr5q14.3-q21.1. We found a missense mutation in the phosphatase domain of PPIP5K2 (c.1255T>G, p.Ser419Ala). We found another missense mutation in the same domain of PPIP5K2 (c.2528A>G, p.Asn843Ser) in a second KC family. PPIP5K2 is a bifunctional enzyme involved in the inositol phosphate metabolic pathway. In vitro functional assays indicated the impact of the identified mutations on the enzymatic activity of PPIP5K2. PPIP5K2 expresses at a higher level than its homolog PPIP5K1 in both human and mouse corneas. A transgenic mouse model with the loss of phosphatase activity and elevated kinase activity of Ppip5k2 exhibited corneal structural abnormalities emphasizing the important role of PPIP5K2 in the homeostasis of corneal integrity. This study advances our knowledge of KC genetic etiology and helps in identifying a potential therapeutic target for KC.
    • STRONGER TOGETHER: A CASE-STUDY ANALYSIS OF THE IMPLEMENTATION OF A SCHOOL-BASED MENTORING PROGRAM FOR MIDDLE SCHOOL STUDENTS

      Crouch, John Jeffrey; Department of Advanced Studies and Innovation (Augusta University, 2019-05)
      Mentoring has been shown to have a positive impact on student outcomes such as attendance, behavior, and overall connectedness to school. Through strengthening relationships with a non-familial adult, mentoring has also been shown to have a positive impact on student interactions with other adults within the school environment. However, there are many logistical considerations that can adversely impact the implementation of a school-based mentoring program. This study began as a mixed methods study intended to examine the impact of a community-based mentoring program on student discipline referrals and absences. During the course of the study, the scope and methods shifted to become a qualitative study that focused on the implementation of an after-school mentoring program for middle school students. The authors employed a case-study methodology using a variety of data collection methods including interviews with mentors and administrators, a focus group with the mentees, and repeated observations of the mentoring sessions. Thematic content analysis revealed six themes: goals, experiences, perceptions, relationships, challenges to implementation, and sustainability and improvement. Findings suggest that the faculty and staff had a high level of confidence in their leadership which was likely to positively impact the mentoring program, as they were more likely to trust his decisions and work diligently to ensure that his goals for the program were met. Should a mentoring program be implemented, our findings indicated that time and prioritization are imperative to its success. Keywords: mentoring, relationships, leadership, school-based, improvement
    • Targeting cyclic GMP signaling for the treatment of gastrointestinal diseases

      Sharman, Sarah Kristen; Department of Biochemistry and Molecular Biology / Cancer Center (2017)
      Continual renewal of the luminal epithelium in the gut is essential for the maintenance of a healthy intestine as it sustains the barrier that protects underlying tissue from infiltration of material passing through the lumen. Dysregulation of homeostatic processes involved in maintenance of the barrier have been implicated in numerous gastrointestinal diseases. The cGMP signaling axis has emerged as an important regulator of homeostasis in the intestinal mucosa, and has been implicated in the suppression of visceral pain, colitis, and colon cancer. While there is considerable interest in exploiting this pathway, until recently the approaches used to increase cGMP have been limited. The present study sought to test the hypothesis that elevation of cGMP in the intestinal epithelium using PDE5 inhibitors will alter epithelial homeostasis and be therapeutic for constipation and preventative for colon cancer. Healthy mice treated with the PDE5 inhibitor sildenafil or the GC-C agonist linaclotide exhibited reduced proliferation and apoptosis, and increased numbers of differentiated secretory cells in the intestinal epithelium. In addition to these homeostatic effects, both drugs normalized intestinal transit and fecal water content in two mouse models of constipation. Furthermore, administration of sildenafil to mice treated with dextran sulfate sodium tightened the disrupted epithelial barrier. Treatment of ApcMin/+ mice with sildenafil or linaclotide significantly reduced the number of polyps per mouse (67% and 50%, respectively). The effect of these cGMP-elevating agents was not on the polyps themselves but was rather on the pre-neoplastic tissue, which was less proliferative and more apoptotic in the presence of the drugs. Taken together, the results of this study demonstrate that increasing cGMP with a pediatric dose of PDE5 inhibitors could be a potential alternative to GC-C agonists for the treatment of gastrointestinal diseases.
    • Oxidation of Dietary Amino Acids Disrupts their Anabolic Effects on Bone Marrow-Derived Mesenchymal Stem Cells

      El Refaey, Mona M.; Department of Cellular Biology and Anatomy (2016-07)
      Age-dependent bone loss has been well documented in both human and animal models. Since it has been proposed that aging is associated with an increase in the generation of damaging reactive oxygen species (ROS), our hypothesis was that the oxidized products of dietary amino acids could play a role in age-induced bone loss by altering osteoprogenitor cell differentiation and function or activating osteoclastic activity. We first examined the effects of the oxidized nutrients on the bone marrow-derived mesenchymal stem cells and our data showed a decrease in the protein and gene expression of osteogenic markers normally stimulated by nutrients. Aromatic amino acids activated signaling pathways involved in protein synthesis in vitro, and thus, in contrast, the oxidized metabolites of these aromatic amino acids had no effect on the activation of these anabolic pathways. We then examined the bone marrow concentration of the oxidized aromatic amino acids in mature (12 months) vs. aged (24 months) C57BL/6 mice and found that kynurenine, the oxidized product of the aromatic amino acid tryptophan, was found in the highest concentration in 12 months mice. Thus, we tested the effects of kynurenine, fed as a dietary supplement, on the bone mass of twelve-month-old C57BL/6 mice compared to a normal protein diet to see if the oxidized amino acid would induce a pattern consistent with age-related bone loss. Twelve-month-old, male C57BL/6 mice were fed one of four diets; 18% protein diet (normal protein diet); 8% protein diet + tryptophan; 8% protein diet + kynurenine (50 μM) and 8% protein diet + kynurenine (100 μM) for 8 wks. Bone densitometry and micro-CT analyses demonstrated bone loss following the kynurenine diet. Histological and histomorphometric studies showed a decreased bone formation and an increased MONA M. EL REFAEY Oxidation of Dietary Amino Acids Disrupts Their Anabolic Effects on Bone Marrow-Derived Mesenchymal Stem Cells (Under the direction of DR. CARLOS M. ISALES) osteoclastic activity in the kynurenine groups; these animals also exhibited an increase in serum pyridinoline, a marker of bone breakdown. Thus, these data demonstrate that feeding an oxidized product of an essential amino acid induces bone loss in a pattern consistent with accelerated aging, and we propose that one of the mechanisms involved in age-induced bone loss may be from alterations of dietary nutrients by the increased generation of ROS associated with aging.
    • Rapamycin, an evolving role in up-regulation of autophagy to improve stroke outcome and increase neuronal survival to stroke type injuries

      Buckley, Kathleen; Department of Cellular Biology and Anatomy (2015-10)
      Rapamycin was shown to reduce infarct size in a non-reperfusion and a slow reperfusion model of murine stroke; it also improved neurological score and survival in the slow-reperfusion model. The rapamycin improvement was 50 percent greater than that observed with chloroquine. In HT22 mouse hippocampal neurons, rapamycin was shown to improve survival to an oxidative/reperfusion injury with H2O2 and a hypoxic/ischemic injury with oxygen and glucose deprivation to a larger degree than chloroquine. Rapamycin treatment increased punctate microtubule light chain associated protein 3, LC3, in the HT22 neurons in an uninjured and oxygen and glucose deprivation injured HT22 neurons compared to untreated neurons. Finally, genetic knockdown of autophagy with shRNA to autophagy protein 5, ATG5, abrogated the rapamycin’s positive effect on survival to injury.
    • Role of microtubules and motor proteins in mRNA localization

      Sanghavi, Paulomi; Department of Cellular Biology and Anatomy (2015-08)
      Establishment of polarity is essential for many cell types to perform their functions. A common mechanism that is used to establish polarity is localization of mRNAs at specific sites. This results in spatial restriction of protein expression. mRNA localization is a widespread phenomenon, occurring in most species. However, the mechanism by which mRNAs are localized is poorly understood. Using Drosophila as the model system, we investigated the localization of one such localized transcript, oskar mRNA. Studying the mechanism by which oskar mRNA is localized is important because many factors involved in localizing this transcript also function in localizing mRNAs in mammalian neurons. oskar mRNA localizes at the posterior pole of the Drosophila oocyte. This results in the posterior restriction of Oskar protein, which is turn functions in establishment of polarity in the oocyte and the future embryo. Localization of oskar mRNA is microtubule-dependent. We, therefore, characterized the polarity of microtubules in the oocyte. Our findings suggest that the posterior region is highly enriched in microtubule plus ends. However, this polarization is not essential for oskar mRNA localization. Secondly, the posterior localization of oskar mRNA was shown to be mediated primarily by the Kinesin-1 motor. Our findings demonstrate the role of an additional motor, Dynein, in this pathway. We found that Dynein associates with oskar mRNA in vivo and depletion of Dynein caused a significant delocalization of oskar mRNA. Next, we examined the role of a Dynein adaptor, Egalitarian (Egl), in the oskar mRNA localization pathway. Egl has been shown to recruit localized mRNAs to the Dynein motor in Drosophila embryos. Our results suggest that Egl associates with oskar mRNA in vivo and is required for the posterior localization of this transcript. Interestingly, one of the mechanisms by which Egl affects the localization of oskar mRNA is by affecting the microtubule polarity in the oocyte. Additionally, depletion of Egl caused precocious translation of oskar mRNA in the oocyte. Thus, our findings revealed a novel function for Egl in organizing oocyte microtubules and in regulating the translation of a localized mRNA.
    • Characterization of the Retinal Phenotype In Methylene Tetrahydrofolate Reductase (Mthfr) Deficient Mice, A Model Of Mild Hyperhomocysteinemia

      Markand, Shanu; Department of Cellular Biology and Anatomy (2015-05)
      (First Paragraph) The visual system is the primary source by which information is acquired by the human brain (Fernald, 1997). The eye is the organ responsible for vision. Blindness is a devastating condition as it affects the quality of life severely. In addition, there are financial consequences associated with blindness. Most untreatable disorders of vision that lead to blindness are due to disorders/degeneration of the retina. Retina is the photosensitive layer of the eye responsible for vision (Young et al, 2006). Several factors: genetic, environmental, systemic disease have been explored in the pathophysiology of various retinal disorders. One factor implicated in retinal diseases is excess levels of the amino acid homocysteine (hcy) (Selhub et al, 1999). The purpose of these studies was to analyze the expression of Cbs and Mthfr, key enzymes of hcy pathways in the mouse retina and to characterize the retinal phenotype in Mthfr deficient mice. To lay the foundation for this thesis, this introduction is organized in the following way: The eye, retina and retinal in-vivo diagnostics are described first. This is followed by description of hcy metabolism and its association with retinal diseases and mitochondrial dysfunction as a possible mechanism of hyperhomocysteinemia (Hhcy)-mediated retinal damage.
    • Bisphosphonate-Related Osteonecrosis of the Jaw: From Mechanism to Treatment

      Howie, Rebecca; Department of Cellular Biology and Anatomy (2015-04-20)
      With 55 million prescriptions issued each year, bisphosphonates are the second most prescribed class of drug in the United States. They are widely used to treat diseases with excessive osteoclastic resorption, including post-menopausal osteoporosis, Paget’s disease, and tumor metastasis to bone. Unfortunately, with long term intravenous administration of nitrogen-containing bisphosphonates some patients develop bisphosphonate-related osteonecrosis of the jaw (BRONJ). This debilitating disease has limited treatment options once it has manifested and no mechanism for its development has been elucidated. This dissertation explores the novel concept that bisphosphonates cause osteonecrosis of the jaw by impairing osteocyte-induced osteoclastogenesis and, through the physical accumulation of bisphosphonates in bone, impairing the ability of recruited osteoclasts to attach thereby arresting bone healing. Furthermore, it explores the possibility that chelating agents can be used for the removal of bisphosphonate attachment from bone systemically and locally during extractions, potentially leading to a future preventive treatment. It was found that 13 weeks of 80µg/kg intravenous tail vein injections of Zoledronate followed by two mandibular molar extractions caused the clinical presentation of BRONJ as analyzed by the gross, radiographic, and histological methods. Bone dynamic parameters and TRAP staining suggested an impaired ability for the bone to remodel and defective osteoclast attachment in treated groups that persisted eight weeks after the cessation of treatment. Additionally, it was found through the use of a fluorescently tagged bisphosphonate, that the decalcifying agents cadmium, EDTA, and citric acid all had the ability to cause the significant release of bound bisphosphonate from bone. Finally, this dissertation showed that the migration of monocytes treated with low doses of Zoledronate had increased migration, while their migration to conditioned media of osteocytes treated with Zoledronate was impaired. Collectively, these data suggest that invasive trauma by itself consistently precipitated massive bone necrosis in Zoledronate treated animals, possibly through a bisphosphonate driven alteration of monocyte migration and that the use of decalcifying agents could acutely remove bisphosphonate from bone both systemically and locally. This study establishes and effective rodent model for BRONJ and a possible preventive strategy for the side-effects of bisphosphonates during high-risk situations.
    • Role of Autophagy and Apoptosis in the Pathogenesis of Murine Cytomegalovirus Retinitis

      Mo, Juan; Department of Cellular Biology and Anatomy (2014-05)
      This study focused on the roles o f autophagy and apoptosis in the pathogenesis of murine cytomegalovirus (MCMV) retinitis. An overview of MCMV retinitis and the literature concerning autophagy, apoptosis and viral infection are given below, followed by detailed descriptions o f the eye, the retina, MCMV, autophagy and apoptosis.
    • Dynamic Bayesian Network Analysis Reveals Unique and Conserved Elements of Genetic Circuitry Governing Two Different Cell-Specific Regenerative Paradigms in the Retina

      Walker, Steven L.; Department of Cellular Biology and Anatomy (2014-03)
      Regeneration—the capacity to replace lost body parts—has fascinated scientists since the time of Aristotle. Regenerative phenomena became popular with observationalists during the 18th and 19th centuries and a fertile ground for experimentation. In fact, early regenerative biology practitioners such as Abraham Trembley (1710-1784) and his colleagues have been credited with establishing the foundations of modem experimental biology. However, interest faded during the 20th century in part due to the rise of Genetics and later reinforced by the dominance of mammalian model species which have a limited capacity for tissue replacement. At the start of the 21st century, a confluence of “stem cell promise” and new genetic manipulation techniques applicable to a broad range of species has rekindled the field. (Rosenthal, 2003) One aspect, however, remains a consistent and somewhat limiting theme: the emphasis on large-scale injury paradigms (e.g., limb loss). Conversely, the diseases most often cited as potential therapeutic beneficiaries of stem cell/regenerative research advances are typically linked to the loss of specific cell types (e.g., Parkinson’s disease, Type 1 diabetes). We and others have begun to explore cell-specific regenerative paradigms in order to increase understanding of how the loss of individual cell types is detected, how the response to cell loss is regulated, and ultimately how cell-type specific regeneration can be promoted. The goal of this project is to identify genetic networks that regulate the 12 regeneration of individual retinal neuron subtypes. Visual impairment is cited as the second most feared disease following cancer (Office, 2004). While currently considered ‘irreversible’ in mammals, including humans, we posit that retinal cell loss can be ‘cured’ by stimulating dormant regenerative capacities of adult neural stem cells located in the eye (Das et al., 2006); i.e., that reparative therapeutic strategies can be developed that restore visual function to patients by replacing cells lost to degenerative disease or ocular trauma. We developed a system for studying cell-specific loss and replacement in the zebrafish retina, a highly regenerative species (Poss, Wilson, & Keating, 2002), as a means of understanding how the regenerative potential retinal stem cells is regulated. Mammals, including humans, have a limited innate capacity for retinal regeneration (Das et al., 2006). However, recent data suggests that the potential for regeneration is retained in mammals; treatments with discrete molecular factors can enhance retinal cell replacement in mammalian disease models and human retinal stem cells can give rise to new neurons in cell culture. In addition, key cellular and molecular mechanisms governing retinal regeneration appear to be conserved between fish and mammals (e.g., Muller glia acting as injury-induced retinal stem cells) (Reh & Fischer, 2006). Accordingly, we sought to identify genes and genetic networks which regulate the regeneration of individual retinal cell subtypes using temporally resolved differential expression assays combined with cutting-edge statistical methods for establishing connectivity patterns in genetic circuits. By defining pathways which stimulate retinal stem cells to respond to cell losses in a regenerative manner, we aspire to further the development of novel therapies aimed at reversing vision loss in humans.
    • Characterization of the Thioredoxin System in the Diabetic Retina

      Lamoke, Folami; Department of Cellular Biology and Anatomy (2013-07)
      Diabetes is a group of diseases, which are characterized by high blood glucose levels that are a consequence of the inability to produce and/or utilize insulin. Type 1 diabetes (T1D, previously referred to as juvenile diabetes) is typically diagnosed in children and young adults. In this form, the body does not produce insulin primarily due to autoimmune-mediated destruction of pancreatic - cells, leading to insulin deficiency. Type 2 diabetes (T2D, adult onset or noninsulin dependent diabetes) is a chronic condition where the body either resists the effects or does not produce sufficient insulin. This form is more common in African Americans, Latinos, Native Americans, Asian Americans, Pacific Islanders, as well as the aged population.
    • DRP-1 and BIF-1 Regulations in Mitochondrial Dynamics During Apoptosis

      Cho, Sung Gyu; Department of Cellular Biology and Anatomy (2013-06)
      Recent studies have revealed that mitochondrial fragmentation is a critical event in apoptosis. Mitochondria become fragmented and notably, the fragmentation causes the permeabilization o f the mitochondrial outer membrane and consequently contributes to mitochondrial dysfunction and apoptotic cell death. In apoptosis, mitochondrial fragmentation involves the activations of D rpl, a key fission protein, and Bif-1, a protein originally identified to interact with Bax. However, the molecular mechanisms by which Drpl and Bif-1 regulate mitochondrial dynamics during apoptosis remain unclear. In the first study o f my thesis work, I investigated Drpl regulation and its role in apoptosis o f rat proximal tubular cell (RPTC) following ATP depletion. During ATP depletion, Drpl was shown to be dephosphorylated at serine-637. The dephosphorylation could be suppressed by cyclosporine A and FK506, two calcineurin inhibitors, which also prevented mitochondrial fragmentation, Bax accumulation, cytochrome c release and apoptosis in RPTC. The results suggest that Drpl is activated by calcineurin-mediated dephosphorylation at serin-637. Upon activation, Drpl stimulates mitochondrial fragmentation and the permeabilization of outer membrane, resulting in the release o f apoptogenic factors and apoptosis. In the second study, I detected Bif-1 translocation to mitochondria during apoptosis o f RPTC. Notably, apoptotic events including mitochondrial fragmentation, Bax insertion and oligomerization, and cytochrome c release were all suppressed in Bif-1 deficient cells. Mechanistically, we showed that during apoptosis, Bif-1 bound to prohibitin-2 (PHB2), a mitochondrial protein implicated in mitochondrial inner membrane regulation. Furthermore, PHB2 was shown to form hetero-oligomeric complex with prohibitin-l (PHB1) in control cells and the complex broke down upon apoptosis, which was accompanied by the proteolysis of optic atrophy 1 (OPA1), the mitochondrial inner membrane fusion protein. In Bif-1 deficient cells, the breakdown o f PHB complexes and OPA1 proteolysis were both inhibited, supporting a critical role o f Bif-1 in mitochondrial inner membrane fragmentation by regulating PHB2 and OPA1. Our studies have shed new light on the critical molecular mechanisms responsible for the alteration o f mitochondrial dynamics upon cell stress, resulting in mitochondrial fragmentation, injury and apoptosis.
    • Role of NEK1 in VHL and Cell Cycle Regulation

      Patil, Mallikarjun; Department of Cellular Biology and Anatomy (2013)
      Nekl is the member of NIMA (Never in mitosis gene A) related protein kinase family that is widely expressed in mammals. Nekl is an essential protein because loss of function in Nekl gene causes polycystic kidney disease in mice, which is similar to ADPKD (Autosomal Dominant Polycystic Kidney Disease) in humans. In Humans Nekl mutations also cause short rib polydactyl syndrome characterized by renal cysts and other developmental defects. At the cellular level Nekl thought to regulate ciliogenesis, centrosome duplication and DNA damage response.Nekl mutations leading to PKD have long been attributed to its role in ciliogenesis. Interestingly, VHL (Von hippel lindau) protein a known tumor suppressor is also involved in ciliogenesis.VHL mutations cause cystic kidney disease and renal clear cell carcinoma. Since Nekl and VHL are involved in ciliogenesis and cystic kidney disease, my overall goal was to investigate if Nekl and VHL are part of common regulatory pathway and also to investigate the role of Nekl in cell cycle regulation. My results indicate that Nekl phosphorylates VHL and this has important role in cilia regulation. Nekl phosphorylates VHL on multiple sites and S168 of VHL a site phosphorylated by Nekl significantly affects its stability. Importantly renal cells expressing S168A VHL that cannot be phosphorylated by Nekl grow cilia that are resistant to serum stimulation and Nocodazole treatment. Surprisingly I also found that Nekl is an essential regulator of S phase. Nekl knockdown in HEK cells blocks cell cycle progression. Further characterization Nekl showed that Nekl is needed for S phase progression and DNA replication. Nekl deficient cells have replication stress and activate cell cycle check point. Nekl loads on to chromatin and this increases during replication stress. We have also identified that Nekl interacts with and affects Ku80 loading on to chromatin. These findings have provided novel insights into the Nekl functions, which help in understanding the pathophysiology and development of polycystic kidney disease in mice and short rib polydactyl syndrome mejawski in humans.
    • microRNA Regulation of Acute Kidney Injury

      Bhatt, Kiri; Department of Cellular Biology and Anatomy (2011-08)
      Acute kidney injury (AKI) is caused by an injury or insult to the kidneys resulting in abrupt loss of renal function. Acute kidney injury is a highly prevalent disease characterized by high rates of morbidity and mortality mainly due to the absence of effective therapeutic options. Dissecting the molecular basis of AKI is vital not only for understanding the mechanisms of disease pathogenesis, but also for designing effective treatments. The small regulatory non-coding RNAs, microRNAs, are vital regulators of normal cellular function and critical modulators of various pathological conditions. An intense focus has recently emerged on the study of microRNA regulation in the maintenance of kidney function and the development of renal diseases. Our laboratory demonstrated the first evidence that microRNAs play a pathogenic role during ischemiainduced AKI by utilizing a conditional Dicer knockout mouse model. The focus of my work was to identify and functionally characterize novel microRNAs that contribute to AKI. Firstly, using a cisplatin-induced nephrotoxicity model of AKI, we showed that miR-34a is up-regulated in a p53 dependent manner and contributes to renal cell survival. Secondly, we identified a novel microRNA, miR-687, as the most significantly upregulated microRNA during ischemia-induced AKI. Mechanistic studies showed that miR-687 is up-regulated in a hypoxia-inducible factor 1 (HIFIndependent manner and subsequently negatively regulates PTEN expression under hypoxic conditions. These studies have unearthed an important HIFl-miR-687-PTEN signaling pathway that regulates cell cycle progression during hypoxia. Thirdly, we show that inhibiting miR-687 significantly ameliorates ischemia-induced AKI. These studies have identified a pivotal signaling mechanism involved in cellular response to hypoxia that may be targeted for renoprotection during ischemic AKI.
    • Molecular and Biochemical Characterization and Regulation of Folate Transport Proteins in Retinal Muller Cells

      Bozard, B. Renee; Department of Cellular Biology and Anatomy; Georgia Regents University (2011-07)
      Of the special sensory systems of vision, audition, olfaction, and tactile, the sense of vision is by far the most utilized (by sighted persons) in providing information about our surroundings. The visual pathway involves the retina as well as the brain and maintenance of retinal function is paramount for normal vision. Among the cells required to maintain the retina are Müller cells. This project was undertaken to examine the process by which retinal Müller cells acquire the essential vitamin folate. To lay the foundation of this work, this introductory chapter will describe (1) the retina, the tissue being studied, (2) the retinal Müller cell, the specific cell type examined, (3) folate and the known mechanisms of folate uptake described for other cells, (4) diseases associated with folate deficiency, (5) mechanisms of folate uptake in retina, and (6) factors that may regulate folate uptake in retinal Müller cells.
    • Signaling Mechanism of Blood-Retinal Barrier Regulation: Role of Mitogen-Activated

      Yang, Jinling; Department of Cellular Biology and Anatomy (2011-03)
      Breakdown of the blood-retinal barrier (BRB) is an early hallmark of diabetic retinopathy. A critical component in retinal vascular hyper-permeability is increased production of vascular endothelial growth factor (VEGF). VEGF is a potent permeability factor that activates mitogen-activated protein (MAP) kinases. Pigment epithelium-derived factor (PEDF), an endogenous anti-permeability factor, blocks VEGF-induced vascular permeability increase. However, the mechanisms underlying the actions of VEGF and PEDF in regulating endothelial permeability are not yet clear. Previous studies in our laboratory have shown that VEGF induces paracellular permeability via beta-catenin nuclear translocation/transcriptional activation and subsequent upregulation of urokinase plasminogen activator receptor (uPAR). This current study tests the role of two MAP kinases, p38 and extracellular-signal regulated kinase (ERK), in regulating VEGFinduced beta-catenin signaling, uPAR expression and BRB breakdown. We also evaluate the effects of PEDF on this VEGF permeability inducing pathway. The role of MAP kinase in this VEGF permeability inducing pathway was first evaluated using inhibitors of p38 and ERK. These inhibitors preserve the endothelial barrier function upon VEGF treatment. In confluent endothelial cells, cytosolic beta-catenin is phosphorylated by glycogen synthase kinase (GSK) then ubiquitinated and degraded. With VEGF treatment, GSK is phosphorylated/inactive followed by beta-catenin cytosolic accumulation, nuclear translocation and subsequent uPAR expression. These effects were blocked by MAP kinases inhibitors. This indicates p38 and ERK as mediators of VEGF-induced beta-catenin signaling, uPAR expression and endothelial barrier breakdown. Next, it was found that PEDF not only blocks VEGF-induced endothelial permeability increase and MAP kinase activation but also prevents the activation of GSK/beta-catenin signaling as well as uPAR expression. However, PEDF did not block VEGF receptor-2 (VEGFR-2) phosphorylation suggesting that PEDF acts downstream of VEGFR-2 and upstream of MAP kinase level. To further evaluate the role of p38 in regulating VEGF-induced permeability, adenovirusmediated delivery of p38alpha mutants was used. One p38alpha mutant has an altered ATP-binding site thus looses its activity. It is more efficient in blocking VEGF-induced GSK/beta-catenin signaling, uPAR expression and paracellular permeability increase. This study identifies p38alpha and ERK as mediators of VEGF permeability-inducing signaling. They could also serve as potential therapeutic targets for diseases featured by blood-retinal barrier dysfunction.