Os camarões do gênero Atya Leach, 1816 são os maiores camarões da família Atyidae, sendo que as 13 espécies reconhecidas estão distribuídas em rios e riachos das regiões tropicais e subtropicais da América (vertentes atlântica e pacífica) e oeste da África. O primeiro relato de uma Atya ocorreu no séc. XVII e, desde então, novas espécies foram descritas e descrições prévias revisadas, produzindo um histórico de instabilidade e reclassificações. Embora ao longo do séc. XX revisões taxonômicas tenham estabilizado a sistemática do gênero, a variabilidade morfológica e distribuição geográfica trans-ístmica da espécie A. innocous gerou questionamentos. Além disso, mais recentemente trabalhos de filogenia molecular da família Atyidae que incluíram representantes de Atya suscitaram questões em relação à sistemática do gênero (possível não monofilia) e de algumas espécies como A. gabonensis, A. margaritacea e A. scabra. Visto que o uso de marcadores moleculares nunca foi empregado para a delimitação das espécies de Atya e que seu uso de forma complementar à morfologia poderia aperfeiçoar a sistemática do gênero, o objetivo do presente estudo foi avaliar por meio de dados moleculares as hipóteses taxonômicas das espécies A. gabonensis, A. innocous, A. margaritacea e A. scabra. Sequências dos genes mitocondriais 16S e Citocromo Oxidase I e gene nuclear Histona 3 foram geradas por meio de protocolos de extração e sequenciamento de DNA a partir do tecido de espécimes obtidos em empréstimos/doações. Potenciais espécies evidenciadas pelas análises de similaridade nucleotídicas (distâncias genéticas), compartilhamento de caracteres em um contexto evolutivo (reconstruções filogenéticas), Automatic Barcode Gap Discovery, Poisson Tree Processes e Generalized Mixed Yule Coalescence foram confrontadas com as hipóteses taxonômicas específicas atuais. A avaliação sistemática com dados moleculares aqui realizada, adicionalmente às informações morfológicas existentes na literatura sustentaram A. gabonensis como uma espécie de distribuição anfi-atlântica, mas não corroborou a hipótese de A. innocous como uma espécie trans-ístmica. Assim, o uso do nome A. innocous para as populações do Mar do Caribe e A. tenella para aquelas restritas ao Pacífico é sugerido. A espécie A. margaritacea, distribuída ao longo da costa pacífica da América foi considerada uma espécie válida e distinta de A. scabra, amplamente distribuída na vertente atlântica da América do Sul, África e Mar do Caribe. Contudo, é discutida a possibilidade de uma espécie críptica restrita no Golfo do México existir. Adicionalmente, o conhecimento existente e pertinente para futuros estudos de sistemática e taxonomia sobre os camarões do gênero Atya foram sumarizados e são apresentados. / The genus Atya Leach, 1816 shrimps are the largest of the Atyidae family, and the 13 acknowledge species are geographically distributed in rivers and stream in the tropical and subtropical regions of America (Atlantic and Pacific drainages) and West Africa. The first registry of an Atya was in the XVII century and since then new species were described and previous description revised in an eventful taxonomic historic. Although throughout the XX century taxonomic revisions stabilized the genus systematics, the morphological variability and the trans-isthmic geographic distribution of A. innocous caused questioning. Moreover, molecular phylogenetic studies that included Atya representatives raised doubt on the genus systematics (possibly non-monophyletism) and some species A. gabonensis A. margaritacea and A. scabra hypothesis. As molecular markers have never been used concerning Atya species delimitation complementary to the morphology and it could improve the genus systematics, the goal of this study was to evaluate with molecular markers the taxonomic hypothesis of the species A. gabonensis, A. innocous, A. margaritacea e A. scabra. Sequences of the mitochondrial genes 16S and Cytochrome Oxidase I and nuclear gene Histone 3 were generated by means of DNA extraction and sequence protocols from specimens obtained in loans/donations. Putative species evidenced by the analysis of nucleotide similarity (genetic distances), character sharing (phylogenetic reconstitutions), Automatic Barcode Gap Discovery, Poisson Tree Processes and Generalized Mixed Yule Coalescence were compared to the prevailing taxonomic hypothesis. The systematic evaluation with the molecular data of this study, in addition with the morphological information in the literature sustain A. gabonensis as an amphi-atlantic distributed species, but do not corroborated A. innocous hypothesis as an trans-isthmian species. In this sense, the use of A. innocous stricto sensu for the Caribbeans Sea populations and A. tenella to that restricted to the pacific drainage of America is suggested. Atya margaritacea, distributed along the pacific drainage of America, is considered a valid species distinct from A. scabra, widespread distributed in the Atlantic drainage of America and Africa, besides Caribbean Sea. However, the possibility of a cryptic species in the Gulf of Mexico population is discussed. Aditionally, the relevant knowledge to future systematic and taxonomy studies about the shrimps of the genus Atya were summarized and are shown.

O presente estudo avaliou a capacidade de invasão de hepatócitos de rato por Shigella flexneri (S. flexneri) nas condições de normóxia e hipóxia. O estudo do microambiente de hipóxia tem grande importância, por estar presente em muitas doenças hepáticas, além de aumentar a translocação quando presente no lúmen intestinal. Bactérias invasivas como S. flexneri podem romper a barreira intestinal e chegar ao fígado através da circulação portal. O efeito da invasão bacteriana das células hepáticas é pouco conhecido. Neste trabalho buscamos pesquisar as alterações morfológicas e funcionais de hepatócitos de rato após infecção por S. flexneri na presença e na ausência de hipóxia. Para esta finalidade foram utilizados hepatócitos de rato cultivados pela técnica de cultura primária. Vários parâmetros foram analisados, tais como: taxa de invasão celular pela bactéria, quantificação da produção e liberação de DHL, produção de TNF-, taxa de morte celular por apoptose e a expressão do fator de transcrição HIF-1a. Os resultados mostraram que a metodologia empregada para a obtenção do microambiente hipóxico foi satisfatória, com redução de 70% da pO2 inicial (atingindo 43.2 mmHg in vitro ou 6.5% O2). A invasão de hepatócitos de rato por S. flexneri foi menor nas células previamente expostas à hipóxia quando comparada com a invasão das células cultivadas em normóxia. A viabilidade dos hepatócitos não apresentou diferenças significativas entre os grupos experimentais, variando entre 74 e 86%. A liberação de TNF- nas situações de normóxia e hipóxia foi similar, embora as células infectadas em normóxia tenham aumentado a liberação desta citocina. Na condição de hipóxia + infecção a liberação de TNF- foi menor do que na condição de normóxia + infecção, porém ambos os grupos produziram aumento significativo da citocina em relação aos controles normóxicos e hipóxicos. Este resultado sugere que a presença da bactéria no interior das células aumenta significativamente a liberação de TNF-pelos hepatócitos. A produção de DHL também foi maior de forma significativa no grupo hipóxico em relação ao grupo normóxico, porém não apresentou alteração nos grupos infectados por S. flexneri após uma hora. As taxas de apoptose aumentaram nos grupos hipóxia e nos grupos infectados com S. flexneri de maneira similar, variando entre 24 e 31%, quando comparados aos grupos controle em normóxia. A expressão do fator de transcrição HIF ocorreu nos grupos: hipóxia, normóxia + infecção e hipóxia + infecção, evidenciando que a infecção por S. flexneri induz a expressão deste fator. Em seu conjunto, nossos resultados buscam contribuir para o maior conhecimento da interação entre S. flexneri e hepatócitos em condição de hipóxia e normóxia. Tal conhecimento poderá ser útil na construção de futuras estratégias para auxiliar no combate a esta importante bactéria invasiva, principalmente nos casos de septicemia / This study evaluated the invasiveness of rat hepatocytes by Shigella flexneri (S. flexneri) in normoxia and hypoxia conditions. The study of hypoxia microenvironment is of great importance, since hypoxia is present in many liver diseases and increases bacterial translocation when present in intestinal lumen. Invasive bacteria such as S. flexneri can disrupt the intestinal barrier and reach the liver through portal circulation. The effect of bacterial invasion in liver cells is poorly understood. In this study we investigated the morphological and functional changes of rat hepatocytes after infection with S. flexneri in the presence and absence of hypoxia. For this purpose we used primary cultures of rat hepatocytes. Several parameters were analyzed, such as: bacterial invasion cell rate, quantification of LDH production and release, TNF-a production, cell death rate by apoptosis and expression of the transcription factor HIF-1a. The results showed that the methodology used to obtain the hypoxic microenvironment was satisfactory, with 70% reduction of initial pO2 (to 43.2 mmHg in vitro or 6.5% O2). The invasion of rat hepatocytes by S. flexneri was lower in cells previously exposed to hypoxia compared with the invasion of cells grown in normoxia. The viability of hepatocytes showed no significant differences between experimental groups, ranging between 74% and 86%. The release of TNF-a in situations of normoxia and hypoxia was similar, although the infected cells in normoxia have increased the release levels of this cytokine. In hypoxia + infection condition the release of TNF-a was lower than normoxia + infection condition, but both groups produced a significant increase in cytokine release when compared to normoxic and hypoxic controls. This result suggests that the presence of bacteria inside the cells significantly increases the release of TNF-a by hepatocytes. DHL production was also significantly greater in the hypoxic group compared to the normoxic group, but had no change in the groups infected with S. flexneri after an hour. The apoptosis rates increased in hypoxia and infected groups in a similar way, varying between 24% and 31% when compared with control group in normoxia. The expression of HIF- 1a transcription factor occurred in hypoxia, normoxia + infection and hypoxia + infection groups, indicating that infection with S. flexneri induces the expression of this factor. Overall, our results sought to contribute to a greater understanding of the interaction between S. flexneri and hepatocytes under hypoxia and normoxia conditions. Such knowledge may be useful in building future strategies to assist in combating these major invasive bacteria

La biogenèse du ribosome est un processus complexe et dynamique qui nécessite de nombreuses étapes de maturation et de modification des ARNr ainsi que l’assemblage et le transport des RNPs précurseurs. Un ribosome mature contient une centaine de pièces, ARN et protéines confondus, mais son assemblage requiert l’intervention de plus de 400 facteurs de synthèse. De part le coût énergétique important de ce processus, plusieurs voies de régulation interviennent pour contrôler la biogenèse des ribosomes en fonction des conditions nutritives. L’une des voies les plus connue est la voie TOR (Target of rapamycin). Cette voie de régulation agît principalement au niveau de la transcription des différents intervenants de la biogenèse :les ARNr, les protéines ribosomiques mais aussi les facteurs de synthèse. Ces facteurs, ayant une action transitoire dans la maturation des ribosomes, sont, par économie, recyclés pour la synthèse de nouveaux ribosomes. Nous nous sommes donc intéressés au devenir de ces facteurs, plus particulièrement de ceux intervenants dans la biogenèse de la petite sous unité, lorsque les conditions environnementales sont inadaptées à la croissance cellulaire. Ainsi, nous avons pu montré, pour quatre facteurs particuliers :Dim2, Rrp12, Hrr25 et Fap7, que leur localisation est dépendante de la synthèse ribosomique. Ainsi, lors de carence en sources nutritives, l’inhibition de la synthèse et de l’activité ribosomique entraîne un confinement de ces facteurs ribosomiques dans le nucléole ou dans des corps cytoplasmiques. En outre, la localisation particulière des facteurs ribosomiques Hrr25 et Fap7 dans les P-bodies en phase de croissance saturée laisse penser que ces corps cytoplasmiques sont le lieu de dégradation des pré-ribosomes lorsque les carences nutritives perdurent. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Abstract Heterotopic ossification is a pathological condition in which bone forms outside the skeletal system. It can also occur in skin, which is the case in some genetic disorders. In multiple miliary osteoma cutis (MMOC), tiny bone fragments develop in the dermis and nearby subcutaneous tissue of the face and upper chest region during middle age. The etiology of the disease is poorly understood. The origin of the osteoma-forming cells is not known and also unknown are the signaling factors that direct the skin cells towards an osteogenic lineage. The purpose of this study was to investigate MMOC and the pathogenesis of ectopic bone formation by combining patient study and cell biology methods. The results from an extensive review of the literature and five new cases revealed MMOC as a distinct disease entity, where heterotopic bone formation is intramembranous. No correlation was found between MMOC and acne scars, hormonal disturbances or GNAS gene mutations. In cell culture studies mouse and human dermal fibroblasts and mouse dermal papilla (DP) cells were found to differentiate into osteoblast-like matrix mineralizing cells. The bone morphogenetic protein 4 (BMP-4) homodimer and BMP-2/7 heterodimer had significant effects on the osteogenic differentiation of the above mentioned cells. Interestingly, the BMPs enhanced the differentiation of mouse cells but reduced it in human cells. In mouse DP cells and human fibroblasts BMP-2/7 was more potent than BMP-4. The skin area affected by osteomas in patients was compared to their unaffected skin and also to the corresponding skin areas in controls with regard to osteogenic differentiation and gene expression studies. MMOC patients’ skin differs from controls both in osteoma and unaffected skin areas, which suggests MMOC is not only a local but also a systemic skin disease. The results confirm the previous findings that gene expression in skin is different in different parts of the body, which could explain why the osteomas develop in certain skin areas. The results of this study provide new information about MMOC and heterotopic ossification in skin and could be useful when developing treatments for MMOC. This study also presents new information about BMPs and their different effects in mouse and human cells, which may stimulate discussion about the generalization of mouse studies in humans and the clinical use of BMPs. / Tiivistelmä Virhesijaintinen luutuminen on patologinen tila, jossa luuta muodostuu luisen tukirangan ulkopuolelle. Tätä voi tapahtua myös ihossa, kuten käy tietyissä sairauksissa. Ihon lukuisat jyvämäiset osteoomat on tauti, jossa pieniä luujyväsiä ilmaantuu verinahkaan ja ihonalaiskudokseen keski-iässä. Taudin syytä, osteoomia muodostavien solujen alkuperää tai sitä, mitkä viestinvälittäjät saavat esiastesolut siirtymään luusolulinjalle, ei tiedetä. Tässä työssä tutkittiin ihon lukuisia jyvämäisiä osteoomia ja virhesijaintista luutumista yhdistämällä kliinisiä ja solubiologisia menetelmiä. Laajasta kirjallisuuteen perehtymisestä ja viidestä omasta potilaasta saadut tulokset osoittivat ihon lukuisten jyvämäisten osteoomien olevan oma erillinen tautinsa, jossa virhesijaintinen luutuminen tapahtuu suoran luutumisen mekanismilla. Tauti ei näytä olevan yhteydessä aknearpiin, hormonihäiriöihin tai GNAS-geenin mutaatioihin. Soluviljelykokeissa hiiren ja ihmisen verinahan fibroblastien ja hiiren karvatupen nystyn solujen havaittiin erilaistuvan osteoblastityyppisiksi soluväliainetta mineralisoiviksi soluiksi. Luun morfogeneettisillä proteiineilla (BMP) 4 ja 2/7 oli merkitsevä vaikutus yllä mainittujen solujen erilaistumisessa. Yllättävää kyllä, ne edistivät hiiren solujen, mutta vähensivät ihmisen solujen erilaistumista. Hiiren karvatupen soluille ja ihmisen fibroblasteille BMP-2/7 oli tehokkaampi kuin BMP-4. Potilaiden osteoma-aluetta verrattiin heidän terveeseen ihoalueeseensa samoin kuin vastaaviin ihoalueisiin kontrollihenkilöillä käyttäen menetelminä solujen erilaistamista luuta muodostavaan suuntaan sekä geenien ilmentymisen tutkimista. Potilaiden iho erosi kontrollien ihosta sekä osteooma-alueella että terveellä ihoalueella, mikä viittaa taudin olevan koko elimistöön vaikuttava. Tulokset vahvistavat aikaisempia löydöksiä siitä, että geenien ilmentyminen ihossa on erilaista eri puolilla kehoa. Tämä voisi selittää osteoomien esiintymisen vain tietyllä alueella. Tämän tutkimuksen tulokset antavat uutta tietoa ihon lukuisista jyvämäisistä osteoomista ja virhesijaintisesta luutumisesta ja saattavat olla hyödyksi kehitettäessä taudin hoitoa. Tutkimus antaa uutta tietoa luun morfogeneettisten proteiinien erilaisesta käyttäytymisestä hiirellä ja ihmisellä, mikä herättänee keskustelua hiirikokeiden yleistämisestä ihmiseen ja luun morfogeneettisten proteiinien kliinisestä käytöstä.

Les canaux ioniques sont des complexes macromoléculaires clés exprimés dans tous les types de cellules et sont impliqués dans divers processus physiologiques, y compris la génération et la propagation de potentiels d'action. Des canaux défectueux conduisent à des maladies graves, notamment l'épilepsie, des arythmies et des syndromes douloureux, ce qui en fait une cible potentielle intéressante pour le développement de médicaments. Pour améliorer notre compréhension de ces assemblages biologiques et éventuellement trouver des traitements spécifiques pour les canalopathies, il est crucial d'étudier la structure et la fonction des canaux ioniques. L'objectif principal de cette thèse a été d'étudier ce type de détails structurels et fonctionnels pour trois canaux ioniques associés aux domaines des capteurs de douleur et des canaux potassiques voltage-dépendants en utilisant des techniques de fluorescence et d'électrophysiologie. Dans le premier projet, nous avons étudié la stœchiométrie des canaux hétéromères Kv2.1 / 6.4 (chapitre trois). La technique du décompte de sous-unités isolées (single subunit counting :ssc) permet de compter les sous-unités marquées par fluorescence d’un complexe isolé en déterminant le nombre d'événements de photoblanchiment, qui apparaissent en sauts irréversibles vers le bas sur les traces de fluorescence. Pour désigner la stœchiométrie la plus probable, nous avons utilisé des calculs de probabilités pondérées et avons constaté que les canaux Kv2.1 / 6.4 s'expriment dans un arrangement 2 : 2. Plus précisément, les études fonctionnelles des canaux concatémériques montrent que les sous-unités Kv6.4 et 2.1 doivent être disposées de manière alternée. Le deuxième projet était également basé sur des expériences de SSC et visait à déterminer l'état oligomérique du nouveau canal ionique TACAN (chapitre quatre). Nous avons trouvé une portion significative de canaux intracellulaires, ce qui a provoqué une fluorescence de fond dans les expériences de SSC traditionnelles réalisées avec les cellules mammifères. Pour améliorer le rapport du signal sur bruit de fond, nous avons effectué des expériences de SSC sur des canaux purifiés qui ont été immobilisés sur des lamelles de verre fonctionnalisées Ni-NTA. En utilisant la méthode de calcul décrite dans le premier projet, nous avons trouvé différents états oligomériques et proposons que les canaux TACAN natifs s'assemblent en tétramères qui sont instables lorsqu'ils sont solubilisés dans un détergent. Dans le dernier projet, nous avons étudié la relation structure-fonction de la sous-unité auxiliaire DPP6 pour les canaux Kv4.2 (chapitre cinq). Ici, nous avons progressivement tronqué le grand domaine extracellulaire de 700 acides aminés de DPP6 et étudié son effet sur les courants macroscopiques en utilisant la technique du cut-open voltage clamp. Nous avons constaté que les sous-unités DPP6 avec un domaine extracellulaire court ne parviennent pas à moduler les propriétés du canal aussi efficacement que la DPP6 pleine longueur. Plus précisément, la seconde moitié du domaine extracellulaire b-propeller de DPP6 est responsable d'une inactivation du canal considérablement accélérée. Sur la base de la structure cristalline du domaine extracellulaire, nous avons proposé qu'un domaine b-propeller stable et possiblement la formation de dimères DPP6 sont responsables de la déstabilisation efficace de l'état du canal ouvert. / Ion channels are key macromolecular complexes expressed in all cell types and are involved in various physiological processes including the generation and propagation of action potentials. Defective channels lead to severe diseases including epilepsy, arrhythmias and pain syndromes making them an interesting potential drug target. To improve our understanding of these biological assemblies and eventually find specific treatments for channelopathies, it is crucial to study the structure and function of ion channels. The main purpose of this thesis has been to investigate such structural and functional details of three ion channel complexes from the field of pain sensors and voltage-gated potassium channels using fluorescence and electrophysiological techniques. In the first project, we studied the stoichiometry of heteromeric Kv2.1/6.4 channel complexes (chapter three). Single subunit counting (SSC) allows to directly count the number of fluorescently labeled subunits by determining the number of irreversible, step-wise photobleaching events. To determine the most probable stoichiometry, we used weighted likelihood calculations and found that Kv2.1/6.4 channels express in a 2:2 arrangement. More precisely, functional studies of concatemeric channels (performed by our collaborators) illustrate that Kv6.4 and 2.1 subunits need to be arranged in an alternating fashion. The second project was also based on SSC experiments and aimed at determining the oligomeric state of the novel ion channel TACAN (chapter four). We found a significant amount of channels in the intracellular which caused background fluorescence in traditional SSC experiments performed in cells. To improve the signal to background ratio, we performed SSC experiments on purified channels that were immobilized on Ni-NTA functionalized glass coverslips. Using the model selection method described in the first project, we found different oligomeric states and propose that native TACAN channels assemble as tetramers which are unstable when solubilized in detergent. In the last project, we investigated the structure-function relation of the auxiliary DPP6 subunit in Kv4.2 channel complexes (chapter five). Here, we progressively truncated DPP6’s 700 amino acids long extracellular domain and studied its effect on macroscopic currents using the cut-open voltage clamp technique. We found that DPP6 subunits with a short extracellular domain fail to modulate the channel properties as efficiently as the full length DPP6. More precisely, the second half of the extracellular b-propeller domain of DPP6 is responsible for drastically accelerated channel inactivation. Based on the crystal structure of the extracellular domain, we proposed that a stable b-propeller domain and possibly DPP6 dimer formation is responsible for destabilizing the open channel state efficiently.

Culicoides (Diptera: Ceratopogonidae) are small (<3mm) blood feeding flies. These flies are biological vectors of viruses, protozoa and filarial nematodes affecting birds, humans, and other animals. Among the viruses transmitted those causing bluetongue (BT), African horse sickness (AHS) and epizootic haemorrhagic disease (EHD) are of major veterinary significance. Culicoides (Avaritia) imicola Kieffer, a proven vector of both AHS and BT viruses, is the most abundant and wide spread livestock-associated Culicoides species in South Africa. Field isolations of virus and oral susceptibility studies, however, indicated that a second Avaritia species, C. bolitinos Meiswinkel may be a potential vector of both BT virus (BTV) and AHS virus (AHSV). Differences in oral susceptibility, which are under genetic control, of populations from different geographical areas to viruses may be an indication of genetic differences between these populations, which may be the result of limited contact between these populations. A good knowledge of the distribution, spread and genetic structure of the insect vector is essential in understanding AHS or BT disease epidemiology. In the present study, an effort was made to gather field specimens of both C. imicola and C. bolitinos from different areas within their natural distribution in South Africa. The aim was to partially sequence two mitochondrial genes from these specimens and to analyse the sequence data making use of phylogenetic trees to clarify the genetic relationships between individuals or groups collected from geographically distinct sites. The two species were collected from four geographically separated areas in South Africa viz. Gauteng Province, Eastern Cape Province, Western Cape Province as well as the Free State Province. DNA was extracted from a total of 120 individual midges of the two Culicoides species using DNA extraction kits. Extracted DNA was analysed using PCR, sequencing as well as phylogenetic methods. A total of 117 mitochondrial DNA COI and 104 mitochondrial 16S ribosomal RNA Culidoides</i. sequences were analysed. DNA sequence polymorphism and phylogenetic relationships of various groups of C. imicola and C. bolitinos midges were determined. The results of the phylogenetic analysis of Culicoides populations using mitochondrial COI gene fragment showed that, at least one subpopulation of C. imicola and two distinct genotypes of C. bolitinos species do exist in South Africa, and further analysis is necessary. This study showed that COI has the potential to separate Culicoides midges based on their geography / Dissertation (MSc)--University of Pretoria, 2010. / Veterinary Tropical Diseases / unrestricted

Introdução: A identificação de variantes genéticas que predispõem a maior susceptibilidade à dependência à nicotina pode ser importante para a prevenção e o tratamento do tabagismo. No contexto de medicina personalizada, os principais objetivos do presente estudo foram avaliar se polimorfismos nos genes CHRNA2, CHRNA3, CHRNA5 e CHRNB3 estão associados com o nível de dependência em indivíduos fumantes e com o resultado do tratamento antitabágico. Métodos: Estudo de coorte com 1049 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona e/ou terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. A escala de conforto PAF foi utilizada para avaliar o conforto do paciente durante o tratamento. Os polimorfismos CHRNA2 rs2472553, CHRNA3 rs1051730, CHRNA5 rs16969968, CHRNA5 rs2036527 e CHRNB3 rs6474413 foram genotipados pela análise da curva de melting. Resultados: As mulheres portadoras dos genótipos GA e AA para os polimorfismos CHRNA5 rs16969968 e rs2036527 obtiveram maior taxa de sucesso no tratamento antitabagismo: 44,0% e 56,3% (rs16969968), 41,5% e 56,5% (rs2036527), respectivamente; em comparação com as mulheres portadoras do genótipo GG: 35,7% (rs16969968) e 34,8% (rs2036527), (P=0,03; n=389; P=0,01; n=391). Os genótipos GA ou AA para os rs16969968 e rs2036527 foram associados com maior OR para o sucesso em mulheres (OR=1,63; IC 95%=1,04-2,54; P=0,03 e OR=1,59; IC 95%=1,02-2,48; P=0,04; respectivamente), em um modelo multivariado. Não foi encontrada associação dos polimorfismos no gene CHRNA5 com o escore de FTND. Para os polimorfismos CHRNA2 rs2472553, CHRNA3 rs1051730 e CHRNB3 rs6474413 não foram encontradas associações significativas com os fenótipos estudados. Conclusão: Os polimorfismos rs16969968 e rs2036527 no gene CHRNA5 foram associados com maior taxa de sucesso no tratamento antitabagismo em mulheres. Estes resultados podem contribuir com avanços na terapêutica baseada em medicina personalizada / Background: The identification of genetic variants that predispose increased susceptibility to nicotine dependence becomes increasingly important for the prevention and smoking treatment. In the context of personalized medicine, the main aims of this study were to evaluate whether the CHRNA2, CHRNA3, CHRNA5 and CHRNB3 polymorphisms are associated with the level of dependence in smokers and the result of smoking treatment. Methods: This cohort study enrolled 1049 smoking patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion plus/or nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. PAF comfort scale was used to evaluate the comfort of the patient during treatment. The CHRNA2 rs2472553, CHRNA3 rs1051730, CHRNA5 rs16969968 and rs2036527 and CHRNB3 rs6474413 polymorphisms were genotyped by high resolution melting analysis. Results: Females with GA and AA genotypes for CHRNA5 rs16969968 and rs2036527polymorphisms had higher success rate in smoking cessation treatment: 44.0% and 56.3% (rs16969968), 41.5% and 56.5% (rs2036527), respectively; compared with carriers of the GG genotypes: 35.7% (rs16969968), 34.8% (rs2036527), (P=0.03, n=389; P=0.01, n=391). The GA or AA genotypes to the rs16969968 and rs2036527 were associated with higher odds ratio for success in women (OR=1.63; 95%CI=1.04 to 2.54; P=0.03 and OR=1.59, 95%CI=1.02 to 2.48; P=0.04; respectively), in a multivariate model. We found no association of these polymorphisms with FTND score for nicotine dependence. For the CHRNA2 rs2472553, CHRNA3 rs1051730 and CHRNB3 rs6474413 polymorphisms no significant associations were found with phenotypes studied. Conclusion: The CHRNA5 rs16969968 and rs2036527 were associated with higher success rate in the smoking cessation treatment in women. These results can contribute to major advances in personalized medicine based therapy

Introdução: A identificação de variantes genéticas que predispõem a maior susceptibilidade à dependência à nicotina pode ser importante para a prevenção e o tratamento do tabagismo. No contexto de medicina personalizada, os principais objetivos do presente estudo foram avaliar se polimorfismos nos genes CHRNA2, CHRNA3, CHRNA5 e CHRNB3 estão associados com o nível de dependência em indivíduos fumantes e com o resultado do tratamento antitabágico. Métodos: Estudo de coorte com 1049 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona e/ou terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. A escala de conforto PAF foi utilizada para avaliar o conforto do paciente durante o tratamento. Os polimorfismos CHRNA2 rs2472553, CHRNA3 rs1051730, CHRNA5 rs16969968, CHRNA5 rs2036527 e CHRNB3 rs6474413 foram genotipados pela análise da curva de melting. Resultados: As mulheres portadoras dos genótipos GA e AA para os polimorfismos CHRNA5 rs16969968 e rs2036527 obtiveram maior taxa de sucesso no tratamento antitabagismo: 44,0% e 56,3% (rs16969968), 41,5% e 56,5% (rs2036527), respectivamente; em comparação com as mulheres portadoras do genótipo GG: 35,7% (rs16969968) e 34,8% (rs2036527), (P=0,03; n=389; P=0,01; n=391). Os genótipos GA ou AA para os rs16969968 e rs2036527 foram associados com maior OR para o sucesso em mulheres (OR=1,63; IC 95%=1,04-2,54; P=0,03 e OR=1,59; IC 95%=1,02-2,48; P=0,04; respectivamente), em um modelo multivariado. Não foi encontrada associação dos polimorfismos no gene CHRNA5 com o escore de FTND. Para os polimorfismos CHRNA2 rs2472553, CHRNA3 rs1051730 e CHRNB3 rs6474413 não foram encontradas associações significativas com os fenótipos estudados. Conclusão: Os polimorfismos rs16969968 e rs2036527 no gene CHRNA5 foram associados com maior taxa de sucesso no tratamento antitabagismo em mulheres. Estes resultados podem contribuir com avanços na terapêutica baseada em medicina personalizada / Background: The identification of genetic variants that predispose increased susceptibility to nicotine dependence becomes increasingly important for the prevention and smoking treatment. In the context of personalized medicine, the main aims of this study were to evaluate whether the CHRNA2, CHRNA3, CHRNA5 and CHRNB3 polymorphisms are associated with the level of dependence in smokers and the result of smoking treatment. Methods: This cohort study enrolled 1049 smoking patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion plus/or nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. PAF comfort scale was used to evaluate the comfort of the patient during treatment. The CHRNA2 rs2472553, CHRNA3 rs1051730, CHRNA5 rs16969968 and rs2036527 and CHRNB3 rs6474413 polymorphisms were genotyped by high resolution melting analysis. Results: Females with GA and AA genotypes for CHRNA5 rs16969968 and rs2036527polymorphisms had higher success rate in smoking cessation treatment: 44.0% and 56.3% (rs16969968), 41.5% and 56.5% (rs2036527), respectively; compared with carriers of the GG genotypes: 35.7% (rs16969968), 34.8% (rs2036527), (P=0.03, n=389; P=0.01, n=391). The GA or AA genotypes to the rs16969968 and rs2036527 were associated with higher odds ratio for success in women (OR=1.63; 95%CI=1.04 to 2.54; P=0.03 and OR=1.59, 95%CI=1.02 to 2.48; P=0.04; respectively), in a multivariate model. We found no association of these polymorphisms with FTND score for nicotine dependence. For the CHRNA2 rs2472553, CHRNA3 rs1051730 and CHRNB3 rs6474413 polymorphisms no significant associations were found with phenotypes studied. Conclusion: The CHRNA5 rs16969968 and rs2036527 were associated with higher success rate in the smoking cessation treatment in women. These results can contribute to major advances in personalized medicine based therapy

The small ribosomal subunit gene of the Chaetomium thermophilum DSM 1495 is invaded by a nested intron at position mS1247, which is composed of a group I intron encoding a LAGLIDADG open reading frame interrupted by an internal group II intron. The first objective was to examine if splicing of the internal intron could reconstitute the coding regions and facilitate the expression of an active homing endonuclease. Using in vitro transcription assays, the group II intron was shown to self-splice only under high salt concentration. Both in vitro endonuclease and cleavage mapping assays suggested that the nested intron encodes an active homing endonuclease which cleaves near the intron insertion site. This composite arrangement hinted that the group II intron could be regulatory with regards to the expression of the homing endonuclease. Constructs were generated where the codon-optimized open reading frame was interrupted with group IIA1 or IIB introns. The concentration of the magnesium in the media sufficient for splicing was determined by the Reverse Transcriptase-Polymerase Chain Reaction analyses from the bacterial cells grown under various magnesium concentrations. Further, the in vivo endonuclease assay showed that magnesium chloride stimulated the expression of a functional protein but the addition of cobalt chloride to the growth media antagonized the expression. This study showed that the homing endonuclease expression in Escherichia coli can be regulated by manipulating the splicing efficiency of the group II introns which may have implications in genome engineering as potential ‘on/off switch’ for temporal regulation of homing endonuclease expression . Another objective was to characterize native homing endonucleases, cytb.i3ORF and I-OmiI encoded within fungal mitochondrial DNAs, which were difficult to express and purify. For these, an alternative approach was used where two compatible plasmids, HEase.pET28b (+)-kanamycin and substrate.pUC57-chloramphenicol, based on the antibiotic markers were maintained in Escherichia coli BL21 (DE3). The in vivo endonuclease assays demonstrated that these homing endonucleases were able to cleave the substrate plasmids when expressed, leading to the loss of the antibiotic markers and thereby providing an indirect approach to screen for potential active homing endonucleases before one invests effort into optimizing protein overexpression and purification strategies. / October 2016

La présente thèse par articles aborde différentes facettes du dépistage prénatal de certaines aneuploïdies au premier trimestre de la grossesse. L’introduction retrace l’historique du dépistage prénatal et énonce les différents marqueurs biochimiques et échographiques associés aux aneuploïdies. La première publication démontre que le tabagisme maternel abaisse significativement les niveaux sanguins maternels de PAPP-A et de la fraction libre de la β-hCG et augmente significativement la clarté nucale, confirmant la nécessité de contrôler cette co-variable dans le calcul de risque final, du moins pour la trisomie 18. Le deuxième article identifie des seuils de clarté nucale au-delà desquels la biochimie génétique n’apporte aucune valeur additionnelle au dépistage prénatal de la trisomie 21 et de la trisomie 18. Pour les fœtus avec clarté nucale supérieure aux seuils établis, un diagnostic prénatal intrusif devrait être offert sans délai. Le troisième et dernier article porte sur la première détermination des niveaux plasmatiques maternels de la protéine FLRG (follistatin-related gene) au premier trimestre de grossesse et sur son rôle potentiel à titre de marqueur biochimique dans le dépistage prénatal de la trisomie 21. Bien que détectables, les niveaux plasmatiques maternels de FLRG ne sont pas significativement altérés en présence de fœtus avec syndrome de Down. Dans la discussion générale, les trois articles sont abordés sous un angle plus spécifique au Québec. Des données complémentaires et originales y sont présentées. Une discussion sur l’évolution future du dépistage prénatal est entamée et des axes de recherche sont proposés. / In this thesis by articles, we explore different facets of first trimester prenatal screening of aneuploidy. Introduction retraces the origin of prenatal screening and enunciates current biochemical and ultrasound markers associated with aneuploidy. In the first article, impact of maternal smoking on first-trimester prenatal screening results is assessed for Down syndrome and trisomy 18. Both maternal blood levels of PAPP-A and free β-hCG are significantly decreased by maternal smoking while fetal nuchal translucency (NT) thickness is significantly increased. Without adjustment, this results in an increase of false positives, at least for trisomy 18. Based on these results, adjustment for smoking should be mandatory in first-trimester prenatal screening. In the second article, we identify NT threshold values above which biochemical screening provides no additional benefit. In pregnancies in which NT is above the proposed upper cut-offs, invasive prenatal screening should be offered without undue delay. In the third and last article, maternal plasma levels of follistatin- related gene protein (FLRG) are determined for the first time in first trimester of pregnancy. Its potential role as a new marker for Down syndrome is assessed. Although FLRG can be successfully detected in maternal plasma, its levels are not significantly altered by the presence of Down syndrome fetuses. In the general discussion, articles are mainly addressed under a Quebec standpoint. Additional and complementary original data are presented and different clinical research avenues are proposed.