以下に掲載:Biochemical and Biophysical Research Communications. November 2012, Volume 428, Issue 4, Pages 512–517, doi:10.1016/j.bbrc.2012.10.073.

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Growth hormone (GH), a 22 kD polypeptide primarily produced in the anterior pituitary gland, is a key regulator of postnatal growth and affects carbohydrate, protein and lipid metabolism. Growth hormone receptor (GHR) is a single membrane-spanning type ‡T glycoprotein of the cytokine receptor family and is initially synthesized as a precursor and undergoes carbohydrate processing during transport through the Golgi. It has no intrinsic kinase activity. One molecule of GH binding to predimerized GHR on the cell surface induces the receptor conformational change activating the cytoplasmic domainassociated tyrosine kinase, Janus Kinase 2 (JAK2), which phosphorylates GHR, followed by the activation of downstream pathways, including MAPK, STATs and PI3K. Thus, GH sensitivity is largely determined by cell surface GHR abundance. Previous studies have demonstrated that JAK2, in addition to its role in GH signaling, enhanced GHR maturation, mature GHR stability and surface availability. By reconsititution of ƒÁ2AGHR or ƒÁ2A-JAK2 cells with mutant JAK2 or mutant GHR respectively, we found that the membrane proximal region of GHR called Box1 element and intact N-terminus of JAK2 are necessary and sufficient for the JAK2-dependent stabilization of the GHR in the absence of its ligand. In contrast, neither the kinase domain nor the kinase-like domain of JAK2 is required for this effect. Furthermore, we investigated the effect of JAK2 on GH-induced GHR loss. Our data suggested that in the presence of JAK2, GH treatment markedly enhances GHR degradation in a dose-dependent manner. However, in contrast to constitutive GHR loss, GH-induced receptor downregulation relies not only on GHR-JAK2 association but also on JAK2 kinase activity and GHR tyrosine(s). Tyrosine mutation of GHR diminished GH-induced GHR internalization. Mutation of the serine residues in DSGRTS motif of GHR did not impair GH-induced GHR degradation. Both lactacystin (a proteasome inhibitor) and chloroquine (a lysosome inhibitor) blocked GHinduced GHR loss. Further experiments also indicate that, like downregulation, JAK2 kinase activity and GHR tyrosine(s) are also critical for GH-dependent ubiquitination of GHR.

ix, 118 p. : ill., digital, PDF file

Cell Biology

Joint Health Sciences

GHR Downregulation Determinants

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Advisors/Committee Members: Frank, Stuart J., Collawn, Jim<br&#62;Messina, Joseph L.<br&#62;Chang, Chenbei<br&#62;Lin, Weei-Chin.

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Maintenance of immune homeostasis involves a balance between T cell effector responses to antigen stimulus and reciprocal downregulation of this response through peripheral tolerance mechanisms. Upon exposure to pathogen, cytokine production and signaling serve to tightly coordinate cell-mediated clearance of antigen followed by contraction of the immune response. Interkeukin-2 (IL-2) is a type I family cytokine critical for expansion of activated T cells in vitro and enhancement of T cell memory responses in vivo. Deficiency of IL-2 in vivo also revealed a critical role for IL-2 in immune tolerance through the maintenance of T regulatory cell populations (Treg) in peripheral lymphoid tissues. Thus, regulation of Il2 gene expression and autocrine signaling are central to the balance of immune homeostasis. Defining the regulatory mechanisms associated with Il2 gene expression is crucial for defining the integral role for IL-2 in tolerance and effector immunity. In order to evaluate the role of IL-2 production during the immune response we have engineered a BAC transgenic as well as a gene-targeted knock-in reporter mouse model system to mark Il2 gene activation. Our studies indicate that both mouse models exhibit reporter expression with high fidelity to endogenous Il2 expression patterns while stably marking IL-2-producing cells. Using our Il2 BAC transgenic system (2BiT) system to model inhibition of IL-2 production in responder CD4 T cells by Tregs we describe a model in which Tregs prevent initial activation of Il2 transcription that also subsequently associates with increased responder cell death of non-IL-2 producers. Suppression of naïve activated CD4 T cells by Tregs followed a mechanism consistent with competition for co-stimulation and preventing a threshold of activation rather than cytokine deprivation. Utilizing an Il2 GFP knock-in model we show evidence that IL-21 enhances proliferation and the frequency of IL-2-producing cells in activated naïve CD4 T cells. These results are suggestive of a coordinate relationship between Il2 expression and IL- 21 production and signaling in effector T cell function and fate. Future Il2 expression studies using our novel mouse reporter systems will enable a more in-depth understanding of the role of this cytokine in effector function and immune tolerance.

1 online resource (xvi, 141 p.) : ill., digital, PDF file.

Microbiology;

Joint Health Sciences;

Interleukin-2 T regulatory cells immune tolerance T cell activation gene transcription BAC transgenic and knock-in reporter mice

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Advisors/Committee Members: Weaver, Casey T., Burrows, Peter D.<br&#62, Chaplin, David D.<br&#62, Hatton, Robin D.<br&#62, Raman, Chander<br&#62, Ryan, Thomas M..

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Introduction: Cardiovascular disease (CVD) is the number one killer of women in the United States (US). Increasing rates of CVD have been associated with the epidemic rates of type 2 diabetes mellitus (T2DM) in the US. Minority women, particularly American Indian women, experience a greater burden of health risk factors and significant disparities in health status related to CVD and T2DM. Furthermore, women who have had gestational diabetes mellitus (GDM) during pregnancy are at greater risk for these diseases than women who have not had GDM. However, these women often do not perceive themselves to be at risk, or, if they do, they often do not possess the selfefficacy to carry out lifestyle behaviors that would decrease their risk. Article Synthesis: The first published article included in this dissertation, titled Type 2 Diabetes – Fueling the Surge of Cardiovascular Disease in Women (Jones & Appel, 2008), describes the cardiometabolic risk conferred by T2DM and CVD and provides a foundational review for the two articles that follow. With a more narrow scope, the second published article included, titled, A Review of the Health Beliefs and Lifestyle Behaviors of Women with Previous Gestational Diabetes (Jones, Roche, & Appel, 2009) examines the health beliefs, risk perceptions, and health behaviors related to diet and physical activity of women with previous GDM (pGDM). Finally, the third article, titled, A Mixed Methods Investigation of Cardiometabolic Risk and Risk Perception among Oklahoma American Indian Women with Previous Gestational Diabetes describes a pilot study which examines the cardiometabolic risk factors, risk perceptions, and self-efficacy beliefs of Oklahoma American Indian women with pGDM. The final article addresses gaps in the literature related to the estimation of cardiometabolic risk among American Indian women with pGDM and the description of knowledge, risk perceptions, and selfefficacy beliefs related to prevention of T2DM and CVD. Findings from the descriptive mixed methods study presented in this final article contribute to the growing body of nursing knowledge related to health disparities and women’s cardiometabolic health.

1 online resource (xi, 159 p. : ill., digital, PDF file)

Nursing;

cardiometabolic risk gestational diabetes type 2 diabetes cardiovascular disease risk perception mixed methods

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Advisors/Committee Members: Appel, Susan, Eaves, Yvonne<br&#62, Moneyham, Linda<br&#62, Oster, Robert<br&#62, Ovalle, Fernando.

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Insulin resistance commonly occurs following injuries or critical illness independent of previous diabetic status. The development of insulin resistance and hyperglycemia is associated with increased mortality and morbidity in critically ill patients. However, the molecular mechanisms underlying the acute insulin resistance following injuries remain poorly understood. With an animal model of trauma and hemorrhage, we have previously demonstrated the rapid development of insulin resistance in liver, and a critical role of TNF? in hemorrhage-induced defects in insulin signaling following resuscitation. However, hepatic insulin signaling is impaired prior to a significant increase in plasma TNF?, suggesting the initial development of hemorrhage-induced hepatic insulin resistance may be TNF? independent. By inhibiting the activity of TNF? or reactive oxygen species (ROS), our current studies further demonstrated that elevated ROS is responsible for the initial development of hepatic insulin resistance following hemorrhage, while TNF? has a dominant role later, following resuscitation. The mechanisms by which ROS induces the rapid development of hepatic insulin resistance were next investigated. Both Inhibitory-?B kinases (IKK) and c-Jun Nterminal kinase (JNK) can be activated by ROS and inhibit insulin signaling, potentially by increasing serine phosphorylation of insulin receptor (IR) or insulin receptor substrates (IRSs). Activation of hepatic IKK and JNK was observed as early as 15 minutes after trauma and hemorrhage, which was concomitant with the rapid impairment of hepatic insulin signaling. The important roles of IKK and JNK in the early development of hemorrhage-induced insulin resistance were further demonstrated by direct inhibition of the IKKs (IKK? and ?) and JNK1 with adenovirus-mediated expression of dominant negative mutants of IKKs and JNK1 in liver. Using two approaches of Kupffer celldepletion, we further demonstrated that the early development of hemorrhage-induced hepatic insulin resistance and activation of IKK and JNK are Kupffer cell independent. Thus, acute activation of IKK and JNK likely occurs within hepatocytes where they directly interact with IR or IRS proteins and inhibit insulin signaling. In conclusion, our studies provide mechanistic insights into the acute onset of hepatic insulin resistance following injury. IKK and JNK are potential therapeutic targets for treating hyperglycemia in the Intensive Care Unit.

1 online resource (xii, 199 p.) :ill., digital, PDF file.

Joint Health Sciences

Acute insulin resistance, Injury, Liver, TNF?, Serine kinase, Kupffer cell

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Advisors/Committee Members: Joseph L. Messina, Additional advisors: Jeonga Kim, John D. Mountz, Alexander Pereboev, Selvarangan Ponnazhagan..

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Successful treatment of breast cancer directly correlates with tumor stage and grade at diagnosis. Early diagnosis leads to a five-year relative survival rate of 96.8% as opposed to only a 22.5% five-year survival rate when diagnosed at late stage. Cancerspecific mechanisms need to be extended to enable cancer detection in an individual patient, in particular, the metastatic burden that currently cannot be assessed by conventional imaging. This approach for cancer detection would remove the biases of patient characteristics, permit immediate diagnostic feedback, improve sensitivity for early detection of small metastatic lesions, and be cost effective. Toward this goal, the cancer-specific diagnostic adenovirus (Ad) vector (Ad5/3-Id1-SEAP-Id1-mCherry) was produced that includes a human secreted embryonic alkaline phosphatase (SEAP) reporter for blood-based screening and a fluorescent reporter (mCherry) for localized imaging. Importantly, the cancer-specific promoter Id1 was selected to drive both reporters due to the correlation between aggressive cancer phenotypes and high Id1 expression. Safety and efficacy issues have limited the use of recombinant Ad for routine clinical applications. To overcome this impediment, ultrasound microbubbles (MB) were developed to improve Ad delivery to the tumor vasculature. This was achieved by targeting the Ad packaged MB to receptors overexpressed in the tumor, namely αVβ3 integrin, P-selectin, and VEGFR2. During in vitro assessment, both SEAP and mCherry reporter expression was shown to correlate with cancer cell phenotype. In a tumorbearing mouse model, SEAP reporter expression was found to be 14-fold over background when as little as 2.5% of tumor cells were infected. Triple-targeted MBs displayed enhanced affinity for tumor vasculature when assessed in vitro and in vivo. Packaging of the targeted MBs with diagnostic Ad was confirmed and the approach was tested in a tumor-bearing mouse model. Groups receiving the targeted Ad packaged MBs displayed a 4-fold increase in SEAP reporter expression over groups receiving equivalent unpackaged Ad dosing. In addition, the mCherry fluorescence reporter was localized in the tumor. Safe delivery of a non-invasive diagnostic reporter system using FDA approved MBs has the potential for immediate application, allowing this diagnostic system to impact populations currently in need of more advanced and sensitive detection modalities.

1 online resource (xi, 111 p. : ill., digital, PDF file)

Pathology

Joint Health Sciences;

breast cancer screening gene therapy microbubbles ultrasound contrast agent adenovirus delivery

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Advisors/Committee Members: Sanderson, Ralph D., Borovjagin, Anton V.<br&#62, Feng, Xu<br&#62, Frost, Andra R.<br&#62, Lopez, Richard D.<br&#62, Ponnazhagan, Selvarangan<br&#62, Zinn, Kurt R..

▼ Behavior Screeners are important tools for early identification of children’s social, emotional, and behavioral problems in schools. This study evaluated the concurrent and predictive validity of the BASC-2 Behavioral and Emotional Screening System (BASC-2 BESS). It was compared to the Behavior Assessment System for Children, Second Edition (BASC-2) to determine the congruent validity between the instruments. Predictive validity was examined by comparing the instruments to office referrals. The rating scales were administered to parents (96% mother, 4% father) of 8 identified and 15 non-identified students (mean age of 10.3 years; 52% male, 48% female). Pearson correlation coefficients examining the consistency between the two instruments were generally strong and positive, supporting the use of both instruments for assessing a child for behavioral or emotional problems. An analysis of hits and misses found the BASC-2 BESS accurate for predicting office referrals and therefore usable for screening students for possible behavioral problems.

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Ovarian cancer remains the most lethal gynecological malignancy in the U.S. Conventional therapies have limited therapeutic value due to advanced stage of the dis-ease at diagnosis. Conditionally-replicating adenoviruses (CRAds) are promising, novel anti-cancer agents that are designed to selectively replicate in and lyse tumor cells. In clinical trials, CRAds exhibited limited efficacy thus far. Second generation CRAds are being developed to express a therapeutic protein to further enhance antitumor efficacy. One attractive target in ovarian tumor microenvironment is the matrix metalloproteinases (MMPs) that degrade the extracellular matrix. Thus, tissue inhibitor of metalloproteinase 2 (TIMP2), an endogenous inhibitor of MMPs, is a promising candidate to arm a CRAd intended to treat ovarian cancer. We hypothesize that a CRAD armed with TIMP2 will inhibit ovarian cancer progression through two distinct mechanisms. First, specific repli-cation in tumor cells should lead to oncolysis. Second, secretion of TIMP2 into the tu-mor microenvironment should inhibit excess extracellular MMPs and activate other MMP-independent signaling pathways to inhibit tumor growth, angiogenesis and metas-tasis. A targeted and armed CRAd, Ad5/3-CXCR4-TIMP2 was developed using the CXCR4 promoter for enhanced replication, expressing the TIMP2 transgene. First, we confirmed the secretion and functional activity of TIMP2, as demonstrated by the inhibi- tion of gelatin degradation by MMPs. In addition, arming with TIMP2 did not inhibit viral replication nor oncolytic potency, as the TIMP2 armed viruses showed enhanced killing of cancer cells when compared to the unarmed viruses. Examination of viral rep-lication in primary stage III and IV ovarian cancer samples revealed a consistent high level of viral replication with Ad5/3-CXCR4-TIMP2. The therapeutic efficacy of the TIMP2-armed CRAd was then evaluated in an orthotropic model of ovarian cancer, in which athymic mice were intraperitoneally injected with human ovarian cancer cells. The results demonstrated that the Ad5/3-CXCR4-TIMP2 delayed tumor growth and sig-nificantly increased survival when compared to the unarmed CRAd. This effect was me-diated through inhibition of MMPs. Collectively, the present study demonstrated the en-hanced therapeutic efficacy of the dual-action, TIMP2-armed CRAd in vivo and the trans-lational potential of using Ad5/3-CXCR4-TIMP2 for treatment in patients with advanced ovarian cancer.

1 online resource (xiv, 113 p.) : ill., digital, PDF file.

Pathology;

Joint Health Sciences;

CRAds CXCR4 TIMP2 ovarian cancer

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Advisors/Committee Members: Ponnazhagan, Selvarangan, Alvarez, Ronald D.<br&#62, Bedwell, David M.<br&#62, Lorenz, Robin<br&#62, Siegal, Gene P.<br&#62, Strong, Theresa V..

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Identification and characterization of ethnic differences in insulin dynamics, insulin sensitivity (SI), and inflammation may shed light on the greater risk for type 2 diabetes in African Americans (AA), relative to European Americans (EA). Ideally, such studies should take into account the potential confounding roles of adiposity, fat distribution, genetic background, and socioeconomic status (SES). The objectives of this study were, 1) to evaluate ethnic differences in insulin dynamics in AA and EA children and adults using robust measures of insulin secretion and clearance; 2) to identify the contributions of genetic admixture and SES to insulin dynamics; 3) to examine ethnic differences in markers of inflammation (CRP, IL6, TNF-[alpha], and TNFRII) in AA and EA adults; and 4) to examine the association of markers of inflammation with SI after adjusting for measures of adiposity and fat distribution. Measures of SI and insulin dynamics were obtained via intravenous glucose tolerance test and mathematical modeling. Body composition was assessed with dual-energy X-ray absorptiometry, and fat distribution with computed tomography scanning. Subjects were genotyped for African and European genetic admixture. In children, African genetic admixture was independently associated with greater first-phase insulin secretion, and tended to be associated with lower insulin extraction. Total body fat masked the effect of genetic admixture on firstphase secretion. Children with lower SES have greater total insulin secretion. In adults, AA vs. EA women had greater early-phase [beta]-cell responsivity, compensated by lower total-[beta]-cell responsivity, and similar insulin extraction. AA had lower TNF-[alpha] and TNFRII, relative to EA. Significant independent associations of SI with IL6 and TNFRII were present, after adjusting for confounding variables. The greater first phase secretion in AA may serve as a risk factor for development of type 2 diabetes, by facilitating early [beta]-cell exhaustion. While this study highlights the independent association between SI and inflammation, it is unclear whether the role of inflammation in chronic disease is similar in AA and EA. Future studies should aim at understanding the physiologic significance of greater first phase insulin secretion, and lower TNF-[alpha] and TNFRII, in AA with respect to implication for chronic disease.

viii, 89 p. : digital, PDF file.

Nutrition Sciences

Health Professions

Ethnicity Insulin Secretion Insulin Clearance Inflammation African American

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Advisors/Committee Members: Gower, Barbara, Ard, Jamy D.<br&#62, Arnett, Donna K.<br&#62, Fernandez, Jose R.<br&#62, Ovalle, Fernando.

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Type 2 diabetes (T2D) is a complex disease with genetic and environmental components. An increased prevalence of T2D in patients with schizophrenia (SCZ) has been observed. Exposure to psychiatric medications such as antipsychotics (APs) have been shown to induce weight gain and T2D in some patients providing a unique opportunity to study gene and treatment related risk for T2D. Linkage and association studies have identified important candidate genes for T2D that may interact with AP medication to induce T2D with biological plausibility including TCF7L2, CAPN10, and ENNP1. The PAARTNERS study is a unique population (N~3000) of African American families with at least one SCZ or schizoaffective (SAD) member in addition to 412 mentally healthy African American controls recruited at 8 sites throughout the eastern US. The current study of PAARTNERS SCZ and SAD cases (N=891), exposed to similar psychiatric medication regimens, examined select genetic and treatment related risk factors for T2D. It was hypothesized that AP treatment may interfere with cellular, signal transduction pathways important in metabolism, specifically the WNT and insulin signaling pathways. Among SCZ/SAD cases, results show main effect associations with T2D of TCF7L2 (rs7903146) with OR=2.6 (p=0.003) and CAPN10(rs3792267) with OR=1.65 (p=0.05), both involved in the WNT signaling pathway. Current AP treatment was associated with increased odds of T2D with OR=2.4 (p=0.02) and results for specific current AP medications were consistent with the extensive literature on antipsychotic induced weight gain and T2D (clozapine, OR=3.0, p=0.01). Current valproic acid treatment increased the odds of having T2D three fold after adjustment for AP treatment (p=0.002). Interestingly, for stratified analysis that included controls results were suggestive of a TCF7L2 by current AP treatment interaction on the multiplicative scale for increased risk for T2D (OR=1.7, p=0.3 for TCF7L2 risk genotype, no AP treatment; OR=2.7 p<0.0001 for AP treatment, wild type TCF7L2; OR=6.3, p<0.001 for both and 1.7*2.7<6.3). A major limitation of the pilot study is that PAARTNERS is a crosssectional study and we were unable to measure specific AP treatment at T2D onset, dose, duration and even that AP treatment preceded T2D onset.

xii, 134 p. : ill., digital, PDF file.

Epidemiology

Public Health

Type 2 Diabetes TCF7L2 CAPN10 ENPP1 Schizophrenia Antipsychotics

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Advisors/Committee Members: Go, Rodney C.P., Arnett, Donna K.<br&#62;Kabagambe, Edmond K.<br&#62;Tiwari, Hemant K.<br&#62;Savage, Robert M..

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Defining the epitope specificities of the neutralizing antibody response to the HIV-1 envelope protein (Env) during natural human infection and in response to vaccination by HIV-1 immunogens could provide insight into the mechanisms of virus immune containment and facilitate vaccine design. Transplantation of discrete HIV-1 neutralizing epitopes into HIV-2 Env scaffolds may provide a sensitive, biologically functional context in which to quantify specific antibody reactivities in complex sera. Here we describe the development of a novel HIV-2 proviral scaffold (pHIV-2KR.X7) into which we have substituted the env V3 region of several laboratory and primary HIV-1 strains to yield a panel of chimeric viruses. The HIV-2KR.X7HIV-1 V3 chimeric viruses were found to be infectious and replication competent, and each maintained receptor engagement, coreceptor selection, and virus-cell membrane fusion kinetics typical of primary viruses. The HIV-2KR.X7HIV-1 V3 chimeras and parental viruses, HIV-2KR.X4 and HIV-2KR.X7, were each found to be resistant to neutralization by anti-HIV-1 monoclonal antibodies (mAbs) directed at the CD4 binding site, CD4 induced (CD4i) epitopes, and the membrane proximal external region (MPER). Notably, the HIV- 2KR.X7HIV-1 V3 chimeras were exquisitely sensitive to neutralization by the HIV-1 V3 specific mAbs 447-52D and F425 B4e8 (IC50 < 0.005 μg/ml for each), while the parental viruses remained resistant. Evaluation of V3-specific reactivities in plasma specimens from clade B and clade C HIV-1 infected individuals and from subjects vaccinated with iv HIV-1 immunogens indicated that potent, broadly reactive V3-specific antibodies are elicited early during natural HIV-1 infection and after vaccination with HIV-1 immunogens, prior to the development of antibodies targeting CD4i epitopes, the MPER, or epitopes that mediate autologous or heterologous neutralization. However, V3- specific antibodies exhibited low potency against autologous or heterologous primary HIV-1 strains because V3 epitopes are effectively masked within the trimeric Env spike. We conclude that V3-specific antibodies possess potent and broad neutralizing potential and constrain the HIV-1 Env to structure(s) in which the V3 is occluded prior to CD4 engagement. Otherwise, V3 antibodies fail to contribute to neutralization breadth or potency against primary viruses including in the setting of vaccination.

viii, 231 p. : ill., digital, PDF file

Microbiology

Joint Health Sciences

HIV-1 HIV-2 Neutralizing Antibodies V3

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Advisors/Committee Members: Hoxie, James A., Kearney, John F.<br&#62, Kutsch, Olaf<br&#62, Lorenz, Robinna G.<br&#62, Luo, Ming.

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Hematopoiesis is a highly regulated process directed by the microenvironment or niche in the bone marrow and the transcription factors those signals activate. Gene knockout experiments have identified critical cytokine signals and transcription factors required for promoting the differentiation of a hematopoietic stem cell to a B cell, but the regulatory mechanisms have yet to be fully elucidated. In early B cell development, a transcriptional hierarchy exists wherein E2A proteins activate early B-cell factor (Ebf1), which in turn activates expression of the B cell commitment factor, Pax5. In our studies, we use IL-7 receptor alpha chain (IL-7R[Alpha]) knockout animals that are arrested in adult B cell development at the earliest B-specified stage. We found that increasing the expression level of E47 through use of a retroviral vector in IL-7R[Alpha]-deficient bone marrow is not sufficient to induce Ebf1 expression. Rather, E47 expression resulted in significant upregulation in the mRNA levels of inhibitors of differentiation proteins, Id2 and Id3. In contrast, enforced expression of Ebf1 in IL-7R[Alpha]-/- bone marrow cells restores B cell differentiation in vivo, potently downregulates expression of both Id2 and Id3 mRNA, and it further upregulates the expression of E47 mRNA. This suggests that one major function of Ebf1 in B-lineage specification is to down-regulate Id levels to allow for activation of E2A protein activity. Additionally, we investigated the role of cytokine signaling in regulating the induction of Ebf1 expression. Our studies show a correlation between down-regulation of expression of c-Kit and Flt3 receptors from the cell surface and developmental progression from the common lymphoid progenitor (CLP) to the earliest B cell progenitor in adult bone marrow. Additionally, in vitro stimulation of the receptors with recombinant SCF or Flt3L was sufficient to block IL-7 induction of Ebf1 expression. Therefore, c-Kit and Flt3 are responsible for inhibiting the B cell promoting activity of IL-7 signaling and for maintaining the full lymphoid potential of common lymphoid progenitors. Collectively, these studies suggest a model in which the balance between positive and negative environmental cues determines maintenance of the lymphoid progenitor population versus Ebf1 induction and B cell specification.

1 online resource (ix, 97 p. : ill., digital, PDF file)

Microbiology;

Joint Health Sciences;

EBF E2A Id proteins early B cell development c-Kit Flt3

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Advisors/Committee Members: Klug, Christopher, Chaplin, David<br&#62, Burrows, Peter<br&#62, Weaver, Casey<br&#62, Kearney, John.

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Self-management behaviors are the cornerstone for control of type 2 diabetes mellitus (T2DM). Patients living with T2DM manage their own care on a daily basis using a variety of self-care strategies. Rural people living with diabetes are diagnosed later and receive less than optimal care compared to people living with T2DM in urban areas. Research evidence supports a relationship between self-efficacy and selfmanagement behaviors of people living with T2DM. Determining mediators of the relationship between self-efficacy and self-management can provide direction for development of interventions to improve participation in self-management behaviors, which can in turn improve diabetes outcomes. The purpose of the current study was to examine whether social support and social problem-solving were mediators of the relationship between self-efficacy and diabetes self-management behaviors in rural Alabamians living with T2DM. Additionally, relationships among self-efficacy, social support, social problem-solving, and diabetes self-management behaviors in rural Alabamians living with T2DM were examined in this mediational study. A descriptive, correlational design was used to explore the relationships examined in the study, based on the theory of stress, appraisal, and coping by Lazarus and Folkman. A convenience sample of 152 rural Alabamians living with T2DM participated in the study. Participants ranged in age from 19 to over 81 with the majority (56.6%) being between 51 and 70. The majority of the sample was African American (58.6%), female (65.8%), and had at least a high school education (57.9%). Concerning marital status, 33.6% were single and 36.8% were married. Most (59.3%) of the participants had been diagnosed with T2DM for 10 years or less. Self-efficacy was found to be a strong predictor of diabetes self-management. The effect of social support on diabetes self-management differed among men and women in the sample. Social support and social problem-solving were significantly associated with diabetes self-management in men in this sample. Multiple regression was used in four steps of mediation testing. Social support and social problem-solving were not mediators of the relationship between self-efficacy and diabetes self-management in this sample of people living with T2DM in rural Alabama.

2011

1 online resource (xiii, 162 p.) : ill., digital, PDF file.

Nursing;

Nursing;

Type 2 diabetes mellitus self-efficacy social support social problem-solving self-management

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Advisors/Committee Members: Grant, Joan S., Moneyham, Linda D.<br&#62, Pryor, Erica R.<br&#62, Steele, Michael M.<br&#62, Wilder, Barbara F..

▼ Global amphibian declines pose a major threat to the world’s biodiversity. We examined the observation bias associated with volunteer based anuran surveys, such as the North American Amphibian Monitoring Protocol (NAAMP). We followed NAAMP protocol to examine if variation in the persons (1-3) in an observer unit affected observer error. We hypothesized that observation units with multiple observers have less observer bias and would better report anuran assemblages compared to single observers. Larger observer units had fewer incidences of false positive observations. Additionally, we attempted to determine which sampling method for the eastern hellbender (Cryptobranchus a. alleganiensis) had the highest detection rate. We examined the detection probability of three methods: visual encounter surveys (VES), nocturnal spotlighting, and un-baited trapping. After 200 search hours and 300 trap nights, one hellbender was detected during a VES. Due to the small sample size we were unable to determine site occupancy and detection probabilit

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Malignant gliomas are the most common and deadly form of primary brain cancer afflicting adults. Current treatment regimens, including surgical debulking, radiotherapy, and chemotherapy, have limited efficacy, and median patient survival remains only 14 months. Therefore, novel therapies must target different aspects of glioma biology. Two of the most striking features of this cancer are the unusual ability of glioma cells to robustly proliferate and migrate in the brain, and recent evidence suggests that ClC-3, a voltage-gated Cl- channel/transporter is implicated in both of these processes. We hypothesize that ClC-3 may facilitate proliferation and migration by promoting hydrodynamic shape and volume changes; as Cl- efflux occurs, water osmotically leaves the cytoplasm. These shape and volume changes are critical as, for example, a glioma cell divides into 2 daughter cells, or migrates through narrow extracellular spaces in the brain. In this dissertation, we assess upstream signaling to determine how ClC-3 is activated in the context of proliferation and migration. Using a combination of biophysical, biochemical, genetic, and imaging techniques, we identify several mechanisms suggesting that Ca2+/calmodulin-dependent protein kinase (CaMKII) regulates ClC-3 activity. We demonstrate that channels or ligands that increase [Ca2+]i also activate CaMKII, leading to downstream ClC-3 activation and promoting proliferation and migration. CaMKII regulation of ClC-3 is required for a critical cytoplasmic condensation checkpoint at the metaphase-anaphase transition, and inhibition of either protein leads to disrupted volume regulation and proliferation. Additionally, we found that bradykinin, a chemotactic peptide, increases glioma cell migration by activating CaMKII-dependent ClC-3 channels. Inhibition of ClC-3 or CaMKII completely blocked bradykinin-induced migration. We propose that CaMKII activation of ClC-3 is a critical mediator of cellular proliferation and migration and should be integrated into preexisting models. We speculate that [Ca2+]i may be a ""master regulator"" of both proliferation and migration by simultaneously controlling cytoskeletal proteins, kinases, and Ca2+-sensitive ion channels. Finally, our data suggest that targeting Cl- channels or bradykinin receptors on human glioma cells may be a novel therapeutic strategy for the management of malignant gliomas.

PhD

1 online resource (xi, 213 p.) :ill., digital, PDF file.

Neurobiology

Joint Health Sciences

bradykinin cell migration chloride channel ClC-3 Cl- channel

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Advisors/Committee Members: Harald Sontheimer, Bedwell,David Kirk,Kevin Nabors,Burt Wadiche,Jacques.

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In the absence of antiretroviral therapy (ART), the majority of individuals infected with human immunodeficiency virus-1 (HIV-1) will develop AIDS. HIV-1-infected controllers are exceptions to the rule; without the use of ART, these individuals spontaneously control virus replication. A better understanding of the immune mechanisms that mediate delayed disease progression, as seen in controllers, will provide valuable insight to the design and development of an effective HIV-1 vaccine. CD8 T cells are important mediators of the antiviral immune response. However, it is unclear which components of the response are critical for long-lasting protection during HIV-1 infection. We first review methods currently used in the analysis of HIV-1-specific CD8 T cell function. We then discuss our analysis of HIV-1-specific CD8 T cell clonal populations that did or did not produce interleukin-2 (IL-2), an effector function associated with delayed disease progression. Having observed a unique clonotypic profile of IL-2 producing CD8 T cells, we predicted that maintenance of IL-2 production identifies a population of CD8 T cells with enhanced ability to (1) produce antiviral soluble factors as observed using multicolor flow cytometry and (2) restrict HIV-1 replication as detected using an in vitro suppression assay (iVSA). To test this hypothesis, we analyzed epitope-specific CD8 T cell function first during chronic HIV-1 infection and subsequently during primary infection. Using the iVSA, we observed significantly enhanced suppression of HIV-1 replication by CD8 T cells derived from controllers when compared to progressors. Interestingly, the level of suppression correlated with a polyfunctional, IL-2+ CD8 T cell response. Preliminary results of CD8 T cell effector function during primary HIV-1 disease show that functional, epitope-specific CD8 T cell lines can be expanded and these cells are able to suppress HIV-1 replication in vitro, albeit to moderate levels. Both the proliferative and suppressive capacity of CD8 T cells derived from the early stage of disease appeared to increase over time. These studies suggest that suppression of HIV-1 replication and polyfunctional IL-2 production would be promising markers of an effective CD8 T cell response and have important implications for the evaluation of HIV-1 vaccine strategies.

1 online resource (xii, 114 p.) : ill., digital, PDF file.

Microbiology

Joint Health Sciences

UNRESTRICTED

Advisors/Committee Members: Goepfert, Paul A., Britt, William J.<br&#62, Chaplin, David D.<br&#62, Lorenz, Robin G.<br&#62, Prevelige, Peter E..

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Chronic diseases such as type 2 Diabetes Mellitis (T2DM) and cardiovascular disease (CVD) are accompanied by chronic low-grade systemic inflammation and are related to obesity and ethnicity. For instance, African-Americans (AA) have increased risk for developing T2DM and are more likely to be overweight relative to Caucasians (C). However, C have more intra-abdominal adipose tissue (IAAT) and have higher risk for CVD than AA. The influence that ethnicity has on disease risk is obscured by ethnic differences in fat distribution and sensitivity to insulin. Ethnic differences in markers of inflammation (MOI) have not been investigated. It is also unclear if the decreases in MOI seen with weight loss are mediated by fat mass losses in different fat depots, which may differ among the ethnicities. The current study aimed to determine if, independent of fat distribution, there are ethnic differences in MOI. Furthermore, this study was designed to determine the effect of weight loss, with and without exercise interventions, on MOI in AA and C women. 213 overweight, but otherwise healthy women were recruited for a weight loss protocol, with or without exercise. Subjects were randomized into intervention groups: diet only, diet + aerobic training, or diet + resistance training. Body fat distribution was determined by dual-energy x-ray absorptiometry and computed tomography; insulin sensitivity by frequently sampled intravenous glucose tolerance test; and MOI by ELISA. The hypotheses of this study were that C women would have higher MOI than AA. However, after adjusting for IAAT, there would be no ethnic difference in MOI. Furthermore, independent of weight loss modality, MOI would be lower after weight loss and would specifically reflect the amount of fat lost. At baseline, plasma concentrations of TNF-[alpha] and its receptors were higher in C than AA. However, after adjusting for IAAT, the ethnic difference in TNF-[alpha] was attenuated to borderline significance (P=0.054). Among all women combined, all MOI decreased significantly with weight loss. When comparisons were conducted by ethnicity, all MOI decreased with weight loss among C, but only CRP decreased in AA. Overall, weight loss yielded favorable improvements in inflammation regardless of weight loss mechanism.

xi, 62 p. : ill., digital, PDF file.

Nutrition Sciences

Health Professions

Inflammation Fat Distribution Insulin Sensitivity Weight Loss Exercise Ethnicity

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Advisors/Committee Members: Gower, Barbara A., Bamman, Marcas M.<br&#62, Heimburger, Douglas C.<br&#62, Hunter, Gary R.<br&#62, Newcomer, Bradley R.<br&#62, Oster, Robert A..

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The NR4A family of orphan nuclear receptors function as early response genes with numerous stimuli. The NR4A family consists of three known members, Nur77, Nurr1, and Mitogen-inducible nuclear orphan receptor (MINOR). Our lab has previously demonstrated that stable over-expression of MINOR in 3T3-L1 adipocytes enhances insulin-stimulated glucose uptake. In order to assess the in vivo affect of MINOR on adipocytes, we generated transgenic mice with MINOR over-expression driven by the adipocyte- and macrophage-specific AP2 promoter (AP2-MINOR mice). We initially hypothesized that AP2-MINOR mice would display increased glucose tolerance and insulin sensitivity. However, AP2-MINOR transgenic male and female mice have increased fasting glucose and respond poorly to glucose challenge, with males developing insulin resistance late in life. AP2-MINOR males are larger than wild-type due to increases in both lean and fat mass. When males are maintained on high fat chow, transgenic animals are larger, but do not differ from wild-type when challenged with glucose or insulin. These results led us to hypothesize that MINOR over-expression induces adipocyte hypersensitivity to β-adrenergic agonism, leading to increased serum non-esterified fatty acids and impairment of the pancreatic β-cell response to glucose. Indeed, insulin fails to inhibit transgenic NEFA production below the level of wild-type in spite of significantly reduced serum catecholamines. Oddly, fasting serum total cholesterol and LDL were increased and NEFA was decreased in AP2-MINOR animals versus wild-type. Thus, it appears that MINOR over-expression effects both glucose and lipid homeostasis. As a whole, these observations are consistent with a MINOR-mediated down-regulation of Liver X receptor (LXR) and its target, sterol regulatory element binding protein-1c (SREBP-1c).

M.S.

1 online resource (viii, 39 p. : ill., digital, PDF file)

Nutrition Sciences;

Health Professions;

MINOR NOR-1 NR4A adipocyte nuclear receptor type 2 diabetes

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Advisors/Committee Members: Garvey, W. Timothy, Fu, Yuchang<br&#62, Nagy, Timothy R.<br&#62, Wood, Philip A..

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Osteosarcoma is the most predominant primary bone cancer among pediatric patients. The pathogenesis and mechanism of osteosarcoma, however, as yet remains unclear. This study investigated the hypothesis that Ca2+/calmodulin-dependent protein kinase II (CaMK II), activated by Ca2+ and its intracellular receptor, calmodulin, plays a critical role in the growth of human osteosarcoma cells. The inhibition of CaMK II with its pharmacologic antagonist KN-93 was used to investigate the role of CaMK II in the growth of human osteosarcoma cells because our preliminary data showed that =-CaMK II is expressed in several human osteosarcoma cell lines. To examine the in vivo effects of KN-93 on human osteosarcoma cells, MG-63 cells and Saos-2 cells were subcutaneously inoculated in 5-week-old male athymic nude mice. The mice were then treated with either KN-93 or phosphate buffered saline every other day for 6 weeks. The tumor cells were also inoculated in the tibia of the mice at the same time when mice received subcutaneous inoculation. The final total volume of subcutaneous tumors of KN-93-treated group was 40% smaller in MG-63 cells and 41 % smaller in Saos-2 cells than that of each control group at sacrifice. Supporting these findings, micro-computerized tomography of mouse tibia where MG-63 cells were grown provided another line of evidence that pathologic bone formation by osteosarcoma cells was decreased by KN-93 treatment. Further, immunohistochemistry staining on these samples demonstrated that the expression of p21 was significantly increased while that of p-Rb was decreased in the KN-93-treated group. These results suggest that p21-involving signaling cascade mediated by CaMK II may be one of the potential mechanisms controlling the growth of human osteosarcoma cells, which will warrant further investigations aiming at a novel target to treat human osteosarcomas.

M.S.

vii, 40 p. : ill., digital, PDF file.

Orthodontics

Dentistry

Osteosarcoma In Vivo KN-92 CaMK

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Advisors/Committee Members: Sadowsky, P. Lionel, Jacobson, Alex <br&#62, Rahemtulla, Firoz <br&#62, Zayzafoon, Majid.

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Little is known about the potency, breadth and epitope specificities of neutralizing antibody (Nab) responses elicited in natural HIV-2 infection. Analysis of plasma specimens from 64 HIV-2 chronically-infected subjects in a single round infectivity assay (JC53bl-13/TZM-bl) revealed median reciprocal IC50 Nab titers of 1.7x105, 2.8x104 and 3.3x10? against three primary virus strains, HIV-27312A, HIV-2ST and HIV-2UC1, respectively. A subset of 5 plasma samples was tested on 17 additional HIV-2 strains, and similarly high Nab titers were observed against all but four viruses. These Nab titers were higher, by orders of magnitude, than HIV-1 Nab titers in plasma from chronically infected subjects tested against heterologous primary HIV-1 strains. This finding was confirmed by using purified IgG, HIV-2 primary Envs derived by single genome amplification of plasma vRNA, and viruses grown in human PBMCs and tested for neutralization in human PBMCs. We next characterized 15 mAbs that were isolated from subjects chronically-infected with HIV-2. All 15 mAbs bound to the monomeric HIV-2 gp120 and potently neutralized most primary strains of HIV-2, with median IC50 titers ranging from 0.007 to 0.028 ?g/ml. Epitope mapping using cross-competition Env binding, peptide scanning, site-directed mutagenesis, chimeric Env constructions and single-cycle virus neutralization assays revealed three distinct mAb competition groups (CG-I, CG-II and CG-III). CG-I mAbs recognized conformational epitopes centered on the carboxy-terminus of the variable loop 4 (V4). CG-II mAbs bound to linear epitopes in the V3. The epitopes recognized by CG-III mAbs were complex and related to the CD4 or coreceptor binding surfaces. Patient plasmas competed with representative mAbs from each group for binding HIV-2 gp120 in titers that correlated significantly with Nab titers. HIV-2 MPER antibodies were rarely detected, low in titer and did not contribute to the neutralization potency and breadth. These findings indicate that primary HIV-2 strains are highly immunogenic in natural infection and surprisingly sensitive to antibody neutralization. The striking differences in susceptibility to neutralization between primary strains of HIV-2 and HIV-1 suggest fundamental differences in the biophysical properties of the respective Env trimers and mechanisms by which the two viruses persist in vivo in the face of neutralizing antibodies.

1 online resource (ix, 133 p.) :ill., digital, PDF file.

Microbiology

Joint Health Sciences

HIV-2, Neutralizing antibody, envelope

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Advisors/Committee Members: George M. Shaw, Additional advisors: John C. Kappes, Ming Luo, Casey D. Morrow, James E. Robinson, Jamil S. Saad..

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The t(8;21)(q22;q22) AML1-ETO translocation is one of the most frequent translocations in acute myeloid leukemia (AML), occurring in approximately 12% of cases. Our laboratory has generated a mouse model in which AML1-ETO is expressed in hematopoietic progenitor cells using a retroviral vector and assayed using bone marrow transplantation. Animals transplanted with AML1-ETO-expressing cells failed to develop leukemia. However, AML1-ETO promoted significant expansion of immature myeloid cells suggesting a “pre-leukemic” state that requires secondary mutations for disease progression. In addition, analysis of cell cycle kinetics in AML1-ETOexpressing myeloid progenitor cells showed that AML1- ETO slowed G1 to S phase progression compared to control animals. Further analysis using quantitative RT-PCR showed that AML1-ETO increased expression of p15Ink4b and p16Ink4a, which may account for the inhibition of the G1 to S phase transition. Interestingly, p15INK4b is a tumor suppressor that is inactivated by hypermethylation in over 70% of human AML patient samples. In addition, p16INK4a is frequently inactivated in the FAB-M2 subset of AML that expresses the t(8;21). These observations suggest that loss of p15Ink4b or p16Ink4a may cooperate with AML1-ETO in disease progression. To test this hypothesis, p15Ink4b-/-, p16Ink4a-/-, or wild-type bone marrow cells were transduced with AML1- ETO or control retroviruses and then transplanted into lethally irradiated recipient animals. Analysis of reconstituted animals at two months post-transplant showed that p15Ink4b or p16Ink4a deficiency did not accelerate disease progression in the presence of AML1-ETO. However, loss of p15Ink4b alone did cause a modest expansion of hematopoietic stem cells and a 2-fold increase in myeloid progenitor cells based in FACS analysis and differential bone marrow cell counts. These data suggest that the loss of either p15Ink4b or p16Ink4a alone is not a major contributing factor to pathogenesis associated with the AML1-ETO translocation.

vii, 66 p. : ill., digital, PDF file

Biochemistry and Molecular Genetics

Joint Health Sciences

AML1-ETD Leukemia Myeloid Development Hematopoietic Stem Cell

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Advisors/Committee Members: Klug, Christopher A., Chen, Xinbin <br&#62, Emanuel, Peter D. <br&#62, Ruppert, J. Michael <br&#62, Townes, Tim M..

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Lipoteichoic acid (LTA) is an essential bacterial membrane polysaccharide (cell wall component) that is linked to a glycerol backbone with two acyl chains. Since an antibody to LTA has been shown to protect from Gr+ bacterial infections or colonization, and the mutant Gr+ bacteria which produce less amount of LTA have less pathogenic than WT, LTA may be an important role in Gr+ bacterial infection and inflammation. The currently accepted structure of pneumococcal LTA has the pentameric repeating unit bound to the lipid anchor {Glc([beta]1 ? 3)AATGal([beta]1 ? 3)Glc([alpha]1 ? 3)-acyl2Gro}. Unlike the lipid anchor of other Gr+ bacterial LTA, this pneumococcal LTA lipid anchor has not been detected in the bacterial membrane. In addition, the current structure does not explain the Forssman antigen properties of Pneumococcal LTA. Thus we propose here the new model of pneumococcal LTA structure and show the new model has correct structure to explain its functions with a mass spectrometry. LTA can induce the production of various inflammatory molecules via Toll-like receptor 2 (TLR2) like LPS via TLR4. However, the role of LTA in Gr+ induced inflammation is still debated because there are many limitations to study LTA function. For instance, purified LTA is easily contaminated or damaged during purification step and it is impossible to make LTA-deficient Gr+ bacteria, because LTA is an essential component of Gr+ bacteria. Interestingly, its biological function can be altered by removing acyl chains because monoacylated LTA is not active in mouse model. Thus, we examined several phospholipase A2 (PLA2) to develop LTA inactivation methods. We found that PAF-acetylhydrolase, a recombinant human PLA2, is the most efficient and specific enzyme to produce monoacylated LTA that is inactive in mouse model. In early bacterial infection, Gr+ bacteria release/shed TLR2 ligands which are essential PAMPs in early infection and may be important to initiate inflammatory diseases such as sepsis. To define the molecules which are released in early stage, we identified pneumococcal molecules in the early bacteria culture supernatant with antipneumococcal antibody and we found LTA was present and may be dominant in early stage. Since LTA is potential TLR2 ligand, we hypothesis that LTA is an essential inflammatory component in early Gr+ bacterial infection. To investigate the role of LTA in early infection, three LTA inactivation methods were used to examine its role in the culture supernatants. Here we find that LTA is the primary TLR2 ligand in the early phase of Gr+ bacterial infection and remains a major ligand in the late phase when other TLR2 and TLR4 ligand(s) appear. Thus, our studies have established the importance of LTA in Gr+ bacteria induced inflammation. In addition, our findings suggest that LTA is important in the early sepsis process but other components become significant in late stages of bacterial infection.

xvi, 151 p. : ill., digital, PDF file

Microbiology

Joint Health Sciences

Lipoteichoic Acid…

Advisors/Committee Members: Nahm, Moon H., Briles, David E. Pritchard, David G.<br&#62;Waites, Ken B.<br&#62;Hollingshead, Susan K.<br&#62;Michalek, Suzanne M.<br&#62;Benjamine, William B..

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Stroke is the third leading cause of death and the most common cause of neurologic disability for adults in developed nations. Strokes trigger an acute inflammatory response prompted by brain tissue injury at the infarct site and the surrounding ischemic penumbra raising plasma levels of inflammatory markers. Creactive protein (CRP), an acute-phase inflammatory marker, has been significantly correlated with infarct size and post-stroke complications. Lipoprotein-associated phospholipase A2 (Lp-PLA2) may also predict long-term cardiovascular risk in the stroke population. In addition to physiologic changes, up to 60% of all stroke survivors are known to experience depression, which may contribute to decreased physical and cognitive functioning, decreased health-related quality of life, and deterioration of mobility after stroke. The specific aims of this study are to: (1) determine the effect of baseline CRP and Lp-PLA2 levels shortly after first-time stroke on subsequent health outcomes (functionality, neurological impairment, and quality of life) at 3 months post stroke, (2) determine the effect of baseline depression shortly after first-time stroke on subsequent health outcomes at 3 months post stroke, and (3) determine the interactive effect of CRP and Lp-PLA2 and depression on subsequent health outcomes at 3 months post-stroke. A prospective, descriptive cohort design was used. Participants with a first time stroke, over the age of 45, were recruited from the emergency department at a large, hospital in Northern Alabama with 881 licensed beds. The biological markers of CRP and Lp-PLA2 were collected at baseline and at 3 months post-stroke. Depression and health outcomes were assessed on hospital admission, on day 4 of hospital admission or on the day of discharge whichever occurred first, and at 3 months post-stroke. Recurrence of stroke was monitored for 3 months post-stroke. For the specific aims 1-3, data were analyzed using hierarchical multiple regression. Controlling for the effects of the covariates (age, baseline National Institute of Stroke Scale (NIHSS), and t-PA usage), the main effects of CRP, Lp-PLA2, and depression as well as their interactive effects (CRP x depression, Lp-PLA2 x depression, and CRP x Lp-PLA2 x depression) on health outcomes were determined. In this small study, only depression was predictive of functionality and quality of life at 3 months post-stroke.

1 online resource (x, 91 p. : ill., digital, PDF file)

Nursing;

Nursing;

Stroke CRP Lp-PLA2 Depression Health Outcomes

UNRESTRICTED

Advisors/Committee Members: Alexandrov, Anne W., Alexandrov, Andrei V.<br&#62, Pryor, Erica R.<br&#62, Tucker, Diane C.<br&#62, Vance, David E..

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Regulation of neural precursor cell (NPC) death is important for both normal brain development and prevention of brain tumor formation. The tumor suppressor p53 is an important regulator of NPC apoptosis, but the precise mechanism of p53-regulated NPC death remains largely unknown. By using primary cerebellar NPCs and a mouse cerebellar NPC line, we compared the molecular regulation of NPC death produced by staurosporine (STS), a broad-spectrum kinase inhibitor,with that caused by genotoxic agents. We found that both STS and genotoxin-induced NPC death were markedly inhibited by p53 or Bax deficiency. Genotoxin-induced cerebellar NPC death required new protein synthesis and PUMA, a p53 transcriptionally regulated BH3-only molecule. In contrast, STS caused cerebellar NPC death without requiring new protein synthesis or PUMA expression. In addition, genotoxic agents increased nuclear p53 immunoreactivity, whereas STS produced rapid cytoplasmic p53 accumulation, which preceded Bax activation and caspase-3 cleavage. The vast majority of p53 immunoreactivity in STS-treated NPCs was localized in the cytosol and was not associated with mitochondria, lysosomes or endoplasmic reticulum. Following STS administration, p53 co-immunoprecipitated with conformationally altered “activated” Bax suggesting an interaction between these pro-apoptotic molecules in triggering apoptotic death. At baseline, p53 nulcear-cytoplasmic shuttling is dependent on CRM1, a critical mediator of the nuclear export signal (NES)-dependent nuclear export. STS induced cytosolic p53 accumulation occurred in a CRM1-independent fashion and was not due to caspase-induced nuclear membrane disruption. Nucleophosmin (NPM), a CRM1-dependent nuclear chaperone, also translocated from the nucleus to the cytosol after STS treatment, and was co-immunoprecipitated with activated Bax in extracts from STS-treated cells. In summary, our studies indicate that p53 has multiple death promoting mechanisms in cerebellar NPCs. Genotoxin-induced death requires p53-dependent gene transcription of PUMA, whereas STS-induced NPC death requires a direct effect of cytosolic p53 potentially through its association with NPM and activated Bax. In total, these studies highlight the stimulus-specific nature of cell death signaling and clearly demonstrate the importance of p53 in regulating NPC fate.

1 online resource (xvi, 133 p. : ill., digital, PDF file)

Pathology

Joint Health Sciences

p53 neural stem cell apoptosis transcription independent staurosporine

UNRESTRICTED

Advisors/Committee Members: Roth, Kevin A., Carroll, Steven L.<br&#62, Clemens, Thomas L.<br&#62, Welch, Danny R.<br&#62, Wilson, Scott M..

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DYT1 dystonia is an inherited movement disorder caused by a trinucleotide deletion (DeltaGAG) in the DYT1 (TOR1A) gene, which codes for the torsinA protein. Dr. Yuqing Li's laboratory previously reported the characterization of a DYT1 dystonia mouse model, a knock-in carrying DeltaGAG in Dyt1 (KI), which displays a motor learning deficit of motor skill transfer. We report here that this motor learning deficit was reversed with an anticholinergic drug, trihexyphenidyl (THP), a drug commonly used to treat movement problems in dystonia patients. We further show a potential substrate for the pathophysiology, a reduction in D2 receptors in the striatum in KI mice, which may abolish LTD induction in the striatum. KI mice are partially resistant to FPL64176, an agonist of L-type calcium channels involved in LTD induction. These data suggest that altered communication between cholinergic interneurons and medium spiny neurons may be responsible for the LTD deficit. We also show that despite not having visible twisting and repetitive movements similar to dystonia, KI mice show sustained muscle contractions as assessed by electromyographic analysis. Finally, we addressed whether the torsinADeltaE mutation is acting as a loss-of-function or a gain-of-function, or both, using electrophysiological recordings in hippocampal slices. Three mutant mouse lines were tested: heterozygous knock-out mice (+/Delta), conditional homozygous knock-out mice (cKO), and torsinADeltaE KI mice. We discovered that both +/Delta and cKO mice show enhanced LTP in the CA1 region of the hippocampus, while no change was observed in the KI mice. In contrast, KI mice showed significantly enhanced paired pulse ratios (PPRs), which were absent in +/Delta; and cKO mice. In addition, KI mice revealed a decrease in frequency, but not amplitude or kinetics, of spontaneous excitatory post-synaptic currents in CA1 neurons, while these were not altered in +/Delta; and cKO mice. The differences in synaptic alterations between the torsinADeltaE KI and both the heterozygous and conditional homozogous knock-out of torsinA, lend support to a gain-of-function of torsinADeltaE , the mutation which underlies human dystonia.

MS

1 online resource (xiii, 153 p.) :ill., digital, PDF file.

Neurobiology

Joint Health Sciences

dystonia DYT1 gain-of-function pre-synaptic TOR1A torsinA

UNRESTRICTED

Advisors/Committee Members: Yuqing Li, Theibert,Anne Sweatt,David Standaert,David.

▼ Mitochondria are essential organelles that play crucial roles in many aspects of cellular homeostasis. More importantly, the mitochondria are home to the majority of the metabolic pathways within the cell and are responsible for producing most of the cell’s useable energy in the form of adenine triphosphate (ATP) through oxidative phosphorylation (OXPHOS). In mammals, the majority of OXPHOS complex subunits are encoded by nuclear deoxyribonucleic acid (DNA); however, 13 core subunits essential for the function of OXPHOS complexes I, III, IV, and V are encoded in the mitochondrial (mt) DNA (mtDNA) and are synthesized within the mitochondria by its own transcription and translation machinery. Changes in the expression and post-translational modifications (PTMs) of OXPHOS subunits and mitochondrial proteins can be detrimental to mitochondrial energy production. In fact, alterations in mitochondrial functions impact cellular energy metabolism as well as influence whole-body metabolism and have been identified as underlying causes for several diseases including neurological disorders, insulin resistance, type 2 diabetes (T2D), and numerous cancer types. This has led to an extensive search for a better understanding of key players involved in mitochondrial function in disease states, in addition to new preventative and therapeutic strategies that are aimed at exploiting key components of mitochondrial biogenesis and energy metabolism. Our laboratory has shown that differences in mitochondrial biogenesis can be caused by changes in the sequence and/or PTMs of mitochondrial proteins, resulting in altered mitochondrial function and energy metabolism. In the present studies, we investigated changes in mitochondrial energy metabolism in two major metabolic diseases, T2D and liver cancer, and evaluated potential targets and therapies. We first investigated mitochondrial biogenesis and energy metabolism in T2D by studying the differences in the expression and activity of OXPHOS complexes in the liver and kidney of the polygenic T2D model, TALLYHO/Jng (TH), and normal, C57BL/6J (B6), mice. A significant decrease was observed in the expression of both nuclear- and mitochondrial-encoded subunits of complexes I and IV, respectively, in TH mice compared to B6, which coincided with significant reductions in their enzymatic activities. Furthermore, we identified sequence variants in several mitochondrial proteins including OXPHOS complex subunits, a mitochondrial transfer ribonucleic acid (tRNA) synthetase, and mitochondrial ribosomal proteins (MRPs). The sequence variants identified in mitochondrial proteins might contribute to impaired mitochondrial biogenesis and energy metabolism by diminishing OXPHOS expression and activity in TH mice. In addition to T2D, we investigated liver cancer and demonstrated impaired mitochondrial OXPHOS complex expression and activity in cancerous liver biopsies compared to non-cancerous biopsies. The expression of the Src family kinases (SFKs), c-Src and Fyn, were increased in liver cancer cell lines and…

▼  Neurofibromatosis type 2 (NF2) is a benign tumor disease of the nervous system. Development of bilateral vestibular schwannomas is characteristic of NF2; however patients frequently present schwannomas on other nerves, as well as meningiomas and ependymomas. Currently, there are no drug therapies for NF2. There is an urgent need for development of NF2 therapeutics and this dissertation presents two independent potential therapeutic targets. The disease is caused by mutations in the NF2 gene that encodes a tumor suppressor called merlin. Loss of merlin function is associated with increased activity of Rac and p21-activated kinases (PAK) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrates for Cdc42/Rac-PAK, and modulate actin dynamics by phosphorylating cofilin, an actin severing and depolymerizing agent. LIMKs also translocate into the nucleus and regulate cell cycle progression. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2Ex2) exhibited increased levels of LIMK1, LIMK2, and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared to wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared to normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2Ex2 MSC reduced LIMK1 and LIMK2 levels. Pharmacological inhibition of LIMK with BMS-5, decreased the viability of Nf2Ex2 MSCs in a dose-dependent manner, but did not affect viability of iv control MSCs. Similarly, LIMK knockdown decreased viability of Nf2Ex2 MSCs. The decreased viability of Nf2Ex2 MSCs was due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrest cells in early mitosis by decreasing Aurora A activation and cofilin phosphorylation. To increase the search for NF2 therapeutics, we applied an alternative approach to drug discovery with an unbiased pilot high-throughput screen of the Library of Pharmacologically Active Compounds. We assayed for compounds capable of reducing viability of Nf2Ex2 MSC as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2, a mammalian sirtuin, is a NAD+ -dependent protein deacetylase. We show that Nf2Ex2 MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases Nf2Ex2 MSC viability in a dose dependent manner without substantially reducing wildtype MSC viability. Inhibition of SIRT2 activity in Nf2Ex2 MSC causes cell death accompanied by release of the necrotic markers lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest. Overall this work uncovered two novel potential therapeutic targets, LIMK and SIRT2 for NF2 and tumors associated… Advisors/Committee Members: Fernandez-Valle, Cristina.

▼ The purpose of this work is to explore and exploit the principles of Displacement Electrochemical Immunosensing (DEI) and Indirect Competitive ELISA (ICE) to detect 2,4,6-trichloroanisole (TCA). The rational design of indirect competitive ELISA for TCA detection is attempted. The developed assay detects TCA at concentrations from 1ppt to 1 ppm, with a limit of detection of 4.2 ppt. The assay can be commercially useful in situations where less than 80 minutes total assay time is required. A mathematical model (MM) is developed for the rational design of an electrochemical displacement immunosensor (DEI). Despite the low affinity constants of the antibodies obtained for this work a functional DEI is developed with the predicted by the MM high limit of detection for TCA (0.2 ppm). The non-specific adsorption (NSA) of proteins is identified as a critical problem inhibiting further optimization of the DEI. The use non-insulating Cu UPD as NSA controller or electrochemically compatible blocking, together with amperometric displacement detection are proposed as a platform that could permit further development of reagentless and labelless immunosensors. Advisors/Committee Members: [email protected] (authoremail), true (authoremailshow), Katakis, Ioanis (director), Lozano Sánchez, Pablo (codirector).

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La Partie I présente l’obtention du développement asymptotique topologique pour une classe d’équations elliptiques quasilinéaires. Un point central réside dans la possibilité de définir la variation de l’état direct à l’échelle 1 dans R^N. Après avoir défini un cadre fonctionnel approprié faisant intervenir les normes L^p et L², et avoir justifié la classe d’équations considérée, la méthode se poursuit par l’étude du comportement asymptotique de la solution du problème d’interface non linéaire dans R^N et par une mise en dualité appropriée des états direct et adjoint aux différentes étapes d’approximation.La Partie II traite d’estimations et de développements asymptotiques de p-capacités de condensateurs, dont l’obstacle est d’intérieur vide et de codimension &#62; ou = 2. Après les résultats préliminaires, les condensateurs équidistants permettent de donner deux illustrations de l’anisotropie engendrée par un segment dans l’équation de p-Laplace, puis d’établir une minoration de la p-capacité N-dimensionnelle d’un segment, qui fait intervenir les p-capacités d’un point, respectivement en dimensions N et (N-1). Les condensateurs elliptiques permettent d’établir que le gradient topologique de la 2-capacité n’est pas un outil approprié pour distinguer les courbes des obstacles d’intérieur non vide en 2D

Part I deals with obtaining topological asymptotic expansions for a class of quasilinear elliptic equations. A key point lies in the ability to define the variation of the direct state at scale 1 in R^N. After setting up an appropriate functional framework involving both the L^p and the L² norms, and then justifying the chosen class of equations, the approach goes on with the study of the asymptotic behavior of the solution of the nonlinear interface problem in R^N and by setting up an adequate duality scheme between the direct and adjoint states at each step of approximation. Part II deals with estimates and asymptotic expansions of condenser p-capacities and focuses on obstacles with empty interiors and with codimensions &#62; ou = 2. After preliminary results, equidistant condensers are introduced to point out the anisotropy caused by a segment in the p-Laplace equation, and to provide a lower bound to the N-dimensional condenser p-capacity of a segment, by means of the N-dimensional and of the (N-1)-dimensional condenser p-capacities of apoint. Introducing elliptical condensers, it turns out that the topological gradient of the 2-capacity is not an appropriate tool to separate curves and obstacles with nonempty interior in 2D

Advisors/Committee Members: Masmoudi, Mohamed (thesis director), Amstutz, Samuel (thesis director).

▼  Fiber based ultrashort pulse laser sources are desirable for many applications; however generating high peak powers in fiber lasers is primarily limited by the onset of nonlinear effects such as self-phase modulation, stimulated Raman scattering, and self-focusing. Increasing the fiber core diameter mitigates the onset of these nonlinear effects, but also allows unwanted higher-order transverse spatial modes to propagate. Both large core diameters and single-mode propagation can be simultaneously attained using photonic crystal fibers. Thulium-doped fiber lasers are attractive for high peak power ultrashort pulse systems. They offer a broad gain bandwidth, capable of amplifying sub-100 femtosecond pulses. The longer center wavelength at 2 ?m theoretically enables higher peak powers relative to 1 [micro]m systems since nonlinear effects inversely scale with wavelength. Also, the 2 [micro]m emission is desirable to support applications reaching further into the mid-IR. This work evaluates the performance of a novel all-fiber pump combiner that incorporates a thulium-doped photonic crystal fiber. This fully integrated amplifier is characterized and possesses a large gain bandwidth, essentially single-mode propagation, and high degree of polarization. This innovative all-fiber, thulium-doped photonic crystal fiber amplifier has great potential for enabling high peak powers in 2 [micro]m fiber systems; however the current optical-to-optical efficiency is low relative to similar free-space amplifiers. Further development and device optimization will lead to higher efficiencies and improved performance. Advisors/Committee Members: Richardson, Martin.