The Department of Cellular Biology and Anatomy has as its core mission the advancement of outstanding research and education. We work collaboratively to discover new knowledge through innovative biomedical research, to transmit that knowledge to students, and to train future researchers, educators and health care professionals.

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  • 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
  • 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.
  • Increased Membrane Thiol Oxidation in Sickle Erythrocytes

    Hill, Benjamin Albert; Department of Cell and Molecular Biology (1988-06)
  • EFFECTS OF VITAMIN D3 DEFICIENCY, VITAMIN D RECEPTOR KNOCKOUT, AND DIABETES ON CORNEAL EPITHELIAL NERVE DENSITY

    Vick, Sarah; Department of Biological Sciences; Department of Cellular Biology and Anatomy; Lu, Xiaowen; Watsky, Mitchell; Augusta University (2019-02-13)
    It is estimated that 41.6% of the US population suffers from vitamin D deficiency, with Blacks (82.1%) and Hispanics (69.2%) at even greater risk�Vitamin D deficiency can be caused by a variety of sources and given the wide range of causes, it is important to understand what measures this population might take to proactively prevent greater harm, or to reverse harm that might have already occurred. This project is designed to test the general hypothesis that Vitamin D deficiency exacerbates preexisting primary corneal pathologies. Previous research has established that the corneal epithelium in diabetic mice heals at a faster rate than the epithelium in diabetic vitamin D receptor (VDR) knockout (KO) mice. It is known that within diabetic mice, the corneal nerve density is decreased. However, it is unknown how VDR KO mice or vitamin D deficient with diabetes will affect corneal nerve density. In order to identify variabilities within the nerves that indicate slow wound healing, the mouse corneas will be collected, stained for confocal microscope observation, and analyzed through image processing to determine nerve density.
  • 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.
  • 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.
  • Salubrinal Mediated Fetal Hemoglobin Induction Through The PERK-eIF2α-ATF4 Signaling Pathway

    Lopez, Nicole; Department of Cellular Biology and Anatomy (2016-03)
    Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene affecting ~100,000 people in the United States. These individuals suffer from hemolytic anemia, pain, and progressive organ damage. The best therapeutic intervention in SCD is fetal hemoglobin (HbF) induction by pharmacologic agents, however, Hydroxyurea is the only FDA-approved drug with proven efficacy. The goal of this project is to discover drugs that induce HbF by novel mechanisms for SCD treatment. Salubrinal (SAL), a selective inhibitor of eukaryotic initiation factor 2α (eIF2α), was shown to increase HbF levels by enhancing γ-globin mRNA translation. These findings lead us to test the hypothesis that SAL activates the PERK-eIF2α-ATF4 stress response, as a mechanism of HbF induction in erythroid progenitors. Studies were conducted in K562 and erythroid progenitor generated from CD34+ stem cells treated with SAL (5, 12, and 18µM) for 48hr. RT-qPCR and western blot were used to measure γ-globin mRNA and HbF protein levels respectively. Preliminary data revealed a dose-dependent increase for HbF levels in K562 and erythroid progenitors treated with SAL. Flow cytometry showed an increase in the number of cells producing HbF (%F-cells). Furthermore, eIF2α and ATF4 levels were increased by SAL in K562 cells. These findings suggest SAL mediates HbF induction through eIF2α/ATF-4 signaling; future studies using the preclinical sickle cell mouse model will be investigated.
  • Deletion of the Mammalian Homolog of Yeast Vacuolar Protein Sorting 34 Inhibits Compensatory Nephron Hypertrophy

    Liu, Ting; Department of Cellular Biology and Anatomy (2016-03)
    Reduction of functioning nephrons stimulates all components of the remaining nephrons, particularly the proximal tubule, to undergo compensatory nephron hypertrophy (CNH). Recent studies in our lab revealed activation of the mammalian homolog of yeast vacuolar protein sorting 34 (mVPS34) in the remaining kidney within 30 min in response to uninephrectomy (UNX). Interestingly, mVPS34 has been reported to be an upstream mediator of mTORC1 activation in cultured cells. However, whether mVPS34 activation is essential in mediating mTORC1 signaling to CNH in vivo remains unknown. We crossed mVPS34flox/flox mice with SG.Cre mice expressing tamoxifen-inducible Cre recombinase mainly in the S1 and S2 segments of the proximal tubule and generated proximal tubule-specific mVPS34 knockout (mVPS34ptKO) mice. The body weight and kidney/body weight ratio (K/Bwt) of mVPS34ptKO mice were similar to those of wild type control (mVPS34Ctrl) littermates. 8-week-old mVPS34Ctrl and mVPS34ptKO mice were uninephrectomized. UNX-induced CNH in mVPS34ptKO mice was blocked by 40-55%, as indicated by inhibition of increases in K/Bwt ratio compared to that of mVPS34Ctrl mice (15.81±2.82 vs. 33.15±1.97%; p<0.001, n=7-9). There was no change in BUN levels in mVPS34ptKO and mVPS34ctrl mice with or without UNX. This study provided the first genetic evidence that mVPS34 mediates 40-55% of CNH. Further studies will determine the interactions between mVPS34 activation and mTORC1 signaling in regulating CNH.
  • 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.
  • Androgenic Maintenance of Rat Penile Erection

    Reilly, Christopher M.; Department of Cellular Biology and Anatomy (1997-06)
    Prior studies from this laboratory, using untreated-castrated rats (CASTRATE) and testosterone-treated castrated rats (TESTO), have shown that the magnitude of the intracavemosal pressure increase during erection is androgen dependent. Studies from this and other laboratories have also presented evidence suggesting that penile erection is mediated principally by nitric oxide (NO). The present report was designed to confirm that androgens maintain the availability of cavemosal NO, and to determine if this androgenic action is exerted at the genomic level modulating the expression of the neuronal form of nitric oxide synthase (nNOS) gene. The results showed that administration of supplemental L-arginine failed to augment the erectile response in either group, suggesting that substrate availability is not a cause o f the reduced response in CASTRATE animals. Inhibition of NO synthesis with a nitro-arginine competitive inhibitor of NOS resulted in strong inhibition of erection in both TESTO and CASTRATE rats. When given in conjunction with ganglionic stimulation to induce erection, the NO releasing drug, sodium nitroprusside (SNP), increased intracavemosal pressure in CASTRATE but not in TESTO rats suggesting a deficiency of the available NO in CASTRATE animals. Finally, reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that mRNA levels for the enzyme nNOS in the penis were greater in TESTO animals than in CASTRATE rats. These results support the hypothesis that androgens mediate the erectile response in the rat penis by stimulating the expression of the neuronal isoform of nitric oxide synthase, thus maintaining an adequate supply of NO.
  • Venous Contraction to Endothelin-1 in Congestive Hearth Failure

    Reddy, Vikram; Department of Cellular Biology and Anatomy (2003-04)
    Endothelin-1 (ET-1) is produced by endothelial cells and can stimulate either the ETa or the ETB receptors. The role o f ET-1 and the identity of the endothelin receptors involved in mediating tone in the mesenteric small veins of the Golden Syrian hamster are not known. ET-1 induces venoconstriction, thereby increasing the preload to the heart in congestive heart failure. However, mechanisms mediating contraction to ET-1 in the mesenteric small veins of the cardiomyopathic hamsters in the early and late stages of CHF are not known. Therefore, mechanisms mediating ET-1 induced contraction were determined in the mesenteric small veins o f the Golden Syrian and cardiomyopathic hamsters in the early and late stages of CHF. Baseline intraluminal diameter of small veins was measured before and after treatment with either ETa or ETB receptor antagonists. ET-1 induced contraction was higher in the early stage o f CHF, while it was decreased in the late stage of CHF. Blockade of the ETA receptor decreased ET-1 induced contraction in the mesenteric small veins from the control and cardiomyopathic hamsters in both the early and late stage o f CHF. ETB receptor blockade decreased the ET-1 induced contraction in the control and cardiomyopathic hamsters in the early, but not late, stage o f CHF. Therefore, ET-1 induced contraction in the mesenteric small veins is mediated by the ETa receptors alone in the late stage of CHF, while both the ETa and ETB receptors mediate vasoconstriction in the controls and in the early stage o f CHF. Stimulation of ET-1 receptors is associated with an increase in calcium levels within the vascular smooth muscle cells. It is not known whether the increase in reactivity to ET-1 in the early stage o f CHF or the decrease in reactivity to ET-1 in the late stage of CHF is due to problems with mobilization o f the intracellular calcium levels within the vascular smooth muscle cell. Following ET-1, calcium levels within the vascular smooth muscle cell were increased to a larger extent in the early stage o f CHF, than in the late stage of CHF, in agreement with the vascular reactivity data. Calcium levels were also measured before and after treatment with either ETa or ETb receptor antagonists. Blockade o f the ETa receptor inhibited the ET-1 induced increase in calcium levels in the mesenteric small veins from the control and cardiomyopathic hamsters in both the early and late stage of CHF. However, ETB receptor blockade inhibited the ET-1 induced increase in calcium levels in only the control and cardiomyopathic hamsters in the early stage o f CHF. These results indicate the absence of a functional responses mediated by the ETb receptor in the late stage of CHF. Studies have shown that NO can modulate the contraction to ET-1 in the vasculature. Baseline intraluminal diameter o f small veins were measured before and after treatment with N-nitro-L-arginine (LNA), a specific inhibitor o f nitric oxide synthase. LNA decreased the contraction to ET-1 in the early stage o f CHF, but increased contraction to ET-1 in the late stage of CHF. This indicates that NOS mediates a vasodilatory effect that counteracts contraction to ET-1 in the late stage, but contributes to the vasoconstrictor effect of ET-1 in the late stage o f CHF. NOS activity was measured to identify the NOS isoforms contributing to the modulation o f ET-1 induced vascular reactivity. Total NOS activity was significantly increased in the cytosolic fraction of small veins from hamsters in the late stage o f CHF and in the particulate fraction in hamsters in the early stage of CHF. In the late stage, the increase in NOS activity was inhibitable by 1400W, an iNOS selective inhibitor, suggesting that an increase in iNOS decreases the contraction to ET-1. In summary, in the early stage of CHF, there is an increase in the vascular reactivity to ET-1 associated with an increase in intracellular calcium levels and partially mediated by NOS. This may increase preload and impair myocardial function in CHF. There is an absence o f ETb receptor-mediated responses in the late stage of CHF, associated with very high plasma ET-1 levels and impaired intracellular calcium signaling. NOS activity is significantly enhanced in the mesenteric small veins from the cardiomyopathic hamsters in the late phase of CHF, and this increase in NOS activity is at least partially dependent on iNOS and may contribute to impaired venous contraction to ET-1 in cardiomyopathic hamsters. This may serve as a compensatory mechanism to decrease the preload to the failing heart.
  • 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.
  • 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.
  • 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.
  • 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.
  • Characterization of Cardiac L-Type and T-Type Calcium Channels During Normal and Defective Chick Heart Development

    Nichols, Carol A.; Department of Cellular Biology and Anatomy (2000-03)
    (First Paragraph) The human heart is vital for survival from early in embryonic development throughout life. It begins developing around the third week of gestation from a pair of endocardial tubes that fuse to form a single primitive heart tube. The single-lumen heart tube develops a series of expanded areas and infoldings that divide it into four presumptive chambers. As the embryo grows, the heart begins looping. This looping process serves to bring the four presumptive chambers into the appropriate orientation for septation. The developing heart remodels itself into four separated chambers (two atria or holding chambers, two ventricles or pumping chambers) which provide for separate systemic and pulmonary circulation at birth. In most mammals, oxygenated blood enters the left atrium through four pulmonary veins. The blood is forced into the left ventricle when the left atrium contracts. When the left ventricle contracts, blood is pumped through the aorta and carried throughout the body. Deoxygenated blood returns to the right atrium via the superior and inferior vena cavae. Blood is forced into the right ventricle by contraction of the right atrium. Blood is then pumped through the pulmonary trunk and arteries to the lungs to be re-oxygenated. The four- chambered heart is formed by the eighth week o f gestation. (Larsen, 1997; de la Cruz & Markwald, 1998).
  • 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.
  • The Effects of pp60v-src Expression on the Development of the Chicken Optic Tectum

    Mogan, John C.; Department of Biology and Anatomy (1999-03)
    The chicken optic tectum (OT) develops from the dorsal mesencephalon (midbrain) and processes crossed input from each retina. Previous experiments using a replicationdeficient retrovirus that contained the marker gene lacZ have demonstrated the normal pattern of development for the OT. Clonal cohorts derived from a single neuroepithelial stem cell migrate both radially and tangentially and differentiate into many types of neurons and at least three types of glia (radial glia and two types o f astrocytes). The goal of our laboratory is to identify important proteins involved in tectal development by: (1) directly altering expression of endogenous proteins through senseor antisense-containing retroviruses, or (2) indirectly altering endogenous protein expression or function by retroviral expression of an exogenous protein. These two approaches will allow us to determine which proteins are important in the normal and abnormal development of tectal clones. Many processes are involved in the development of the OT: proliferation, migration, differentiation, and synapse formation. Four non-receptor tyrosine kinases of the Src family (c-src, c-src+, fyn, and yes) are expressed in a spatially and temporally regulated manner in the nervous system. Their expression patterns in neural cells in vivo and in vitro have implicated these Src family members in all four of the major developmental processes mentioned above. Knockout mice of these three Src family members individually (c-src, fyn, or yes), however, show few or no overt neural developmental abnormalities. These unexpected results indicated that other members of the Src family can assume the roles of the missing kinase. Knockout mice for the major known negative regulator of Src family kinases, Csk (c-src kinase), however, show severe developmental abnormalities and defects in neural tube closure. These mice died around E9-E10 and showed elevated kinase activity for at least three Src family members (c-src, fyn, and Iyn). This fact makes it impossible to conclude that the overactivity of any one Src family tyrosine kinase is responsible for the developmental defects observed, and the early death of these embryos prevents the study of neural cell lineage, migration and differentiation in vivo. Given these results, the use of antisense to reduce expression of c-src would yield little information about the role of this kinase in neural development due to functional redundancy among Src family kinases. I decided to express in tectal clones an unregulated member of the Src family, pp60“'src, to determine how its expression alters normal tectal development. The v-src oncogene of Rous sarcoma virus was the first member of the Src family to be discovered, v-src encodes an activated tyrosine kinase (pp60,'"irc) which has lost a critical regulatory tyrosine present in the carboxy terminus of all other Src family members. Consequently, pp60'fcsrc expression affects the proliferation, migration and differentiation of many cell types in vitro and in vivo, but the effects of its expression on neural development in vivo are not well characterized. Expression of this kinase in tectal clones will provide an excellent system to study how a single unregulated Src kinase influences development of the nervous system. Expression o f a protein (pp60v'src) known to affect many different processes (proliferation, migration/cell adhesion and differentiation) in tectal clones will allow us to answer many questions of biological significance: (1) Is the proliferative potential of neuroepithelial stem cells restricted in vivo, or can stem cells generate clones of larger size?, (2) If multiple cell adhesion systems are presumptively inactivated in pp60*N,rc - expressing tectal neuroblasts, then how will clonal migration patterns differ from the norm?, (3) Is clonal differentiation in the OT controlled by only extracellular influence (e.g., growth factors, gradients) or can the developmental fate of stem cell progeny be altered by expression of pp60*’'src. In the first set of experiments I wanted to determine how wild-type pp60w'5rc expression alters the development of tectal clones in vivo. I used a replication-deficient retrovirus (LZIS), which efficiently coexpresses both LacZ and pp60*fc*rc, to determine the effects of pp60,,'*rc expression on several clonal parameters: cell number, migration pattern, and differentiation. In the next set of experiments I constructed and tested retroviral vectors which efficiently coexpress LacZ and mutated pp60w‘src proteins with deleted SH2 or SH3 domains (LZISASH2 and LZISASH3). These domains normally allow the pp60u'*rc tyrosine kinase to associate with certain cellular proteins which contain phosphotyrosines or a proline-rich stretch of amino acids, respectively. Mutation or deletion of these domains alters the biochemical and biological function of pp60^rc. I hoped to determine if the SIC or SID domains of pp60v'src are necessary for the wild-type pp60ltsrc phenotype, and to determine if they afford a unique but altered clonal phenotype compared to wild-type pp60v^rc. These experiments are novel in that they demonstrate that the overexpression of activated forms of Src family kinases influences development of the vertebrate brain. I conclude from my results that: (1) the proliferative potential of neuroepithelial stem cells in the OT is not restricted, (2) tangential migration of neuroblasts in the developing OT appears enhanced with pp60*fc*rc expression, and (3) the proper differentiation of radial glia is hindered but not prevented by pp60lHirc

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