Thesis (Ph.D. in Analytic Health Sciences) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 57-60). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 133-142). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis--University of Oklahoma. / Includes bibliographical references.

Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 98-112). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

München, Techn. Univ., Diss., 2004.

Universiẗat, Diss., 2004--Bielefeld. / Erscheinungsjahr an der Haupttitelstelle: 2003.

Data de aprova??o ausente. / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-06-08T22:19:37Z No. of bitstreams: 2 luana_martins_santos.pdf: 1170564 bytes, checksum: 215e11cc17c654f8760cc6085cca3464 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-06-22T15:18:34Z (GMT) No. of bitstreams: 2 luana_martins_santos.pdf: 1170564 bytes, checksum: 215e11cc17c654f8760cc6085cca3464 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-06-22T15:18:34Z (GMT). No. of bitstreams: 2 luana_martins_santos.pdf: 1170564 bytes, checksum: 215e11cc17c654f8760cc6085cca3464 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A produ??o de muda ? uma etapa primordial e decisiva para a implanta??o de uma floresta. No entanto, devido a diversos fatores de ordem t?cnica, as perdas no setor de produ??o s?o bastante relevantes. Dentre estes fatores est? o descarte do substrato, que quando feito de forma err?nea favorece a prolifera??o de micro-organismos fitopatog?nicos em viveiro. Isso pode desencadear em perdas significativas e ainda se tornar um passivo ambiental. Raramente o substrato ? reutilizado nos viveiros comerciais, uma vez que presume-se que as caracter?sticas f?sica, qu?mica e biol?gicas formam perdidas durante a produ??o das mudas. Contudo, h? poucos estudos sobre micro-organismos em substratos. A import?ncia deste estudo fundamenta-se na escassez de pesquisa sobre essa problem?tica que afeta o setor de produ??o de mudas, bem como buscar alternativas sustent?veis que pressup?e a demanda de uso desse insumo. A aplica??o de t?cnicas moleculares vem sendo empregadas para detectar e identificar os micro-organismos em diversos ambientes. Neste contexto, o objetivo deste estudo foi detectar a diversidade microbiol?gica em amostras de substratos descartados pela t?cnica de PCR-RFLP. Foram coletados dez amostras de substratos em diferentes viveiros no estado de Minas Gerais em diferentes fases de produ??o e estado de tecnifica??o. O DNA gen?mico total foi extra?do e amplificado pela t?cnica de PCR com oligonucleot?deos espec?ficos para fungos, bact?ria e archaea. Os produtos amplificados foram submetidos ? clivagem com enzimas de restri??o HaeIII, BamHI, TaqI e HindIII, para detec??o de poss?veis polimorfismos entre as amostras por meio da t?cnica de PCR-RFLP. Foi poss?vel verificar diferen?as entre as amostras de substratos, tanto em rela??o ao tamanho da regi?o do DNA amplificada, bem como em rela??o ? presen?a de s?tios de restri??o. Com base no ?ndice de similaridade, detectou-se uma maior varia??o da diversidade dentro da amostra em diferentes fases do que nas amostras entre os diferentes viveiros. Portanto, estes resultados podem ser relevantes para o conhecimento da diversidade microbiol?gica em substrato. Por?m mais estudos se fazem necess?rios para maior compreens?o destes micro-organismos e sua fun??o no substrato. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Ci?ncia Florestal, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / The production of changes is a crucial and decisive step for the implementation of a forest. However, due to several technical factors, losses in the production sector are quite relevant. Among these factors is the disposal of substrate, which when done wrongly favors the proliferation of phytopathogenic microorganisms in nursery. This can trigger significant losses and still become a environmental liabilities. Rarely the substrate is reused in commercial nurseries, since it is assumed that the physical, chemical and biological characteristics were lost during the production of seedlings. However, there are few studies on microorganisms on substrates. The importance of this study is based on the scarcity of research on this issue that affects the production of seedlings, as well as seek sustainable alternatives that assumes the use of this raw material demand. The application of molecular techniques have been employed to detect and identify microorganisms in various environments. In this context, the objective of this study was to detect microbiological diversity in substrate samples dropped by PCR-RFLP technique. Ten samples were collected of substrates in different nurseries in the State of Minas Gerais in different stages of production and State of modern farms. Total genomic DNA was extracted and amplified by the PCR technique with specific oligonucleotides to fungi, bacteria and archaea. The amplified products were submitted to cleavage with HaeIII restriction enzymes, BamHI, HindIII, and TaqI for detection of possible polymorphisms between the samples by PCR-RFLP technique. It was possible to check differences between samples of substrates, both in relation to the size of the amplified DNA region as well as in relation to the presence of restriction sites. Based on the index of similarity, if a greater variation of diversity within the sample at different stages than in samples between different nurseries. Therefore, these results may be relevant to the knowledge of microbiological diversity in substrate. However more studies are needed to better understanding of these microorganisms and their role in the substrate.

No presente trabalho foi investigado o efeito do oligonucleotídeo-modelo 5-AAAAAAAAAA-3(ODN) sobre a estabilidade e estrutura de vesículas catiônicas de brometo de dioctadecildimetilamônio (DODAB), extrusadas através filtros de 100 nm, em dispersão aquosa, com as técnicas de espalhamento de luz dinâmico (DLS), medidas de potencial de superfície (potencial zeta), calorimetria diferencial de varredura (DSC), espalhamento de raios-X a baixos ângulos (SAXS), espectroscopias de absorção óptica e de fluorescência do estado estacionário da sonda Laurdan incorporada a vesículas de DODAB-ODN. Este fluoróforo monitora a polaridade e estrutura da superfície da membrana de DODAB. Variando a concentração de ODN, três diferentes regimes foram observados. Para baixas concentrações de ODN, ([ODN]/[DODAB]) 0.025 mM, a dispersão é estável, límpida, apesar do diâmetro médio das vesículas aumentar, um aumento de turbidez ser observado por medidas de Absorbância, e o SAXS já acusar a presença de algumas poucas multilamelas. As vesículas mistas apresentam potencial de superfície positivo, semelhante ao potencial medido para vesículas de puro DODAB. A calorimetria mostra a coexistência de regiões da bicamada de puro DODAB, e regiões mistas, com DODAB-ODN, sendo estas últimas mais estáveis, apresentando maior temperatura de transição gel-fluido. A forma e posição da banda de fluorescência do Laurdan incorporado às vesículas não são alteradas pela presença do oligonucleotídeo, indicando pouca variação na polaridade e estrutura da superfície da membrana mista monitorada pela sonda. Um segundo regime é observado para ([ODN]/[DODAB]) 0.05 mM, onde não é mais observado por calorimetria a presença significativa de domínios de puro DODAB, e a dispersão mostra-se instável, turva, com agregação/fusão das vesículas. Finalmente, o terceiro regime, para altas concentrações de ODN, ([ODN]/[DODAB]) 0.075 mM, onde é observado um potencial de superfície negativo, portanto, com predominância da carga do oligonucleotídeo, e a dispersão volta a ser estável, apresentando baixa turbidez. Neste regime, a calorimetria indica uma grande estabilidade da fase gel, medidas de SAXS mostram a formação de estruturas multilamelares, porém DLS indica a presença de vesículas pequenas, com dimensões às observadas para DODAB puro. Neste regime, a sonda Laurdan monitora variações na superfície da membrana, possivelmente indicando a diminuição da quantidade de moléculas de água na superfície e/ou um enrijecimento da bicamada. Os estudos aqui apresentados fazem parte de um amplo esforço para entender as características estruturais de agregados lipídio-material genético, com o objetivo de seu uso futuro em terapias gênicas. / In the present work the effect of 5\'-AAAAAAAAAA-3 \'oligonucleotide model (ODN) was investigated on the stability and structure of dioctadecyldimethylammonium bromide (DODAB) cationic vesicles, extruded through 100 nm filters in aqueous dispersion, with dynamic scattering techniques (DLS), surface potential measurements (zeta potential), differential scanning calorimetry (DSC), small angle X-ray scattering (SAXS), optical absorption spectrometry and stationary-state fluorescence spectroscopy of Laurdan incorporated into DODAB-ODN vesicles. This fluorophore monitors the polarity and surface structure of the DODAB membrane. Varying the ODN concentration, three different behaviors were observed. For low concentrations of ODN, ([ODN] / [DODAB]) 0.025 mM, the dispersion is stable, clear, although the mean diameter of the vesicles increases, an increase in turbidity is observed by Absorbance measurements, and SAXS already shows the presence of a few multilamellar structure. The mixed vesicles present positive surface potential, similar to the potential measured for pure DODAB vesicles. Calorimetry shows the coexistence of regions of the pure DODAB bilayer, and mixed regions, with DODAB-ODN, the latter being more stable, presenting a higher gel-fluid transition temperature. The shape and position of the Laurdan fluorescence band incorporated into the vesicles are not altered by the presence of the oligonucleotide, indicating minor variation in the polarity and surface structure of the mixed membrane monitored by the probe. A second behavior is observed for ([ODN] / [DODAB]) 0.05 mM, where the presence of pure DODAB domains is no longer detected by calorimetry, and the dispersion is unstable, cloudy, displaying vesicle aggregation/fusion. Finally, the third behavior is detected at high concentrations of ODN, ([ODN] / [DODAB]) 0.075 mM, where a negative surface potential is observed, therefore, with predominance of the charge of the oligonucleotide, and the dispersion is stable, exibiting low turbidity. In this regime, the calorimetry indicates a great stability of the gel phase, SAXS measurements show the formation of multilamellar structures, however DLS indicates the presence of small vesicles, with dimensions to those observed for pure DODAB. In this region, the Laurdan probe monitors variations at the surface of the membrane, possibly indicating the decrease in the amount of water molecules on the surface and/or a stiffening of the bilayer. The studies presented here are part of a broad effort to understand the structural characteristics of \"lipid-genetic material\", aggregates, with the aim of their future use in gene therapies.

L’ARN est sans aucun doute la molécule biologique la plus versatile qui soit. Tout comme l’ADN, il peut contenir et transmettre de l’information génétique. Tout comme les protéines, il peut accomplir une multitude de fonctions biologiques. De plus, son rôle le plus connu demeure celui d’intermédiaire entre l’ADN et les protéines. L’ARN est donc au cœur d’un bon nombre de processus biologiques. Ceci lui confère un immense potentiel thérapeutique qui jusqu’à présent demeure largement inexploité. Pour accomplir ses fonctions, l’ARN doit adopter une structure tridimensionnelle précise qui est dépendante à la fois de sa séquence et de son environnement. Ainsi, en modifiant la structure d’un ARN, il est possible d’en moduler sa fonction. C’est l’objectif global des travaux présentés dans cette thèse. Pour y parvenir, de courts oligonucléotides antisens (OA) ont été utilisés. Cette stratégie revêt plusieurs avantages. Comme les OA s’apparient à leur cible en formant des paires de bases Watson-Crick, ils offrent une grande spécificité et leur design est facile. De plus, en se fiant aux données structurales et aux logiciels de prédictions de structures des ARN, on peut aisément identifier les régions à cibler avec les OA. Enfin, cette technique est versatile puisqu’on peut cibler différents motifs d’ARN. La première cible a été le ribozyme du virus de l’hépatite D. Cet ARN, qui catalyse une réaction d’auto-coupure, a été modifié afin que son activité devienne dépendante à la liaison d’OA. Plusieurs modules ont ainsi été créés et combinés afin d’obtenir des ribozymes qui répondaient à la présence d’un ou plusieurs OA. En insérant ces interrupteurs moléculaires dans les régions non traduites d’un ARNm, nous avons ainsi modulé l’expression de ce gène avec les OA. Cet outil a des applications intéressantes pour la régulation de gènes en biologie synthétique. Un autre motif ciblé a été le G-quadruplex (G4). Cette structure non canonique exerce de nombreuses fonctions biologiques et représente donc une cible thérapeutique intéressante. Lorsque présent dans la région 5’ non traduite d’un ARNm, le G4 mène généralement à une diminution de la traduction. En utilisant des OA qui empêchent la formation du G4, nous avons été en mesure d’augmenter la traduction du gène ciblé. De plus, il a été possible de développer des OA qui favorisent la formation d’un G4 dans le but de diminuer l’expression de la cible. Finalement, dans le dernier chapitre de cette thèse, il est démontré que les G4 présents dans les microARN primaires influencent leur maturation en microARN matures. Des OA ciblant ces G4 ont été utilisés afin de favoriser la maturation de microARN suppresseurs de tumeurs, ce qui présente un potentiel thérapeutique intéressant. En bref, les travaux présentés dans cette thèse démontrent clairement que les OA sont un outil de choix pour cibler et modifier la structure de motifs d’ARN spécifiques. / Abstract : RNA is a versatile biological molecule. Like DNA, it can contain and transmit genetic information. Like proteins, it can accomplish multiple biological functions. Also, its most known role remains that of intermediary between DNA and proteins. RNA is thus a key player in many biological processes. This gives it an immense therapeutic potential which remains largely untapped. To fulfill its functions, RNA must adopt a precise threedimensional structure that is dependent on both its sequence and its environment. Thus, by modifying the structure of an RNA, it is possible to modulate its function. This is the overall objective of the work presented in this thesis. To achieve this, small antisense oligonucleotides (ASO) have been used. This strategy has several advantages. As ASO bind their target with Watson-Crick base pairs, they offer great specificity and their design is easy. Moreover, reliance on structural data and RNA structure prediction softwares makes it easy to identify the regions to be targeted with ASO. Finally, this technique is versatile since it is possible to target different RNA motifs. The first target was the HDV self-cleaving motif. This RNA, which catalyzes a self-cleaving reaction, has been modified so that its activity became dependent on the binding of ASO. Several modules were thus created and combined in order to obtain ribozymes which responded to the presence of one or more ASO. By inserting these molecular switches into an mRNA’s UTR, the expression of this gene was modulated with the ASO. This has interesting applications for the regulation of genes in synthetic biology. Another target motif was the G-quadruplex (G4). This non-canonical structure exerts many biological functions and therefore represents an interesting therapeutic target. When present in the mRNA’s 5’UTR, G4 generally lead to a decrease in translation. Using ASO that prevent G4 formation, we were able to increase the translation of the target gene. In addition, it has been possible to develop ASO which promote the formation of a G4 in order to decrease the expression of the target. Finally, in the last chapter of this thesis, it is demonstrated that the G4 present in the primary microRNAs influence their maturation in mature microRNAs. ASO targeting these G4 have been used in order to promote the maturation of tumor suppressor microRNAs, which has an interesting therapeutic potential. The work presented in this thesis clearly demonstrates that ASO are ideal for targeting and altering the structure of specific RNA motifs.

Selon les estimations de l'OMS, le bilan mondial du paludisme a atteint 212 millions de cas et 429 000 décèsen 2015 (OMS, 2016). Cette gravité est principalement due à Plasmodium falciparum. A l’heure actuelle, P. falciparum présente des résistances à tous les antipaludiques donnés en monothérapie.Par conséquent, pour réduire le risque d’échec thérapeutique, l'OMS a recommandé depuis 2001 l’utilisation de bithérapie, notamment d'Artemisinin Combination Therapy (ACT), comme traitement de première intention.Les ACT sont composés essentiellement d’un dérivé d’artéminisine, à demi-vie courte et un autre antipaludique à demi-vie longue, connu en monothérapie.Le parasite a également montré des signes de résistance aux ACT, principalement en Asie du Sud-est, menaçant les programmes d’éradications contre le paludisme.La découverte de nouveaux composés à activité antipaludique ou de nouvelles procédures de traitement sont urgentes.La valorisation d’anciennes molécules est également au cœur des études afin d’améliorer notamment leur biodisponibilité et réverser les mécanismes de résistance du parasite. Ainsi, des études prouvent l’intérêt de l’utilisation de nanotechnologies pour l’amélioration de l’efficacité d’antipaludiques. L’atovaquone en est un exemple, cette modification a notamment permis d’améliorer sa biodisponibilité. Notre étude a porté sur une de ces formulations, de l’atovaquone encapsulée dans une nanoémulsion cationique (NE) appelée ATQ. Une deuxième génération a ensuite été testée par association d’oligonucléotides antisens anti-ARNm de topoisomérase II(AST) de P. falciparum. En effet, des stratégies antisens thérapeutiques font leur preuve en santé humaine et présentent un intérêt croissant en parasitologie. Les NE/AST ont montré une activité anti-palustre spécifique contre P. falciparum in vitro. Leur spécificité a permis d’aboutir à l’arrêt du cycle cellulaire et une forte diminution du taux d’ARNm de la topoisomérase II. Ce phénomène a montré être dépendant de l’action de la RNase H. Un effet synergique de ces NE/AST a également été montré en association avec la chloroquine, l’atovaquone et la dihydroartémisinine sur une souche sensible de P. falciparum et des souches résistantes aux antipaludiques précédemment cités.L’ATQ a également montré une forte efficacité sur une souche résistante à l’atovaquone d’un facteur 5. En présence d’ATQ, la mitochondrie a rapidement été altérée conduisant à une mort précoce du parasite. Un traitement à l’ATQ a abouti à la guérison de souris Swiss infectée par P. berghei après deux injections en i.v. en 5 jours. Enfin, l’ATQ/AST a montré une efficacité in vitro contre P. falciparum et P. berghei in vivo. Un test de cytoadhérance des hématies parasitées a des cellules endothéliales a révélé un fort pourvoir d’inhibition de la cytoadhérance de l’ATQ/AST.Un résultat prometteur dans le cadre de traitement du neuropaludisme. / According to the estimations of theWHO, in 2015, 212million cases ofmalariahave been reported(WHO,2016). These figuresmakemalariathe most deadlyparasitic diseasein the world, with429.000deaths per year. Some treatments against Plasmodium falciparum exist. However, no really good treatment option can be found in monotherapy due to the resistance emergency. Therefore To reduce the risk of resistance, WHO has recommended since 2001 combination therapies, which is basically an Artemisinin Combined Therapy (ACT), as first-line treatment. The main problem of commercialized bi-therapy is that they are composed of two molecules with individual resistance which leaded to the emergence of resistance to the latest ACTs such as a dihydroartemisinin /piperaquine combinationmainly in South-East Asia.Thus the use of new therapeutic combination strategy that can bypass the parasites' mechanisms of resistance is urgent to effectively treat malaria. As the pathway from drug discovery to drug commercialization is both long and very expensive, it is essential to develop ways to improve existing antimalarial treatments. In the first place it’s necessary to find a new antimalarial formulation based on an already commercialized drug to modify its biodisponibility and its mechanism of action in order to revert the resistance. In the second place its necessary to associate this formulation with a novel none commercialized antimalarial strategy such as the antisens oligonucleotides already usedinhumanhealth. In our lab we have developed nanoemulsions containing atovaquone and antisense oligonucleotides anti topoisomerase II against P. falciparum.Nanoemulsionsvectoringantisens oligonucleotidesandused againstP. falciparum topoisomerase II(NE/AST) showed encouraging anti-parasite killing results.Additionalresultshave showna synergistic in vitro effectwithantimalarial drugs(chloroquine, dihydroartemisinin and atovaquone) in sensitive and resistances strains. Moreover NE/ASTrestricted Topoisomerase II gene expression and blocked the cell cycle in G2/M phase leading to parasite’s death by mitophagy.As Drug delivery systemscan improve the efficacy ofcommon antimalarial drugs by delivering the drug to its target, while protecting it from degradation in biological environment and increasing its biodisponibility, our nanoemulsions containing atovaquone (ATQ) leaded to reversion of atovaquone resistance with 5 fold decrease in its IC50. Observations made with confocal microscopy have shown mitochondrial alteration after ATQ treatment.Our novel and original bi-therapy is focused on the association ofATQ with NE/AST (ATQ/AST).We obtained an IC50 8-fold lower than atovaquone’s IC50with total inhibition of parasites’ capacity to reinfect new red blood cells. A cytoadherence test of parasitized erythrocytes to endothelial cells revealed a strong capacity of cytoadherence inhibition of ATQ / AST, a promising result in the treatment of cerebral malaria.