No description available.

Evidence has accumulated that the voltage dependent anion channel (VDAC), located on the outer membrane of mitochondria, plays a central role in apoptosis. The involvement of VDAC oligomerization in apoptosis has been suggested in various studies. However, it still remains unknown how exactly VDAC supra-molecular assembly can be regulated in the membrane. Previous studies suggested the possible influence of various proteins on the formation of VDAC oligomers, but the important issue of the VDAC oligomeric state regulation by lipids has not been studied so far. Nevertheless, the effect of lipids on the oligomerization of several membrane proteins has been mentioned in the literature and in general, protein-lipid interactions are under extensive investigation. In the present work, I addressed the influence of lipids on VDAC oligomerization experimentally by reconstituting the fluorescently labelled VDAC in giant unilamellar vesicles (GUVs)—a chemically well defined, cell-free minimal model system. Fluorescence cross-correlation spectroscopy was performed to determine the oligomeric state of VDAC. I investigated the effect of important for apoptosis anionic lipids, phosphatidylglycerol and cardiolipin, on VDAC oligomerization. I demonstrated that phosphatidylglycerol significantly enhances VDAC oligomerization in the membrane, whereas cardiolipin disrupts VDAC oligomers. These results suggest that up- or down- regulation of these lipids in mitochondria during apoptosis can tune VDAC oligomerization in the membrane. Thus, this study sheds light on the role played by the above-mentioned lipids in the regulation of VDAC oligomerization during apoptosis and provides additional information on the molecular mechanisms of the programmed cell death. Another objective of this work was to investigate the partitioning of VDAC into liquid disordered or liquid ordered lipid phases. The existence of lipid domains or the lipid rafts in mitochondria and VDAC enrichment in these rafts is still under debate. Additionally, mitochondrial VDAC was recently found in the plasma membrane. The role of this VDAC is not known, however, it was shown to be located in caveolae (specialized lipid rafts) and play an important role in neuronal apotosis and Alzheimer’s disease. Therefore, VDAC partitioning to the lipid rafts is an interesting question for investigation. The possibility to reconstitute VDAC into minimal model systems–GUVs with phase separation, allowed to reveal the preferential partitioning of VDAC into liquid disordered lipid domain, which suggests either non-raft localization of VDAC or the requirement of the other factors for the recruitment of VDAC into lipid rafts.

Submitted by Pedro Henrique Rodrigues (pedro.henriquer@ufpe.br) on 2015-03-05T18:28:52Z No. of bitstreams: 2 Dissertação_Hígor Monteiro.pdf: 2083546 bytes, checksum: 1daef94a46b98eef13d85cda74e4ac8c (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-05T18:28:52Z (GMT). No. of bitstreams: 2 Dissertação_Hígor Monteiro.pdf: 2083546 bytes, checksum: 1daef94a46b98eef13d85cda74e4ac8c (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012-02 / Esta pesquisa tem como objetivo principal analisar iniciativas de Gestão de Processos de Negócio (Business Process Management – BPM) no setor público com a proposta de identificar fatores que são críticos para o seu sucesso. O contexto é definido por organizações públicas brasileiras que possuem iniciativas de BPM. A motivação deste trabalho designa-se pelos altos índices de falhas na indústria e pela baixa investigação empírica no contexto público sobre os fatores críticos de sucesso. As particularidades e o crescente interesse do setor público por iniciativas de BPM também contribuíram para a motivação. Caracterizada como uma pesquisa empírica, esta dissertação utilizou procedimentos que focaram em coletar dados em organizações a fim de gerar conhecimento a partir deles e alcançar os objetivos da pesquisa. Dessa forma, foram realizados dois estudos de casos múltiplos, um questionário de avaliação e por fim uma discussão com especialistas. Nos estudos de caso, participaram das entrevistas 31 profissionais de seis organizações que desempenhavam papéis de patrocinadores, líderes, analistas de processos, clientes internos e consultores externos nas iniciativas estudadas. O questionário obteve um total de 50 respostas significativas e a discussão contou com 5 especialistas. A partir da análise dos dados coletados, foi possível obter um conjunto de Fatores Críticos de Sucesso (FCS) em iniciativas de BPM focado no setor público. O ambiente estudado confirmou muitos dos FCS descritos na literatura atual. No entanto, como resultado principal, esse ambiente de governo permitiu encontrar novos fatores que não estão evidenciados na literatura, mas que merecem bastante atenção ao conduzir uma iniciativa de BPM em organizações públicas. Além desse resultado, a pesquisa também definiu, através de sugestões de especialistas, estratégias com a finalidade de gerenciar cada fator crítico encontrado. Como principal limitação, deve-se observar que o número de organizações pesquisadas não permite que os resultados sejam generalizados para todas as organizações públicas brasileiras. Como conclusão, deve-se ressaltar que esta dissertação contribui para preencher a lacuna existente na literatura para os fatores críticos de sucesso em iniciativas de BPM no setor público, e serve como base para novos estudos empíricos nesta área.

No description available.

No description available.

Evidence has accumulated that the voltage dependent anion channel (VDAC), located on the outer membrane of mitochondria, plays a central role in apoptosis. The involvement of VDAC oligomerization in apoptosis has been suggested in various studies. However, it still remains unknown how exactly VDAC supra-molecular assembly can be regulated in the membrane. Previous studies suggested the possible influence of various proteins on the formation of VDAC oligomers, but the important issue of the VDAC oligomeric state regulation by lipids has not been studied so far. Nevertheless, the effect of lipids on the oligomerization of several membrane proteins has been mentioned in the literature and in general, protein-lipid interactions are under extensive investigation. In the present work, I addressed the influence of lipids on VDAC oligomerization experimentally by reconstituting the fluorescently labelled VDAC in giant unilamellar vesicles (GUVs)—a chemically well defined, cell-free minimal model system. Fluorescence cross-correlation spectroscopy was performed to determine the oligomeric state of VDAC. I investigated the effect of important for apoptosis anionic lipids, phosphatidylglycerol and cardiolipin, on VDAC oligomerization. I demonstrated that phosphatidylglycerol significantly enhances VDAC oligomerization in the membrane, whereas cardiolipin disrupts VDAC oligomers. These results suggest that up- or down- regulation of these lipids in mitochondria during apoptosis can tune VDAC oligomerization in the membrane. Thus, this study sheds light on the role played by the above-mentioned lipids in the regulation of VDAC oligomerization during apoptosis and provides additional information on the molecular mechanisms of the programmed cell death. Another objective of this work was to investigate the partitioning of VDAC into liquid disordered or liquid ordered lipid phases. The existence of lipid domains or the lipid rafts in mitochondria and VDAC enrichment in these rafts is still under debate. Additionally, mitochondrial VDAC was recently found in the plasma membrane. The role of this VDAC is not known, however, it was shown to be located in caveolae (specialized lipid rafts) and play an important role in neuronal apotosis and Alzheimer’s disease. Therefore, VDAC partitioning to the lipid rafts is an interesting question for investigation. The possibility to reconstitute VDAC into minimal model systems–GUVs with phase separation, allowed to reveal the preferential partitioning of VDAC into liquid disordered lipid domain, which suggests either non-raft localization of VDAC or the requirement of the other factors for the recruitment of VDAC into lipid rafts.

Este trabajo de Tesis Doctoral se ha centrado en comprender el comportamiento de electrocatalizadores basados en materiales carbonosos para la reacción de reducción de oxígeno. Con el fin de profundizar en el conocimiento de la naturaleza de los sitios activos de catalizadores basados en materiales carbonosos para esta reacción, se han seleccionado o preparado muestras con diferentes composiciones, texturas porosas y estructuras. De estos resultados se han conseguido importantes avances en el conocimiento del papel que los sitios activos de catalizadores basados en materiales carbonosos desempeñan en dicha reacción. Estos conocimientos y los materiales derivados pueden utilizarse en el desarrollo de cátodos para pilas de combustible en medio alcalino. / Heiwa Nakajima Foundation

In this thesis the deployment of a methodological combination of two single molecule techniques, the planar bilayer technique and fluorescence fluctuation spectroscopy, is presented. The newly devised electro-optical setup will serve as a sophisticated model system for electrical excitable biological membranes. The expectation on a combined electro-optical setup is to be able to correlate the function of membrane channels (electrical activity) with its structural properties (fluorescence assays). The thesis is grouped into four chapters: A general introduction, providing the biological and methodological background, is followed by two studies on the application of the electro- optical setup in the field of membrane biophysics. In the first study the electrical and diffusion properties of planar bilayer membranes made of simple and ternary lipid mixtures are characterized. Additionally, the influence of temperature dependent lipid phase separation on the electrical activity of the ion channel gramicidin A is studied. The second study addresses the conformational changes of the pore-forming toxin Colicin A during membrane binding and ion channel formation. Finally, the potentials and the limitation of the presented setup are discussed.

<p>Bid and Bax are proteins that play a key role in mitochondrial outer membrane permeabilization during apoptosis. Elucidating the molecular mechanisms regulating the function of these proteins is essential to understanding how apoptosis is altered in diseases and for developing therapies. In this thesis, a mitochondria-like supported lipid bilayer (SLB) system is established and used to characterize the membrane binding behaviour of cBid and Bax with single molecule resolution. The formation of SLBs on mica substrate was characterized and lipid diffusion in the plane of the membrane was measured by fluorescence correlation spectroscopy to be 2-6 µm²/s depending on the fluorescent probe used. The binding of cBid to lipids in supported bilayers occurred with an apparent K_D of 121 µM. Combining fluorescence intensity distribution analysis and confocal imaging, cBid binding to SLBs revealed 2 populations of molecules in the membrane: monomers which primarily diffuse in the plane of the membrane and higher order oligomers which are predominantly immobile. In the presence of Bax, the distribution of cBid molecules shifted towards more mobile protein and smaller complexes, while in the presence of Bcl-XL an opposite shift towards immobile protein and larger complexes occurred. This suggests a mechanism for cBid function where the mobility of cBid in the membrane and its oligomeric state can alter its propensity to activate Bax and influence the progression of apoptosis. Bax binding to SLBs was also observed as two populations of mobile and immobile protein. Both populations comprised mostly Bax monomers. Immobile Bax oligomers up to decamers were also observed, and the formation of higher Bax oligomers appeared to be a cooperative processes once a dimer was formed. In comparison to Bax binding to liposomal membranes, its affinity for SLBs appeared to be 40-fold less with a measured K_D of 381 µM.</p> / Master of Science (MSc)

No description available.