Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-10-07T13:45:39Z No. of bitstreams: 1 DissMOG.pdf: 9813481 bytes, checksum: d5fdf73e1fcd764903f71fe58d1c80c8 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T19:35:08Z (GMT) No. of bitstreams: 1 DissMOG.pdf: 9813481 bytes, checksum: d5fdf73e1fcd764903f71fe58d1c80c8 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T19:35:14Z (GMT) No. of bitstreams: 1 DissMOG.pdf: 9813481 bytes, checksum: d5fdf73e1fcd764903f71fe58d1c80c8 (MD5) / Made available in DSpace on 2016-10-20T19:35:21Z (GMT). No. of bitstreams: 1 DissMOG.pdf: 9813481 bytes, checksum: d5fdf73e1fcd764903f71fe58d1c80c8 (MD5) Previous issue date: 2014-09-08 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Studies focusing on genetic diversity and population structure of naturally fragmented taxa are of conservation concern. Such fragmented populations are more subject to endogamy and genetic diversity erosion due to the pronounced effects of isolation and genetic drift. Pilosocereus jauruensis is a columnar cactus species restricted to patches of xeric vegetation on rock outcrops occurring around the Pantanal biome in southwestern South America. This species has been recently resurrected from synonymy with P. machriisi in the P. AURISETUS species group, and three taxa (P. paraguayensis, P. saudadensis e P. densivillosus) are synonymized with P. jauruensis in the current taxonomy. The present study aims to investigate the genetic population structure of P. jauruensis and identify possible distinct population segments within this species. We used data from four SSR markers in 157 individuals of six P. jauruensis populations and 49 individuals of two populations of P. vilaboensis, a related species occurring close to P. jauruensis populations in central Brazil. Furthermore, nucleotide variation in two plastid intergenic spacers (trnSGCU-trnGUCC and trnT-trnL) was investigated in four to five samples of each population. The indices FST e G”ST for SSR data in P. jauruensis was 0.201 and 0.559 respectively, revealing high levels of genetic differentiation among populations. Three haplotypes (Hd 0.663) of P. jauruensis with six polymorphic sites were found in the cpDNA. NJ dendograms showed similar relationships using SSR and cpDNA markers. Two populations, occurring in the same geographic region of the invalid taxa P. saudadensis were highly differentiated from other P. jauruensis populations. The cpDNA haplotype found in the northernmost P. jauruensis population was closely related with P. vilaboensis haplotypes. Also, two populations, occurring in the same geographic region of the invalid taxa P. densivillosus were highly differentiated from other P. jauruensis populations. These results were incongruent with the current taxonomic circumscription of P. jauruensis and suggest the presence of distinct taxa within this species. / Os estudos sobre a diversidade genética e estrutura populacional de espécies naturalmente fragmentada são de grande interesse para a conservação. Essas populações fragmentadas estão mais sujeitas a endogamia e perda da diversidade genética devido aos efeitos pronunciados de isolamento e deriva genética. Pilosocereus jauruensis é uma espécie de cacto colunar restrita a manchas de vegetação seca sobre afloramentos rochosos que ocorrem em torno do bioma Pantanal, no sudoeste da América do Sul. Esta espécie foi recentemente retirada de sinonímia com P. machriisi no grupo de espécies P. AURISETUS e três táxons desse grupo (P. paraguayensis, P. saudadensis e P. densivillosus) são atualmente sinonimizados a ela. O presente estudo tem como objetivo investigar a estrutura genética de populações de P. jauruensis e identificar possíveis segmentos populacionais distintos dentro desta espécie. Foram utilizados dez marcadores microssatélites em 157 indivíduos de seis populações de P. jauruensis e 49 indivíduos de duas populações de P. vilaboensis, uma espécie do grupo P. AURSETUS que ocorre em regiões próximas às populações de P. jauruensis no Brasil central. Além disso, a variação nucleotídica em dois espaçadores plastidiais intergênicos (trnSGCUtrnGUCC e trnT-trnL) foi investigada em quatro a cinco amostras de cada população. Os índices de FST e G"ST para dados SSR em P. jauruensis foi 0,201 e 0,559, respectivamente, revelando elevados níveis de diferenciação genética entre as populações. Três haplótipos plastidiais (Hd 0,663) de P. jauruensis com seis sítios polimórficos foram encontrados. As relações de similaridade entre as populações foram semelhantes entre os dois tipos de marcadores estudados. Duas populações, que ocorrem na mesma região geográfica do táxon inválido P. saudadensis, foram altamente diferenciadas em relação às outras populações de P. jauruensis e proximamente relacionadas às populações de P. vilaboensis. As duas populações de P. jauruensis que ocorrem na mesma região geográfica do táxon inválido P. densivillosus também foram diferenciadas em relação às populações restantes de P. jauruensis. Estes resultados foram incongruentes com a circunscrição taxonômica atual de P. jauruensis e sugerem a presença de táxons distintos dentro desta espécie.

Previous small-scale methods for plasmid DNA (pDNA) purification fail to meet theindustry’s demand for sufficient quantities. Greater volumes of bacterial lysates are a consequence of larger volumetric fermentations and traditional large-scale down-stream purification processes have some disadvantages and limitations. The market is believed to continue to expand, hence the need for efficient, cost-effective, andscalable purification processes becomes apparent. A crucial trade-off exists between pDNA yield and purity, necessitating careful consideration in chromatographic pu-rification steps. Each step enhances purity while likely sacrificing yield. In order to achieve a higher degree of pDNA yield, optimal purification entails a single chro-matographic step, specifically anion-exchange chromatography (AEX) in combina-tion with filtration. Alternatively, a two-step purification approach involving AEX followed by hydrophobic interaction chromatography (HIC) is recommended to elim-inate complementary impurities and achieve a high level of purity. Furthermore, the utilization of monolithic chromatographic supports is suggested to facilitate the sug-gested purification strategies. This is due to monoliths promoting higher binding capacities, ensuring robust and consistent results even at high flow rates.

<p>In this thesis, chitosan, a biocompatible polysaccharide that has been approved as a food additive was selected as a platform for the development of safe, efficient non-viral gene delivery systems to mammalian cells. Previously, chitosan-based gene formulations had been generally associated with high molecular weight chitosans, which were poorly characterised in terms of molecular weight distribution and degree of acetylation. Therefore, in order to improve the properties of chitosan-based gene formulations, the research associated with this thesis focused on establishing the structure-property relationships of well-defined, low molecular weight chitosans (chitosan oligomers) as delivery systems for nucleic acids (pDNA and siRNA)<i> in vitro</i> and after lung administration <i>in vivo</i>. pDNA dissociated more easily from chitosan oligomers than from conventional high molecular weight chitosans, resulting in a faster onset and higher levels of<i> in vivo</i> gene expression, comparable to those mediated by polyethyleneimine (PEI), one of the most efficient non-viral delivery systems. Coupling of a trisaccharide branch to the chitosan oligomers so as to target extracellular lectins resulted in a significant improvement in transfection efficiency because of enhanced cellular uptake and colloidal stability. In contrast to pDNA, longer linear chitosan oligomers were required to form physically-stable nanoparticles with siRNA that mediated efficient, sustained gene silencing <i>in vitro</i>. Finally, the use of an optimised catheter device for the nebulisation of small volumes of pDNA formulations resulted in improved dose precision and lung distribution<i> in vivo</i> compared with conventional intratracheal instillation. In conclusion, chitosan oligomers are interesting and viable alternatives to other non-viral gene delivery systems.</p>

In this thesis, chitosan, a biocompatible polysaccharide that has been approved as a food additive was selected as a platform for the development of safe, efficient non-viral gene delivery systems to mammalian cells. Previously, chitosan-based gene formulations had been generally associated with high molecular weight chitosans, which were poorly characterised in terms of molecular weight distribution and degree of acetylation. Therefore, in order to improve the properties of chitosan-based gene formulations, the research associated with this thesis focused on establishing the structure-property relationships of well-defined, low molecular weight chitosans (chitosan oligomers) as delivery systems for nucleic acids (pDNA and siRNA) in vitro and after lung administration in vivo. pDNA dissociated more easily from chitosan oligomers than from conventional high molecular weight chitosans, resulting in a faster onset and higher levels of in vivo gene expression, comparable to those mediated by polyethyleneimine (PEI), one of the most efficient non-viral delivery systems. Coupling of a trisaccharide branch to the chitosan oligomers so as to target extracellular lectins resulted in a significant improvement in transfection efficiency because of enhanced cellular uptake and colloidal stability. In contrast to pDNA, longer linear chitosan oligomers were required to form physically-stable nanoparticles with siRNA that mediated efficient, sustained gene silencing in vitro. Finally, the use of an optimised catheter device for the nebulisation of small volumes of pDNA formulations resulted in improved dose precision and lung distribution in vivo compared with conventional intratracheal instillation. In conclusion, chitosan oligomers are interesting and viable alternatives to other non-viral gene delivery systems.

<p>Over the past decade, the development of macromolecular drugs based on peptides, proteins and nucleic acids has increased the interest in microparticulate drug delivery, i.e., the delivery of drug systems in the nanometer and micrometer ranges. However, little is known so far about the effect that microparticulate systems have on various tissues after administration. Additionally, the knowledge of mechanisms responsible for the uptake and transport of microparticles across the human intestine is incomplete and requires further investigation to improve both the safety profiles and the efficiency of these drug delivery systems.</p><p>This thesis is comprised of two parts. The first one investigates gene expression responses obtained from DNA arrays in local and distal tissues after microparticulate drug delivery. The second part focuses on the mechanisms responsible for the transport of microparticles across epithelial cells lining the intestine.</p><p>The results presented in the first part demonstrated that gene expression analysis offers a detailed picture of the tissue responses after intramuscular or pulmonary administration of microparticulate drug delivery systems compared to the traditional techniques used for such evaluations. In addition, DNA arrays provided a useful and sensitive tool for the initial characterization and evaluation of both local and distal tissue responses, making it possible to distinguish between gene expression patterns related to each studied delivery system.</p><p>The results presented in the second part demonstrated that the surface properties of the microparticle were important for the extent of transport across an <i>in vitro</i> model of the follicle-associated epithelium (FAE), comprised of intestinal epithelial cells specialized in particle transport (M cells). Another important finding was that the enteropathogen bacterium, <i>Yersinia pseudotuberculosis</i>, induced microparticle transport across the normal intestinal epithelium, represented by Caco-2 cells and excised human ileal tissue. This transport was most probably mediated by an increased capacity for macropinocytosis in the epithelial cells.</p>

Over the past decade, the development of macromolecular drugs based on peptides, proteins and nucleic acids has increased the interest in microparticulate drug delivery, i.e., the delivery of drug systems in the nanometer and micrometer ranges. However, little is known so far about the effect that microparticulate systems have on various tissues after administration. Additionally, the knowledge of mechanisms responsible for the uptake and transport of microparticles across the human intestine is incomplete and requires further investigation to improve both the safety profiles and the efficiency of these drug delivery systems. This thesis is comprised of two parts. The first one investigates gene expression responses obtained from DNA arrays in local and distal tissues after microparticulate drug delivery. The second part focuses on the mechanisms responsible for the transport of microparticles across epithelial cells lining the intestine. The results presented in the first part demonstrated that gene expression analysis offers a detailed picture of the tissue responses after intramuscular or pulmonary administration of microparticulate drug delivery systems compared to the traditional techniques used for such evaluations. In addition, DNA arrays provided a useful and sensitive tool for the initial characterization and evaluation of both local and distal tissue responses, making it possible to distinguish between gene expression patterns related to each studied delivery system. The results presented in the second part demonstrated that the surface properties of the microparticle were important for the extent of transport across an in vitro model of the follicle-associated epithelium (FAE), comprised of intestinal epithelial cells specialized in particle transport (M cells). Another important finding was that the enteropathogen bacterium, Yersinia pseudotuberculosis, induced microparticle transport across the normal intestinal epithelium, represented by Caco-2 cells and excised human ileal tissue. This transport was most probably mediated by an increased capacity for macropinocytosis in the epithelial cells.

Le microscope à force atomique (AFM) fait partie des microscopies de champ proche dites à sonde locale. De par sa versatilité, un grand nombre de domaines des nanosciences tant en physique, que chimie ou biologie utilisent cette technique. Cependant, le champ d’investigation de la microscopie AFM classique est restreint temporellement et spatialement. En effet, en raison de sa limite de vitesse d’acquisition d’image et sa limite de caractérisation des interactions en surface, des études de dynamique moléculaire ou d’éléments sub-surface ne sont pas envisageables. Nous montrons donc que la caractérisation en volume est permise en utilisant une méthode d’imagerie non destructive, la microscopie de champ proche holographique ultrasonore (SNFUH). Cette méthode développée pour étudier à l.air et en liquide, a fourni des informations localisées en profondeur avec une haute résolution spatiale, en utilisant des fréquences de résonance dans la gamme du MHz. Une calibration a été effectuée sur des échantillons de structures enterrées ou non, réalisés par lithographie e-beam. Ces échantillons ont été utilisés pour ajuster les fréquences de résonance et comprendre la formation des images en mode acoustique (profondeur investiguée et inversion de contraste). Cet outil non invasif et innovant de caractérisation a donc été développé. Il présente un énorme potentiel pour des échantillons biologiques en termes de résolution et d’information. Les microscopes AFMclassique et acoustique SNFUH sont soumis à des contraintes de temps. Pour s’en affranchir, un prototype, le microscope à force atomique haute-vitesse (HS-AFM) a été développé par l’équipe du Professeur T. Ando à l’Université de Kanazawa (Japon). Il autorise ainsi le balayage à vitesse vidéo, i.e. 25 images/s, en milieu liquide. Nous avons amélioré le prototype avec une nouvelle génération de boucle d’asservissement et augmenté la zone de caractérisation. La résolution dépend fortement du levier utilisé. De plus une qualité d’image supérieure est obtenue grâce à l’utilisation de surpointes en carbone sur ces mêmes leviers. Finalement, nous montrons des résultats obtenus avec ces deux techniques de microscopies sur différents édi.ces biologiques en milieu liquide. Ainsi, avec le microscope AFM haute-vitesse, des dynamiques biomoléculaires ont pu être visualisées (ex : structures protéine-ADN) avec une résolution nanométrique. Puis une étude des changements conformationnels intracellulaires de kératinocytes vivantes dans leur milieu physiologique a été réalisée par microscopie acoustique SNFUH et montre la dégradation du matériel biologique. L’ensemble de ces résultats ouvre un nouveau champ d’investigation dans le domaine de la biologie. / The atomic force microscope (AFM) made part of scanning near-field probe microscopy. Thanks to its versatility, many fields as physics, chemistry or biology use this technique. However, the field of investigation of the classical AFM microscope is limited temporally and spatially. Indeed, due to his scan speed limitation and surface interaction caracterisation limitation, studies of molecular dynamics and sub-surface elements are not possible. We show that the volume caracterisation is permitted using a non-destructive imaging method, called Scanning Near-Field by Ultrasound Holography (SNFUH). This tool developed for study in air and liquid has provided depth information as well as spatial resolution at the nanometer scale using resonant frequencies of about range of MHz. Calibration has been performed on samples of buried or not structures made by e-beam lithography and have been used to adjust the resonant frequency and understand the acoustic image formation (depth investigation and contrast in-version). We have developed a non-invasive and innovative tool of characterization for biology : he presents a huge potential for biological samples in terms of resolution and information. Classical AFM and acoustic SNFUH microscopes are time resolution limited. To overcome this time constraint, a prototype, High Speed Atomic Force Microscope (HS-AFM), has been developed by the team of Prof. T. Ando, Kanazawa University (Japan). It allows a scan rate at video speed, i.e. 25 frames/s, in liquid medium. We have improved the prototype, through a new generation of feedback control and increased the scan area. The resolution depends strongly of the probe used. Moreover a better image quality is obtained through the use of carbon tips on these cantilevers. Finally, we show our results obtained with these two microscopy techniques about biological buildings in liquid environment. Thereby, with the HS-AFM microscope, biomolecular dynamics have been visualized (e.g. protein-DNA structures) with nanometric resolution. Then a study about intracellular conformational changes of keratinocytes living cells in their physiological medium has been realized by acoustic microscopy SNFUH and show deterioration of biological components. All of these results provide new insights in biology field.