Orientador: Marcelo Lancellotti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T11:46:02Z (GMT). No. of bitstreams: 1 Mattos_IvesBernardellide_M.pdf: 15060536 bytes, checksum: 32a633419d15c105fec735cb5753d5b2 (MD5) Previous issue date: 2012 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital quando liberada / Abstract: The abstract is available with the full electronic document when available / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Les vésicules sont gouttes de rayon de quelques dizaines de micromètres, limitées par une membrane lipidique imperméable d'environ 4 nm d'épaisseur, et immergées dans un fluide visqueux externe. Les propriétés spécifiques de la membrane de la vésicule rendent le système très déformable et très contraint dans le même temps. Les vésicules représentent également un modèle simplifié intéressant pour les globules rouges, car ils partagent aussi certains comportements mécaniques similaires.Ce manuscrit s’intéresse à la modélisation d'une vésicule soumise à des contraintes externes d'origine hydrodynamique, dans le régime Stokes et dans des domaines confinés. À partir d'un modèle BEM déjà existant pour des fluides infinis, des méthodes numériques originales sont développés pour faire face au calcul des interactions entre la membrane de la vésicule et les frontières solides. Une attention particulière est accordée à la situation d'une vésicule qui sédimente vers un mur horizontal et une vésicule soumise à un écoulement de Poiseuille dans un capillaire étroit. / Vesicles are drops of radius of a few tens micrometers, bounded by an impermeable lipid membrane of approximately 4 nm thickness, and embedded in an external viscous fluid. The specific properties of the vesicle membrane make the system very deformable and very constrained at the same time. Vesicles represent also an interesting simplified model for red blood cells, since they also share some similar mechanical behaviours.This manuscript deals with the modeling of a vesicle subjected to external stresses of hydrodynamical origin, in the Stokes regime and in confined domains. Starting from an existing BEM model for free space flows, original numerical methods are developed to deal with the computation of interactions between the vesicle membrane and solid boundaries. A particular attention is paid to the situation of a vesicle sedimenting towards a planar wall and a vesicle submitted to a Poiseuille flow in a narrow capillary.

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.

Single-stranded positive-sense RNA viruses commonly rearrange host cell organelle membranes into neo-organelles which are involved in virus replication and assembly. These organelles serve to concentrate viral and host factors as well as to conceal viral RNA replication activities from host cell surveillance. To date, many virus-induced membrane rearrangements have been studied by targeted electron tomographic (ET) imaging of specific viral structures at timepoints of known interest. However, the broad cellular context within which these membrane modifications occur and how they change over time are not well understood. A question spanning many virus families is the morphological mechanism of formation of membrane rearrangements. Additionally, it is largely unknown how the membrane modifications affect the morphology of the organelle of origin. In this study, we address specific questions about virus-derived organelles induced by two positive-sense RNA viruses: the coronavirus mouse hepatitis virus (MHV) and the alphavirus Venezuelan equine encephalitis virus (VEEV). Utilizing serial sectioning and montage imaging for ET, volumes representing approximately 10% of virus-infected cells were imaged and detailed organelle analysis was performed. Using MHV-infected cells, we demonstrate that coronavirus-induced double-membrane vesicles (DMVs) are formed by budding from the endoplasmic reticulum (ER) and are trafficked to lysosomes for degradation. The ER remains largely morphologically normal early in infection despite the presence of hundreds of DMVs; however, late in infection, virus envelopment in the ER lumen leads to loss of cisternal morphology. For the alphavirus VEEV, we analyze the structure and origin of virus-derived cytopathic vacuoles II (CPVII). We identify four distinct morphological forms of CPVII and provide evidence that all four forms are derived from the Golgi apparatus. Additionally, a protocol is outlined for a newly-developed method for improved cell ultrastructure during genetically-encoded peroxidase tagging of membrane-proteins. This method is also amenable to ET. Overall, this work provides morphological cellular context for virus-induced membrane rearrangements from two families of positive-sense RNA viruses. Analysis of virus-host cell interactions from this large-scale ultrastructural perspective has the potential to lead to new approaches and strategies to combat current and future viral diseases.<br>

The silica-based cell walls of diatoms are outstanding examples of nature’s capability to synthesize complex porous structures with genetically controlled patterns from the nanometer scale to the range of hundreds of micrometers. Formation of the cell wall building blocks (valves and girdle bands) occurs in membrane-bound compartments, termed silica deposition vesicles (SDVs), which are unique organelles in silica-forming protists. Isolation of the SDVs has not yet been achieved, which has severely hampered the efforts to understand the mechanisms of biological silica morphogenesis. The present thesis aimed to address this shortcoming. The foundation was the development of an improved cell cycle synchronization and a fluorescence labeling method for the model diatom Thalassiosira pseudonana that enabled rapid identification of valve SDVs in a cell lysate. Correlative fluorescence and electron microscopy allowed visualizing the development of valve silica with unprecedented spatio-temporal resolution. Elemental analysis and demineralization of immature valves provided the first direct chemical evidence that silica morphogenesis is an interplay of inorganic and organic molecules inside the valve SDVs. Cryo TEM imaging of valve SDVs indicated the formation of organic patterns that precede silica depostion. From these observations, an organic biomolecule dependent, liquid-liquid phase separation based model for pore formation in the diatom T. pseudonana was proposed. The second part of this thesis was focused on the enrichment of valve SDVs from T. pseudonana and the subsequent proteomics based identification of more than 40 potential valve SDV proteins. Among these, three diatom-specific proteins contained conserved protein protein interaction domains (ankyrin-repeats) and were surprisingly predicted to be located in the cytoplasm. The fluorescent tagging of the three proteins (termed dANK1-3) confirmed their association with the valve SDVs. When the respective dank genes were knocked out by CRISPR/Cas9, the valves displayed permanent anomalies in the quantity and the pattern of ~22 nm sized pores. Double knockout mutants lacking both dank1 and dank3 were almost completely devoid of pores. The analysis of valve morphogenesis in the single and double knockout mutants revealed phenotypic changes that were consistent with the liquid-liquid phase separation based model for pore pattern formation in diatom biosilica. The work of this thesis has provided for the first time direct access to valve SDVs, which has opened entirely new possibilities for studying the composition, properties, and working mechanism of an organelle that forms a complex shaped mineral.

Of the challenges facing the improvement of human health, none has taken the forefront quite like the endeavor to discover novel treatments for heart disease. As heart disease has now become the leading cause of death throughout the world [1], the medical community has made incredible strides in the mission to treat atherosclerosis which is the major contributor to heart disease. Very Low Density Lipoproteins (VLDL) are secreted by the liver and subsequently converted to Low Density Lipoproteins (LDL). Many factors contribute to the narrowing of the arterial walls, however oxidized LDL is the main factor that leads to the deposition of plaque, leading to atherosclerosis pathologies. Recently, a main focus of research into atherosclerotic processes has been the synthesis and trafficking of VLDL in hepatocytes. The rate-limiting step for the secretion of VLDL from the liver has been determined to be the transport of VLDL from the endoplasmic reticulum (ER) to the Golgi apparatus. VLDL molecules are transported in a specialized transport vesicle the Very Low Density Lipoprotein Transport Vesicle (VTV) [2]. VLDL’s core protein, apolipoproteinB-100 (apoB100), is initially lipidated in the ER, and then subsequently delivered to the Golgi apparatus where the VLDL molecule undergoes maturation involving further lipidation and glycosylation of apoB100. Oxysterol Binding Proteins (OSBP) and the sub family OSBP Related Proteins (ORP) have been implicated in many different trafficking processes, mainly the trafficking of sterols, cholesterol, and lipids. Recently, ORP 10 was shown to be a negative regulator of apoB100 secretion in growth medium [3]. Using co-immunoprecipitation, the current study shows that ORP 10 interacts with VLDL’s core protein apoB100 directly. Employing an in vitro budding assay, we show that the blocking of ORP 10 with a specific antibody against ORP10 increases VTV formation from the ER. Given that the ER to Golgi pathway is the rate-limiting step in overall VLDL secretion, these findings support the conclusion that ORP 10 is a negative regulator of VLDL trafficking between the ER and Golgi, and that this process is mediated by the ORP 10 protein binding with apoB100.

Extracellular vesicles (EVs) are lipid bilayer encapsulated nanoparticles which have emerged as an excellent source of biomarkers for multiple diseases, including cancer. However, they are highly heterogeneous in their molecular compositions which remains a major challenge hindering the utilization of their biomarker potential. A single-EV analysis is essential to both discovery and detect EVs that carry disease-specific signature. In this work, we designed plasmonic nanohole array for capturing single EVs and perform fluorescence detection of their membrane proteins by exploiting plasmonic amplification of the fluorescence signal. The design of the array was optimized using COMSOL Multiphysics-based simulation. Nanohole arrays with three different periodicities were fabricated on aluminum thin film on glass substrate. The substrates were then functionalized with three different methods for investigation of antibody-free capturing techniques, which are electrostatic interaction, hydrophobic interaction, and size-selective capturing. After surface functionalization with each of the techniques, genetically engineered EVs expressing mNeonGreen (mNG) were incubated and their capture efficiency were compared. The presence of single-EVs within plasmonic nanoholes was verified through both fluorescence analysis and atomic force microscopy (AFM). Fluorescence intensities of mNG-EVs recorded with the plasmonic chip with different periodicities showed intensity variations in agreement with the simulation results. Furthermore, the EVs were immunostained with R-phycoerythrin (R-PE) conjugated CD-9 to demonstrate the possibility of general and multimarker fluorescence detection. In a separate experiment, DOPC liposomes were synthesized and their deformability was analyzed by using AFM. The nanohole array provides a basis for a future platform of EV analyses, promising to capture the signature arising from low expressing proteins. / Extracellulära vesiklar (EV) är lipadmembranförsedda nanopartiklar som har dykt upp som en utmärkt källa till biomarkörer för flera sjukdomar, däribland cancer. De är dock mycket heterogena i sina molekylära sammansättningar, vilket skapar en stor utmaning och hindrar utnyttjandet av deras potential som biomarkörer. EV-analys på enpartikelnivå är nödvändig både för att upptäcka och detektera vesiklar som har en sjukdomsspecifik signatur. I detta arbete designade vi en plasmonisk uppsättning av nanohål för att fånga enstaka EVs och utföra fluorescensdetektion av deras membranproteiner genom att utnyttja plasmonisk amplifiering av fluorescenssignaler. Designen av uppsättningen optimerades med hjälp av COMSOL Multiphysics-baserad simulering. Nanohålsuppsättningar med tre olika periodiciteter tillverkades på tunn aluminiumfilm på glassubstrat. Substraten funktionaliserades sedan enligt tre olika metoder för undersökning av antikroppsfria bindningsmetoder. De tre metoderna är elektrostatisk interaktion, hydrofob interaktion och storleksselektiv bindning. Efter ytfunktionalisering med var och en av teknikerna inkuberades vesiklar genetiskt modifierade att uttrycka mNeonGreen (mNG) och deras bindningseffektivitet jämfördes. Närvaron av individuella EVs i plasmoniska nanohål bekräftades genom både fluorescensmikroskopi och atomkraftsmikroskopi (AFM). Fluorescensintensiteter för mNG-EVs registrerades med plasmonchipet med olika periodiciteter och visade intensitetsvariationer i överensstämmelse med simuleringsresultaten. Dessutom immunfärgades vesiklarna med R-fykoerytrin (R-PE) konjugerad CD-9 för att påvisa möjligheten till allmän och multimarkör fluorescensdetektion. I ett separat experiment syntetiserades DOPC-liposomer och deras deformerbarhet analyserades med AFM. Nanohåluppsättningen lägger grund för en framtida plattform för EV-analys, som lovar att fånga signaturen som uppstår från låguttryckande proteiner.

No description available.

<p>Vesicle trafficking is an important process critical for secretory and endocytic purposes, but it is also crucial for cell homeostasis, <i>e.g.,</i> for maintenance of organelle identity and recycling of membrane components.</p><p>The endomembrane-located adaptor protein Epsin R (Epsin-Related protein) is believed to be important for recycling of SNARES like Vti1b from endosomes to the trans Golgi network (TGN), although its involvement in TGN to endosome transport has been also proposed. Further highlighting its impact in cellular and organismal physiology, certain <i>EPSIN R</i> SNPs have been linked to schizophrenia and Epsin R deficiencies correlate with other pathological conditions related to epidermis homeostasis such as psoriasis and eczema.</p><p>Epsin R belongs to the conserved Epsin family of adaptors and as such it presents a characteristic Epsin N-Terminal Homology (ENTH) domain and a largely unstructured C-terminus. The latter contains binding motifs for important elements of the vesicle trafficking machinery.</p><p>Here we identified a C-terminal region of Epsin R with prion-like characteristics (Prion Forming Region or PFR). We found that GFP-Epsin R is localized in intracellular punctate structures colocalizing with different intracellular markers; however, in contrast to other epsin family members, Epsin R displayed puncta of different size and with different protein content with a substantial contribution of large/bright particles. Importantly, the C-terminal Epsin R’s PFR was required for Epsin R localization and for the formation of large and bright puncta. Further, these structures displayed characteristics shared with other prion-like proteins. Our results therefore suggest that Epsin R possesses PFR-dependent prion properties that play an important role in this adaptor’s localization and function.</p><p>We propose a model in which prion-like proteins like Epsin R can rapidly and stably self-assemble at vesicle budding sites. These proteins would accelerate the formation of vesicle trafficking machinery and the recruitment of cargo. We also speculate that oligomerizing, self-templating reactions would occur under strict control of several cellular factors such as chaperones and post-translational modifications (<i>e.g.,</i> phosphorylation, ubiquitination, etc.) to assure quick and <i>reversible</i> association of prion-like proteins.</p>

In-depth characterization has introduced new molecular subtypes of gastric cancer (GC). To identify these, new approaches and techniques are required. Liquid biopsies are trendsetting and provide an easy and feasible method to identify and to monitor GC patients. In a prospective cohort of 87 GC patients, extracellular vesicles (EVs) were isolated from 250 µL of plasma. The total RNA was isolated with TRIZOL. The total RNA amount and the relative mRNA levels of CD44, PTEN, and FASN were measured by qRT-PCR. The isolation of EVs and their contained mRNA was possible in all 87 samples investigated. The relative mRNA levels of PTEN were higher in patients already treated by chemotherapy than in chemo-naïve patients. In patients who had undergone neoadjuvant chemotherapy followed by gastrectomy, a decrease in the total RNA amount was observed after neoadjuvant chemotherapy and gastrectomy, while FASN and CD44 mRNA levels decreased only after gastrectomy. The amount of RNA and the relative mRNA levels of FASN and CD44 in EVs were affected more significantly by chemotherapy and gastrectomy than by chemotherapy alone. Therefore, they are a potential biomarker for monitoring treatment response. Future analyses are needed to identify GC-specific key RNAs in EVs, which could be used for the diagnosis of gastric cancer patients in order to determine their molecular subtype and to accompany the therapeutic response.