• A Data-Mining Strategy That Identifies Drugs and Genes Associated With Anti-Cancer Drug Sensitivity

      Schleifer, Robert John; Department of Pharmacology and Toxicology (1/25/2018)
      The success of cancer therapy for patients often hinges on the responsiveness of the cancer cells to therapeutics. Drug resistance to anti-cancer therapeutics, both intrinsic and acquired, has important clinical and scientific significance. Identification of drug resistance genes using traditional methodologies and translation of those findings to the clinic has proven challenging. We developed a predictive data mining-based bioinformatic framework using public patient data and high-throughput cancer cell drug screening data. This information was used for genome-wide rankings of putative drug resistance genes. Prominent drug resistance genes (e.g. ABCB1, EGFR, and AXL) were successfully identified by the pipeline, additional genes hypothesized to be novel drug resistance genes were then investigated. Experimental confirmation of the novel genes using knockdown technologies indicated a propensity for of decreased proliferation/viability of cancer cells and increased sensitivity for anticancer compounds after knockdown much like known drug resistance genes. We then assessed the potential of each gene as an anti-cancer therapeutic target by exploring how gene knockdown behaved with clinical anticancer compounds. A second arm of the data-mining pharmaco-genomic strategy involved identification of candidate compounds that decrease expression of drug resistance genes. Using the drug resistance gene AXL as a proof-of-concept, three compounds were identified that decreased AXL expression at sub-micromolar concentrations. These compounds were characterized using microarray and cell signaling studies and found to decrease cell cycle signaling as well as activity of the Akt, mTOR, and ERK pathways. This study illustrates a novel approach for rapid and efficient identification of drug sensitivity genes or gene expression altering compounds utilizing bioinformatic data-mining.
    • Deciphering mechanisms of DNA methylation regulation by depletion of the DNA methyltransferases and SETD2

      Tiedemann, Rochelle Lee; Department of Biochemistry and Molecular Biology / Cancer Center (2015)
      DNA methylation (5mC) is a stabile epigenetic mark that confers differential function for gene expression and chromatin accessibility dependent on the context and locality of the mark. Promoter regions populated by CpG islands (CGIs) are highly unmethylated while the remaining ∼80% of CpGs are methylated and distributed across gene bodies, repetitive and transposable elements, and intergenic regions of the genome. The presence and/or absence of particular histone modifications also dictate the patterning of 5mC genome-wide. In cancer, a reversal of 5mC patterns occur in which hypermethylation of tumor suppressor gene CGIs confers gene silencing, and hypomethylation of repetitive and transposable elements contribute to genomic instability. The mechanisms by which 5mC becomes aberrantly regulated in cancer remain unknown. In this study, direct and indirect mechanisms of 5mC regulation were investigated. To understand the direct regulation of 5mC genome-wide, we depleted cell line models of the DNA methyltransferases (DNMTs) that are responsible for establishing (DNMT3A, DNMT3B, DNMT3L) and maintaining (DNMT1) 5mC patterns. Profiling of 5mC patterns on the Illumina HumanMethylation450 BeadChip revealed a unique antithetical relationship between DNMT1 and DNMT3B for the regulation of both 5mC and DNA hydroxymethylation (5hmC) across gene bodies. DNMT3B mediated nonCpG methylation, while DNMT3L influenced the activity of DNMT3B toward nonCG versus CpG site methylation. DNMT3B depletion induced 5mC patterns that closely resemble those observed during cellular differentiation and occurred across gene bodies of highly expressed, H3K36me3-marked genes. SETD2, the histone methyltransferase responsible for H3K36me3 establishment across active gene bodies, was determined to influence the guidance of DNA methylation genome-wide through an indirect mechanism. SETD2 knockout induced widespread loss of H3K36me3 that did not coincide with changes in 5mC. However, paradoxical gains in H3K36me3 significantly induced hypermethylation and upregulation of underlying genes. Genes marked exclusively by the poised enhancer mark, H3K4me1, were commonly targeted for this epigenetic phenotype. DNA methylome profiling of loss-of-function SETD2 mutated clear cell renal cell carcinoma, papillary renal cell carcinoma, and lung adenocarcinoma tumors confirmed the predominance of the hypermethylation phenotype upon loss of SETD2. Collectively, these studies provide novel insight to understanding the regulatory mechanisms by which 5mC patterns are conferred.
    • A descriptive study of hospital nurses' valuation of quality assurance

      Showman, Lonnie; School of Nursing (1984-05)
      The purpose of this study was to describe hospital nurses' valuation of quality ~ssurance. It replicateq Edwardson and Anderson's (1983) study through use of their questionnaire. One nursing department located in a southern community was studied. A total of 78 nursing employees supplied data regarding valuation of quality assurance. Three distinct groups supplied the data: staff nurses who worked Monday through Friday; management nurses; and nurses who worked a special staffing option, the Baylor Plan, on weekends. The Metropolitan Nurses for Quality Assurance Nursing Survey Questionnaire, developed by members of the Metropolitan Nurses in Quality Assurance of Greater Minneapolis and St. Paul, was used to measure valuation of quality assurance. Data were analyzed descriptively~ The study hypotheses proposed by Edwardson and Anderson did not appear. to be supported. Nurses who had participated in formal quality assurance activities within the last year were not more likeiy to want to write care standards for their specialty area or engage in peer review.
    • A Descriptive Study of Student Attrition Patterns in Georgia Nursing Programs

      Brandon, Marsha Ann; Department of Physiological and Technical Nursing (1975-05)
    • Design, Synthesis, and Initial Evaluation of D-Glyceraldehyde Crosslinked Gelatin-Hydroxyapatite as a Potential Bone Graft Substitute Material

      Florschutz, Anthony V; Institute of Molecular Medicine and Genetics (2012-07)
      Utilization of bone grafts for the treatment of skeletal pathology is a common practice in orthopaedic, craniomaxillofacial, dental, and plastic surgery. Autogenous bone graft is the established archetype but has disadvantages including donor site morbidity, limited supply, and prolonging operative time. In order to avoid these and other issues, bone graft substitute materials are becoming increasingly prevalent among surgeons for reconstructing skeletal defects and arthrodesis applications. Bone graft substitutes are biomaterials, biologies, and guided tissue/bone regenerative devices that can be used alone or in combinations as supplements or alternatives to autogenous bone graft. There is a growing interest and trend to specialize graft substitutes for specific indications and although there is good rationale for this indication-specific approach, the development and utility of a more universal bone graft substitute may provide a better answer for patients and surgeons. The aim of the present research focuses on the design, synthesis, and initial evaluation of D-glyceraldehyde crosslinked gelatin-hydroxyapatite composites for potential use as a bone graft substitutes. After initial establishment of rational material design, gelatinhydroxyapatite scaffolds were fabricated with different gelatin:hydroxyapatite ratios and crosslinking concentrations. The synthesized scaffolds were subsequently evaluated on the basis of their swelling behavior, porosity, density, percent composition, mechanical properties, and morphology and further assessed with respect to cell-biomaterial interaction and biomineralization in vitro. Although none of the materials achieved mechanical properties suitable for structural graft applications, a reproducible material design and synthesis was achieved with properties recognized to facilitate bone formation. Select scaffold formulations as well as a subset of scaffolds loaded with recombinant human bone morphogenetic protein-2 were implanted ectopically in a rodent animal model and histologically evaluated for biocompatibility, degradation, and bone formation in vivo. The gelatin-hydroxyapatite scaffolds retained dimensional structure over 28 days and did not elicit any undesirable systemic or local effects. Distinct areas of mineralization and osteoid/bone were noted in all the implanted scaffolds and quantitative differences were primarily dependent on the presence of hydroxyapatite.
    • Detection of Single Base Substitutions in DNA

      Hallowes, William Cannon; Department of Cell and Molecular Biology (1987-12)
    • Determinants of Participant and Health Promotion Activities in Rural Caregivers

      Easom, Leisa R.; Department of Physiological and Technological Nursing (2003-05)
      Rural elderly caregivers, a rapidly growing segment of our population, are particularly at risk for adverse health outcomes. Caregiving is difficult work and can be exhausting physically, mentally, and spiritually for the caregiver. The purpose of this study was to examine the potential relationships between perceived self-efficacy, perceived barriers and personal factors (functional/physical health, emotional health, folk home remedy behavior, and spirituality) and engagement in health promotion practices in rural, elderly caregivers. The probability sample consisted of 80 rural, elderly caregivers (71 female; 9 male) ranging in age from 65 to 84 years. Each was interviewed via telephone using a self-report questionnaire. A model of health promotion for rural, elderly caregivers based on Pender’s Health Promotion model was tested with multiple regression analyses. The proposed model accounted for 19% of the explained variance of health promotion activities. An interesting finding was the high level of frequency of engagement in health promotion activities except for exercise. Surprisingly, folk home remedy behavior did not impact engagement in health promotion activities in these rural elderly caregivers. While 99% of the caregivers in this sample reported using at least one folk home remedy in the last year, these data suggest that the caregivers utilized these remedies in addition to the reported health promotion activities. Emotional health was a significant positive correlate of engagement in health promotion activities suggesting that emotional health is an important factor for a healthy lifestyle in the rural, elderly caregiver. Future research should include the examination of specific health promotion domains in rural elderly caregivers. Additionally intervention studies with longitudinal designs are needed for this population. Implications for theory development and community-based interventions are presented.
    • Determinants of Quality of the Caregiving Relationship

      Sauter, Maranah A.; Department of Physiological and Technological Nursing (1996-10)
      The purpose of this study was to examine selected caregiver and care receiver characteristics which may influence perceived quality of the dyadic caregiving relationship. The theoretical model evolved from reciprocity and social exchange theories and the theoretical and empirical caregiving literature. A purposive sample of 100 elderly care receivers and their family caregivers was selected for this study. Caregiving dyads were narrowed to community residing, cognitively intact care receivers, age 60 and over, and their spouse or adult daughter/daughter-inlaw caregivers. Care receivers were assisted with two or more activities of daily living three times per week or more. Approximately half of the study dyads were spouses. Caregiver characteristics included social and economic resources, physical and emotional health, and length of time as caregiver. Care receiver characteristics included social and economic resources, physical, functional, and emotional health, and length of time as care receiver. The concept of perceived reciprocity was used as a measure of quality of the caregiving relationship. Four dimensions of reciprocity were examined. These were caregiver perceived reciprocity, care receiver perceived reciprocity, intradyadic congruence of perceived reciprocity, and dyadic perceived reciprocity. Regression analyses revealed that lower caregiver depression, higher caregiver social and economic resources, and higher care receiver depression predicted caregiver perceived reciprocity. When examining dyadic perceived reciprocity, high care receiver depression was again a significant predictor of reciprocity. However, an interaction (cross-product) of both caregiver and care receiver depression showed that when both members of the dyad had low levels of depression, dyadic perceived reciprocity increased. Dyadic perceptions of reciprocity were congruent between caregivers and care receivers in the study; however, with-in dyad levels of perceived reciprocity were different. Dyadic descriptions of caregiving relationships reflected strong commitments to caregiving relationships and strong bonds of attachment. Quality of these relationships provides benefits which may help sustain caregiving. Longitudinal research is needed to determine the effects of caregiver and care receiver depression over time on quality of the caregiving relationship. Studies are also needed to investigate how quality of the caregiving relationship influences decisions to maintain or terminate caregiving.
    • Determining the Molecular Basis for Depressed Ventricular Contractile Function in Cardiac Neural Creat-Ablated Chick Embryos

      Hatcher, Cathy J; Department of Biochemistry and Molecular Biology (1998-01)
      Ablation of the cardiac neural crest (CNC A) in embryonic chicks at Hamburger- Hamilton stages 8-10 results in a high incidence of persistent truncus arteriosus, a congenital heart defect characterized by a single arterial trunk leaving the heart. Decreased ventricular contractility, which could be due to defects at the level of the contractile apparatus or in the excitation-contraction coupling (ECC) process, has been documented in this model. The first hypothesis that the decrease in isometric force produced per cross-bridge by Triton-skinned ventricular muscle preparations was due to inhibition of the contractile apparatus caused by excessive microtubules was not supported by the data: (1) The total microtubule content and the maximum calcium-activated forc generated by ventricular muscle strips of hearts from embryonic day (ED) 15 CNC A and sham-operated control embryos was not significantly different. (2) Destabilization of microtubules in ventricular muscle strips did not improve the force-producing capability of the contractile apparatus in CNCA embryos. Therefore, microtubules do not appear to be the cause for decreased isometric force production. The second hypothesis that the decrease in ventricular contractility was due to an improperly functioning FKBP1 2 .6 , a ryanodine receptor (RyR) modulatory protein which plays a role in ECC, was supported by the data: (1) FKBP1 2 .6 was present in comparable amounts in hearts from ED 15 CNCA and sham-operated embryos, (2) Dissociation of FKBP1 2 .6 from the RyR had significant effects on intact twitch force and calcium-induced calcium release from the sarcoplasmic reticulum in ED IS sham-operated, but not CNCA embryos. Therefore, FKBP1 2 .6 is present, but not functional in hearts from CNCA embryos, and, thereby, plays a major role in the impaired ECC in these hearts.
    • THE DEVELOPMENT AND TESTING OF AN OBSERVATION RATING SCALE TO MEASURE BEHAVIORAL DEVELOPMENT OF PREMATURE, VENTILATOR DEPENDENT INFANTS

      Burch, Sharon; School of Nursing (1987-12)
      The purpese of this study was to develop and test an observational a~sessment Seal~ that could be used to measure the behavioral development of premature, ventilator dependent infants. The major goal of this study was to establish interrater reliability and content and criterion-related validity for this.scale. Two research questions guided the development of this instrument: 1) Can certain behaviors reflecting developmental stability of a premature. infant be observed and measured? and 2) Are certain clusters of behaviors indicative of the degree of physiological instability of premature infants? The sample consisted of 20 premature infants: Group I (n=lO) infants of 26-29 weeks of gestation and ventilator dependent, and Group II (n=lO) infants of 32-34 weeks of gestation and not ventilator dependent. These infants were observed prior to a nursing intervention and after a nursing int~rvention. Interrater reliability, content validity and criterion-related validity were obtained. The results indicated that the behaviors characteristic of premature infants of 26-29 weeks of gestation and ventilator dependent can be identified, observed and reliably measured.
    • Development of an Instrument for Assessment of Auditory Hallucinations in Schizophrenia

      Frederick, Jane A; Department of Physiological and Technological Nursing (2000-03)
      The purpose of this study was to develop and assess the psychometrics of an instrument for the assessment of auditory hallucinations in schizophrenia and other psychiatric disorders. Development of the Auditory Hallucination Assessment Scale (AHAS) was guided by Albert Ellis’ reformulated model of Rational Emotive Behavior Therapy, the ABCD model. Applied to auditory hallucinations, the ABCD model asserts that distress and maladaptive coping behaviors are consequences not of the hallucination itself, but of the individual’s beliefs about the hallucination. An initial draft of the AHAS was submitted to an expert panel for assessment of content validity. The revised version of the AHAS is a 32 item self-report. Likert scale items describe characteristics, beliefs and attitudes about hallucinations and consequences of hallucinations. The psychometric properties of the AHAS were studied in a convenience sample of 151 clients in psychosocial rehabilitation outpatient day programs in two Georgia counties. Participants also completed two other hallucination scales, and one depression scale. Exploratory factor analysis was used to determine the underlying factor structure of the items and to assess the construct validity of the AHAS. The factor analysis resulted in a three-factor model which delineated three differing clinical symptom patterns of auditory hallucinations. The factors were labeled Troublesome Voices, Dangerous Voices, and Nurturing Voices. Rather than dividing the hallucinatory experience into three cognitive components as in the theoretical model, the factors reflected connections between the characteristics, beliefs, and consequences of auditory hallucinations. Thus, each of the constructs of the theoretical model operates within each of the resulting factors. The Troublesome Voices Factor consists of items indicating distressing auditory hallucinations and attempts to cope with them. The items on this factor indicate self-awareness and reflect behavioral or emotional reactions to the voices. The items on the Dangerous Voices Factor reflect beliefs that the voices are bad, hostile and powerful. The Nurturing Voices Factor items indicate that the individual experiences soothing and comforting voices. Finally, it is clinically significant that all participants in the sample, ninety-one percent of whom had schizophrenia or schizoaffective disorder, were able to complete the AHAS without difficulty.
    • Development of an Instrument to Measure the Appraisal of Cancer-related Fatigue

      Clark, Jane C.; Department of Biochemistry and Molecular Biology (2001-05)
      Based on a conceptual definition and model of fatigue, a three-phase descriptive study was conducted to develop items for a self-report instrument to measure the appraisal of fatigue. In Phase I, interviews with people experiencing cancer-related fatigue and a review of the literature were used to generate items to sample four constructs of the appraisal of fatigue: Fatigue, meaning, impact, and adaptability to fatigue. In Phase II, items were refined and reduced based on recommendations of a panel of content experts (N=7), instrument development expert, and pilot study results (N=20). In Phase III, reliability and validity estimates of the Fatigue Appraisal Scale were evaluated based on responses of a heterogeneous sample (N=196) of individuals diagnosed with cancer. Acceptable estimates of internal consistency reliability (Cronbach’s alpha) were determined for subscales of Fatigue (a =.91), Impact of Fatigue (a =.89), and Adaptability to Fatigue (a =.74). The internal consistency reliability of the Meaning of Fatigue subscale did not meet the required .60 for a new measurement instrument. To assess construct validity, hypotheses were generated and tested about the relationships of the subscales of the Fatigue Appraisal Scale and selected subscales of the Profile of Mood States-Shortened Form, Fatigue Assessment Instrument, and Functional Assessment of Cancer Therapy-Anemia. Results indicated low to moderate correlations (r = .20 to .70) among the subscales in the directions hypothesized with the exception of the correlation of the Adaptability to Fatigue subscale and the FACT-An Emotional and Functional Well-being subscales and the Impact of Fatigue subscale and the POMS Tension-anxiety subscale. Construct validity was examined further through factor analysis, using principal components analysis with varimax rotation. A four-factor solution with item loadings of .40 or greater on each factor was determined. Four items did not load on any of the four factors and were deleted. The factors were named: Global Fatigue, Impact of Fatigue, Adaptability to Fatigue, and Challenge of Fatigue. Items from the original Meaning of Fatigue subscale loaded on the Global Fatigue, Impact of Fatigue subscale or on the new factor, Challenge of Fatigue. Findings supported the factor structure for the instrument. Recommendations for future validation studies of the 34-item Revised Fatigue Appraisal Scale were offered.
    • Development of Novel Inhibitors of HSP90

      Patwardhan, Chaitanya A.; Department of Biochemistry and Molecular Biology (2014-02)
      Pharmacological inhibition of the Hsp90 machinery is an exciting option for cancer therapy. Clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to Hsp90, and induced overexpression of anti-apoptotic proteins, Hsp70 and Hsp27, is thought to contribute to this undesired outcome. We therefore face an urgent need to develop much better inhibitors of the Hsp90 machinery that can effectively kill cancer cells with minimal side effects. The goal of this dissertation is to identify novel inhibitors of Hsp90 chaperoning machinery to efficiently kill cancer cells with minimal side effects on normal cell survival. First, we report that the natural product, gedunin, may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to the co-chaperone p23 and inactivates it, without inducing over-expression of Hsp27 and only a relatively modest induction of Hsp70. Using molecular docking and mutational analyses; we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits p23 chaperoning activity, blocks its cellular interaction with Hsp90 and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the Cterminus. These results provide important insight into the molecular mechanism of action of this promising lead compound. Second, we report the development of a novel semi-high-throughput drugscreening assay to identify small molecule inhibitors of Hsp90 and its cochaperones. Our assay quantitatively measures the ability of Hsp90 and its cochaperones to refold the progesterone receptor (PR), a physiological client of Hsp90, in an in vitro assay performed in a 96-well plate format. We tested the NIH clinical collection drug library of 446 compounds and identified capsaicin as a “hit”. Our data show that capsaicin targets the Hsp70-Hsp90 chaperone complex in cells and alters Hsp70 multi-chaperone complexes. It induces cellular destabilization of Hsp90-Hsp70 client proteins and causes degradation of the Hsp70 (induced form) but not the Hsc70 (constitutive form) protein through lysosome-autophagy pathway. Cell survival assays showed that capsaicin selectively kills cancer cells by inducing mitophagy. Taken together, our data suggest that capsaicin could be used in combination with Hsp90 inhibitors for cancer treatment.
    • The Development of the Human Field Image Metaphor Scale

      Johnston, Linda W.; Department of Physiological and Technological Nursing (1993-05)
      The purpose of this study was the theoretical development and operationalization of the concept, Human Field Image, within the framework of the Rogerian Science of Unitary Human Beings. Initial stages involved a review of the relevant literature, followed by a process of theoretical development and definition. Human Field Image was defined as an individual awareness of the infinite wholeness of the human field. Two areas of content domain, identified as field manifestations of Human Field Image, were specified; (1) one's individual perception of potential, (2) the perception of the integral nature of one's human and environmental fields. The process of definition was followed by a process of item development and refinement for the Human Field Image Metaphor Scale (HFIMS). The metaphor was chosen as the appropriate item form for this instrument. An initial pool of 105 metaphors was reviewed by prominent Rogerian scholars and reduced to 32 items for the pilot form of the instrument. A pilot study with a sample of 50 adults preceeded the major study and led to the deletion of two items. This thirty item form of the HFIMS was administered to a sample of 3 58 adults ranging in age from 17 to 85. Factor Analysis revealed the presence of five factors, with a total scale Cronbach's Alpha of .9211. Additional items were eliminated and the number of factors was decreased. Revision of the instrument resulted in a final form of 25 items with three factors and a Cronbach's Alpha of .9131. The three factors were labeled "expressions of clear images of human field", "expressions of blurred images of human field", and "Integrality." Content validity was established through consultation with Dr. Martha Rogers and other Rogerian scholars. Construct validity was established through the correlation of scores on the HFIMS with scores on the Pictorial Form of the Human Field Motion Tool (r .6647, p < .01). Results of this study indicate that the HFIMS is a valid and reliable instrument which will make a significant contribution to the Science of Unitary Human Beings.
    • Developmental and Behavioral Analyses of clarin-2: A Novel Somatosensory Neuron Subtype-Enriched Gene

      Roberts, Rachel; Department of Neuroscience and Regenerative Medicine (2017-06)
      The trigeminal ganglion (TG) is a somatosensory organ that relays stimuli in the head to the hindbrain and spinal cord, and it comprises multiple subtypes of sensory neurons that respond to different somatosensory stimuli and establish distinct neuronal circuits. The Trpa1b subtype of TG sensory neurons (TGSNs) are responsible for sensing noxious chemicals, but the molecular cues that specify the development of this neuronal subtype remain poorly understood. Zebrafish were previously established as a robust model for studying the development of TGSNs due to its small size, translucency, and robust somatosensory behaviors. A previous microarray study in zebrafish found a novel four transmembrane-domain protein, clarin-2, to be enriched in Trpa1b-expressing cells. Nothing is known about the function of clarin-2, but a close homolog, clarin-1, is one of the causative genes for Usher Syndrome Type 3, a disorder characterized by progressive hearing and vision loss. We hypothesize that clarin-2 may play a role in the development and sensory function of TGSNs. To test this hypothesis, we examined the expression of clarin-2 within the TG during development and used clarin-2 knockout (KO) fish to study the genesis and neurite outgrowth of Trpa1b TGSNs. We found that clarin-2 is indeed enriched in a subset of TGSNs but is not required for the morphogenesis of the TG or the specification of nociceptive sensory neurons. Furthermore, axon projections from Trpa1b neurons were normal in clarin-2 KO fish, compared to control siblings. To test whether clarin-2 is required for the function of TGSNs, we tested somatosensory behaviors in larval zebrafish, including chemo-, thermo-, and mechanosensation. Behavioral analyses showed that clarin-2 is not required for the ability of Trpa1b neurons to detect the chemical irritant mustard oil. Additionally, the detection of heat or vibration was not affected in clarin-2 KO fish. Together, these results suggest that although clarin-2 is enriched in a subset of TGSNs, it is not required for the general morphogenesis of TGSNs or for somatosensation.
    • Diabetes-Induced Cholinergic Supersensitivity of Rat Heart

      Edwards, Ashley; Department of Pharacology & Toxicology (1984-11)
    • 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.
    • The Differential Roles of PI3K P110 Isoforms in Regulating CD4 T Cell Subset Polarization

      Webb, Mason James; Department of Biochemistry and Molecular Biology (5/12/2017)
      Class IA phosphatidylinositol-4,5-bisphosphate 3-kinases, or PI3K’s, are one of the earliest bottlenecks for T cell receptor signaling transduction, without which phosphorylated phosphatidylinositides cannot be generated and the T cell activation cascade becomes impaired. Of the catalytic class IA PI3K subunits, there are three isoforms designated as p110α, p110β, and p110δ. The Khleif laboratory has discovered that these catalytic subunits display unique roles in T regulatory cells and non-polarized activated CD4+ T cells. This thesis aims to determine what differential control these p110 isoforms have upon distinct polarized CD4+ T cell subsets.