published_or_final_version / abstract / toc / Surgery / Master / Master of Philosophy

HER2 overexpression in breast cancer tumors predicts lower overall survival. Because of the aggressive nature of HER2 tumors and the association with metastatic disease, the HER2 receptor holds great promise as a therapeutic target in metastatic breast cancer. We are developing small molecule inhibitors that bind to the ATP binding site of the tyrosine kinase domain in order to inhibit tyrosine auto-phosphorylation. This process controls biological pathways that mediate the cell growth. In normal cells this process is highly controlled. We are targeting the modification of the side chain of the hydroxy methyl group of 2-Hydroxy methyl-5,8-dimethoxy-1,4-naphthaquinone. These compounds should inhibit the tyrosine kinase cascade of reactions thereby suppressing the overexpression of HER2 shutting down the tumor growth. The synthesis and characterization of a series of substituted naphthaquinone analogs with different increasing chain lengths will be reported.

Thesis (M.Sc.). / Written for the Dept. of Chemistry. Title from title page of PDF (viewed 2009/06/23). Includes bibliographical references.

Thesis (M.S.)--Mississippi State University. Department of Biological Sciences. / Title from title screen. Includes bibliographical references.

Thesis (Ph.D.)--Memorial University of Newfoundland, Faculty of Medicine, 2001. / Typescript. Bibliography: leaves 188-241.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 139-193).

PTP69D is a receptor protein tyrosine phosphatase (RPTP) with two intracellular catalytic domains (Cat1 and Cat2), which has been shown to play a role in axon outgrowth and guidance of embryonic motorneurons, as well as targeting of photoreceptor neurons in the visual system of Drosophila melanogaster. Here, we characterized the developmental role of PTP69D in the giant fiber (GF) neurons; two interneurons in the central nervous system (CNS) that control the escape response of the fly. In addition to guidance and targeting functions, our studies reveal an additional role for PTP69D in synaptic terminal growth in the CNS. We found that inhibition of phosphatase activity in catalytic domain (Cat1) proximal to the transmembrane domain did not affect axon guidance or targeting but resulted in stunted terminal growth of the GFs. Cell autonomous rescue and knockdown experiments demonstrated a function for PTP69D in the GFs, but not its postsynaptic target neurons. In addition,complementation studies and structure-function analyses revealed that for GF terminal growth, Cat1 function of PTP69D requires the immunoglobulin and the Cat2 domain but not the fibronectin type III repeats nor the membrane proximal region. In contrast, the fibronectin type III repeats, but not the immunoglobulin domains, were previously shown to be essential for axon targeting of photoreceptor neurons. Thus, our studies uncover a novel role for PTP69D in synaptic terminal growth in the CNS that is mechanistically distinct from its function during earlier developmental processes. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

This dissertation addresses the role for Janus Kinase 3 (Jak3) in CD4+ T cell homeostasis and function. Jak3 is a protein tyrosine kinase whose activity is essential for signals mediated by the γc dependent cytokines IL-2, -4, -7, -9, -15, and -21. Previous data have demonstrated that peripheral CD4+ T cells from Jak3-deficient mice have a memory phenotype and are functionally impaired in both proliferative and IL-2 responses in vitro. Interestingly, Jak3/γc activity has been previously shown to play a role in the prevention of T cell anergy. These studies were initiated to more precisely define the role for Jak3/γc cytokines in the prevention of T cell anergy and the maintenance of functional CD4+ T cell responses. We began to address this question by assessing global gene expression changes between wild type and Jak3-/- CD4+ T cells. These data indicate that Jak3-/- CD4+ T cells have an increase in gene expression levels of inhibitory surface receptors as well as immunosuppressive cytokines. Further analyses confirmed that Jak3-deficient T cells express high levels of PD-1, secrete a Trl-type cytokine profile following direct ex vivo activation, and suppress the proliferation of wild type T cells in vitro. These characteristics indicate that CD4+ Jak3-/- T cells share properties with regulatory T cell subsets that have an important role in peripheral tolerance and the prevention of autoimmunity. We next addressed whether these regulatory characteristics were T cell intrinsic or rather the result of expanding in a Jak3-deficient microenvironment characterized by a number of immune abnormalities and a disrupted splenic architecture. Jak3-/- CD4+ T cells proliferate in vivoin a lymphopenic environment and selectively acquire regulatory T cell characteristics in the absence of any additional activation signals. While the precise mechanism by which Jak3-deficient T cells acquire these characteristics remains unclear, our data indicate that one important component is a T cell-intrinsic requirement for Jak3 signaling. These findings indicate several interesting aspects of T cell biology. First, these studies, demonstrate that the homeostatic proliferation of CD4+ T cells is not dependent on signaling via γc-dependent cytokine receptors. And, second, that the weak activation signals normally associated with homeostatic expansion are sufficient to drive Jak3-/- T cells into a non-conventional differentiation program. Previous data indicate that, for wild type T cells, signaling through both the TCR as well as γc-dependent cytokine receptors promote the homeostatic proliferation of T cells in lymphopenic hosts. Since Jak3-/- T cells are unable to receive these cytokine signals, their proliferation is likely to be wholly dependent on TCR signaling. As a consequence of this TCR signaling, Jak3-/- T cells proliferate, but in addition, are induced to up regulate PD-1 and to selectively activate the IL-10 locus while shutting off the production of IL-2. Since this fate does not occur for wild type T cells in a comparable environment, it is likely that the unique differentiation pathway taken by Jak3-/- T cells reflects the effects of TCR signaling in the absence of γc-dependent cytokine signaling. Interestingly, wild type T cells undergoing homeostatic expansion in lymphopenic hosts show many common patterns of gene expression to freshly-purified unmanipulated Jak3-/- T cells. For instance, micro array analysis of gene expression in wild type CD4+ T cells after lymphopenia induced homeostatic expansion show a similar pattern of upregulation in surface markers (PD-1 and LAG-3), and cytokine signaling molecules (IL-10 and IFN-γ cytokine, receptors, and inducible gene targets) to that of Jak3-/- CD4+ T cells immediately ex vivo. These data suggest that the process of homeostatic proliferation normally induces immune attenuation and peripheral tolerance mechanisms, but that full differentiation into a regulatory T cell phenotype is prevented by γc-dependent cytokine signals. Taken together these data suggest that Jak3 plays an important role in tempering typical immune attenuation mechanisms employed to maintain T cell homeostasis and peripheral tolerance.

Introduction and aims. South Africa is the epicenter of the HIV/AIDS pandemic. Hypertensive disorders of pregnancy (15.7%) are the second cause of maternal deaths of which pre-eclampsia represents 83%. Normal pregnancy requires a balance between pro- and anti-angiogenic factors to necessitate effective vasculogenesis, angiogenesis and placental development, however, pre-eclampsia is characterised by an excess anti-angiogenic state. The hypoxic placenta releases excess anti-angiogenic factors into the maternal circulation causing endothelial dysfunction. However, there is no data to verify if HIV infection affects pre-eclampsia, or if the angiogenic imbalance is affected. Contradictory data exists on the association between HIV infection and pre-eclampsia. In an attempt to assess the role of HIV infection in pre-eclampsia, this study examined the immunolocalisation of sFlt-1, sEng, PlGF and VEGF in placentae of HIV negative and positive normotensive and pre-eclamptic pregnancies at term using immunohistochemistry (IHC) and immunoelectron microscopy (IEM). Additionally, we estimated the placental expression of sFlt-1, sEng, PlGF and VEGF to verify if the HIV negative differed from the HIV positive cohorts. We further evaluated the maternal serum to determine if variations existed in the circulating levels of these factors in HIV negative and positive normotensive and pre-eclamptic pregnancies. Methods. Following institutional ethical approval and informed consent, placental biopsies and maternal serum were collected post-delivery. For IHC and IEM, 130 and 25 placentae were evaluated, respectively. Following conventional immunohistochemical processing, 5μm sections were immunostained & immunoexpression of the various antibodies were evaluated with the Zeiss Axioscope A1 interfaced with an AxioVision Image analysis software package (version 4.8.3) in combination with the auto-measurement module (Carl Zeiss, Germany). Post-conventional immunoelectron processing, ultra-thin sections were immunolabelled. Sections were post-fixed, contrast enhanced with uranyl acetate and Reynolds lead citrate and viewed on a Jeol 1011 Transmission Electron Microscope. Additionally, the placental expressions of these factors were assessed using RT-PCR. In an attempt to confirm if maternal circulating levels of these factors differed, we quantitatively evaluated these factors in serum from HIV negative normotensives, HIV negative pre-eclamptics, HIV positive normotensives, and HIV positive pre-eclamptics using ELISA techniques. Results and Discussion. The expression of sFlt-1, sEng, PlGF and VEGF was confirmed using immunohistochemistry, RT-PCR and ELISAs. Irrespective of the HIV status, sFlt-1 and sEng was elevated with the concomitant reduction in PlGF in pre-eclamptic compared to normotensive pregnancies. The levels of VEGF were however undetectable across all study groups. It is plausible that this lack of effect of HIV status on the factors under study may be attributed to the treatment regimen as HAART is known to restore the immune response of HIV positive preeclamptic women. However, a concise anti-retroviral treatment history in our study was unavailable. Additionally, this study is novel in that it ultrastructurally immunolocalises sFlt-1, sEng, PlGF and VEGF within the placenta. This immunoelectron localisation data corresponds to our immunohistochemical data. Our study further demonstrates strong immunoreactivity of both placental sFlt-1 and sEng in pre-eclampsia with concurrent elevations in the maternal circulation. A qualitative increase in the occurrence of syncytial knots in the pre-eclamptics compared to the normotensive pregnancies was noted. These observations support the detachment of antixxx angiogenic rich microparticles from syncytial knots in the pre-eclamptics compared to the normotensive pregnancies was noted. These observations support the detachment of antiangiogenic rich microparticles from syncytial knots and their subsequent deportation and elevation in the maternal circulation. Moreover, their consequent antagonistic effects on VEGF, PlGF and TGF-β, disrupts the vascular endothelial maintenance. The strong immunoreactivity of sFlt-1, sEng, PlGF and VEGF was observed in villous endothelial cells. Moreover, a strong sFlt-1 and sEng but a weak PlGF and VEGF immunoreactivity was noted in syncytio- and cytotrophoblasts. This immunoexpression within trophoblasts is suggestive of their autocrine mode of action on normal trophoblast functions including invasion, differentiation and production. It is plausible that the angiogenic imbalance observed in our study, will impact on placental function, by modifying trophoblast activity thereby contributing to abnormal placentation. Conclusion. Our study supports the hypothesis that pre-eclampsia is characterized by an imbalance between pro- and anti-angiogenic factors. Whether the pregnancy is complicated by immune insufficiencies or not, does not affect the role of the anti-angiogenic factors in pre-eclampsia development. Nevertheless, the neutralising effect of HIV infection on the immune system may be insufficient in the development of pre-eclampsia. To our knowledge, the quantification of serum pro-/anti-angiogenic factors in HIV-associated pre-eclampsia is novel. In conclusion, our data reinforces the hypothesis that increased concentrations of sFlt-1 and sEng are involved in the pathogenesis of pre-eclampsia and indicates their possible use as discriminatory factors between diseases. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 135-155.