A L-Asparaginase (ASNase) é um importante agente quimioterapêutico utilizado para o tratamento da leucemia linfoblástica aguda (ALL) há mais de 40 anos. No entanto, devido à origem biológica da ASNase, enzima produzida por Escherichia coli, problemas como a imunogenicidade e baixa meia vida-plasmática devem ser considerados. Com o objetivo de minimizar essas desvantagens, várias ASNases homólogas bem como formulações de ASNase de E. coli foram investigadas. Nenhuma das formulações desenvolvidas, entretanto, foi capaz de resolver definitivamente esses problemas associados à sua origem. Nesse sentido, considerando os recentes avanços na ciência de polímeros com a possibilidade do obtenção de vesículas poliméricas usando copolímeros, este trabalho concentrou-se no desenvolvimento de polimerossomos de poli(etileno glicol)-b-poli(ε-caprolactona) (PEG-PCL) para encapsular a ASNase. Diversas condições experimentais foram investigadas e, ao final, os polimerossomos foram produzidos pela técnica de hidratação do filme polimérico utilizando a centrifugação como técnica de pós-filme para remoção de copolímero precipitado, produzindo assim vesículas polímericas de 120 a 200nm com PDI de aproximadamente 0,250. A eficiência de encapsulação da ASNase, utilizando as metodologias de centrifugação ou cromatografia de exclusão molecular, revelou taxas de encapsulação de 20-25% e 1 a 7%, repectivamente. Esses resultados apontam a importância de se determinar a eficiência de encapsulação por cromatografia de exclusão molecular ou método direto no caso de nanoestruturas auto-agregadas formadas por copolímeros, devido a valores superestimados com o emprego da centrifugação. Ainda que estudos complementares se façam necessários para liberação da enzima encapsulada ou penetração da L-asparagina nas vesículas, nossos resultados demonstram o potencial de polimerossomos para veiculação de ASNase, bem como de outras proteínas terapêuticas. / L-Asparaginase (ASNase) is an important chemotherapeutic agent used for the treatment of acute lymphoblastic leukemia (ALL) for more than 40 years. However, due to the biological origin of ASNase (produced by Escherichia coli) some drawbacks such as immunogenicity and low plasma half life are present. In order to minimize the disadvantages, several ASNases proteoforms and formulations of E. coli ASNase were investigated. However, none of this formulations completely solved the main drawbacks of ASNase. In this sense, considering the recents advances in polymers science with the possibility to develop polymeric vesicles using copolymers, this work aimed at the development of poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-PCL) vesicles to encapsulate ASNase. Different experimental conditions were investigated and, the final polymersomes formulation was prepared by film hydratation using centrifugation as a post-film technique to remove the bulky coplymer. Polymeric vesicles of 120 to 200nm with PDI of approximately, 0.250 were obtained. The encapsulation efficiency of ASNase was determined indirectly by centrifugation and directly by size exclusion chromatography, resulting in encapsulation rates of 20-25% and 1 to 7%, respectively. These results indicate the importance of determining the efficiency of encapsulation by size exclusion chromatography or direct method in the case of self-aggregated nanostructures formed by copolymers, due to values overestimated with the use of centrifugation. Our results point to the potential of polymersomes for ASNase delivery, as well as other therapeutic proteins. Nonetheless, complimentary studies are still necessary for ASNase release or L-asparagine penetration into the vesicles.

A acne vulgar é uma das doenças cutâneas mais comuns, apresentando como um de seus fatores fisiopatológicos primários a colonização pelo microrganismo Propionibacterium acnes. Atualmente, têm-se buscado terapias alternativas para o combate ao P. acnes, destacando-se alguns ácidos graxos, como o ácido laúrico (LA). O LA é uma molécula pouco solúvel em água, sendo possível sua incorporação em lipossomas. Os lipossomas apresentam capacidade de encapsulação/ liberação de ativos e impedem a desidratação da pele, tornando-se ingredientes inovadores na área de cosméticos. Foram preparados lipossomas de dipalmitoilfosfatidilcolina (DPPC) contendo diferentes concentrações de LA, que variaram de 0 a 50% da concentração total em mol, em quatro pHs: 9,0, 7,4, 5,0 e 3,0. Nestes pHs o estado de protonação do LA muda variando de 0 a -1. Os lipossomas foram extrusados por filtros com poros de 100 nm de diâmetro visando à obtenção de vesículas unilamelares grandes (LUV). As LUV foram caracterizadas quanto a sua estabilidade em condições de prateleira, temperatura de transição de fase da bicamada, encapsulamento no interior aquoso, liberação do LA, difusão das vesículas na pele e seus aspectos morfológicos foram caracterizados por espalhamento de raios-X a baixo ângulo (SAXS) e crio-microscopia eletrônica de transmissão. Estudos de estabilidade mostraram que independentemente da concentração de LA, as formulações são mais estáveis em pHs mais altos, quando LA está em sua maioria na forma de laurato. Os experimentos de DSC revelaram que em pHs 3,0 e 5,0 e concentrações maiores de LA, a interação deste ácido graxo com as bicamadas é favorecida, havendo um aumento da temperatura de transição de fase (Tm) e diminuição da cooperatividade. Análises de taxa de incorporação de sondas hidrofílicas confirmaram a presença de um compartimento aquoso interno para as vesículas de DPPC:LA. O LA conseguiu permear a pele no período avaliado e pouco LA foi liberado das vesículas em condições de temperatura ambiente. A morfologia das LUV se mostrou bem diferente da esperada e se observaram vesículas com mais de uma bicamada e outros formatos que não o esférico. Estes resultados podem auxiliar na otimização das condições para uma formulação que poderá ser usada no tratamento da acne, aumentando a eficácia do LA no sítio alvo. / Acne vulgaris is one of the most common skin diseases, presenting as one of its causes the microorganism Propionibacterium acnes colonization. Currently, it has been sought alternatives therapies against P. acnes, especially some fatty acids, like the lauric acid (LA). LA is a molecule with low water solubility, allowing its incorporation in liposomes. Liposomes have encapsulation/release capacity of drugs and promote skin lipids regeneration, becoming an innovative ingredient in cosmetics area. Dipalmitoylphosphatidylcholine (DPPC) vesicles containing different LA concentrations were prepared at pHs: 9.0, 7.4, 5.0 and 3.0. At these pHs the LA protonation changes and its charge varies from 0 to -1. The vesicles were extruded through filters containing pores of 100 nm to obtain large unilamellar vesicles (LUV) that were characterized for stability in shelf conditions, bilayer phase transition temperature, aqueous internal compartment encapsulation, LA release and in vitro skin permeation. Its morphological features were characterized by small angle X-ray scattering (SAXS) and cryo-electron transmission microscopy. Stability assays showed that, regardless LA concentration, formulations were more long-term stable in higher pHs, when LA is mostly in the form of laurate. The differential scanning calorimetry, DSC, experiments showed that, at pHs 3.0 and 5.0 and higher LA concentrations, the interaction between the bilayer and LA is favored, increasing phase transition temperature (Tm) and reducing cooperativity. Incorporation of hydrophilic probes confirmed the presence of an internal aqueous compartment in DPPC:LA vesicles. The LA managed to permeate the skin on the period evaluated and, in ambient conditions, low LA concentration was released from the vesicles. LUV containing more than one bilayer and non-spherical structures were observed. The obtained results may help in the optimization of the conditions for a formulation that can be used in the treatment of acne and improving the effectiveness of LA delivery to the target site.

La polarité cellulaire est une caractéristique commune à tous les organismes. Jusqu’à récemment, il était assumé que la sécrétion de protéines vers des domaines polaires de la cellule végétale se faisait de façon non polarisée, mais ce point de vue a été re-étudié, la sécrétion est polarisée mais la dynamique, les voies de traficempruntées et les mécanismes sont toujours inconnus. Précédemment, mon laboratoire d’accueil a caractérisé un enrichissement en sphingolipides contenant des acides gras à très longues chaines (VLCFAs) au niveau d’un sous-domaine du trans-Golgi Network (TGN) appelé Vésicules de Sécrétions (SVs). Plus précisément, il a été montré que la longueur des acides gras des sphingolipides jouait un rôle critique dans la sécrétion du transporteur d’auxine PIN2 des SVs vers des domaines polaires de la membrane plasmique. Pendant ma thèse, je me suis intéressé à la question suivante : comment les sphingolipides agissent-t-ils au TGN? En identifiant le protéome des SVs, ainsi qu'en utilisant des outils génétiques et pharmacologiques en combinaison avec la visualisation de marqueurs lipidiques, j'ai pu identifier que les sphingolipides agissent sur l’homéostasie des phosphoinositides en mettant en avant un lien fonctionnel entre ces deux classes de lipides au sein de la cellule végétale. En utilisant un set de marqueurs des phosphoinositides (PIPs), j’ai pu montrer que les sphingolipides ciblent principalement le phosphatidyl-inositol-3-phosphate, PI(3)P et le phosphatidylinositol- 4-phosphate, PI(4)P. De plus, mon analyse protéomique a montré que la localisation d'un ensemble de protéines liées aux PIPs était diminuée dans les SVs/TGN immunopurifiées quand la composition des sphingolipides est altérée. Mes résultats nous forcent à revoir notre vision de la dynamique des lipides au niveau des membranes, et suggère l’idée que la dynamique de remodelage de la composition d’une classe de lipide, les phosphoinositides, peut être modulée par une autre classe de lipide, les sphingolipides. / Cell polarity is a defining feature of all organisms. Until very recently, it was thought that delivery of proteins to polar domains of root epidermal cells plasma membrane was non-polar, but this view has been re-examined, the delivery is polar but the dynamics, the paths taken, and the mechanisms are unknown. My host team previously characterised an enrichment of Very-Long-Chain-Fatty-Acids (VLCFAs)-containing sphingolipids at the site of secretory vesicles (SVs) sub-domain of the trans-Golgi Network (TGN). Moreover, the length of sphingolipids acyl-chain was found to play a critical role in secretory sorting of the auxin carrier PIN2 from SVsassociated TGN to apical polar domain of the plasma membrane (PM). During my PhD, I addressed the following question: how sphingolipids act at SVs/TGN? Using proteomics of SVs, genetics and pharmacological tools in combination with visualisation of lipid probes we could identify that sphingolipids act on phosphoinositides (PIPs) homeostasis establishing a new functional link between these two lipids in plant cells. Using a set of multi-affinity fluorescent PIPs probes I could show that sphingolipids target phosphatidylinositol-3-phosphate (PI3P) and phosphatidylinositol-4-phosphate (PI4P). Moreover, my proteomic analyses show that several PIPs-related proteins are downregulated in immuno-purified TGN-associated SVs when the sphingolipid composition is altered pharmacologically. My results force the reassessment of our view of lipid membranes dynamics and highlight the idea that dynamic remodelling of the composition of one lipid class, the phosphoinositides, can be modulated by another lipid class, the sphingolipids.

A extensão relativa da dissociação iônica (α) de micelas de cloreto de hexadecildimetilamônio (CTACI), brometo de hexadecildimetilamônio (CTAB), vesículas de cloreto de dioctacecildimetilamônio (DODAC) e brometo de dioctadecildimetilamônio (DODAB) foram determinadas usando um método de dediazonização. O método de dediazonização consiste na determinação de produtos resultantes da lenta decomposição espontânea de tetrafluorborato de 2,4,6-trimetilbenzenodiazônio, seguida pela reação rápida do cátion fenílico com nucleófilos presentes no meio. Foi demostrado que este método determina a composição relativa da pseudofase aquosa interagregado. Os valores de α obtidos para CTACI e CTAB foram, respectivamente, 0,29 e 0,26, em excelente concordância com valores previamente publicados. O coeficiente de troca iônica para brometo/cloreto, determinado através do mesmo método, foi de 2,65, coincidindo com dados publicados. Estes valores confirmam o uso deste método para se obter tanto grau de dissociação iônica relativo como coeficientes de seletividade. O método foi também aplicado para a determinação do grau de dissociação iônica externo (αe e interno (αi) de vesículas de DODAC e DODAB. Para vesículas de DODAB sonicadas por tip obteve-se αe de 0,20; para vesículas de DODAC sonicadas em banho αe foi 0,20; para vesículas de DODAC sonicadas por tip αe foi 0,25 e para vesículas de DODAC grandes αe foi 0,11. Foi também determinado, pela primeira vez, o valor de 0,07 para o αi de vesículas grandes de DODAC. O conjunto de valores obtidos para αi e αe permitiram a análise quantitativa da catálise pelo compartimento aquoso interno e externo de vesículas sem a utilização de α como parâmetro ajustável. Foi modelado com sucesso a decomposição espontânea do íon 6-nitrobenzisoxazol-3-carboxilato e a hidrólise alcalina do ácido 5,5\'-ditiobis(2-nitrobenzóico). Este trabalho é a primeira análise quantitativa de reações modificadas por vesículas nos dois compartimentos usando dados independentes para dissociação iônica das superfícies vesiculares. / The relative extents of ion dissociation (α) from micelles of hexadecyltrimethylammonium (CTA) chloride (CTACl) and bromide (CTAB) and vesicles of dioctadecyldimethylammonium (DODA) chloride (DODAC) and bromide (DODAB) were determined using a dediazoniation method. The dediazoniation method consists in the determination of the products resulting from the slow spontaneous decomposition of 2,4,6-trimethylbenzene diazonium tetrafluoroborate, followed by fast reaction of the resulting phenyl cation with neighboring nucleophiles. This method was shown to determine the relative composition of the interaggregate aqueous pseudophase and, consequently, the degree of ion dissociation of the aggregate. The values of α obtained for CTACl and CTAB were, respectively, 0.29 and 0.26, in excellent agreement with previously published values. The ion exchange selectivity coefficient for bromide/chloride exchange, determined using the same method was 2.65, coinciding with published data. These values lend confidence to the use of this method for obtaining both relative dissociation degrees and ion selectivity coefficients. The method was also applied for the determination of the external (αe)as well as the internal (αi) α of DODAC and DODAB vesicles. For tip-sonicated DODAB vesicles αe was 0.20, for bath-sonicated DODAC αe was 0.20, for tip-sonicated DODAC αe was 0.25 and for large DODAC vesicles a was 0.11. We also determined, for the ftrst time, the a value of 0.07 for αi of large vesicles. The set of values obtained for αi and αe permitted quantitative analysis of two reaetions in both the internal and external aqueous compartments of vesicles without recourse to ion dissociation adjustable parameters. We successfully modeled the spontaneous decomposition of 6-nitrobenzisoxazol-3-carboxylate and the alkaline hydrolysis of 5,5\'-dithiobis(2-nitrobenzoate). This work is the first quantitative analysis of vesicle-modified reactions in both vesicle compartments using independent data for ion dissociation from vesicle\'s surfaces.

At the neuronal synapse, neurotransmitter-filled synaptic vesicles (SVs) fuse with the presynaptic plasma membrane during activity. Following exocytosis, SVs must be retrieved for neurotransmission to be maintained. Several modes of SV recycling have been identified. During mild neuronal activity, clathrin-mediated endocytosis has been regarded as the dominant SV retrieval mode, however the recently identified ultrafast endocytosis mode may also be important in this condition. During elevated activity, activity-dependent bulk endocytosis (ADBE) is the dominant SV retrieval pathway. In ADBE, large invaginations are formed from the plasma membrane, which then undergo scission to create bulk endosomes. In a second distinct step, SVs bud from these endosomes and specifically repopulate the reserve SV pool. However, since its first identification, only few molecules have been shown to participate in ADBE. The aim of this PhD was to identify novel molecules and elucidate the molecular mechanism of ADBE. To achieve this, two independent biochemical approaches were designed to purify and enrich bulk endosomes from primary neuronal cultures. In the first approach, bulk endosomes and SVs were labelled with a dye, FM1-43, using a strong stimulus. Cells were broken mechanically and the post nuclear supernatant, that contains all intracellular organelles, was collected. The supernatant was then subjected to subcellular fractionation using discontinuous Nycodenz gradients. This stimulated sample was always processed in parallel with a basal sample, where no neuronal stimulus was applied, in order to visualise activity dependent FM loading. After different fractionation protocols were applied, bulk endosomes were efficiently separated from SVs, as revealed by tracking fluorescence in different fractions. The fractionation results were further validated by electron microscopy, where bulk endosomes and SVs were labelled with horseradish peroxidase and purified using the established protocol. Immunoblotting against selected SV cargo proteins from stimulated bulk endosome and SV samples, indicated the specific and preferential localisation of VAMP4 on bulk endosomes, in contrast to other SV cargo. The molecular identity of bulk endosomes was also approached by submitting the bulk endosome fractions to semi-quantitative mass spectrometry. This analysis revealed many different proteins that were identified in bulk endosome samples and quantification approaches further indicated proteins that can be localised on bulk endosomes and have a potential role in ADBE. A second magnetic isolation approach was designed, to purify bulk endosomes using a completely different methodology. In this case, bulk endosomes were specifically labelled with iron nanoparticles, which are preferentially taken up by bulk endosomes since they are larger than SVs. The cells were broken as before and post nuclear supernatant was acquired. In this case, the supernatant was submitted to magnetic isolation that separated iron beads labelled structures from all other intracellular organelles. An extensive immunoblotting analysis of magnetic bulk endosomes validated that VAMP4 and syndapin I, two essential ADBE proteins, were enriched in these purified samples. These magnetic bulk endosomes were also analysed using semi-quantitative MS and revealed many proteins with a potential role in ADBE. Significant overlap between the two independent methods was observed, further validating these approaches. Combining these two methods with bioinformatics tools allowed the identification of the molecular signature of ADBE as well as novel key candidates for this process. Specific molecules were investigated for their role in ADBE and SV recycling using a variety of different real-time fluorescent imaging assays. A major focus was on rab small GTPases. High molecular weight dextran uptake was used to specifically study the role of these proteins in ADBE, as it preferentially reports uptake via larger bulk endosomes. A pH sensitive chimeric protein, synaptophysin-pHluorin, was used to investigate the role of these proteins in CME. Additional imaging assays were used to answer emerging questions regarding the function and localisation of these targets in the presynapse. Using these approaches, rab11A and rab35 were found to promote ADBE and accelerate clathrin-mediated endocytosis. This effect was specific to high intensity stimulation, while SV exocytosis was not affected. Further research on the role of both novel and established ADBE molecules will provide key future insights into the mechanism of both bulk endosome generation/scission and subsequent SV reformation. A very promising group is rab proteins and now evidence for their implication in SV recycling is presented here. Identification and characterisation of new targets will allow to investigate the role of ADBE in neurotransmission in both physiology and pathophysiology.

The parasite secretome has been shown to play a key role in both pathogenicity and the regulation of host defence, allowing pathogens, such as helminths, to establish a chronic infection within the host. The recently discovered presence of extracellular vesicles within parasite-derived excretory-secretory products introduces a new mechanism of potential cross-species communication. Extracellular vesicles (EVs), such as exosomes, facilitate cellular communication through the transfer of small RNAs, lipids and proteins between cells and organisms across all three kingdoms of life. In addition to their roles in normal physiology, EVs also transport molecules from pathogens to hosts, presenting parasite antigens and transferring infectious agents. Here, I examine secreted vesicles from the murine gastrointestinal nematode Heligmosomoides polygyrus, and their potential role in the host-helminth interactions. Transmission electron microscopy reveals vesicle-like structures of 50- 100 nM in the ultracentrifuged secretory product, and potential evidence of multi-vesicular bodies in the worm intestine. This, coupled with information from the exoproteome, helped support the hypothesis that exosomes originate from the parasite intestinal tract. I have completed a series of studies looking at the fundamental properties of exosome-cell interactions, providing comparative studies between mammalian and H. polygyrus-derived exosomes. I have determined some of the key factors influencing exosome uptake, including time of incubation, cell type and exosome origin. Through microarray analysis of H. polygyrus exosome-treated small intestinal epithelial cells, we see significant gene expression changes, including those involved in the regulation of signalling and the immune response, such as DUSP1 (dual-specificity phosphatase) and IL1RL1 (the receptor for IL-33). The modest reduction of inflammatory cytokine responses by exosomes in small intestinal cell lines was amplified in immune cells, such as macrophages. Exosomes can significantly reduce expression of classical activation markers, as well as inflammatory cytokine production in the macrophage cell line RAW 264.7, and this is further supported by similar responses in bone marrow-derived macrophages. Owing to their suppressive nature, I demonstrate that immunization of mice with an exosome/alum conjugate generates significant protection from a subsequent H. polygyrus larval challenge, as seen through a reduction in egg counts and worm burden. I have investigated the role of the IL33 receptor (IL-33R); a key molecule associated with parasitic resistance that is suppressed by exosomes in type-2 associated immune responses. Uptake of H. polygyrus-derived exosomes by alternatively activated macrophages caused the suppression of type 2 cytokine/protein release and the reduction of key genes associated with this phenotype. In addition, there was also significant repression of both transcript and surface T1/ST2, a subunit of the IL-33R). Using a model of lung inflammation, in vivo studies demonstrate that, in both prophylactic and co-administration experiments, exosomes modulate the innate cellular response. This is represented by changes in the number of innate lymphoid cells (ILCs), bronchoalveolar lavage eosinophils and type-2 cytokine output. In this system, the expression of T1/ST2 on type 2 ILCs was also significantly reduced. I have extended the investigation on exosome-IL-33R responses by using T1/ST2 knockout mice. Despite generating strong antibody responses, vaccination against exosomes could not protect T1/ST2 knockout mice against a subsequent infection. This work suggests that exosomes secreted by nematodes could mediate the transfer and uptake of parasite products into host cells, establishing cross-species communication to suppress the host ‘danger’ or inflammatory response.

Copolímeros, macromoléculas orgânicas ou inorgânicas com alta massa molar, consistem de unidades monômericas repetidas, unidas por ligações covalentes e que apresentam mais de um tipo de monômero. Neste trabalho estudamos três copolímeros constituídos por dois blocos distintos, isto é copolímeros dibloco, onde um dos blocos é constituído pelo polimetacrilato de metila, PMMA e o outro pelo polimetacrilato de N,N dimetilamino, PDMAEMA, e o homopolímero de PDMAEMA. A fórmula geral dos copolímeros dibloco estudados é PMMAm-b-PDMAEMAn onde o número de monômeros de cada bloco é representado pelos índices m e n. Os copolímeros PMMA1-b-PDMAEMA6,3 (MM, 54.200), PMMA1-b-PDMAEMA3,0 (MM 27.558), o PMMA1-b-PDMAEMA1,1 (MM 25.555) e o homopolímero PDMAEMA (MM 41.614,9) tiveram suas massas molares e a razão entre os monômeros de cada bloco caracterizados por cromatografia de gel permeação, GPC, e NMR. Determinamos a concentração micelar crítica (CMC) dos copolímeros e verificamos, por RMN e fluorescência, o efeito da concentração e do pH na sua agregação. A interação dos copolímeros com vesículas unilamelares grandes, LUVs, preparadas com misturas de fosfatidilcolina, PC, e fosfatidilglicerol, PG, foi estudada em diferentes condições de pH e força iônica. A interação dos polímeros com as LUVs também foi estudada medindo-se o diâmetro hidrodinâmico e potencial zeta das LUVs na presença dos polímeros. Observou-se que, quando a razão polímero/lipídio é alta, os polímeros se ligam às LUVs, neutralizando completamente a carga das vesículas. Quando a concentração de LUVs é próxima à dos polímeros, forma-se uma rede que conduz à agregação e precipitação dos complexos. Estudamos o efeito dos polímeros na permeabilização de LUVs e vesículas unilamelares gigantes (GUVs) preparadas com misturas de PC:PG. Os copolímeros permeabilizaram as LUVs, dependendo da razão polímero/lipídio, do pH, força iônica e das características hidrofóbicas e hidrofílicas de cada copolímero. Quanto maior a porcentagem de PG nas LUVs maior a interação com os copolímeros. A ligação dos copolímeros às LUVs deve produzir segregação dos fosfolipídios negativos na bicamada das LUVs (i.e., separação lateral de fases) facilitando a permeabilização das vesículas. Demonstramos que os copolímeros se ligam à superfície das GUVs modificando a sua forma e levando ao rompimento das vesículas. Estes efeitos foram modulados pelo pH e a força iônica do meio. O efeito de permeabilização dos copolímeros foi correlacionado com as razões entre os blocos hidrofóbicos e hidrofílicos. Quando o copolímero tem uma maior fração de PDMAEMA, o bloco mais hidrofílico do copolímero, este efeito é mais evidente. A porcentagem de permeabilização, após um tempo definido, para copolímeros de mesma massa molar, como o PMMA1-b-PDMAEMA3,0 (MM 27.558) e o PMMA1-b-PDMAEMA1,1 (MM 25.555), é muito maior com o copolímero com maior porção hidrofílica, o PMMA1-b-PDMAEMA3,0. O homopolímero DMAEMA também se mostrou eficiente na interação com as LUVs, porém menos que o PMMA1-b-PDMAEMA6,3.que possui maior numero de unidades DMAEMA e uma sequencia de monômeros de MMA. Demonstramos, nesta Tese, que o efeito de copolímeros sintéticos contendo regiões hidrofóbicas e hidrofílicas são bons modelos de peptídeos e proteínas permitindo avaliar quantitativamente o efeito dessas interações em modelos de membranas. / Copolymers are organic or inorganic macromolecules with high molecular weight, consisting of repeated monomer units joined by covalent bonds and exhibit more than one type of monomer. Here, we studied three copolymers consisting of two different blocks, i.e. diblock copolymers, where one block is constituted by methyl polymethacrylate, PMMA and other by polymethacrylate N, N-dimethylamino, PDMAEMA and a homopolymer PDMAEMA. The general formula of the diblock copolymers studied was PMMAm-b-PDMAEMAn where the number of monomers of each block is represented by indexes m and n. The PMMA1-b-PDMAEMA6,3 copolymers (MW, 54,200), PMMA1 PDMAEMA3,0-b (MW 27,558), the PMMA1 PDMAEMA1,1-b (MW 25,555) and PDMAEMA homopolymer (MW 41614.9) had their molecular weights and the ratio between the monomers of each block characterized by gel permeation chromatography, GPC, and NMR. The critical micelle concentration (CMC) of the copolymers was determined by fluorescence and the aggregation of the copolymers verified by NMR. The effect pH on the copolymers CMC was also determined. The interaction of copolymers with large unilamellar vesicles, LUV, prepared with mixtures of phosphatidylcholine, PC, and phosphatidylglycerol, PG, was studied under different conditions of pH and ionic strength. The interaction of the polymers with the LUVs was also studied measuring the hydrodynamic diameter and zeta potential of the LUVs in the presence of the polymers. It was observed that when the ratios polymer / lipid are high, the polymers bind to the LUVs, completely neutralizing the charge of the vesicles. When the concentration of LUVs is close to that of the polymers, it forms a network that leads to aggregation and precipitation of the complexes. We studied the effect of polymers in permeabilization LUVs and giant unilamellar vesicles (GUVs) prepared with PC:PG. The copolymers permeabilized the LUVs, depending on the ratio polymer / lipid, pH, ionic strength and the hydrophobic and hydrophilic characteristics of each copolymer. The higher the percentage PG LUVs lead to greater interaction with the copolymers. The binding of the copolymers to the LUVs with negative charge induced phase separation of phospholipids in the bilayer of LUVs (ie, lateral phase separation) facilitating the permeability of the vesicles. We demonstrate that copolymers bind to the surface of GUVs changing its shape and leading to rupture of vesicles. These effects are modulated by pH and ionic strength of the media. The permeabilization effect of the copolymers was correlated with the ratios of the hydrophobic and hydrophilic blocks. When the copolymer has a larger fraction of PDMAEMA, the more hydrophilic block copolymer, this effect is more evident. The percentage of permeabilization, after a set time, for the same molar mass copolymers such as b-PDMAEMA3,0 PMMA1 (MW 27,558) and PMMA1 PDMAEMA1,1-b (MW 25,555), is much more efficient with copolymer with greater hydrophilic portion, the b-PMMA1 PDMAEMA3,0. The DMAEMA homopolymer also proved efficient in interacting with the LUVs. We demonstrated that synthetic copolymers containing hydrophobic and hydrophilic regions are good models of peptides and proteins allowing quantitatively evaluate the effects of these interactions on model membranes

Made available in DSpace on 2014-06-11T19:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-03-03Bitstream added on 2014-06-13T20:01:04Z : No. of bitstreams: 1 santos_ccm_dr_sjrp.pdf: 1848361 bytes, checksum: 77ec9eb274e716c7917ddf552f845a66 (MD5) / Poli(etilenoglicol) (PEG) é um polímero neutro hidrossolúvel, e brometo de dioctadecildimetilamônio (DODAB) e diododecildimetilamônio (DDAB) são surfactantes catiônicos dialquilados, C18 e C12, respectivamente, derivados da amônia quaternária, formadores de vesículas. As suas propriedades físicas, em solução aquosa, são bem conhecidas. No entanto, as propriedades de misturas desse polímero com esses surfactantes não o são. Investigamos a interação de PEG com DODAB e DDAB em solução aquosa, numa faixa de concentração total dos componentes até 1% em peso, e construímos diagramas de fases, com especial destaque para a fase vesicular. Utilizamos PEG com massa molecular entre 200 Da e 2 MDa e os diagramas de fases foram construídos a 25oC, isto é, acima da temperatura de transição gel-líquido cristal (Tm) de DDAB (Tm = 16oC) e abaixo da Tm de DODAB (Tm = 45oC). DODAB e DDAB têm a característica comum de formar vesículas unilamelares em baixas concentrações do surfactante e vesículas multilamelares em concentrações mais elevadas; em regiões intermediárias, vesículas uni e multilamelares coexistem em equilíbrio na solução. A fronteira entre essas regiões de vesículas não é muito bem definida e observamos, neste estudo, que o efeito de PEG na estrutura de agregados de DODAB e DDAB em água depende da massa molecular do polímero. Além disso, PEG favorece a formação de vesículas unilamelares em concentrações mais elevadas de DODAB e DDAB, quando predominam as vesículas multilamelares de DODAB puro (sem o polímero), possibilitando a formação de vesículas mistas de DODAB/PEG e DDAB/PEG em água e concentrações relativamente grandes desses surfactantes. Dentre os métodos experimentais empregados nesse estudo, destacamos, turbidimetria, fluorescência de estado estacionário, calorimetria diferencial de varredura (DSC) e por titulação isotérmica (ITC) e análise visual por polarizadores cruzados. / Poly (ethyleneglycol) (PEG) is a water soluble neutral polymer, and dioctadecyldimethylammonium (DODAB) and diododecyldimethylammonium (DDAB) bromide are double chain cationic surfactants derived from the quaternary ammonium that have been widely investigated due to their high application potential in different areas of the science and technology. The physical properties of PEG, DODAB and DDAB in aqueous solution are well-known. However, the properties of mixtures of the polymer with those surfactants are not. We investigated the interaction of PEG with DODAB and DDAB in aqueous solution, within a range of total concentration of the components of 0-1 wt%, and the phase diagrams built up, with special interest for the vesicular phases. We used PEG with molecular mass between 200 Da and 2 MDa and the phase diagrams were built up at 25°C, that is, above the melting temperature (Tm) of DDAB (Tm = 16°C) and below Tm of DODAB (Tm = 45°C). DODAB and DDAB have the common characteristic of forming unilamellar vesicles at low surfactant concentrations and umultilamellar vesicles at higher concentrations; at intermediate concentrations, uni- and multilamellar vesicles coexist in solution. The borders of these different vesicle phases are not well defined. We observed in this Thesis that the effect of PEG on the structures of DODAB and DDAB aggregates in water depends on the molecular mass of the polymer. Besides, PEG stabilizes the unilamellar vesicles at high concentrations of DODAB, where the multilamellar vesicles are the dominant structures present in solution, thus allowing the formation of mixed DODAB/PEG and DDAB/PEG vesicles in water at relatively high concentrations of these surfactants. The experimental methods used in this investigation include turbidimetry, steady-state fluorescence, differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC), and visual analysis by crossed polaroids.

The work presented in this thesis deals with the structural and functional properties of peptides at surfaces. The interaction of peptides with surfaces is an ever so common occurrence in our every day life, from the bug squashed on the windshield of our car to the barnacle on our boat, and from the blood plasma used in the hospital to the proteins in our cells. The effect these occurrences has on our lives is diverse, the bug is annoying whereas the barnacle settlement of ship hull is costly for marine transportation, the blood plasma contains components of vital importance for our immunological defense system and the proteins in our cells are crucial for regulatory processes and life.One part of this thesis, performed as a part of the EU-founded project AMBIO, deals with the concept of marine biofouling. A number of short peptides have been designed, synthesized, and used to investigate their effect on the settlement on marine biofoulers, such as the Ulva linza algae and the Navicula diatom, on template surfaces coated with thin layers of these molecules. The surfaces have been thoroughly investigated with respect of their physio-chemical properties before and after submersion in artificial seawater and ultimately in suspensions containing the organisms. The most interesting results were obtained with an arginine-rich peptide coating that when introduced to Ulva linza zoospores, displayed extensive settlement, compared to reference surfaces. In addition, a large fraction of the settled spores had an abnormal morphology.The other part of this thesis is focused on designed peptides that when adsorbed on a negatively charged surface adopts a well-defined secondary structure, either α-helical or β-sheet. Precisely placed amino acids in the peptides will strongly disfavor structure in solution, primarily due to electrostatic repulsion, but when the peptides are adsorbed on the negatively charged surfaces, they adopt a well-defined secondary structure due to ion pair bonding. These interactions have been thoroughly investigated by systematic variations of the side-chains. In order to determine the factors contributing to the induced structure, several peptides with different amino acid sequences have been synthesized. Factors that have been investigated include 1) the positive charge density, 2) distribution of positive charges, 3) negative charge density, 4) increasing hydrophobicity, and 5) incorporating amino acids with different helix propensities. Moreover, pH dependence and the effect of different interaction partners have also been investigated. It has also been shown that the system can be modified to incorporate a catalytic site that is only active when the helix is formed. This research will increase our understanding of peptide-surface interactions and might be of importance for both bionanotechnology and medicine.

O Trypanosoma cruzi libera para o meio, vesículas contendo material de sua superfície e aparentemente estas vesículas seriam uma maneira de ativar e preparar a célula hospedeira para a invasão, além de induzir um aumento na expressão de alguns componentes da matriz extracelular. Neste trabalho demonstramos que essas vesículas aumentam a expressão de fibronectina na matriz, sendo este aumento dose-dependente e linear ao longo do tempo. Porém, para laminina não conseguimos observar o mesmo comportamento. Nossos resultados também mostram que os constituintes lipídicos das vesículas podem ser os responsáveis pelo aumento da expressão de fibronectina em cultura de células epiteliais. Os dois grandes grupos de glicoproteínas encontradas na superfície do parasita e nas vesículas, mucinas e Tc85 não parecem estar envolvidos no processo. As culturas celulares tratadas com os lipídeos extraídos das vesículas apresentaram um aumento de fibronectina também dose-dependente, porém, com uma resposta linear ao longo do tempo e uma expressão máxima atingida em tempos menores / Trypanosoma cruzi releases to the environment plasma membrane vesicles. Apparently, these vesicles could be a signal released by the parasite to prepare the host cell for the invasion. This study demonstrated that these vesicles induce a dosisdependent expression of fibronectin, linear over time. The same behavior has not been observed for laminin. Our results also show that lipids from the vesicles are involved in the increase of expression of fibronectin by epithelial cells. The two major surface membrane glycoproteins, Tc85 and mucins, also present in the vesicles do not participate in this phenomenon. Cell cultures that have been treated with lipids extracted from T. cruzi membrane vesicles also provoked a dosis-dependent increase in fibronectin and linear over time