Nucleobindins are a class of secreted, multi-domain Ca2+ binding proteins that interact with nucleic acids. Two nucleobindins, nucleobindin-1 (NUCB1) and nucleobindin-2 (NUCB2) have been identified so far. In 2006, nesfatin-1, an 82 amino acid peptide encoded in NUCB2 was discovered. Nesfatin-1 is an anorexigenic and insulinotropic peptide found abundantly in hypothalamus, pancreas and stomach. Meal responsive insulin secretion is regulated by glucagon like peptide-1 (GLP-1), glucose dependent insulinotropic polypeptide (GIP), peptide YY (PYY) and cholecystokinin (CCK) secreted by intestinal mucosal cells. Since both nesfatin-1 and intestinal hormones modulate insulin secretion, nesfatin-1 could regulate intestinal hormones to elicit its insulinotropic action. Nucleobindin-1 primarily regulates Ca2+ homeostasis. Like NUCB2, NUCB1 is also present in the pancreas, stomach, intestine and pituitary. NUCB2 has a high similarity (62% in humans) to NUCB1. Both proteins also retain their prohormone convertase cleavage sites. However, no information exists on whether NUCB1 encodes bioactive peptides. The fact that NUCB1 is a secreted protein suggests an endocrine function for NUCB1 and/or its encoded peptide. This research hypothesizes that nesfatin-1 is enterotropic, and NUCB1 encodes an insulinotropic nesfatin-1-like peptide (NLP). Nesfatin-1 protein expression was found in STC-1 cells and it co-localized GLP-1, GIP, CCK and PYY in mouse enteroendocrine cells. Treatment of STC-1 cells with nesfatin-1 stimulated GLP-1, GIP, CCK mRNA expression and protein secretion, while opposite effects were found for PYY. In silico analysis of the NUCB1 amino acid sequence found a 77 amino acid NLP. Mouse pancreatic islets and MIN6 cells express NUCB1 mRNA and protein. NUCB1 was co-localized with insulin in mouse pancreatic islets. While treatment of cells with synthetic NLP increased preproinsulin mRNA expression and secretion, a scrambled peptide based on NLP was ineffective, indicating that the specific amino acid sequence is crucial for its insulinotropic action. Overall, the data presented supports the hypotheses. The studies reaffirm NUCB2 expression in intestine and provide the first set of evidence for nesfatin-1 regulation of enteric hormones. It also found a novel NUCB1 encoded insulinotropic NLP that could elicit other functions of nesfatin-1.

Parkinson's disease (PD) is the most common neurodegenerative motor disorder, whose clinical features are rest tremor, bradykinesia, muscular rigidity and postural instability. Although most reported cases are sporadic, a handful of familial cases and their causative genes have been identified. Loss-of-function mutations in DJ-1, one of these genes, are responsible for 1% of familial PD cases. Our laboratory has previously reported that DJ-1- lacking neurons are sensitive to oxidative stress, induced by hydrogen peroxide or the neurotoxin MPTP. To investigate the possible mechanisms through which DJ-1 protects against oxidative stress, we performed a proteomic screen and identified Von Hippel Lindau (VHL) and Paraoxonase2 (PON2) as potential DJ-1 interacting partners. VHL is an E3 ubiquitin ligase which, in normal conditions, poly-ubiquitinates HIF-1 , a subunit of a master hypoxic/oxidative stress transcription factor, whose function is protective in oxidative and hypoxic stresses. In the present study, we provided further evidence of interaction of DJ-1 with VHL. We also demonstrated that HIF-1 protein level, as an indicator of VHL activity, is lower in cells lacking DJ-1, suggesting the inhibitory role of DJ-1 on VHL. Our in vitro studies also showed that DJ-1 inhibits ubiquitin ligase activity of VHL on HIF-1 by reducing the VHL-HIF-1 interaction. Importantly, accumulation of HIF-1 protects embryonic cortical neurons against MPP+ induced neuronal death. Finally, we confirmed the impairment of HIF-1 response to oxidative stress in human lymphoblastoids of DJ-1-linked PD cases. In the second part of this study, we demonstrated the interaction of DJ-1 and PON2. Interestingly, PON2 lactonase activity is reduced in DJ-1 deficient cells which could be rescued by re-introduction of DJ-1, suggesting a modulating role of DJ-1 on PON2 activity. In addition, PON2 deficiency, like DJ-1 deficiency, hypersensitizes neurons to MPP+, which could be rescued by over-expression of PON2 in both cases. Taken together, our data provide evidence that DJ-1 exerts its protective role by inhibiting VHL activity, enhancing HIF-1 stability, and increasing PON2 pro-survival function in PD models.

Myeloid Cell Leukemia-1 (Mcl-1) is a widely expressed anti-apoptotic member of the Bcl-2 family that is elevated in a variety of tumour types including breast cancer. Mcl-1 promotes tumour cell survival and drug resistance and was a mechanism of resistance to first generation Bcl-2 family inhibitors. To determine the significance of Mcl-1 in breast cancer, we evaluated the regulation of Mcl-1 by signalling via the epidermal growth factor receptors (EGFRs). EGFR signalling is frequently deregulated in breast cancer and leads to increased proliferation and survival of tumour cells. We aimed to determine whether Mcl-1 is a critical downstream effector of this pathway and therefore an important therapeutic target. We found that Mcl-1 protein and messenger RNA levels were rapidly induced upon stimulation of breast cancer cells with epidermal growth factor. This induction was blocked by inhibitors of the Ras/Raf/Mek/Erk signalling cascade and was dependent upon activation of the transcription factor Elk-1. We found Mcl-1 to be an essential survival protein, as targeted knock-down with small interfering RNA alone was sufficient to induce apoptosis. Mcl-1 may be critical for the survival advantage conferred by EGFR activation, as prevention of its up-regulation by Mek/Erk inhibitors significantly reduced the drug resistance conferred by EGF. Furthermore, we found a correlation between phosphorylated Elk-1 and Mcl-1 protein levels in breast tumour samples. Therefore, we conclude that Mcl-1 is an important downstream effector of survival and drug resistance mediated by elevated EGF signalling, making it an important therapeutic target in breast cancer.

Universiẗat, kommulative Diss.--Bremen, 2006.

Thesis (Ph. D.)--University of Toronto, 1999. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Toronto, 1999. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thèse (Ph. D.)--Université Laval, 2005. / Titre de l'écran-titre (visionné le 15 décembre 2005). Bibliogr.

Zugl.: Kassel, Universiẗat, Diss., 2005.

Considerando os efeitos da radiação gama sobre proteínas e a capacidade do sistema imune de reconhecer macromoléculas modificadas, decidimos pesquisar as principais citocinas envolvidas no padrão de resposta de células Th1 ou Th2 em camundongos (B10.PL, BALB/c ou Knockout-IFN-/-) imunizados com a Bothropstoxina-1 (BTHX-1), uma miotoxina do veneno de Bothrops jararacussu, na forma nativa ou irradiada, na presença ou não de substâncias scavengers. Para avaliar prováveis modificações estruturais na molécula da proteína após o processo de irradiação (radiação de 60Co), foi realizada a eletroforese em gel de poliacrilamida 15% para a BTHX-1, na forma nativa e irradiada, na presença ou não de substâncias scavengers. Os resultados mostraram que a radiação foi capaz de promover alterações na molécula da BTHX-1. Na presença dos scavengers, mesmo após a irradiação da toxina, houve a preservação da banda principal (14 kDa). Com relação aos aspectos imunológicos, foram realizados ensaios de produção e identificação de anticorpos, nos quais, os animais foram imunizados com a BTHX-1, na forma nativa ou irradiada, na presença ou não dos scavengers. Observou-se que a proteína nativa induziu, preferencialmente, uma resposta do tipo Th2, enquanto que a proteína irradiada induziu uma resposta do tipo Th1. A toxina irradiada na presença do tbutanol induziu menor produção de IgG2b, quando comparada a toxina irradiada sem scavengers. Para a quantificação da expressão de citocinas em células esplênicas obtidas dos camundongos, foi realizado o Real-time PCR para a detecção das citocinas IFN-, IL-2, IL- 4 e IL-10. As células dos camundongos B10.PL e dos BALB/c imunizados com a BTHX-1 nativa e estimuladas in vitro com a toxina irradiada, apresentaram maior expressão de IFN- e IL-2 (padrão de resposta Th1). As células dos Knockout-IFN-/- imunizados com a BTHX-1 nativa apresentaram maior expressão de IL-4 (padrão de resposta Th2). As células dos camundongos B10.PL e dos BALB/c imunizados com a BTHX-1 + t-butanol apresentaram maior expressão de IL-4 e IL-10, respectivamente. Estes fatos reforçam a participação do OH na modulação da resposta imune contra a toxina irradiada. / Considering the effects of gamma radiation on proteins and the ability of immune system to recognize modified macromolecules, we have identified the major cytokines involved in immune response of B10.PL, BALB/c and Knockout- IFN-/- mice exposed to native or irradiated bothropstoxin-1 (BTHX-1), in the presence and absence of scavengers substances. In order to evaluate possible molecule structural modifications after being irradiated (60Co gamma rays), bothropstoxin-1 was submitted to SDS-PAGE analyses. Our results indicated that irradiation process has promoted modifications in the BTHX-1 molecule, however, in the presence of scavengers and even after irradiation process, the main band of toxin was preserved (14 kDa). Sera of animals immunized with the native or irradiated toxin, in the presence or not of scavengers, were analyzed in order to quantify specific isotypes. While the native BTHX-1 induced a predominant Th2 response, the irradiated toxin apparently promoted a switch towards a Th1 pattern. The toxin, when irradiated in the presence of t-butanol, induced to a lower production of IgG2b (Th1 response) if compared with the irradiated toxin without scavengers. We also performed a Real-time PCR to quantify the expression of cytokines IFN-, IL-2, IL-4 and IL-10 in spleen cells from mice. The cells of B10.PL and BALB/c mice immunized with native BTHX-1 and in vitro stimulated with irradiated toxin, showed higher expression of IFN- and IL-2 (Th1 response) than the control sample. The cells of Knockout-IFN-/- mice immunized with native BTHX-1 showed higher expression of IL-4 (Th2 response). The cells obtained of B10.PL and BALB/c mice immunized with BTHX-1 + t-butanol, showed higher expression of IL-4 and IL-10, respectively. These facts reinforce the involvement of OH in the modulation of immune response against the irradiated toxin.

Made available in DSpace on 2015-12-06T23:05:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2005 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O Vírus da Imunodeficiência Humana (HIV) exibe uma enorme variabilidade genética. Tal característica é criticamente importante para que o vírus possa se adaptar às mudanças ambientais e escapar ao sistema imune do hospedeiro, desenvolver resistência à drogas e impedir o sucesso de eventuais vacinas. Portanto, entender esta diversidade é fundamental para se desenvolver vacinas e drogas eficientes, extremamente necessárias para se conter a epidemia de HIV/AIDS. Neste estudo, nós investigamos a magnitude da diversidade das principais variantes de HIV-1 circulante no Brasil, através de seqüênciamento e análise de genoma completo. O DNA foi extraído de 22 amostras previamente classificadas em nosso laboratório como sendo 6 subtipos B, 8 subtipos C e 8 subtipos F, com base no seqüênciamento de pequenos amplicons. A reamplificação do DNA destas amostras foi realizada por PCR de fragmentos sobrepostos, seguido por seqüênciamento direto. Duas de seis amostras identificadas parcialmente como subtipo B e seis de oito amostras identificadas parcialmente como subtipo F foram então caracterizadas como BF recombinantes pela análise de seus genomas completos. Todas as 8 amostras previamente classificadas como subtipo C apresentaram-se como cepas não recombinantes através da análise de genoma completo. Dois recombinantes BF possuem estrutura de recombinação genômica idêntica, porém diferente da cepa Argentina CRF 12_BF, e provavelmente representam uma nova forma recombinante circulante no Brasil. As análises das seqüências dos subtipos C e F revelaram uma linhagem distinta altamente suportada pela filogenia, cada qual correspondendo a sua cepa de referência brasileira. Nossos dados indicam fortemente que a atual epidemia Brasileira de HIV-1 com as cepas dos subtipos C e F foi introduzida no país por um único evento, e não por fontes múltiplas. Além disso, a evidencia da recombinação BF obtida pela análise do genoma completo do HIV, indica uma disseminação dos recombinantes BF em nossa população de infectados pelo HIV-1 que pode representar força significativa na evolução do vírus. / BV UNIFESP: Teses e dissertações