Thèse (M.Sc.)--Université Laval, 2004. / Bibliogr.: f. 77-91. Publié aussi en version électronique.

Thèse (Ph. D.)--Université Laval, 2004. / Bibliogr. Publié aussi en version électronique.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Includes bibliographical references.

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

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 94-115).

The relationship between human erythrocyte glucose transporter (GLUT1) oligomeric structure and function was studied. GLUT1 was purified from human erythrocytes in the absence (GLUT1-DTT) or the presence (GLUT1+DTT) of dithiothreitol. Chemical cross-linking studies of lipid bilayer-resident purified GLUT1 and hydrodynamic studies of cholate-solubilized GLUT1 support the view that GLUT1-DTT is a homotetramer and GLUT1+DTT is a homodimer. Parallel studies on human erythrocyte, and studies employing conformation-specific antibodies (anti-GLUT1-DTT antibodies, ∂-IgGs), indicate that erythrocyte-resident GLUT1 resembles GLUT1-DTT (a homotetramer). While the D-glucose binding capacities of GLUT1-DTT and GLUT1+DTT are indistinguishable, GLUT1-DTT presents at least two population of binding sites to D-glucose whereas GLUT1+DTT presents only one population of sugar binding sites. The cytochalasin B (CCB) binding capacity of GLUT1-DTT (0.4 sites/monomer) is one half of that of GLUT1+DTT. GLUT1-DTT and GLUT1+DTT contain 2 and 6 free sulfhydryls per monomer respectively. The subunits (monomers) of tetrameric and dimeric GLUT1 are not linked by disulfide bridges. Erythrocyte resident GLUT1 presents at least two binding sites to D-glucose and binds CCB with a molar stoichiometry of 0.55 sites per GLUT1 monomer. Following treatment with high pH plus dithiothreitol, the sugar binding capacity of erythrocyte membrane resident transporter is unaltered but the transporter now presents only one population of binding sites to D-glucose, binds CCB with molar stoichiometry of 1.3 sites per GLUT1 monomer and displays significantly reduced affinity for ∂-IgGs. These findings demonstrate that erythrocyte resident glucose transporter is GLUT1-DTT (a GLUT1 tetramer) and that GLUT1 oligomeric structure determines GLUT1 functional properties. A model which rationalizes these findings is proposed.

Made available in DSpace on 2014-06-11T19:32:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-10-07Bitstream added on 2014-06-13T18:43:19Z : No. of bitstreams: 1 marqui_abt_dr_sjrp.pdf: 1283437 bytes, checksum: 0fe3659e1058875d6800b1e4a6048ab1 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A Neurofibromatose Tipo 1 (NF1) é uma doença autossômica dominante causada por mutações no gene NF1, responsável pela síntese da proteína neurofibromina. Muitos estudos publicados sobre NF1 têm focado as alterações desse gene e de seu produto em indivíduos afetados, mas as análises de expressão protéica são escassas. No presente estudo, nós investigamos diferenças quantitativas e qualitativas da expressão de proteínas entre amostras de neurofibroma e pele adjacente histologicamente normal, utilizando abordagem proteômica. As proteínas de neurofibroma e pele normal foram separadas por eletroforese bidimensional (2-DE) e identificadas por peptide mass fingerprinting, utilizando espectrometria de massas por dessorção e ionização a laser auxiliada por matriz com base no tempo de vôo (MALDI-TOF). Cinco proteínas foram identificadas: a caspase 14 e a proteína de choque térmico 27/HSP 27, que exibiram expressão reduzida em neurofibromas; a imunoglobulina, a flavina redutase e a proteína de ligação a fosfatidiletanolamina/PEBP, com expressão elevada em neurofibromas. Do nosso conhecimento, este é o primeiro relato de análise comparativa de neurofibromas e pele normal de pacientes com neurofibromatose tipo 1. Das proteínas identificadas, a HSP27 e a PEBP estão conectadas com as vias de sinalização celular p21ras ou cAMP, também relacionadas com a atuação da neurofibromina. A caspase 14 não exibe um elo conhecido com essas cascatas e tal fato pode abrir novos caminhos para o estudo da neurofibromatose. Estudos adicionais ainda são necessários para elucidar o papel dessas proteínas no desenvolvimento da neurofibromatose. Nosso estudo é um passo inicial na descoberta de mecanismos moleculares desta doença e mostra o valor da utilização da análise proteômica na identificação de novos parceiros da neurofibromina relacionados com o desenvolvimento da NF1. / Neurofibromatosis Type 1 (NF1) is a common autosomal dominant disorder caused by mutations in the NF1 gene. Many of the studies published on NF1 have focused attention on the gene level, but protein expression analyses are scarce. In the present study, we investigated quantitative and qualitative differences in neurofibroma and histologically normal surrounding skin protein expression of NF1 patients, using a proteomic approach. Proteins from neurofibroma and normal skin were separated by two-dimensional electrophoresis (2-DE) and identified by peptide mass fingerprinting, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Five proteins were identified: caspase 14 and heat shock protein 27 kDa protein/HSP 27 (downregulated in neurofibroma), immunoglobulin, flavin reductase and phosphatidylethanolamine binding protein/PEBP (upregulated in neurofibroma). To our knowledge, this is the first report of a comparative analysis of neurofibromas and normal skin from neurofibromatosis type 1 patients. Of the proteins identified, HSP27 and PEBP have a connection with p21ras or cAMP signaling. Caspase 14 has no known link with these pathways and may open a new avenue for studying neurofibromatosis. Further studies are still needed to elucidate the actual roles of the differentially expressed proteins. Our work is an initial step toward uncovering the molecular mechanism of this disease and shows the value of using proteomic analysis to identify novel partners of neurofibromin related to the development of NF1.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / A Diabetes mellitus à uma doenÃa de causa mÃltipla, ocorrendo quando hà falta de insulina ou quando a mesma nÃo atua de forma eficaz, causando um aumento da taxa de glicose no sangue (hiperglicemia). Ainda nÃo se sabe precisamente o mecanismo de aÃÃo da insulina, alguns trabalhos sugerem que pode ser possivelmente mediado atravÃs do fosfoglicano inositol (IPGs), cujas algumas formas foram identificadas como: mioinositol e D-quiroinositol. Hà estudos que relacionam a reduÃÃo da glicemia em indivÃduos diabÃticos com o aparecimento desses inositÃis nas secreÃÃes corpÃreas, embora ainda nÃo haja registro de identificaÃÃo dessas molÃculas na composiÃÃo salivar. O objetivo deste estudo foi determinar a relaÃÃo salivar do mioinositol e quiroinositol em crianÃas com diabetes tipo 1 e comparar a presenÃa e concentraÃÃo dessas substÃncias com um grupo de crianÃas sadias (nÃo diabÃticas). Um total de 45 (quarenta e cinco) voluntÃrios, 25 com diabetes tipo 1 descompensados e 20 sadios (nÃo diabÃticos), de ambos os sexos, com idades de 3 a 12 anos, foram selecionados e convidados a participar do estudo. Amostras de saliva foram coletadas e centrifugadas. Os sobrenadantes foram separados, liofilizados e purificados. Logo em seguida, foram analisados por cromatografia lÃquida de alta eficiÃncia (HPLC) para a identificaÃÃo do mioinositol e quiroinositol. A partir dessa anÃlise, foi observado uma menor concentraÃÃo de quiroinositol (p=0,001, Kruskal- Wallis ANOVA seguido por mÃtodo de Dunnâs) e uma maior da concentraÃÃo de mioinositol (p=0,001, Kruskal- Wallis ANOVA seguido por mÃtodo de Dunnâs) nas crianÃas afetadas em comparaÃÃo com as crianÃas saudÃveis. Os pacientes com diabetes tiveram a razÃo mio/quiroinositol maior que do grupo controle (p=0,001, Kruskal- Wallis ANOVA seguido por mÃtodo de Dunnâs) e apresentaram uma correlaÃÃo entre sua proporÃÃo o DM1(p= 0,001). O resultado desse estudo sugere que o mioinositol e o quiroinositol encontrado na saliva de crianÃas com DM1 podem influenciar no controle metabÃlico e desempenhar um papel de marcadores da DM1. / Diabetes mellitus is a disease of multiples causes that occurs either when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces, causing a rise in blood glucose levels (hyperglycemia). It is not clear the action mechanism of insulin but it has been suggested that inositol phosphoglicans, such as myoinositol and D-chiro-inositol, can be important secondary messengers in insulin signal transduction. Although there are some studies linking a reduction in blood glucose levels in diabetic patients with the presence of these inositols in body secretions, there are not reports about the presence of these molecules in salivary composition. Thus, this study aimed to determinate the myoinositol and D-chiro-inositol salivary relation in children with type 1 diabetes and to compare the presence and concentration of these molecules with healthy children (non-diabetic). It has been selected and invited 45 volunteers of both sexes aged 3-12 years, 25 with decompensate type 1 diabetes and 20 healthy children. Saliva samples were collected and centrifuged. The supernatants were separated, purified and lyophilized. The identification of myoinositol and D-chiro-inositol were carried out by means of high-performance liquid chromatography (HPLC). The results showed that children with type 1 diabetes have a lower concentration of D-chiro-inositol and a higher concentration of myoinositol than healthy children. Consequently, the myo/chiro-inositol rate was higher in type 1 diabetes children and there is a correlation between the rate and type 1 diabetes incidence. In conclusion, our data suggests that myoinositol and chiroinositol found in the saliva of children with type 1 diabetes may influence in metabolic control and plays an important role as markers of type 1 diabetes.

Type 1 Diabetes (T1D) is an autoimmune disease affecting around 0.1-0.8% of the population worldwide and is characterized by a progressive destruction of insulin-producing beta cells. Pro-inflammatory cytokines released by immune cells around the islets contribute for the “first wave” of beta cell apoptosis. Cytokine-mediated activation of the transcription factor nuclear factor kappa (NF-κB) contributes to beta cell demise in T1D. This is unusual, since NF-κB has anti-apoptotic effects in other cells. NF-κB is activated in most cells via the canonical pathway, while its activation via the non-canonical NF-κB pathway is restricted to few cell types, such as maturing/differentiating immune cell and osteoclasts. We have now observed that IL-1β+IFN-γ induces an atypical activation of the non-canonical NF-κB pathway in beta cells. This activation depends on different crosstalk mechanisms between the canonical and non-canonical NF-κB pathways, including the down-regulation of the E3 ligase Fbw7, which targets the p100 for proteasomal degradation, and up-regulation of another E3 ligase, βTrCP, which in turn induces cleavage of p100 to p52, a hallmark step in the non-canonical NF-κB activation. Importantly, cytokine-mediated activation of the non-canonical pathway regulates the expression of late NF-κB dependent genes, such as Ccl5, Ccl19, Ccl12, Fas that regulate both pro-inflammatory and pro-apoptotic responses and are implicated in beta cell loss in T1D. This atypical activation of the non-canonical NF-κB pathway by pro-inflammatory cytokines in beta cells constitutes a novel “feed-forward” mechanism that may explain the particular pro-apoptotic effect of this transcription factor in beta cells. Besides regulation of pro-death responses, NF-κB activation in beta cells triggers the expression of the ubiquitin-editing protein A20, encoded by TNFAIP3. A20 restricts NF-κB signalling and possess anti-apoptotic activities in beta cells. Importantly, genome-wide association studies have identified TNFAIP3 as a candidate gene for T1D. We presently demonstrated that A20 effects in beta cells are not restricted to inhibition of NF-κB. Thus, A20 also suppresses the pro-apoptotic mitogen-activated protein kinase c-Jun N-terminal kinase (JNK), and activates the survival signaling mediated via the v-akt murine thymoma viral oncogene homolog (Akt), thus inhibiting the intrinsic pathway of apoptosis. Finally, a cohort study of T1D children indicated that the risk allele of the rs2327832 single nucleotide polymorphism of TNFAIP3 predict lower C-peptide and higher hemoglobin A1c (HbA1c) levels 12 months after disease onset, indicating that this candidate gene contributes for reduced residual beta-cell function and impaired glycemic control in early T1D. In conclusion, our results indicate a critical role for A20 in the regulation of beta cell survival and unveil novel mechanisms by which A20 controls beta-cell fate. Moreover, we identified the single nucleotide polymorphism rs2327832 of TNFAIP3 as a prognostic marker for diabetes outcome in children with T1D.We have also observed that A20 protects beta cells against the pro-apoptotic effects of cytokines by preventing the degradation of the anti-apoptotic protein Mcl-1. Mcl-1 belongs to the Bcl-2 family of proteins that regulate the intrinsic apoptotic pathway. It was previously shown that Mcl-1 depletion contributes to apoptosis in rat beta cells and that its expression is downregulated in islets from T1D individuals infected by enteroviruses. We have now confirmed in human beta cells that decreased Mcl-1 expression contributes to cytokine-mediated beta cell death. We generated a beta cell specific Mcl-1 knockout mouse line (βMcl-1 KO) and observed that islets derived from these mice were more susceptible to pro-apoptotic stimuli exposure ex vivo. Of note, βMcl-1 KO mice were more vulnerable to multiple low dose streptozotocin-induced diabetes than their wild type littermates. One of the mechanisms by which cytokines mediate Mcl-1 degradation is via its phosphorylation by GSK3β. Overexpression of A20 increased AKT phosphorylation, providing a negative feedback on GSK3β activity and preventing Mcl-1 degradation. Cytokines also increase Mcl-1 ubiquitination, a process regulated by the E3 ligases Mule and βTrCP and the deubiquitinase USP9X. The present findings indicate that pro-inflammatory cytokines trigger post-translational modifications of Mcl-1 leading to its degradation. This contributes to exacerbation of pro-death responses and beta cell demise in T1D, but it can be prevented, at least in part, by A20. As a whole, our data unveil novel post-translational mechanisms and different ubiquitin editing proteins that regulate beta cell fate in T1D and are modulated by pro-inflammatory cytokines. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Background: Diabetes is one of Sweden's national diseases. Around 7000 children live with type 1 diabetes in Sweden today. The diagnosis requires planning of everyday life and revaluation of routines which places great demands on the family. Aim: The aim of this study was to describe parents and siblings' experiences of living with a child or young adult diagnosed with type 1 diabetes. Method: The study was designed as a literature-based study to contribute to evidence-based nursing based on analysis of qualitative research to reach a deeper understanding of families' experiences. An analysis of thirteen qualitative articles resulted in four main themes and ten subthemes. Results: The results of the study showed that the whole family is affected when a child gets diabetes. That the child was diagnosed was a shock that caused many feelings with both parents and siblings. There was a constant concern for complications in both the long and short term, resulting in a fear to hand over responsibility, causing a huge pressure on parents who could feel isolated. Conclusion: The families experienced strong feelings when the child was diagnosed. The families needed support from the healthcare professionals to be able to handle the disease.