Exocyst positive organelle (EXPO) is a newly discovered double membrane organelle involved in exocytosis and likely other vesicle trafficking processes. EXPO is likely generated from the ER, fused with plasma membrane and released a single membrane vesicle to cell exterior. The Arabidopsis protein Exo70E2 was found to be associated with EXPO and therefore is considered as a marker of EXPO and might play a role in EXPO-mediated vesicle trafficking. Understanding the biological function of AtExo70E2 (abbreviated as E2 in this thesis) will be very helpful in unraveling the function of EXPO. The aim of this work was to use various molecular, genetic and physiological approaches to determine the possible role of Arabidopsis Exo70E2 in biological pathways. By using the Exo70E2pro:GUS line, the expression pattern of Exo70E2 was determined. Exo70E2 was expressed mainly in roots, especially in root tips and epidermal cells in the division and elongation zones of roots. Its expression level was induced when the seedlings were treated with Flg22, a peptide derived from bacterial flagillin protein that induces the plant defense response. The tissue subcellular localization of Exo70E2 was also studied using the 35S:Exo70E2-eYFP and Exo70E2pro:Exo70E2-GFP reporter lines. The GFP fusion protein was found primarily in the epidermal cells of roots even in the 35S:Exo70E2-eYFP lines. For phenotypic analysis resulting from mutations of the Exo70E2 gene, I obtained three T-DNA insertion mutant lines and generated its overexpression lines. The two mutant alleles, e2-2 and e2-3 are in the Columbia ecotype background and further characterized. e2-2 which has a T-DNA insertion in an exon is likely a knock out line as Exo70E2 gene transcript could not be detected. e2-3, which carries a T-DNA insertion in its promoter region, was found to accumulate a higher level of the transcript, suggesting that the insertion causes its enhanced expression of Exo70E2. There was no obvious difference between wild type and e2-2 in their phenotypes under different conditions tested in this study. However, e2-3 had a retarded growth phenotype when grown in soil or on MS medium. The seedlings of e2-3 on MS medium also had a yellowish color although such a phenotype was not obvious when they were grown in soil. When supplementing the MS medium with sucrose, glucose or mannitol, the growth of e2-3 was more reduced compared to wild type under these conditions. However, on the medium with NaCl or under phosphate deficiency, the yellowish phenotype of e2-3 was rescued and the mutant seedlings became relatively healthier than the seedlings under the regular MS medium. A proteomics approach was taken to compare protein secreted from the seedlings of wild type and the mutants. Proteins secreted by seedlings to the liquid medium were collected, concentrated and subjected to MS analysis. Comparison of the profiles of secreted proteins between the wild type and the mutants leaded to identification of candidate proteins whose secretion might be affected by the mutation. My study indicates that Exo70E2 and EXPO are involved in transporting proteins (likely also metabolites) to the exterior of cells and the rhizosphere and might play an important role in stress responses.

The thesis aims to gain a better understanding on the mechanisms of the complicated macromolecular fouling in ultrafiltration (UF). The work is divided into three main parts. Firstly, comprehensive literature reviews on both membrane fouling and cleaning were carried out for a better overview on this problem. The findings such as the identification of the main foulants and the current knowledge on fundamental fouling mechanisms, directly contributed to further parts of the thesis. Secondly, a multiscale approach was developed to form generalised framework for modelling complex fouling scenarios. Two complex fouling models combining multi- ple fouling mechanisms were derived accordingly. The models were then applied to the filtration data collected from UF experiments (constant-pressure and dead-end) on three individual macromolecular solutes, i.e., dextran blue (DB), polyethylene oxide (PEO), and humic acid (HA), respectively. During the experiments, the effect of macromolecular concentration and transmembrane pressure was investigated. Using the appropriate combined model, the overall and initial fouling behaviours and the predominant fouling mechanisms at different stages of filtration were identified. The fouling parameters in the combined models were determined and found to be consistent with the existing theories. The switch points between the dominant fouling mechanisms were assessed using two methods (integral and differential), respectively. Comparing all the information together gave a comprehensive understanding of the physics involved in the macromolecular fouling. Finally, the effect of the deformability of a macromolecule on membrane fouling was studied. The sieving results from the experiments indicated a flux-dependent permeation during UF of the DB solution, hypothetically due to the elongational deformation of the large DB molecule (a linear polymer at 2000 kDa MWCO) under high velocity gradient at the pore entrance, allowing the molecule to adapt a smaller transversal size to enter pores at nominally 100 kDa cutoff. This not only increased the chances of permeation but also the probability of severe irreversible fouling. Subsequently, a mesoscopic model using dissipative particle dynamics (DPD) was developed to investigate the blocking event at the pore entrance in the presence of a deformable linear macromolecule. The simulation results shed lights on the threshold permeating flux at which the molecular chains start to deform.

Des membranes supports en polymère ont été photo-greffées par des poly(liquide ionique)s (polyLIs) à base d'imidazolium. Les polyLIs permettent de séparer le CO2 d'autres gaz et de stabiliser des nanoparticules. Dans le cas du captage de CO2, les expériences montrent qu'une couche fine homogène de gel réticulé en polyLI gonflé par du liquid ionique (LI) est obtenue sur la surface de fibres creuses. Les fibres ainsi obtenues ont montré des perméances au CO2 plus élevées (600-700 GPU) que des membranes commerciales et des sélectivités de CO2/N2 comparables (13 et 17). Dans le cas de membranes catalytiques, des nanoparticules de palladium (NPPd) servant de catalyseur ont été immobilisées en forte concentration locale au sein d'une couche de polyLI greffée à la surface de membranes. La réactivité des membranes catalytiques a été testée en configuration de contacteur traversé sur différentes réactions (couplage croisé C-C, hydrogénation, etc). Une conversion totale est obtenue pour des temps de séjours de quelques secondes, sans aucun sous-produit formé. Comparée aux NPPd colloïdaux dans un réacteur en batch, la membrane catalytique accélère les réactions d'environ 2000 fois en terme de temps de réaction sans perte de NPPd; la sélectivité est aussi accrue. Le réacteur membranaire catalytique a été modélisé afin d'obtenir les profils de concentration et de température et une meilleure compréhension des performances obtenues. Les membranes catalytiques se révèlent isothermes et les constantes cinétiques sont calculées. Enfin, les capacités de production de ces membranes catalytiques à une échelle industrielle sont estimées à environ 3 t/(hm3) pour le couplage de Suzuki. / Polymeric support membranes were modified via photo-grafting by poly(ionic liquid)s (polyILs), featuring in the capability to separate CO2 from other gases and to stabilize metallic nanoparticles (MNPs). For CO2 capture, a thin polyIL-IL gel layer was homogenously coated on support hollow fibers. The composite fibers show high CO2 permeance and reasonable CO2/N2 selectivity. For the catalytic membrane, palladium NPs were generated inside a grafted polyLI layer. Compared to colloidal palladium system in a batch reactor, the catalytic membrane, as a contactor membrane reactor, is more efficient in terms of reaction time (ca. 2000 times faster), selectivity and MNP retainability. Theoretical study on reactor modeling, concentration & temperature profiles, and production capacity was done for an overall understanding of the catalytic membrane.

Centrifugal membrane and density separation (CMS) is a novel technology proposed for treatment of waste water and industrial process streams. This cross flow filtration process combines the energy recovery inherent to centrifugal reverse osmosis (CRO) with the potential alleviation of membrane fouling and concentration polarization due to the favourable effects of centrifugal and Coriolis accelerations. This dissertation presents a computational study of CMS undertaken to understand the basic hydrodynamics and mass transfer of the processes and to provide insight for the design of CMS devices. Two distinct membrane models were developed, the porous wall model (PWM) and the source term model (STM), and incorporated into Computational Fluid Dynamics (CFD) codes which solve the full Navier-Stokes equations coupled to a scalar transport equation which accounts for dissolved species. These models are used to simulate two and three dimensional laminar flows in both non-rotating and rotating reverse osmosis membrane cartridges and to predict permeate fluxes. Plate and frame geometries are first examined and it is determined that CMS benefits most from channels with streamwise directions directed radially. It is also shown that the benefits of CMS can be attributed largely to the secondary flows and mixing associated with Coriolis acceleration, and the PWM and the STM are found to perform similarly in the case of reverse osmosis. Next, the STM is used to perform a parametric study of the flow and mass transfer in rectangular and square rotating channels. It is shown that while normal rotation is preferable to spanwise rotation, relatively small deviations from the spanwise orientation are adequate to achieve most of the normal rotation performance, and that differences between the two orientations are minimal in the case of square channels. Also, the flow characteristics are again shown to correlate well with the magnitude of the Coriolis acceleration. Flows in triangular and circular channels are also considered, and are shown to perform similarly to rectangular channels. These channel orientations have application in hollow fiber membrane modules and potentially in spiral wound membrane modules. Finally, the flow and mass transfer in channels with periodic streamwise obstacles are considered. Such obstacles are related to feed spacers used in spiral wound membrane elements and impact considerably on the flow characteristics and mass transfer performance. Flow obstacles are shown to increase mass transfer performance in all cases, with alternating surface mounted performing best. A preliminary investigation is undertaken into rotating flows with periodic obstacles, and the flow fields are shown to depend strongly on the blockage ratio and on the Rossby number. In most cases, it is found that mass transfer performance does not necessarily correlate with either wall shear stress or the local flow field. Several general conclusions regarding CMS can be drawn from this work. It is preferable to operate a CMS devices at low flow rates, which is contrary to conventional wisdom in membrane separation. Secondly, the mixing induced by channel rotation is both more effective and more efficient than the mixing induced by the feed spacers considered here. Finally, the magnitude of the Coriolis acceleration is the dominant parameter in determining CMS performance. This means that a CMS device can either operate at relatively low rotational speeds with flow in the radial direction, or at higher speeds but lower angles of inclination with respect to the rotational axis. / Graduate

Charcot-Marie-Tooth disease (CMT) is the commonest inherited neuromuscular disorder, which affects the peripheral nervous system leading to nerve degeneration. CMT is categorised into two forms, ‘axonal’ and ‘demyelinating’, which reflects the main site of pathology as the axon or Schwann cells respectively. Over 90 genes have been identified associated with the disease. Among the genes associated with demyelinating CMT, the focus of my thesis is LITAF, mutations in which lead to an autosomal dominant demyelinating CMT known as CMT type 1C. LITAF is a 17 kDa protein likely to be involved in endocytic degradation and trafficking of specific cargo proteins. It contains an N-terminal proline-rich region mediating protein-protein interactions and a C-terminal LITAF domain consisting of a zinc-ribbon structure with a hydrophobic region incorporated. Most of the CMT1C mutations are clustered in this highly conserved LITAF domain. LITAF was predicted to play roles in recruiting ESCRT components and exosome formation, but the precise function remains unclear. Furthermore, why mutations in LITAF lead to CMT is not known. My work therefore focused on characterising the function of the LITAF protein both in health and disease. In my thesis, I first tried to determine the subcellular localisation of LITAF proteins and also investigated the function of the highly conserved C-terminal LITAF domain in targeting protein to the membrane. With regards to the function of LITAF, potential binding partners were screened using the traditional GST pull-down assay and the in-situ proximity labelling assay, BioID. A number of novel potential binding partners were identified in both assays. Among the list of potential binding partners, BAG3 was captured in both pull-down assays and was chosen for further studies. The interaction with LITAF was characterised and the potential role of this interaction in autophagy was investigated. Integrin, which was also captured in the BioID assay, was another protein chosen for further studies. Internalisation and recycling assay were developed to investigate the function of LITAF in integrin trafficking. The potential of CMT mutations in impairing the internalisation of integrin in A431 cells and the difficulty in performing the assays were discussed. Lastly, with patient fibroblasts available in our lab, disease phenotypes were analysed using two types of imaging technique: transmission electron microscopy (TEM) (in collaboration with J. Edgar) and immunofluorescence microscopy. Swollen vacuoles were observed in the TEM images of the patient fibroblasts only, and various uptake assays were performed to identify these enlarged compartments. In summary, this work offers an insight into the function of LITAF both in health and disease as well as identifying potential intracellular binding partners that might shed light on pathogenesis. Furthermore, the modified trafficking assays described in this thesis can be applied to Schwann cell and patient fibroblasts, providing further tools to probe the underlying membrane trafficking pathways that are dysfunctional in CMT.

Thesis (MTech (Chemical Engineering))--Cape Peninsula University of Technology, Cape Town, 2006 / During recent years, the use of liquid membranes has gained general interest in the treatment of effiuents where solute concentrations are low and large volumes of solutions should be processed, and, if possible, without generating any secondary waste. Liquid membrane processes have been proposed as a clean technology, owing to their characteristics, i.e. high specificity, low energy and utilization. Two liquid membrane processes have been used in metal recovery, which are the liquid surfactant membrane (LSM), which corresponds to double water-in-oil emulsion and solid . supported liquid membranes (SLM), which are made by dispersing or impregnating the extractant within the pores of in.ert solid support. Previously, the recovery of eu (IT) in a SLM system was conducted by other membrane models such as hollow fibre, spiral and flat sheet. Only a small measure of success on scale-up and industrialization of these models has been attained. One of the disadvantages of the hollow fibre system was the small lumen size through which the feed needed to pass. Pores became clogged by suspended particles because the pressure drop over the small diameter augments lower flow rates and therefore, pre-filtering is necessary (Rathore, et al., 2001). In this study the behaviour of a tubular SLM reactor with an inner diameter of the lumen approximately fifty times bigger than that of the hollow fibre are used in order to solve the problem of clogging. This tubular reactor was incorporated in to a bench scale plant and proved successful in copper extraction. By observing transient data, mass transport coefficients were determined and compared to published values.

Thesis (Masters Diploma (Technology)--Cape Technikon, Cape Town,1993 / Various experimental and established membranes were tested on industrial effluents. Ultrafiltration, reverse osmosis and nanofiltration membranes were used in various applications. Research was done on aspects such as the cleaning of fouled membranes, production quality control and process development. Polyvinyl alcohol ultra-thin-film reverse osmosis membranes were manufactured for the desalination of brackish water to a potable standard. The membranes were manufactured in the tubular configuration. Experimental ultrafiltration, reverse osmosis and nanofiltration membranes were tested on cooling water blowdown on a laboratory-scale. On-site testing was done directly on the effluent at a later stage. A s!udy was also conducted to determine the effect of gel-polarization on membrane performance. The gel-layer model was used to predict the limiting flux of specific membranes. Membrane processes were also applied in the fractionation of wine-lees to provide usable by-products such as yeast cells and potassium bitartrate. Ultrafiltration membranes operated in diafiltration mode were used to "wash" the slurry at different solid concentrations. The bitartrate-rich permeate collected from ultrafiltration was then concentrated using reverse osmosis and nanofiltration to allow subsequent precipitation of the product.

Thesis submitted in fulfilment of the requirements for the degree DOCTOR TECHNOLOGIAE: ENGINEERING: CHEMICAL In the FACULTY OF ENGINEERING At the CAPE PENINSULA UNIVERSITY OF TECHNOLOGY 2009 / The white rot fungus, Phanerochaete chrysosporium strain BKMF-1767 (ATCC 24725), produces the extracellular enzymes, Lignin peroxidase (LiP) and Manganese peroxidase (MnP), that constitute the major route for lignin degradation by this organism. LiP and MnP have also been shown to play a major role in aromatic pollutant degradation. Due to the need for continuous production of LiP and MnP, a fixed-film bioreactor, classified as a membrane gradostat reactor (MGR), was developed. The implementation of batch-reactor operational parameters to the MGR system was found to be ineffective, thus creating the need for further research to improve the operational aspects of the MGR system to optimise its capabilities for continuous and industrial-scale operations. The research undertaken in this study, provides information that can be used to classify the dissolved oxygen (DO) transport kinetics into immobilised fixed-films of P. chrysosporium. Operational limitations of the MGR relating to environmental stresses in the bioreactor during operation and to biofilm deterioration, including limitations of DO mass transport, oxidative stress, trace element accumulation and polysaccharide storage in the fungal biomass, were evaluated in single capillary MGR systems (SCMGRs). These conditions were identified as existing in the continuous MGR systems. From DO profiles, the oxygen consumption and flux into the biofilms, including the distribution of DO, was determined to be dependent on the immobilised biofilm’s age. Younger biofilms showed higher DO distribution than older biofilms even when aeration was directed to the extracapillary space (ECS) of the reactor against the biofilm’s surface. An increase in anaerobic zone thickness was observed to be increasing with an increase in biofilm thickness. Although, DO kinetic parameters were comparable with those obtained in submerged mycelia pellets, higher oxygen consumption values were observed in biofilms grown in the SCMGRs. The limitations of MGR were identified as: 1) poor DO distribution in immobilised biofilms because of b-glucan production and storage in the immobilised biomass, resulting in ethanol production; 2) the peroxidation of lipids of the biofilms, which in turn will affect the long-term performance of the biomass caused by oxygenation and 3) trace element ion accumulation enhanced by b-glucan production. Furthermore, trace element ion accumulation was higher in the MGRs than in batch cultures using the same nutrient medium. The development of a perfluorocarbon (PFC) emulsion for the MGRs to counteract these limitations was investigated. The compatibility of the emulsion with oxygen-carrying capacity was shown with an improvement in biomass generation, LiP/MnP production and overall consumption of primary substrates, mainly glucose and ammonium tartrate, in batch cultures. The emulsions investigated were based on the addition of oxygen carriers: Perfluorooctyl bromide (PFOB), Bis-(Perfluorobutyl) ethene (PFBE) and Perfluoropropylamine (PFPA), using Pluronic F 68 (PF 68) as the surfactant. Concentrations of 10 to 30% (w/v) PFC and 8.5% (w/v) PF 68 were tested successfully in batch cultures. The emulsions containing 10% (w/v) PFCs resulted in improved biomass performance as opposed to emulsions with higher PFC oil concentrations. An emulsion containing 10% (w/v) PFOB was used to evaluate its efficacy in the SCMGRs, as the biomass yield and overall enzyme production were superior to PFPA and PFBE-based emulsions with similar oil concentrations. After successfully applying PFOB and PF 68 to the SCMGRs, the following results were obtained: 1) reduced ethanol production; 2) reduced trace element accumulation; 3) lower b-glucan production and 4) improved DO-penetration ratio in immobilised biofilms.

Orientador: Cláudia Valéria Seullner Brandão / Banca: José Joaquim Titton Ranzani / Banca: Antonio Carlos Lottelli Rodrigues / Resumo: A córnea é a estrutura anterior transparente do olho e apresenta-se muito susceptível a traumas e agressões. O objetivo do presente estudo foi avaliar e comparar, clínica e histomorfometricamente, o processo de reparação corneana de úlceras induzidas em coelhos, frente à utilização do plasma rico em plaquetas (PRP) sob a forma de colírio ou tampão. Foram utilizadas 60 fêmeas da espécie leporina, constituindo-se 4 grupos experimentais de 15 animais cada, designados grupo plaquetas (GP), grupo tampão (GT), grupo controle (GC) e grupo controle amniótica (GA). Em todos os animais foi realizada a úlcera experimental, sendo este o único procedimento no GC. No GP, os coelhos foram medicados com colírio autólogo de plasma rico em plaquetas. No GC, foi instilado colírio lubrificante. No GT, foi aplicado tampão sólido rico em plaquetas, revestido por membrana amniótica, para a retenção do mesmo. No GA, foi aplicada apenas a membrana amniótica. Os grupos experimentais foram subdivididos em três subgrupos (M4, M7, M30), de acordo com os períodos finais de avaliação. Os animais foram avaliados por meio de exame clínico e histomorfométrico. Não houve diferenças entre os tratamentos utilizados quanto aos sinais relacionados à sensibilidade ocular, quemose e secreção ocular. Os grupos tratados com PRP, na forma de tampão ou colírio, apresentaram menor opacidade do que os animais tratados apenas com membrana amniótica no momento final de avaliação. Quanto à presença da úlcera corneana, os grupos tratados com PRP (GP e GT) apresentaram menor intensidade de ulceração corneana com relação aos demais grupos (GC e GA). Na avaliação histológica verificou-se maior epitelização corneana na fase inicial da lesão no tratamento à base de colírio de PRP. O uso da membrana amniótica promoveu espessamento do epitélio e estroma corneano, com sinergismo da mesma quando associada ao PRP / Abstract: The cornea is the transparent anterior part of the eye and is very susceptible to trauma and sore. The aim of this work was to evaluate and compare both, clinically and histomorphometrically the process of repair of induced corneal ulcer in rabbits using platelet-rich plasma in the form of eyedrop or clot. Sixty female leporids were divided into four groups of 15 animals, and named as platelet group (PG), clot group (CLG), control group (CG), and amniotic control group (AG). Ocular ulcer was experimentally induced in all the animals. Ulcer induction was the single procedure performed in CG. In PG, autologous platelet-rich plasma as a eyedrop was used five times a day for seven days. In CLG, a platelet-rich clot was covered by amniotic membrane to hold it in place was used. In AG, only the amniotic membrane was used. Experimental groups were then subdivided into three groups (M4, M7, M30), corresponding to the end of the evaluation period. The animals were evaluated through clinical and histomorphometric tests. There were no differences between treatments related to ocular sensitivity (blepharospasm and photophobia), chemosis and ocular secretion. The groups treated with PRP either as eyedrop or a clot, showed less opacity than the animals treated only with amniotic membrane at the moment of the final evaluation. The presence of corneal ulcers in the groups treated with PRP (PG and CLG) showed lower intensity than the other groups (CG and AG). In histological evaluation, corneal epithelization at the initial phase of the lesion was confirmed when using PRP. The use of amniotic membrane promoted corneal epithelial and stromal thickness, as well as synergism when associated to PRP / Mestre

La membrane cellulaire est un objet jouant divers rôles en biologie. Elle sert en particulier de barrière sélective entre l'intérieur et l'extérieur d'une cellule. Une membrane est une bicouche majoritairement composée de lipides, particulièrement de phospholipides, entre lesquels des protéines peuvent s'insérer. Les membranes ont besoin de contrôler l'organisation des protéines pour répondre à différentes fonctions biologiques. En physique de la matière condensée une interface signifie généralement une frontière entre deux phases distinctes, les fluctuations de cette frontière pouvant être étudiées avec les outils de la physique statistique et ceux associés aux phénomènes critiques. C'est dans ce cadre que s'insèrent nos travaux. Dans une première partie, nous nous sommes intéressés à l'organisation bidimensionnelle des lipides dans la membrane. Nous avons développé un modèle analytique de vésicule, objet tridimensionnel constitué d'une membrane fermée, où les lipides sont modélisés comme un fluide binaire en proportions différentes dans les deux feuillets de la bicouche. Un hamiltonien de Landau, qui décrit les interactions entre les lipides dans un feuillet, est couplé à un hamiltonien d'Helfrich qui rend compte des propriétés élastiques du système via une courbure spontanée et un module de courbure élastique qui dépendent de la composition locale. Dans ce modèle, le système présente différentes phases thermodynamiques qui peuvent être associées à des domaines soit épais soit courbés. Les domaines épais sont de bons candidats pour modéliser les radeaux (ou "rafts") lipidiques, qui jouent supposément le rôle de plate-forme de signalisation pour les cellules. La seconde partie porte sur l'impact de ces différentes phases sur la forme globale des vésicules. Pour répondre à cette question nous avons développé un programme numérique qui simule des vésicules composées de différents lipides. Lors de la comparaison de nos premiers résultats avec les solutions du modèle analytique, nous nous sommes aperçus qu'il existe une différence importante entre les paramètres élastiques microscopiques et ceux associés aux spectres de fluctuations mesurés. En effet, deux paramètres sufisent pour décrire le modèle de Helfrich, la tension de surface et le module de courbure élastique. Bien que les variations du module de courbure soient faibles, celles de la tension de surface sont importantes. Nous avons obtenus une formule simple qui relie la tension microscopique à celle du spectre des fluctuations. A l'aide de simulations Monte Carlo extensives et précises nous avons vérifié l'accord de ces résultats. De plus, nous avons étudié la transition de la forme sphérique à la forme "érythrocyte" et montré qu'elle pouvait être associée à l'annulation de la tension de Laplace du système. Nous avons également re-exploré la renormalisation des paramètres du modèle d'Helfrich pour une membrane plane et fait une analogie avec le modèle delta non- linéaire, un modèle de spins bien connu en matière condensée. / The cell membrane is an object playing many roles in biology. It is used in particular as a selective barrier between the interior and the exterior of a cell. A membrane is a bilayer composed mostly of lipids, and in particular of phospholipids, in which proteins can be inserted. The membrane needs to control the spatial organization of proteins to achieve various biological functions. In condensed matter physics, an interface usually means a boundary between two phases, the fluctuations of such border can be studied with the tools of statistical physics and those of critical phenomena. It is in this context that our work is inscribed. In a first part, we are interested in the two-dimensional organization of lipids in the membrane. We have developed an analytical model of a vesicle, a three-dimensional object consisting of a closed membrane, where the lipid bilayer is modeled as a binary mixture with di erent average compositions on both leaflets. A Landau hamiltonian describing the lipid- lipid interactions on each leaflet is coupled to a Helfrich one, accounting for the membrane elasticity, via both a local spontaneous curvature, and a bending modulus which depend on the local composition of lipids. In this model there are different thermodynamics phases that can be associated with thick or curved patches. These thick patches are good candidates for modeling lipidic rafts, which serve as signaling platforms for cells. The second issue concerns the impact of the different phases on the global shape of the vesicles. To answer this, we developed a numerical code that simulates vesicles composed of various lipids. When we compared our first results with analytical solutions, we realized that there is a significant difference between the microscopic elastic parameters and those associated with the fluctuation spectrum in the output of the simulation. Indeed, two parameters are enough to describe the Helfrich hamiltonian, the surface tension and the bending modulus. Although we observe small changes in the bending modulus, those of the surface tension are important. We have obtained a simple formula which connects the microscopic tension with the one associated with the fluctuation spectrum. Using extensive and accurate Monte Carlo simulations we checked the agreement of these results. In addition, we have studied the transition from the spherical shape to the "erythrocyte" one and showed that it could be associated with the cancellation of the Laplace pressure. We also explored the renormalization of Helfrich parameters for a flat membrane and proposed an analogy with the nonlinear delta model, a well known spin model in condensed matter.