• Impact of Genetic Predisposition and Environmental Stress on Measures of Preclinical Essential Hypertension

      Poole, Joseph C.; Department of Cellular Biology and Anatomy (2006-06)
      The main objective of this project was to determine the impact of genetic risk and chronic environmental stress on measures of preclinical essential hypertension (EH) (e.g., exaggerated cardiovascular reactivity, increased resting hemodynamics and increased left ventricular mass [LVM]). A secondary objective was to evaluate the moderating and interactive effects of ethnicity, gender, body mass index [BMI] and anger expression on EH risk indices. Two genes with relevance for blood pressure (BP) control (e.g., beta-2 adrenergic receptor [ADRB2] gene and serotonin transporter [5-HTT] gene) were used to define genetic risk. Chronic environmental stress was assessed by socioeconomic status (SES) and subjective social status (SSS). The project consisted of three sequential studies on a large, multiethnic cohort of young adults (N>500). The first two studies were cross-sectional and based on the analysis of cardiovascular reactivity, resting hemodynamics and LVM data collected at a single visit. The third study was longitudinal and involved the tracking of BP and LVM over a 15-year span from childhood to early adulthood. In the first study, ADRB2 haplotype significantly interacted with anger suppression in African Americans such that high anger suppressing carriers had the highest resting SBP (p<.05) and TPR reactivity to a cold pressor task (p<.01). In European Americans, ADRB2 haplotype significantly interacted with BMI to predict resting hemodynamics, such that carriers who were high in BMI showed the highest SBP (p<.05). In the second study, a significant interaction between the 5-HTT promoter region polymorphism (5-HTTLPR) and social status was found for cardiovascular reactivity, such that S allele homozygotes who were low in SES and high in SSS exhibited the greatest BP and TPR reactivity to the video game stressor (p-values<.05). No significant interaction was found between 5- HTTLPR and social status in the longitudinal study, however a significant 5- HTTLPR by BMI interaction was determined for LVM, such that obese LL homozygotes had the greatest LVM over time (p<.001). Results from this project expand what is currently known with regard to EH etiology and carry implications for the prevention of EH through the early consideration of genetic, environmental and demographic risk factors.
    • 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.
    • 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.
    • Increased Membrane Thiol Oxidation in Sickle Erythrocytes

      Hill, Benjamin Albert; Department of Cell and Molecular Biology (1988-06)
    • Infiltrating Cells, Interferon-gamma and Intraocular Spread of HSV-1 after Anterior Chamber Injection

      Cathcart, Heather M.; Department of Cellular Biology and Anatomy (2009-12)
      Following uniocular anterior chamber (AC) inoculation of HSV-1 (KOS), the anterior segment of the injected eye becomes inflamed and infected; however, virus does not spread from the anterior segment to infect the retina of the injected eye. The overall goal of this study was to identify interferons (IFNs) and early infiltrating cells which may play a role in protecting the retina of the ipsilateral (injected) eye. Female BALB/c, IFNy-/- and macrophage depleted (clodronate, CI2MBP treated) mice were injected in one AC with 3*104 - 6x104 PFU of HSV-1 (KOS). Mice were killed at various time points ranging from 12 to 120 hours post injection (p.i.). The injected eyes were enucleated, snap frozen and frozen sections were stained with antibodies specific for HSV-1, IFNy, Mac-1 (CD11b), Gr-1, CD49b, F4/80, CD4, CD8 and CD11c. The same antibodies were also used to stain freshly isolated single-cell suspensions from the eye or spleen for flow cytometry. Additionally, whole injected eyes were used to determine gene expression levels of IFNs and IFN associated genes. In the anterior segment of the injected eye, the ciliary body and iris were virus infected and inflamed, and infiltrating cells increased during the period of observation. Mac-1 + and F4/80+ cells colocalized with IFNy in the anterior segment and Mac-1 + cells increased in the injected eye beginning at 24 hours p.i. and continuing through 72 hours p.i. Although virus staining was increased in the ciliary body of macrophage depleted mice at 48 and 72 hours p.i., destructive retinitis was not observed in the injected eye of these animals. IFNy gene expression was up regulated in injected eyes of BALB/c mice from 48 to 120 hours p.i., and while HSV-1 infection of IFNy-/- mice resulted in increased virus staining in the ciliary body, destructive retinitis was rarely observed in IFNy-/- mice. Microglia and IFNy play important roles in the immune response to virus infection, but depletion of single cell types or cytokines did not result in early panretinal HSV-1 infection in the injected eye. Taken together, these findings support the idea that the timing and appearance of different cell types and cytokines is critical to protection of the retina of the injected eye from infection due to direct spread of virus; however, it is likely that during the innate immune response in the eye, other cell types and cytokines can compensate for the absence of a single cell type or of a single cytokine.
    • Mechanisms of Homocysteine-Induced Retinal Ganglion Cell Death

      Ganapathy, Preethi S.; Department of Cellular Biology and Anatomy (2010-12)
      The purpose of these studies was to determine the effect of excess homocysteine on retinal ganglion cell viability. An overview of homocysteine metabolism and the literature concerning homocysteine-induced neurotoxicity is given below, followed by detailed descriptions of the eye, the retina, and retinal ganglion cells.
    • The Mechanobiology of Cranial Sutures

      Byron, Craig D.; Department of Cellular Biology and Anatomy (2005-07)
      A central hypothesis that cranial suture growth and modeling vary with respect to the mechanical loading environment is tested in a mouse sagittal suture model using three Specific Aims. Experiments within these aims were designed to elucidate mechanisms of bone formation and bone resorption at the cellular level and to determine how these processes influence the morphology and performance of cranial suture connective tissues. It is argued that suture waveform complexity (measured using fractal analysis) is generated by the positive coupling of osteogenesis along convex bone margins and bone resorption along concave bone margins and that this turnover cycle is regulated in large part by mechanical forces acting on the suture bone-ligament interface. This suture formfunction relationship is believed to operate via mechanosensing mechanisms within skeletal connective tissues. Although mechanically-induced cell wounding appears to be involved in normal suture biology, it does not occur in the fashion predicted. Apoptosis is not directly implicated. Thus, it is predicted that bone resorption in cranial sutures does not localize according to regions of shear-induced cell death but rather to regions adjacent to osteoblastic activity. Tension rather than shear is most likely to be the driving force in this system.
    • Modulation of a Conserved Cathepsin B-Like Extracellular Matrix Protein Impacts Wing and Egg Formation in Drosphila Melanogaster

      Dinkins, Michael B; Department of Cellular Biology and Anatomy (2011-03)
      Conserved in Bilaterian species, the tubulointerstitial nephritis antigen (TIN-ag) family of cathepsin B-like extracellular matrix proteins has been proposed to have roles in cell adhesion and regulation of basement membrane assembly based on in vitro studies of mammalian family members. Here we examined the single Drosophila ortholog, CG3074, and found conservation of its basement membrane localization as well as a role in cell adhesion. RNAi knockdown resulted in wing blistering and held-out wings following eclosion, consistent with defects in adhesion of wing epithelia and tendon cells to the underlying extracellular matrix, but no defects were detected during pupal development. We were unable to demonstrate a genetic or physical interaction with laminin and CG3074 but did detect genetic interactions with integrins and dystroglycan in the wing. A serine substitutes for cysteine in all TIN-ag family members at the 'active site' of the cathepsin B-like domain and is predicted to render the protein inactive as a protease. Overexpression of the mutant CG3074 S213C, in which the 'catalytic' cysteine of cathepsin is restored, resulted in gain-of-function defects in egg formation and larval development. We provide genetic and biochemical evidence that these defects arise from a neomorphic activity of the S213C protein that supports a role of this highly conserved domain in wildtype CG3074 function. These studies broaden our understanding of TINag family function and identify tissue and pathway models for future studies.
    • Murine CD19+ Plasmacytoid Dendritic Cells Expressing Indoleamine 2,3 Dioxygenase

      Kahler, David J.; Department of Cellular Biology and Anatomy (2008-10)
      Indoleamine 2,3 Dioxygenase (IDO) is a potent immunomodulatory enzyme whose role has been described in diverse physiologic states including pregnancy, cancer, tissue transplants, autoimmune disease, chronic inflammation, and depression. IDO suppresses antigen specific T cell proliferation via mechanisms including tryptophan degradation and the production of toxic metabolites, and the activation of resting regulatory T cells (Tregs). IDO expression is tightly regulated in the murine spleen, as only rare dendritic cell (DC) subsets are competent to express IDO. Therefore, an accurate phenotype by which to identify IDO competent DCs in tissues is important when ascribing the role of IDO competent DCs in disease models. Here we show that IDO competent CD19+ pDCs (CD19+ pDCs) express high levels of costimulatory receptors (CD80 / CD86) under homeostatic conditions indicating a mature or activated phenotype and uniquely express the Class I MHC-like molecule CD1d, and the chemokine receptor CCR6. IDO competent pDCs do not share the same lineage as other murine splenic DCs as they were the only DC subset to express Pax5, and were present in reduced numbers in murine models of B cell development indicating that they develop from B cell precursors. Distinct signaling requirements regulate IDO induction in IDO competent pDCs as MyD88 was required for IDO induction and function in inflamed skin draining lymph nodes following phorbol myristate acetate application but not for IDO transcript expression or STAT1 or STAT2 protein phosphorylation following treatment with recombinant cytokines. CD19+ pDCs from WT mice but not mice genetically deficient for the IDO1 gene formed stress granules (SG) following treatment with IFNγ, which were not prevented by inhibitors of IDO activity indicating that SG formation was not IDO dependent. We hypothesize that IDO competent murine splenic pDCs uniquely expressing CD19 are phenotypically and functionally distinct from other splenic DC subsets and respond to inflammatory signals by expressing IDO. We further hypothesize that activated IDO causes distinct yet undefined biochemical changes within IDO competent pDCs following induction most probably by activating the integrated stress response and the eif2a kinases GCN2, PKR, and PERK.
    • NEUROVASCULAR DEGENERATION FOLLOWING RETINAL ISCHEMIA REPERFUSION INJURY: ROLE OF ARGINASE 2

      Shosha, Esraa; Department of Cellular Biology and Anatomy (2017)
      Ischemic retinopathies such as retinopathy of prematurity, central retinal artery occlusion and diabetic retinopathy are leading causes of visual impairment and blindness. These pathologies share common features of oxidative stress, activation of inflammatory pathways and neurovascular damage. There is no clinically effective treatment for these conditions because the underlying mechanisms are still not fully understood. In the current study, we used a mouse model of retinal ischemia reperfusion (I/R) insult to explore the underlying mechanisms of neurovascular degeneration in ischemic retinopathies. The arginase enzyme utilizes the L-arginine amino acid for the production of L-ornithine and urea. Here, we investigated the role of the mitochondrial arginase isoform, arginase 2 (A2) in retinal I/R induced neurovascular injury. We found that retinal I/R induced neurovascular degeneration, superoxide and nitrotyrosine formation, glial activation, cell death by necroptosis and impairment of inner retinal function in wild type (WT) mice. A2 homozygous deletion (A2-/-) significantly protected against the neurovascular degeneration after retinal I/R. That was attributed to decreased oxidative stress and glial activation. A2 deletion protected against I/R induced retinal function impairment. Using Optical coherence tomography (OCT), we evaluated the retinal structure in live animals and found that A2-/- retinas showed a more preserved structure and less retinal detachment. To investigate the underlying mechanisms of A2 induced vascular damage after I/R, we used an in vitro model of oxygen glucose deprivation/ reperfusion (OGD/R) in bovine retinal endothelial cells (BRECs). Analysis of oxidative metabolism showed impaired mitochondrial function. We also found an increase in dynamin elated protein 1 (Drp1), a mitochondrial fission marker. Mitochondria labeling studies showed fragmented mitochondria after OGD/R. Arginase inhibition reduced mitochondrial fragmentation in OGD/R insult. This dissertation presents A2 as a new therapeutic target in reducing neurovascular damage in ischemic retinopathies.
    • 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.
    • PKC and ATR Mediated Regulation of Cisplatin-Induced Renal Tubular Cell Apoptosis

      Pabla, Navjotsingh; Department of Cellular Biology and Anatomy (2009-03)
      Cisplatin is one of the most widely used anti-cancer drug. However, its use and efficacy is limited due to nephrotoxicity. One fourth of patients treated with cisplatin develop varying degree of renal impairment, frequently resulting in acute kidney injury. Due to high mortality associated with acute kidney injury, effort has been made to understand the molecular basis of cisplatin nephrotoxicity and develop effective renoprotective strategies. In kidneys, cisplatin is accumulated in tubular cells; however the uptake mechanism that is responsible for high accumulation of cisplatin in renal cells is unclear. In tubular cell, cisplatin accumulation induces cell death by apoptosis. Mechanistically, our laboratory has demonstrated a critical role of p53 in tubular cell apoptosis during cisplatin nephrotoxicity. However, the proximal events that contribute to p53 activation and related signaling are unknown. The focus of my work was to decipher these early events during cisplatin nephrotoxicity. Firstly, my results suggest that the copper transporter Ctr1 is highly expressed in renal tubular cells and is responsible for renal uptake of cisplatin. Secondly, I show that DNA damage response involving ATR-Chk2 is responsible for p53 activation and consequent apoptosis during cisplatin-induced kidney injury and nephrotoxicity. Thirdly, I have identified that PKCd is a novel regulator of cisplatin nephrotoxicity. During cisplatin treatment PKCd is activated in a Src dependent manner and is responsible for activation of MAPKs, contributing to renal cell death. Most importantly, my results suggest that pharmacological inhibition of PKCd ameliorates renal injury without affecting the anticancer efficacy of cisplatin. These results have not only provided new insights into the 3 molecular mechanism of cisplatin nephrotoxicity, but have also identified a novel strategy to mitigate the side effects of cisplatin in normal renal tissues.
    • Progesterone Regulation of Proliferation and Regression of Rat Decidua Basalis

      Dai, Donghai; Department of Cellular Biology and Anatomy (1998-07)
      During implantation mesometrial cells o f the uterine stroma become decidualized under the coordinate actions of progesterone (P4 ) and estrogen (E) [1,2]. This process is characterized by transformation o f phenotype and stromal cell proliferation between Days 8-12 of gestation, resulting ultimately in the formation o f the decidua basalis (DB) [3,4], By Day 14. however, the DB begins to regress and a reduced layer o f stromal cells persists to the end of pregnancy [5.6]. The regression of DB is accompanied by development o f two other layers, namely junctional zone (JZ) and labyrinth zone (LZ), which are fetal parts of the placenta and morphologically become predominant at the end stages o f pregnancy. Although the morphological changes have been well documented and numerous functions have been revealed for DB [3-8], the mechanism and factors involved in the regulation of proliferation and regression o f DB have not been elucidated. The transition of DB from proliferation to regression occurs in such a delicate way that the morphological integrity and functional competence of the DB and placenta are maintained even though stromal cells are being lost. The objective o f this study was to identify the intracellular signals initially favoring proliferation and synthetic processes and those promoting remodeling and regression as pregnancy progresses.
    • 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.
    • Regulation of Reduced-Folate Transporter-1 in Retinal Pigment Epithelium

      Naggar, Hany A.; Department of Cellular Biology and Anatomy (2003-04)
      (First Paragraph) The purpose of these studies was to analyze the regulation of the folate transport protein, reduced-folate transporter (RFT-1) in the retinal pigment epithelium (RPE) under conditions o f hyperglycemia, hyperhomocysteinemia and folate deficiency. A detailed description o f the retina, followed by information regarding folate and regulation o f RFT-1, is provided below.
    • 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.
    • ROLE OF CLASS III PHOSPHATIDYLINOSITOL 3-KINASE IN THE RENAL PROXIMAL TUBULE

      Liu, Ting; Department of Cellular Biology and Anatomy (Augusta University, 2020-05)
      Yeast only has a single phosphatidylinositol 3-kinase (PI3K) known as vacuolar protein sorting 34 (VPS34); however, mammals have evolved to express three structurally different classes of PI3Ks: class I, II, and III. Although the class III PI3K (Pik3c3) is the only PI3K evolutionarily conserved from yeast to man, its distribution in the mammalian kidney is unknown, and its role in the renal proximal tubule, especially under certain pathophysiological conditions such as nephron loss-induced Compensatory Nephron Hypertrophy (CNH), remains undefined. The goal of Aim 1 was to define the expression pattern and relevant biological function of Pik3c3 in the kidney. We found that the glomerular podocyte expresses the highest level of Pik3c3 in the kidney. Among all renal tubular cells, the specialized distal convoluted tubular epithelial cells called macula densa cells express the highest level of Pik3c3, and the renal proximal tubular cells (RPTC) express the second highest level of Pik3c3. This prompted us to perform additional experiments for Aim 1 that led to the demonstration of an essential function of Pik3c3 in regulating the degradation of epidermal growth factor (EGF) receptor (EGFR) and the termination of EGFR signaling in RPTC following EGF binding with EGFR. The goal of Aim 2 was to determine whether Pik3c3 is essential in mediating uninephrectomy (UNX)-induced compensatory nephron hypertrophy. We generated a global Pik3c3-hypomorphic mouse model and two slightly different proximal tubule-specific Pik3c3 knockout mouse models: Pik3c3Neo-ptKO and Pik3c3ptKO. Interestingly, CNH was markedly inhibited in the global Pik3c3-hypomorphic mouse model and proximal tubule-specific Pik3c3 knockout models. The goal of Aim 3 was to determine the effect and underlying mechanism of complete Pik3c3 deletion in renal proximal tubule cells. We found that complete Pik3c3 deletion in some renal proximal tubule cells resulting in marked cell death that subsequently progressed to tubulointerstitial fibrosis. My project has, for the first time, determined the expression pattern of Pik3c3 in the kidney and provided the first definitive evidence that Pik3c3 controls the degree of CNH and functions upstream of the mTORC1-S6K1-rpS6 pathway in the regulation of CNH. In addition, my project reveals an essential role of Pik3c3 in maintaining the homeostasis and survival of proximal tubule cells.
    • 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.
    • 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.
    • The role of stromal cell-derived factor-1 in cell mobilization, cell homing and neovascularization following stroke

      Walker, Aisha L.; Department of Cellular Biology and Anatomy (2007-11)
      Stroke is the 3rd leading cause of death and the leading cause of long-term disability in the U.S. With only one approved drug presently used in clinics, there is a great need for the development for new therapeutic targets. Stromal cell derived factor-1 (SDF-1) is a small chemokine that may aid in cerebral repair following stroke. Acting primarily through the CXCR4 receptor, SDF-1 is known to be chemotactic for neuroblasts, endothelial cells, and bone marrow derived (BMD) cells including stem and progenitor cells found in the bone marrow. Recently, BMD stem/progenitor cells have become widely studied for their potential role in tissue repair following ischemia. SDF-1 is under hypoxic regulation and is highly expressed in ischemic brain tissue for at least 30 days following ischemia suggesting it may play role in long term repair or remodeling. The goal of these studies is to determine the role of SDF-1 in cerebral repair following stroke. I hypothesize that SDF-1 upregulaton during brain ischemia contributes to tissue repair and neurological recovery by inducing the homing of bone marrow-derived cells to the site of injury and neovascularization. In a mouse middle cerebral artery ligation (MCAL) permanent occlusion stroke model, I investigated mobilization, homing, and differentiation of adult bone marrow derived (BMD) cells in response to SDF-1 induced by cerebral ischemia. Results presented in this dissertation show that SDF-1 induces mobilization of BMD cells following stroke. Once mobilized, BMD cells homed to the brain and either retained their blood cell phenotypes (i.e. monocytes and neutrophils) or they differentiated mostly into microglia cells. Many BMD cells migrated to a perivascular location with a subset becoming pericytes. Additionally, I found that SDF-1 induced neovascularization and this occurs through a combination of angiogenic and vasculogenic processes in the in vivo stroke model as well as in an in vitro tube formation assay. However, we did not detect beneficial preservation of brain tissue or augmented functional recovery with treatment of SDF-1, but it remains to be determined if altering timing, delivery, or isoform-specificity of SDF-1 may be therapeutically beneficial.