by Chan Tak Yee Bonita. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 121-137). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references.

The control of cellular tyrosine phosphorylation levels is of great importance in many biological systems. Among the kinases and phosphatases that modulate these levels, the LAR-RPTPs have been suggested to act in several key aspects of neural development, and in a dysfunctional manner in various pathologies from diabetes to cancer. The aim of this thesis is to describe the physiological functions of one of the members of this subfamily of RPTPs, namely RPTPsigma. First, we showed that glucose homeostasis is altered in RPTPsigma null mice. They are hypoglycemic and more sensitive to exogenous insulin and we proposed that the insulin hypersensitivity observed in RPTPsigma-null mice is likely secondary to their neuroendocrine dysplasia and GH/IGF-1 deficiency. In addition to regulating nervous system development, RPTPsigma was previously shown to regulate axonal regeneration after injury. In the absence of RPTPsigma, axonal regeneration in the sciatic, facial and optical nerves was enhanced following nerve crush. However, myelin-associated growth inhibitory proteins and components of the glial scar such as CSPGs (chondroitin sulfate proteoglycans) have long been known to inhibit axonal regeneration in the CNS, making spinal cord injury irreversible. In collaboration with Dr Samuel David, we unveiled that RPTPsigma null mice are able to regenerate their corticospinal tract following spinal cord hemisections as opposed to their WT littermates. We then isolated primary neurons from both sets of animals and found that the absence of RPTPsigma promotes the ability of the neurons to adhere to certain inhibitory substrates. Finally, in order to better understand the physiological role of RPTPsigma, we used a yeast substrate-trapping approach, to screen a murine embryonic library for new substrates. This screen identified the RhoGAP p250GAP as a new substrate, suggesting a downstream role for RPTPsigma in RhoGTPase signaling. We also identified p130Cas and Fyn as new binding partners. All these proteins have clear functional links to neurite extension. The characterization of RPTPsigma and its signaling partners is essential for understanding its role in neurological development and may one day translate into treatments of neural diseases and injuries.

Recent population studies provide evidence that individuals with high circulating insulin levels have a poor prognosis and/or increased risk of cancer development; however, laboratory studies concerning the role of insulin in breast cancer biology are sparse. We compared the growth of 4T1 murine breast cancer allografts in control mice, alloxan-induced hypoinsulinemic mice, and mice treated with the insulin/insulin-like growth factor-1 receptor tyrosine kinase inhibitor BMS-536924. Both interventions significantly decreased tumor growth versus control and decreased pathway activation downstream of the insulin receptor as reflected by Aktser473 phosphorylation status in the neoplastic tissue. Alloxan-treated mice exhibited signs of insulin deficiency, while BMS-536924-treated animals showed only minor metabolic derangements. Skeletal muscle displayed reduced pAktser473 in alloxan-treated mice. In contrast, BMS-536924 treatment increased pAktser473 in muscle. This raises the possibility that the relative lack of metabolic toxicity of BMS-536924 involves varying tissue levels of the drug. These results support the view that host insulin physiology is a potentially modifiable determinant of breast cancer behaviour.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 90 pages. Includes vita. Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references (leaves 70-84). Also available online.

A paracoccidioidomicose (PCM) é uma doença sistêmica de caráter granulomatoso, causada pelo fungo termodimórfico Paracoccidioides spp. A PCM é endêmica na America Latina e aproximadamente 80% do pacientes vivem no território brasileiro. O tratamento medicamentoso é eficiente, entretanto, é longo e vários pacientes acabam abandonando e recidivas são comuns neste grupo. A utilização de uma vacina terapêutica poderia resultar na redução do tempo de tratamento assim como, recuperar a resposta imune do hospedeiro frente ao fungo. As vacinas de DNA são uma abordagem promissora na imunoterapia e podem ser injetadas por via intramuscular, intradérmica ou via mucosa. As proteínas de choque térmico (HSPs) são proteínas que estão ligadas a homeostase celular e também possuem efeitos imunológicos em diversos casos como doenças infecciosas e autoimunes. No presente trabalho, analisamos o esquema vacinal terapêutico em camundongos BALB/c previamente infectados intratraquealmente com 3x105 leveduras de P. brasiliensis Pb18, 60 dias depois, submetidos a imunização com pcDNA3 contendo sequências codificadoras de PbHSP60 e/ou IL-12 murina e/ou vetor vazio. Foi observada redução significativa no número de unidades formadoras de colônia (UFCs) nos pulmões de camundongos imunizados com PbHSP60. Os grupos que receberam PbHSP60+pcDNA3 vazio ou PbHSP60x2 apresentaram os maiores índices de redução da cargas fúngicas. A inclusão do plasmídeo contendo o inserto de mIL-12, resultou em um efeito deletério. A análise dos cortes histológicos indicou que os animais vacinados apresentavam áreas bem preservadas e com poucos ou nenhum foco de granuloma. Detectamos um perfil de citocinas típico Th1/Th2. Nossos resultados sugerem que a imunização utilizando plasmídeo contendo o inserto HSP60, tem grande potencial vacinal / The paracoccidioidomycosis (PCM) is a systemic granulomatous disease of character, caused by the thermally dimorphic fungus Paracoccidioides spp. The PCM is endemic in Latin America and approximately 80% of patients are living in Brazil. The medical treatment is effective, however, is long and many patients end up abandoning and relapses are common in this group.The use of a therapeutic vaccine could result in the reducing time of treatment as well as recover the host immune response against the fungus. DNA vaccines are a promising approach for immunotherapy and can be injected by intramuscular, intradermal, or mucosal route. The heat shock proteins (HSPs) are proteins that are linked to cellular homeostasis and also have immunological effects in many cases as infectious and autoimmune diseases. In the present study, we analyzed the therapeutic vaccine schedule in BALB/c mice previously infected intratracheally with 3x105 yeast of P. brasiliensis strain 18, and 60 days after, undergoing immunization with pcDNA3 containing coding sequences PbHSP60 and / or murine IL-12 and / or empty vector. Significant reduction was observed in the number of colony forming units (CFU) in the lungs of mice immunized with PbHSP60. The groups that received empty pcDNA3 and PbHSP60 or PbHSP60x2 have higher rates of reduced fungal loads. The inclusion of the plasmid containing the insert mIL-12 resulted in a deleterious effect. The analysis of histological sections indicated that vaccinated animals had wellpreserved, with few or no focus of granuloma areas. It was detected a profile typical Th1/Th2 cytokines. Our results suggest that immunization using plasmid containing the insert HSP60 vaccine has great potential

No description available.

No description available.

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination. / Dissertation