BACKGROUND: Vibrio cholerae and Enterotoxigenic E. Coli (ETEC) are enteropathogens that are global causes of cholera and traveler’s diarrhea which are responsible for millions of diarrhea cases every year. ETEC and cholera are primarily found in Sub-Saharan Africa and Asia, particularly in nations with inadequate sanitation systems or little access to clean water. Infants and children are most vulnerable to these diseases, as severe infections can lead to stunting and death. The incidence of cholera and ETEC diarrhea have increased, due in part to changing weather patterns. At present, robust animal models for studies of ETEC colonization are lacking to study colonization and bottlenecks. The only licensed vaccines against cholera in endemic countries are killed whole cells, however, new live attenuated oral cholera vaccines (OCV) are in development and offer significant advantages. PanChol is a live attenuated OCV entering phase I trials. SPECIFIC AIMS: To propel studies of ETEC pathogenesis, I attempted to create a suckling mouse model of this globally important pathogen. To accomplish this goal, I constructed barcoded ETEC libraries that enabled me to determine founding population sizes along with intestinal ETEC burdens. To better understand PanChol, a new live attenuated OCV, I studied the shedding of the vaccine in the first 3 human volunteers to ingest this novel agent. METHODS: Triparental mating of donor strains MFDλpir pJMP1039 and MFDλpir pSM1 with recipient ETEC strains enabled construction of barcoded libraries. Neonatal CD-1 and C57BL/6 mice were infected with 104-107 CFU of wild-type ETEC to develop an infant mouse model. Founding population sizes of ETEC strains were compared via sequencing and STAMPR analysis while CFU burdens were determined via plating. Shedding of PanChol was done through enumeration of serial dilutions of fecal samples. Serotyping of shed PanChol was carried out using anti-Ogawa and anti-Inaba antisera. RESULTS: There were marked differences in ETEC small intestinal colonization in different mouse strains. Outbred CD-1 suckling mice only colonized with a 107 dose. In contrast, colonization of ETEC was approximately 106 CFU/small intestine at inocula sizes of 105 or greater in C57BL/6 mice. Laboratory studies using simulated bottlenecks made by serial dilutions established that the barcoded libraries accurately reflect founding population sizes up to 105 CFU. There was no difference in founding population sizes at the same inoculum size between WT ETEC and a hypervesiculation ∆mlaE mutant, though the founding population size increased with increasing input. PanChol retained the Hikojima serotype and shedding occurred in all volunteers with maximum colonization occurring 3 days post administration of 106 CFU. CONCLUSIONS: C57BL/6 P5 mice can serve as a new model to study ETEC intestinal colonization. Hypervesiculating ETEC did not produce a difference in founding population or colonization at the same input as WT ETEC strains. PanChol shows great promise as a viable OCV with shedding at 106 input and no serotype reversion.

In this paper we study a methodology for the numerical simulation of stable structures of fluid membranes and vesicles in biological organisms. In particular, we discuss the effects of spontaneous curvature on vesicle cell membranes under the bending energy for given volume and surface area. The geometric modeling of the vesicle shapes are undertaken by means of surfaces generated as Partial Differential Equations (PDEs). We combine PDE based geometric modeling with numerical optimization in order to study the stable shapes adopted by the vesicle membranes. Thus, through the PDE method we generate a generic template of a vesicle membrane which is then efficiently parameterized. The parameterization is taken as a basis to set up a numerical optimization procedure which enables us to predict a series of vesicle shapes subject to given surface area and volume.

La protéine kinase activée par AMP (AMPK) est un senseur et régulateur central de l'état énergétique cellulaire, mais ces voies de signalisation ne sont pour le moment que partiellement comprises. Deux criblages non-biaisés pour la recherche de partenaires d'interaction et de substrats d'AMPK ont précédemment été réalisés dans le laboratoire. Ces derniers ont permis l'identification de plusieurs candidats (protéines), mais leur rôle fonctionnel et physiologique n'était pas encore établi. Ici nous avons caractérisé la fonction de la relation entre AMPK et quatre partenaires d'interaction : gluthation S-transferases (GSTP1 and GSTM1), fumarate hydratase (FH), l'E3 ubiquitine-ligase (NRDP1), et les protéines associées à la membrane (VAMP2 and VAMP3). Chacune de ces interactions parait avoir un rôle différent dans la signalisation AMPK, agissant en amont ou en aval de la protéine AMPK. GSTP1 et GSTM1 contribueraient à l'activation d'AMPK en facilitant la S-glutathionylation d'AMPK en conditions oxydatives moyennes. Cette régulation non-canonique suggère que l'AMPK peut être un senseur de l'état redox cellulaire. FH mitochondrial est l'unique substrat AMPK clairement identifié. Etonnamment le site de phosphorylation se trouve dans le peptide signal mitochondrial, ce qui pourrait affecter l'import mitochondrial. NRDP1, protéine pour laquelle nous avons pour la première fois développé un protocole de production de la protéine soluble, est faiblement phosphorylée par l'AMPK. L'interaction ne sert pas à l'ubiquitination d'AMPK, mais affecte le renouvellement de NRDP1. Finalement, l'interaction de VAMP2/3 avec AMPK n'implique pas d'évènement de phosphorylation ou d'activation d'un des partenaires. Nous proposons un mécanisme de recrutement d'AMPK par VAMP2/3 (" scaffold ") au niveau des vésicules en exocytose. Ce recrutement favoriserait la phosphorylation de substrats de l'AMPK à la surface des vésicules en exocytoses. Une fois mis en commun, nos résultats enrichissent les connaissances sur les voies de signalisation AMPK, et suggèrent une grande complexité de ces dernières. Plus que les kinases en amont et des substrats en aval, la régulation de la signalisation d'AMPK se fait via des modifications secondaires autres que la phosphorylation, via des effets sur le renouvellement de protéines, et probablement via un recrutement spécifique de l'AMPK dans certains compartiments cellulaires. / AMP-activated protein kinase (AMPK) is a central energy sensor and regulator of cellular energy state, but the AMPK signaling network is still incompletely understood. Two earlier non-biased screens for AMPK interaction partners and substrates performed in the laboratory identified several candidate proteins, but functional and physiological roles remained unclear. Here we characterized the functional relationship of AMPK with four different protein interaction partners: gluthatione S-transferases (GSTP1 and GSTM1), fumarate hydratase (FH), an E3 ubiquitin-ligase (NRDP1), and vesicle-associated membrane proteins (VAMP2 and VAMP3). Each of these interaction partners seems to have a different function in AMPK signaling, either acting up- or down-stream of AMPK. GSTP1 and GSTM1 can contribute to AMPK activation by facilitating S-glutathionylation of AMPK under mildly oxidative conditions. This non-canonical regulation suggests AMPK as a sensor of cellular redox state. Mitochondrial FH was identified as the only clear AMPK downstream substrate, but surprisingly the phosphorylation site is present in the mitochondrial targeting prepeptide, possibly affecting mitochondrial import. NRDP1, whose expression as a full-length soluble protein was achieved here for the first time, is phosphorylated by AMPK only at low levels. The interaction does neither serve for AMPK ubiquitinylation, but rather affects NRDP1 turnover. Finally, interaction of VAMP2/3 with AMPK does not involve phosphorylation or activation events of one of the partners. Instead, we propose VAMP2/3 as scaffolding proteins that recruit AMPK to exocytotic vesicles which could favor phosphorylation of vesicular AMPK substrates for exocytosis. Collectively, our results add some new elements to the AMPK signaling network, suggesting that it is much more complex than anticipated. In addition to upstream kinases and downstream substrates, regulation of AMPK signaling occurs by second

O câncer colorretal (CCR) é o terceiro tipo de câncer mais comum no mundo. Apesar dos avanços nos tratamentos convencionais, aproximadamente dois terços dos pacientes com CCR são submetidos à cirurgia potencialmente curativa. Entretanto, grande parte desses pacientes evolui mal, apresentando recidivas e/ou metástases. A busca de novos alvos moleculares para a terapia do CCR revelou a proteína celular Prion (PrPC) como um possível candidato. Trabalhos recentes sugerem participação direta ou indireta de PrPC no crescimento de tumores, na formação de metástases, na composição de complexos multiproteicos e na indução de vias de sinalização envolvidas em diversos processos biológicos, como proliferação. Além disso, PrPC foi descrito como um importante modulador do crescimento de tumor colorretal. Resultados prévios mostraram que a interação da proteína PrPC com a proteína HSP70/HSP90 Organizing Protein (HOP) induz proliferação em glioblastomas. HOP é uma proteína predominantemente citoplasmática, podendo também ser secretada associada às vesículas extracelulares. Assim, o presente estudo objetivou avaliar o papel do complexo PrPC-HOP e das vesículas extracelulares no desenvolvimento e progressão dos tumores colorretais. Os nossos resultados mostram que HOP induziu migração e invasão em linhagens de CCR de maneira dependente de PrPC, uma vez que o uso do peptídeo sem atividade que compete pelo sítio ligação de HOP a PrPC inibiu estes processos. Além disso, nossos dados apontaram que o aumento de migração e invasão das células de CCR induzida pela interação PrPC-HOP é mediada pela ativação da via ERK1/2. Os achados in vitro estimularam a avaliação do perfil de expressão de PrPC e HOP por imuno-histoquímica em tecidos de pacientes com diferentes tipos de tumores colorretais. Nossos resultados sugeriram que essas proteínas são importantes no início do desenvolvimento tumoral e na transição de adenomas para adenocarcinomas, não havendo correlação entre a presença de HOP e/ou PrPC com metástase, linfonodos acometidos, estadiamento, sobrevida ou região tumoral versus tecido normal. Em relação ao papel das vesículas extracelulares na progressão dos tumores colorretais, nossos resultados mostraram que linhagens celulares que apresentam padrões parecidos de agressividade tumoral podem ter perfis de secreção de proteínas e vesículas extracelulares bastante diferentes, induzindo, portanto, processos biológicos com intensidades distintas. O meio condicionado e as vesículas extracelulares da linhagem WiDr apresentaram maior potencial de indução de migração quando comparado com a linhagem HCT8. Além disso, a modulação negativa da proteína VPS4, uma das responsáveis pela formação dos corpos multivesiculares, mostrou-se uma abordagem interessante no estudo da secreção de vesículas por células de CCR, uma vez que o dominante negativo de VPS4 promoveu diminuição do cargo proteico e da secreção de vesículas extracelulares, redução da proliferação celular e do efeito indutor do processo de migração na linhagem WiDr. Assim, em conjunto, o presente trabalho indicou que o complexo PrPC-HOP pode ser um bom alvo terapêutico nos processos de migração e invasão em CCR. Ainda, essas proteínas se mostraram importantes nos estágios iniciais da formação dos tumores. A modulação da secreção de vesículas extracelulares pode contribuir para retardar a progressão dos tumores colorretais. / Colorectal cancer (CRC) is the third most common type of cancer in the world. Despite improvements in conventional treatments, approximately two-thirds of CRC patients undergo potentially curative surgery. However, most of these patients evolve poorly, showing recurrence and/or metastasis. Search of new molecular targets for CRC therapy revealed the cellular protein Prion (PrPC) as a putative candidate. Recent studies have shown that PrPC exhibit direct or indirect participation in tumor growth, formation of metastasis, composition of multiprotein complexes and induction of signaling pathways involved in many biological processes such as proliferation. Moreover, PrPC has been described as an important modulator of colorectal tumor growth. Previous findings showed that the interaction between PrPC and its ligand HSP70/90 heat shock organizing protein (HOP) induces gliobastoma proliferation. It is well known that HOP localizes mainly in the cytoplasm but HOP is also secreted associated with extracellular vesicles. In this way, the present study sought to evaluate the role of PrPC-HOP complex and extracellular vesicles in the development and progression of CRC. We demonstrate that HOP induces the migration and invasion of CRC cell lines in a PrPC-dependent manner because the use of HOP peptide, which is able to bind to PrPC, blocking PrPC-HOP complex formation, inhibited the migration and invasion processes. In addition, our data showed that the enhancement of migration and invasion induced by PrPC-HOP interaction is mediated by ERK1/2 pathway activation. These in vitro results lead us to evaluate the PrPC and HOP expression by immunohistochemistry in tissues from patients with different tumor types. Our data showed that these proteins could be important for the initial steps of tumor development, represented by the transition from adenoma to adenocarcinoma. No correlation was found among HOP and/or PrPC expression and metastasis, lymph node involvement, staging, survival or tumor area versus normal tissue. Regarding the role of extracellular vesicles in the progression of colorectal tumors, our results showed that cell lines exhibiting similar aggressive tumor behavior can have a different protein secretion pattern and a distinct profile of extracellular vesicles release, which could induce biological process with different intensities. The conditioned medium and the extracellular vesicles derived from WiDr cell line showed a higher potential to induce migration than HCT8 cell line. Moreover, the negative modulation of VPS4, one of the proteins responsible for multivesicular body formation, showed to be an interesting approach in the study of extracellular vesicles secretion secreted by CRC cells; the negative dominant of VPS4 promoted in the WiDr cell line a reduction in the protein cargo and secretion of the extracellular vesicles, a decrease of cell proliferation and induction of migration process. Therefore, taken together, our data highlights that PrPC-HOP complex can be considered a new therapeutic target in migration and invasion processes of CRC. Moreover, these proteins appeared to be important at onset of tumor formation. The modulation of extracellular vesicles secretion may contribute for delaying the progression of colorectal tumors.

Neisseria meningitidis B libera vesículas de membrana externa, conhecidas pela sigla OMV. Essas possuem os mesmos componentes da membrana externa da bactéria e podem ser utilizadas como antígenos em vacinas contra a meningite B. As vesículas devem, também, expressar proteínas da membrana externa (OMP) e proteínas reguladoras do íon ferro (IRP). O objetivo deste trabalho é estudar a cinética de crescimento bacteriano, consumo das fontes de carbono e nitrogênio - especialmente os limitantes de crescimento ? e produção de OMV visando melhorar a produção desse antígeno. Realizaram-se cultivos descontínuos em biorreator, com duração de 20 h, empregando meio de Catlin com limitação de ferro e modificações nas concentrações de lactato, aminoácidos e glicerol. As condições do cultivo foram: 4,2 L de meio, temperatura de 36°C, pressão de 0,5 atm, vazão de ar 1 L/min, agitação entre 250-850 rpm, controle de oxigênio dissolvido em 10% de saturação. Constatou-se que o lactato é a principal fonte de carbono limitante, embora somente se tem a hipótese de que o glicerol age como protetor mecânico. O ácido L-glutâmico é a principal fonte de nitrogênio consumida durante o cultivo. As OMV começaram a ser liberadas quantitativamente no início da fase estacionária de crescimento. Sendo que a melhor condição para a produção de OMV, valor 162,3 mg/L, é aquela em que as concentrações iniciais de lactato e aminoácidos foram duplicadas, 15,00 g/L e 2,93 g/L respectivamente. Através da análise do padrão eletroforético, confirmou-se a presença das principais proteínas de superfície, inclusive das IRPs. A integridade da OMV foi constatada por microscopia eletrônica. Assim, o antígeno obtido mostra-se passível de utilização na composição de vacina anti-meningocócica. / Neisseria meningitidis B liberates outer membrane vesicles known by the abbreviation OMV. These vesicles have the same components of the outer membrane of the bacteria and may be used as antigens in vaccines against meningitis B. The vesicles must also express outer membrane proteins (OMP) and iron regulated proteins (IRP). The aim of this paper is to study bacterial growth kinetics, carbon and nitrogen sources consumption ? specially those which limit growth ? and OMV production, seeking to improve the production of this antigen. Discontinuous bioreactor cultivations were carried out for a period of 20 hours in Catlin medium with iron restriction and modifications in lactate, amino acid, and glycerol concentrations. Cultivation conditions were: 4,2 L of medium, temperature at 36ºC, 0,5 atm, air flow rate of 1 L/min, agitation between 250-850 rpm, and dissolved oxygen control at 10% of saturation. It was verified that lactate is the main limiting carbon source, although there is just a hypothesis that glycerol acts as a mechanic protector. The L-glutamic acid is the main source of nitrogen consumed during the cultivation. The OMV started to be liberated quantitatively at the beginning of the stationary phase of growth. The best condition for production of OMV, value 162,3 mg/L, is that where the initial concentrations of lactate and amino acids were duplicated, 15,00 g/L and 2,93 g/L, respectively. Through an analysis of the electroforetic pattern, the presence of the main surface proteins was confirmed, including the IRPs. The integrity of the OMV was testified by electronic microscopy. So, the antigen thus obtained may be used in the antimeningococcal vaccine composition.

O câncer colorretal (CCR) é o terceiro tipo de câncer mais comum no mundo. Apesar dos avanços nos tratamentos convencionais, aproximadamente dois terços dos pacientes com CCR são submetidos à cirurgia potencialmente curativa. Entretanto, grande parte desses pacientes evolui mal, apresentando recidivas e/ou metástases. A busca de novos alvos moleculares para a terapia do CCR revelou a proteína celular Prion (PrPC) como um possível candidato. Trabalhos recentes sugerem participação direta ou indireta de PrPC no crescimento de tumores, na formação de metástases, na composição de complexos multiproteicos e na indução de vias de sinalização envolvidas em diversos processos biológicos, como proliferação. Além disso, PrPC foi descrito como um importante modulador do crescimento de tumor colorretal. Resultados prévios mostraram que a interação da proteína PrPC com a proteína HSP70/HSP90 Organizing Protein (HOP) induz proliferação em glioblastomas. HOP é uma proteína predominantemente citoplasmática, podendo também ser secretada associada às vesículas extracelulares. Assim, o presente estudo objetivou avaliar o papel do complexo PrPC-HOP e das vesículas extracelulares no desenvolvimento e progressão dos tumores colorretais. Os nossos resultados mostram que HOP induziu migração e invasão em linhagens de CCR de maneira dependente de PrPC, uma vez que o uso do peptídeo sem atividade que compete pelo sítio ligação de HOP a PrPC inibiu estes processos. Além disso, nossos dados apontaram que o aumento de migração e invasão das células de CCR induzida pela interação PrPC-HOP é mediada pela ativação da via ERK1/2. Os achados in vitro estimularam a avaliação do perfil de expressão de PrPC e HOP por imuno-histoquímica em tecidos de pacientes com diferentes tipos de tumores colorretais. Nossos resultados sugeriram que essas proteínas são importantes no início do desenvolvimento tumoral e na transição de adenomas para adenocarcinomas, não havendo correlação entre a presença de HOP e/ou PrPC com metástase, linfonodos acometidos, estadiamento, sobrevida ou região tumoral versus tecido normal. Em relação ao papel das vesículas extracelulares na progressão dos tumores colorretais, nossos resultados mostraram que linhagens celulares que apresentam padrões parecidos de agressividade tumoral podem ter perfis de secreção de proteínas e vesículas extracelulares bastante diferentes, induzindo, portanto, processos biológicos com intensidades distintas. O meio condicionado e as vesículas extracelulares da linhagem WiDr apresentaram maior potencial de indução de migração quando comparado com a linhagem HCT8. Além disso, a modulação negativa da proteína VPS4, uma das responsáveis pela formação dos corpos multivesiculares, mostrou-se uma abordagem interessante no estudo da secreção de vesículas por células de CCR, uma vez que o dominante negativo de VPS4 promoveu diminuição do cargo proteico e da secreção de vesículas extracelulares, redução da proliferação celular e do efeito indutor do processo de migração na linhagem WiDr. Assim, em conjunto, o presente trabalho indicou que o complexo PrPC-HOP pode ser um bom alvo terapêutico nos processos de migração e invasão em CCR. Ainda, essas proteínas se mostraram importantes nos estágios iniciais da formação dos tumores. A modulação da secreção de vesículas extracelulares pode contribuir para retardar a progressão dos tumores colorretais. / Colorectal cancer (CRC) is the third most common type of cancer in the world. Despite improvements in conventional treatments, approximately two-thirds of CRC patients undergo potentially curative surgery. However, most of these patients evolve poorly, showing recurrence and/or metastasis. Search of new molecular targets for CRC therapy revealed the cellular protein Prion (PrPC) as a putative candidate. Recent studies have shown that PrPC exhibit direct or indirect participation in tumor growth, formation of metastasis, composition of multiprotein complexes and induction of signaling pathways involved in many biological processes such as proliferation. Moreover, PrPC has been described as an important modulator of colorectal tumor growth. Previous findings showed that the interaction between PrPC and its ligand HSP70/90 heat shock organizing protein (HOP) induces gliobastoma proliferation. It is well known that HOP localizes mainly in the cytoplasm but HOP is also secreted associated with extracellular vesicles. In this way, the present study sought to evaluate the role of PrPC-HOP complex and extracellular vesicles in the development and progression of CRC. We demonstrate that HOP induces the migration and invasion of CRC cell lines in a PrPC-dependent manner because the use of HOP peptide, which is able to bind to PrPC, blocking PrPC-HOP complex formation, inhibited the migration and invasion processes. In addition, our data showed that the enhancement of migration and invasion induced by PrPC-HOP interaction is mediated by ERK1/2 pathway activation. These in vitro results lead us to evaluate the PrPC and HOP expression by immunohistochemistry in tissues from patients with different tumor types. Our data showed that these proteins could be important for the initial steps of tumor development, represented by the transition from adenoma to adenocarcinoma. No correlation was found among HOP and/or PrPC expression and metastasis, lymph node involvement, staging, survival or tumor area versus normal tissue. Regarding the role of extracellular vesicles in the progression of colorectal tumors, our results showed that cell lines exhibiting similar aggressive tumor behavior can have a different protein secretion pattern and a distinct profile of extracellular vesicles release, which could induce biological process with different intensities. The conditioned medium and the extracellular vesicles derived from WiDr cell line showed a higher potential to induce migration than HCT8 cell line. Moreover, the negative modulation of VPS4, one of the proteins responsible for multivesicular body formation, showed to be an interesting approach in the study of extracellular vesicles secretion secreted by CRC cells; the negative dominant of VPS4 promoted in the WiDr cell line a reduction in the protein cargo and secretion of the extracellular vesicles, a decrease of cell proliferation and induction of migration process. Therefore, taken together, our data highlights that PrPC-HOP complex can be considered a new therapeutic target in migration and invasion processes of CRC. Moreover, these proteins appeared to be important at onset of tumor formation. The modulation of extracellular vesicles secretion may contribute for delaying the progression of colorectal tumors.

Made available in DSpace on 2014-06-11T19:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-06-13Bitstream added on 2014-06-13T19:19:37Z : No. of bitstreams: 1 alves_fr_dr_sjrp.pdf: 2029517 bytes, checksum: f737742cea44bc8699abdd17def3823d (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Estudos de calorimetria diferencial de varredura (DSC) e fluorescência de estado estacionário da sonda Vermelho do Nilo indicam a formação de vesículas de DODAX (X = Cl- ou Br-) em concentrações de surfactantes tão baixas quanto 10 µM. Estas vesículas foram denominadas microvesículas (µV), cuja Tm diminui monotonicamente com a concentração de DODAX até valores de Tm das vesículas tradicionais preparadas em 1.0 mM do surfactante. O efeito do contra-íon (Br- e Cl-) no comportamento termotrópico de fase das vesículas mistas de DODAB-DODAC foi investigado por DSC, condutimetria e espalhamento dinâmico de luz (DLS). Observou-se que a Tm aumenta sigmoidalmente de 45.8 a 48.9 oC com a fração molar de DODAC (xDODAC), com um ponto de inflexão no ponto eqüimolar. A condutividade e o diâmetro hidrodinâmico das vesículas variam muito pouco com xDODAB, indicando que a densidade superficial de carga das vesículas de DODAX é semelhante entre si, e o efeito do contra íon na Tm de DODAX se deve a interações específicas desses contra-íons na interface das vesículas. Medidas de DSC, fluorescência e turbidez de misturas de DODAB-DDAB indicam que as vesículas de DODAB têm maior afinidade por DDAB do que o oposto, resultando na formação de duas populações de vesículas mistas de DDAB-DODAB, com propriedades distintas. Além disso, medidas de fluorescência mostraram que a presença de pequena quantia de DODAB incorporado nas vesículas de DDAB causa um efeito significante na emissão da sonda devido ao aumento do tamanho das vesículas, sugerido por medidas de turbidez. O estudo dos sistemas DODAB/CnTAB/água na concentração total de surfactante igual a 1,0 e 5,0 mM, variando a concentração de CnTAB, e também mantendo a concentração de DODAB fixa em 1,0 mM, revelou uma forte dependência do comprimento da cadeia de hidrocarbonetos... / Differential scanning calorimetry (DSC) and fluorescence of the probe Nile Red studies reveal the formation of DODAX vesicles (X = Br- and Cl-) at surfactant concentrations as low as 10 µM. These vesicles were referred to as microvesicles (mV), whose Tm decreases monotonically with increasing DODAX concentration to the value for the ordinary vesicles at 1 mM. The effect of counterion (Br- and Cl-) on the thermotropic phase behavior of mixed DODAB-DODAC vesicles were investigated by differential scanning calorimetry (DSC), conductimetry and dynamic light scattering (DLS). Tm increases sigmoidally from 45.8 to 48.9 oC with DODAC molar fraction (xDODAC), with an inflection point at the equimolarity. The conductivity and the apparent hydrodynamic diameter vary little with xDODAB, indicating that the surface charge density is similar for DODAX, evidencing that the effect of counterion on Tm is due to the counterion specific interactions. DSC, fluorescence and turbidity measurements indicate a higher affinity of DDAB for DODAB vesicles than the reverse, resulting in two populations of mixed DDAB-DODAB vesicles with different properties. Besides, fluorescence measurements show that the presence of a small amount of DODAB in DDAB vesicles causes a pronounced effect on the Nile Red emission, due to the increase in vesicle size, as suggested from turbidity results. The study of DODAB/CnTAB/água systems at 1.0 and 5.0 total surfactant concentration, and varying CnTAB concentrations with constant 1.0 mM DODAB revealed a strong dependence of the chain length n and relative concentration of the surfactante in the properties of mixed DODAB-CnTAB vesicles. This study allowed analyzing the thermotropic phase behavior containing different amount of DODAB, and the mechanism of vesicle-micelle transition with increasing CnTAB concentration, below and above CMC... (Complete abstract click electronic access below)

Synaptic vesicle recycling is the reuse of synaptic membrane material and proteins after vesicles have been exocytosed at the pre-synaptic terminal of a neuronal synapse. The discovery of the mechanisms by which recycling operates is a subject of active research. Within small mammalian central nervous system nerve terminals, two studied mechanisms of recovery are clathrin-mediated endocytosis and activity-dependent bulk endocytosis. Research into the comparative kinetics and mechanisms underlying these endocytosis mechanisms commonly involves time-series fluorescence microscopy of in vitro cultures. Synaptic proteins are tagged with fluorescent markers, or the synaptic vesicles are labelled with fluorescent dye. The change in fluorescence levels of individual synapses over time in response to stimuli is used to understand synaptic activity. The image analysis of these time-series images frequently requires substantial manual effort to extract the changing synaptic fluorescence intensity levels over time. This work focusses on two closely interlinked areas, the development of improved automated image analysis tools to facilitate the analysis of microscopy image data, and computational simulations to leverage the data obtained from these experiments to gain mechanistic insight into the underlying processes involved in synaptic vesicle recycling. The imaged properties of synapses within the time-series images are characterised, in terms of synapse movement during the course of an experiment. This characterisation highlights the properties which risk adding error to the extracted fluorescence intensity data, as analysis generally requires segmentation of regions of interest with fixed size and location. Where possible, protocols to optimise the manual selection of synapses in the image are suggested. The manual selection of synapses within time-series images is a common but time consuming and difficult task. It requires considerable skill on the part of the researcher to select synapses from noisy images without introducing error or bias. Automated tools for either general image segmentation or for segmentation of synapse-like puncta do exist, but have mixed results when applied to time-series experiments. This work introduces the use of knowledge of the experiment protocol into the segmentation process. The selection of synapses as they respond to known stimuli is compared against other current segmentation methods, and tools to perform this segmentation are provided. This use of synapse activity improves the quality of the segmented set of synapses over existing segmentation tools. Finally, this work builds a number of computational models, to allow published individual data points to be aggregated into a coherent view of overall synaptic vesicle recycling. The first is FM-Sim, a stochastic hybrid model of overall synapse recycling as is expected to occur during the course of an experiment. This closed system model handles the processes of exocytosis and endocytosis. It uses Bayesian inference to fit model parameters to experimental data. In particular, it uses the experimental protocol to separate the mechanisms and rates that may contribute to the observed experimental data. The second is a mathematical model of one aspect of synaptic vesicle recycling of particular interest - homoeostasis of plasma membrane integrity on the presynaptic terminal. This model provides bounds on efficiency of the studied endocytosis mechanisms at recovery of plasma membrane area during and after neuronal stimulus. Both the image analysis and the computational simulations demonstrated in this work provide useful tools and insights into current research of synaptic vesicle recycling and the role of activity-dependent bulk endocytosis. In particular, the utility of adding time-dependent experimental protocol knowledge to both the image analysis tools and the computational simulations is shown.

abstract: Measuring molecular interaction with membrane proteins is critical for understanding cellular functions, validating biomarkers and screening drugs. Despite the importance, developing such a capability has been a difficult challenge, especially for small molecules binding to membrane proteins in their native cellular environment. The current mainstream practice is to isolate membrane proteins from the cell membranes, which is difficult and often lead to the loss of their native structures and functions. In this thesis, novel detection methods for in situ quantification of molecular interactions with membrane proteins are described. First, a label-free surface plasmon resonance imaging (SPRi) platform is developed for the in situ detection of the molecular interactions between membrane protein drug target and its specific antibody drug molecule on cell surface. With this method, the binding kinetics of the drug-target interaction is quantified for drug evaluation and the receptor density on the cell surface is also determined. Second, a label-free mechanically amplification detection method coupled with a microfluidic device is developed for the detection of both large and small molecules on single cells. Using this method, four major types of transmembrane proteins, including glycoproteins, ion channels, G-protein coupled receptors (GPCRs) and tyrosine kinase receptors on single whole cells are studied with their specific drug molecules. The basic principle of this method is established by developing a thermodynamic model to express the binding-induced nanometer-scale cellular deformation in terms of membrane protein density and cellular mechanical properties. Experiments are carried out to validate the model. Last, by tracking the cell membrane edge deformation, molecular binding induced downstream event – granule exocytosis is measured with a dual-optical imaging system. Using this method, the single granule exocytosis events in single cells are monitored and the temporal-spatial distribution of the granule fusion-induced cell membrane deformation are mapped. Different patterns of granule release are resolved, including multiple release events occurring close in time and position. The label-free cell membrane deformation tracking method was validated with the simultaneous fluorescence recording. And the simultaneous cell membrane deformation detection and fluorescence recording allow the study of the propagation of the granule release-induced membrane deformation along cell surfaces. / Dissertation/Thesis / Doctoral Dissertation Electrical Engineering 2018

[Truncated abstract] Intracellular communication between osteoclasts and osteoblasts is imperative to maintain bone integrity. A myriad of molecules are responsible for regulating osteoblast and osteoclast activity. In particular, it is well documented that osteoblast-derived factors are crucial in directly controlling osteoclast formation and function. Since bone formation is coupled to bone resorption, it would be expected that osteoclasts also have some role in regulating the growth and function of osteoblast cells. However, despite extensive research upon osteoclast and osteoblast biology, the mechanisms by which osteoclasts regulate osteoblast growth and function is not well understood. In an attempt to further elucidate the mechanisms by which osteoclasts and osteoblasts communicate, the technique of subtractive hybridisation was used to identify a novel osteoclastderived factor identical to that of mouse Seminal Vesicle Secretion VII (SVS VII). Previous characterisation of the gene in bone demonstrated that SVS VII was abundantly and specifically expressed by mature osteoclasts (Phan, 2004). Additional research hinted that SVS VII acted as a novel osteoclast-derived factor, that by paracrine mechanisms, targeted osteoblast function (Phan, 2004). However, it remained open as to whether the SVS VII molecule did uniquely target the osteoblast, and whether this interaction influenced bone formation in vivo. Therefore, this thesis endeavoured to functionally characterise the role of the SVS VII molecule in the bone environment. ... Further work is needed to identigy a clear consensus binding sequence, to determine the specificity of the interaction between SVS VII protein and each phage clone, and to isolate a specific binding partner for SVS VII. In conclusion, the studies of this thesis sought to characterise the significance of SVS VII expression by mature osteoclasts, relative to its effects on osteoblast behaviour, but failed to conclusively determine a role for SVS VII in bone. Given that the effects of SVS VII on in vitro osteoblast activity and function are minimal, it is doubtful that SVS VII primarily acts as a paracrine factor integral to osteoblast function. Therefore, these findings conflict with those presented previously (Phan, 2004). However, it was demonstrated that SVS VII treatment was associated with in vivo effect on the skeleton, suggesting that SVS VII may target other elements of the bone microenvironment. Via mechanisms not yet understood, which possibly involves additional factors of the bone 11 extracellular matrix, SVS VII may target a subset of osteoprogenitor cells within the bone environment and act to regulate their proliferation. Therefore, SVS VII may enhance osteogenic precursor cell number at sites of bone formation which would increase the pool of cells that can differentiate down the osteoblast linage and contribute to bone formation. In this regard, SVS VII might function in a manner homologous to the Ly-6 molecule Sca-1 and act as an important factor that maintains a balance between the bone formation and resorption process. Clearly, more work focusing on alternative facets of bone biology is needed to identify whether there is a significant role for SVS VII in skeletal tissue.