Thesis (Ph. D.)--Duke University, 2007. / Includes bibliographical references.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 163-187.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (p. 155-162).

A família gênica Forkhead P {FOXP) tem sido alvo de muitos estudos envolvendo evolução do cérebro e comportamento animal. Destacam-se particularmente as investigações com o gene FOXP2, que indicam que mudanças neste gene estariam associadas com a evolução da vocalização em algumas espécies de mamíferos, incluindo o Homo sapiens. Recentemente, estudos de desordem intrínseca de proteínas (IDPs) tem ganhado ênfase no contexto evolut ivo, visto que uma correlação posit iva entre regiões de desordem e altas taxas evolutivas tem sido observada. Através de um conjunto de abordagens que inclui predizer o conteúdo de desordem e os motivos lineares de interação, bem como as taxas evolutivas, buscamos desvendar a historia evolutiva dos genes da subfamília FOXP. Concentramos nossas análises sobre regiões desordenadas das proteínas FOXPl, FOXP2, FOXP3 e FOXP4 encontradas em 77 espécies de tetrápodes. Tais regiões proteicas são normalmente negligenciadas em estudos dessa natureza, pois se localizam fora de seus tra dicionais domínios conservados, normalmente associados à função principal da proteína. Sít ios apontados estando sob seleção positiva e relaxamento da restrição seletiva mostraram-se hotspots importantes para mudanças que podem impactar na capacidade de interação das proteínas. Encontramos que os maiores valores de w são mais prevalentes em regiões desordenadas que em ordenadas. Ainda, alto e similar valor de desordem (70%) foi encontrado nas 77 proteínas ortólogas de FOXPl , FOXP2, e FOXP4, indicando a manutenção de um "padrão geral" sobre um longo tempo evolutivo. Portanto, a variabilidade tanto de aminoácidos quanto de motivos lineares dentro das regiões de desordem foi marcante. A proteína FOXP3 apresentou menor nível de desordem (30%), mas signif icante sinal de seleção positiva em alguns sítios. Composição idênt ica de resíduo de aminoácido e/ou motivos lineares em espécies filogeneticamente distantes, indica clara convergência molecular, provavelmente associada a pressões seletivas similares. Sucessivamente, nossos achados mostraram uma clara diferença na composição de motivos lineares entre mamíferos e não mamíferos, dando suporte para a importância dos estudos de evolução da interatividade proteica para as compreensões de características taxa-específicas. / Forkhead Family P (FOXP) has been target of many studies about brain and behavior evo lution among species. FOXP2 receives special attention in academic society, due associations with vocalízation evolution in mammals, including Homo sapiens. Recently, intrinsically disorder proteins studies have gained emphasis in the evolutionary context, as positive correlation between disorder regions and higher evolutionary rate has been observed. Through a set of approaches, including disorder and linear motif predictions, as well as estimate evolutionary rates, we aimed to unveil the evolutionary history of FOXP subfamily genes. We focused our ana lysis over disordered regions of FOXPl, FOXP2, FOXP3 and FOXP4 proteins retrieved in 77 tetrapods. Such protein regions are usually neglected in studies of this nature, for being localized out of the traditional conserved domains, usua lly associated with the main function of the protein. Sites indicated as under relaxation of selective constrains or positive selection have shown to be important hotspots for changes that can impact in protein interaction capability. Higher w va lues are prevalent in disordered regions than in ordered ones. Still, high and similar disorder proportion (~70%) was found among 77 orthologues proteins of FOXPl, FOXP2 and FOXP4, indicating general pattern of disorder maintenance, along tetrapod's evolutionary tree. However, amino acid and linear motifs variability within disordered regions was observed. FOXP3 protein presented lower disorder leveis (~30%), when compared with other paralogues, but signal of positive selection was observed in some sites. ldentical composition of amino acid residues and/or linear motifs is, probably, associated with similar selective pressure. Successively, ou r results showed clear differences in linear motif composition between mammals and non-mammals, supporting the importance of evolutionary studies on protein interaction for the understanding of taxa-specifics characteristics.

A família gênica Forkhead P {FOXP) tem sido alvo de muitos estudos envolvendo evolução do cérebro e comportamento animal. Destacam-se particularmente as investigações com o gene FOXP2, que indicam que mudanças neste gene estariam associadas com a evolução da vocalização em algumas espécies de mamíferos, incluindo o Homo sapiens. Recentemente, estudos de desordem intrínseca de proteínas (IDPs) tem ganhado ênfase no contexto evolut ivo, visto que uma correlação posit iva entre regiões de desordem e altas taxas evolutivas tem sido observada. Através de um conjunto de abordagens que inclui predizer o conteúdo de desordem e os motivos lineares de interação, bem como as taxas evolutivas, buscamos desvendar a historia evolutiva dos genes da subfamília FOXP. Concentramos nossas análises sobre regiões desordenadas das proteínas FOXPl, FOXP2, FOXP3 e FOXP4 encontradas em 77 espécies de tetrápodes. Tais regiões proteicas são normalmente negligenciadas em estudos dessa natureza, pois se localizam fora de seus tra dicionais domínios conservados, normalmente associados à função principal da proteína. Sít ios apontados estando sob seleção positiva e relaxamento da restrição seletiva mostraram-se hotspots importantes para mudanças que podem impactar na capacidade de interação das proteínas. Encontramos que os maiores valores de w são mais prevalentes em regiões desordenadas que em ordenadas. Ainda, alto e similar valor de desordem (70%) foi encontrado nas 77 proteínas ortólogas de FOXPl , FOXP2, e FOXP4, indicando a manutenção de um "padrão geral" sobre um longo tempo evolutivo. Portanto, a variabilidade tanto de aminoácidos quanto de motivos lineares dentro das regiões de desordem foi marcante. A proteína FOXP3 apresentou menor nível de desordem (30%), mas signif icante sinal de seleção positiva em alguns sítios. Composição idênt ica de resíduo de aminoácido e/ou motivos lineares em espécies filogeneticamente distantes, indica clara convergência molecular, provavelmente associada a pressões seletivas similares. Sucessivamente, nossos achados mostraram uma clara diferença na composição de motivos lineares entre mamíferos e não mamíferos, dando suporte para a importância dos estudos de evolução da interatividade proteica para as compreensões de características taxa-específicas. / Forkhead Family P (FOXP) has been target of many studies about brain and behavior evo lution among species. FOXP2 receives special attention in academic society, due associations with vocalízation evolution in mammals, including Homo sapiens. Recently, intrinsically disorder proteins studies have gained emphasis in the evolutionary context, as positive correlation between disorder regions and higher evolutionary rate has been observed. Through a set of approaches, including disorder and linear motif predictions, as well as estimate evolutionary rates, we aimed to unveil the evolutionary history of FOXP subfamily genes. We focused our ana lysis over disordered regions of FOXPl, FOXP2, FOXP3 and FOXP4 proteins retrieved in 77 tetrapods. Such protein regions are usually neglected in studies of this nature, for being localized out of the traditional conserved domains, usua lly associated with the main function of the protein. Sites indicated as under relaxation of selective constrains or positive selection have shown to be important hotspots for changes that can impact in protein interaction capability. Higher w va lues are prevalent in disordered regions than in ordered ones. Still, high and similar disorder proportion (~70%) was found among 77 orthologues proteins of FOXPl, FOXP2 and FOXP4, indicating general pattern of disorder maintenance, along tetrapod's evolutionary tree. However, amino acid and linear motifs variability within disordered regions was observed. FOXP3 protein presented lower disorder leveis (~30%), when compared with other paralogues, but signal of positive selection was observed in some sites. ldentical composition of amino acid residues and/or linear motifs is, probably, associated with similar selective pressure. Successively, ou r results showed clear differences in linear motif composition between mammals and non-mammals, supporting the importance of evolutionary studies on protein interaction for the understanding of taxa-specifics characteristics.

The spun-out silk fiber consists of two fibroin filaments that are cemented together by sericin coating. The serine-rich sericins, which make 20-30% of the cocoon silk proteins in Bombyx mori, are dissolved in hot water during silk fiber reeling from the cocoon. The sericin extract is usually discarded. Only small amounts are currently used in cosmetics and lately also as replacement of bovine serum products in the cell culture media. The use in culture media is hindered by poor standardization of the extracts. To overcome this problem, we attempted preparation of several recombinant proteins based on partial sequences of one out of the three sericin genes of Bombyx mori, sericin 3. Translation product of the Ser3 gene is extremely serine-rich with a relatively high representation of the aspartate, asparagin, glutamate, and glutamine. Using specific primers we have prepared a cDNAs of 267, 279, 525, 672, and 528 bp, respectively, derived from the Ser3 gene. The cDNAs were cloned and expressed as fusion proteins with hexahistidine in Escherichia coli. The proteins were purified by affinity chromatography and analyzed by acrylamide electrophoresis. Ser3 gene contain repetitive motifs rich in serine and including some charged amino acids.

Dans ce document, nous étudions l’évitabilité de différentes formes de répétitions dans les mots. En particulier 3 des 6 chapitres sont dédiés aux répétitions abéliennes en lien notamment avec deux questions d’Erdős de 1957 et 1961. Nous commençons par montrer qu’il existe un algorithme décidant, sous certaines conditions, si un mot morphique évite des puissances abéliennes. Cet algorithme élargit la classe sur laquelle les précédents algorithmes pouvaient décider. Une généralisation de cet algorithme nous permet de montrer que les longs carrés abéliens sont évitables sur l’alphabet ternaire et que les carrés additifs sont évitables sur Z2 . Le premier résultat répond à une question ouverte de Mäkelä datant de 2003 alors que le deuxième rappelle la question ouverte de 1994 concernant l’évitabilité des carrés additifs sur Z.Une autre généralisation de notre algorithme permet d’étudier l’évitabilité des motifs au sens abélien. Nous montrons que les motifs binaires de longueur supérieure à 14 sont évitables sur l’alphabet binaire, améliorant la précédente borne de 118.Nous donnons des conditions suffisantes pour qu’un morphisme soit sans longues puissances nème k-abéliennes. Ce résultat nous permet de calculer, pour tout k ≥ 3, le nombre minimum de carrés k-abéliens qu’un mot binaire infini doit contenir en facteur. Il permet aussi de montrer que les longs carrés 2-abéliens sont évitables sur l’alphabet binaire et qu’il existe un mot ternaire qui ne contient qu’un seul carré 2-abélien en tant que facteur.Enfin, nous proposons une classification complète des formules binaires en fonction de la taille d’alphabet qu’il faut pour les éviter et du taux de croissance (exponentiel ou polynomial) du langage les évitant. / In this document, we study the avoidability of different kind of repetitions in words. We firstshow that under some conditions one can decide whether a morphic word avoids abelian n-thpowers. This algorithm can decide over a wider class of morphism than the previousalgorithms. We generalize this algorithm and use it to show that long abelian squares areavoidable over the ternary alphabet and that additive squares are avoidable over Z2 . The firstresult answers a weak version of a question formulated by Mäkelä in 2003 and the second oneis related to an open question from 1994 about the avoidability of additive squares over Z.Another generalization of this algorithm can be used to study avoidability of patterns in theabelian sense. In particular, we show that binary patterns of length more than 14 areavoidable over the binary alphabet in the abelian sense. This improves considerably theprevious bound of 118.We give sufficient conditions for a morphism to be long k-abelian n-th power-free. This resultallows us to compute for every k ≥ 3 the number of different k-abelian squares that a binaryword must contain. We prove that long 2-abelian squares are avoidable over the binaryalphabet and that over the ternary alphabet there exists a word that contains only one 2-abelian square.We also give a complete classification of binary formulas based on the size of the smallestalphabet over which they are avoidable and on the growth (exponential or polynomial) of theassociated language.

Ce travail porte sur deux thèmes principaux. Le premier est relatif à la drépanocytose et comprend deux études. La première a examiné les connaissances qu’ont 178 hommes et femmes vivant au Bénin sur la maladie, ses causes, ses effets et sa prise en charge. Ils ont répondu à un questionnaire et, leurs réponses ont été comparées à celles d’un groupe d’expert. Il en est ressorti qu’il existait une méconnaissance globale de la maladie, et plus particulièrement de ses symptômes les plus graves et de son traitement. Les résultats révèlent que plus on a un niveau d’éducation élevé, moins on se fait de fausses idées concernant la maladie. En outre, cette méconnaissance était plus accrue chez les personnes se déclarant porteuses du trait génétique (4.83) ou drépanocytaire (5.19) que chez les personnes ayant déclaré ne pas être porteuses (3.72) ou saines (3.62). La deuxième étude a quant à elle cherché à savoir si les 137 personnes interrogées vivant au Bénin, avaient intégré de façon adéquate les règles de transmission génétique de la maladie, en se basant sur la théorie fonctionnelle de la cognition. L’analyse des données a permis de distinguer trois clusters distincts. Dans le premier groupe (n=46), les personnes jugeaient ainsi le risque de transmission de la maladie plus élevé en présence de l’un ou l’autre des deux parents atteints. Dans le deuxième groupe (n=51), le risque était jugé d’autant plus grand que chacun des parents était lui-même gravement atteint. Dans le troisième groupe (n=40), le risque n’avait été jugé élevé qu’en présence des deux parents atteints. Le deuxième thème a donné lieu à une étude, visant à examiner les motifs pouvant sous-tendre un refus d’amputation chez 224 personnes vivant au Bénin, par l’approche de la théorie du renversement. Les résultats montrent que la peur de la perte de l’identité personnelle, la peur des hôpitaux et du personnel médical, la conviction que la chirurgie était inutile, le manque de confiance dans la compétence et le dévouement des médecins, la peur de la perte de l’estime de l’autre et celle de la perte de la force spirituelle, font partie des facteurs principaux pouvant motiver un refus d’amputation. / This thesis focuses on two main themes. The first one is related to sickle cell disease and the second theme is related to limb amputations. For studying the first theme, this research proposes to examine the knowledge of 178 men and women living in Benin, on the disease, its causes, effects and care. They answered a questionnaire and their responses had been compared to those given by an expert group. It had been found that there was an overall lack of awareness on the disease, and especially its most severe symptoms and treatment. The results reveal that the higher level of education they have, the less misconceptions about the disease they have. In addition, this ignorance was increased by people declaring themselves carrying the genetic trait (4.83) or sickle cell (5.19) than those who reported not being carriers (3.72) or healthy (3.62). The second study about the first theme, investigated whether the 137 respondents living in Benin had integrated adequately the rules of inheritance of the disease, based on the functional theory of cognition. Three distinct clusters emerge with data analysis. In the first group (n=46), respondents considered a higher risk of transmission if the one or the other of two parents was/were affected by the disease. In the second group (n=51), the risk was considered even greater as both parents were seriously ill. In the third group (n=40), the risk was considered as high only if both parents were touched. The second theme, Accidents involving physical injury, has motivated a study which aim was to examine the reasons that may underlie a refusal of amputation for 224 people living in Benin, by the approach of the reversal theory. The results show that the fear of losing personal identity, the fear of hospitals and medical staff, the belief that the surgery was unnecessary, a lack of confidence in the competences and dedication of doctors, the fear of losing others esteem and the loss of spiritual strength, are among the main factors motivating a refusal of amputation.

Au cours de notre programme de thèse, nous avons isolé et caractérisé des molécules protéiques à activité antivirale intracellulaire dirigée contre le VIH-1. Ces protéines, appelées aRep, ont été obtenues par criblage d'une banque de protéines artificielles de nouvelle génération, construites de façon combinatoire à partir de protéines naturelles constituées de motifs alpha-hélicoidaux répétés. La cible virale, ou "appât", utilisé pour ce criblage a été une région de la polyprotéine Gag du VIH-1 identifiée comme une cible privilégiée de nouvelles thérapeutiques antivirales, car essentielle à l'assemblage viral, l'empaquetage du génome viral et le clivage de maturation de Gag aboutissant à la formation de virions infectieux. Deux molécules d'aRep à affinité élevée pour la cible virale, l'aRep4E3 (32 kDa; 7 motifs répétés) et l'aRep9A8 (28 kDa; 6 motifs répétés) ont ainsi été identifiées, isolées et clonées. L'étude de l'activité anti-VIH intracellulaire de ces aRep a été réalisée dans différents systèmes d'expression cellulaire, nécessitant la construction de lignées stables de cellules d'insecte et de cellules épithéliales humaines, et leur infection par différents types de vecteurs viraux recombinants, baculovirus ou lentivirus, porteurs du gène rapporteur luciférase. Mais surtout, cette étude a été menée sur des cellules lymphocytaires-T (SupT1), cibles naturelles du virus, exprimant ces aRep et infectées par du VIH-1 naturel infectieux. Nos résultats ont montré que l'aRep4E3 et l'aRep9A8 ont toutes deux un effet négatif significatif sur le cycle réplicatif du VIH-1, mais ciblent des fonctions virales différentes. L'aRep4E3 bloque l'empaquetage du génome viral, tandis que l'aRep9A8 inhibe la maturation et diminue l'infectivité virale. De plus, l'aRep9A8, exprimée de façon constitutive dans les cellules SupT1, leur confère une résistance au VIH: une lignée de SupT1 chroniquement infectée par le VIH-1 a pu être ainsi isolée et maintenue en culture pendant plusieurs semaines, sans effet cytopathique viro-induit apparent. Ces nouvelles données auront des implications non-négligeables dans le choix et la conduite de futures stratégies de thérapie cellulaire anti-VIH / HIV-1 infection is a long-term disease which required a long-life treatment. Besides the standard HAART regiment, HIV gene therapy is a promising alternative strategy which give rise to hope for the better HIV-1 treatment. Protein therapeutics is one another technique that represent high impact results in curing various types of disease. It is already become a significant part of current medical treatments. In this study we first designed aRep, a non-immunoglobulin scaffold protein which target two domains of HIV-1 Pr55Gag, SP1-NC and investigated their roles as an intracellular therapeutic agents. Phage display technology was used for the specific isolation of aRep against a critical C-terminal region of the HIV-1 Pr55Gag precursor from a large and diverse library. The antiviral activity of these two Pr55Gag binders was investigated using different cell systems. Two aRep scaffolds aRep4E3 and aRep9A8 were isolated and characterized. aRep4E3 contains 7 repeat motifs (32 kDa), meanwhile aRep9A8 has 6 repeat motifs (28 kDa). These two scaffold molecules found to be able to display antiviral effects on the late stage of HIV-1 replication, by reducing and delaying the viral progeny production. The difference in the molecular mechanism was observed between these two aRep proteins: aRep4E3 mainly interferes with the packaging of the viral genome, meanwhile aRep9A8 interferes with the proteolytic processing of Gag, and performs as a protease inhibitor to prevent the PR cleavage required for the production of newly infectious mature virus. Interestingly, aRep9A8 is able to survive in the chronical HIV-1 infected cells up to D38 pi with the low level of HIV-1 replication. Taken together, results suggested that aRep, a new type of scaffold protein could serve as a promising alternative agent in protein therapy, not only the HIV-1 infection but also the others pathogens or disorders

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