L’intérêt des peptides comme des médicaments potentiels est en constante augmentation. Des stratégies ont été développées pour améliorer la sélectivité, l’activité, et la stabilité des peptides vis-à-vis de la protéolyse. Dans ce mémoire de thèse, deux nouvelles modifications de peptides sont proposées.Dans le premier chapitre, nous présentons une nouvelle application de la réaction de transfert O-N acylique pour la synthèse de peptides agrafés (ou ‘stapled peptides’). L’introduction d’une ‘agrafe’ dans un peptide est un moyen de stabiliser une structure secondaire hélicoïdale en établissant un pont entre les résidus appropriés des chaînes latérales. Dans notre cas, l'agrafe est formée par un O-acyl isodipeptide. La liaison ester peut être convertie en liaison amide par un transfert O-N acylique. Cette stratégie permet une amélioration de la solubilité d’un peptide hydrophobe agrafé, avant son réarrangement à pH neutre.Dans le dernier chapitre, nous avons développé une méthodologie nouvelle pour la dimérisation de peptides non protégés. Cette méthode repose sur la formation de liaisons siloxane entre des peptides hybrides portant chacun un groupement dimethylchlorosilane. Nous avons ainsi dimérisé une séquence dérivée de la protéine p53, impliquée dans l’apoptose. A titre de comparaison, cette même séquence a été dimérisée en utilisant la cycloaddition d’Huisgen entre deux peptides modifiés possédant un azoture ou un alcyne en position N-terminale. Enfin, plusieurs dimères de la séquence du GHRP-6 (growth hormone releasing peptide) ont été synthétisés, avec des bras dimethylhydroxysilane placés à différentes positions. L’homodimérisation a été effectuée dans l'eau à pH neutre. / The interest in peptides as potential drug candidates was revived and is increasing constantly. Strategies have been developed to improve their selectivity and activity, and their stability toward proteolysis. In this thesis, two new peptide modifications are proposed.In the first chapter, we present a new application of the O-N acyl transfer reaction for the synthesis of stapled peptides. Peptide ‘stapling’ is a way of stabilizing secondary helical structure by establishment of a bridge between the side chains of suitable residues. In our case, the staple is formed by an O-acyl isopeptide which can be converted into amide bond by acyl migration. This strategy allows an improved solubility of the stapled hydrophobic peptide prior to rearrangement at neutral pH.In the last chapter, we developed also a new methodology for the dimerization of unprotected peptides. This method is based on siloxane bond formation between hybrid dimethylhydroxysilane peptides. A peptide derived from the tumor suppressor protein p53 was dimerized in water, at neutral pH using this methodology. The method was compared with the homodimerization carried out by Cu(I) azide-alkyne cycloaddition (CuAAC). For that purpose, p53 peptide derivative was synthesized with azide and alkyne linkers at the N-terminus. At last, several homodimers of growth hormone releasing hexapeptide (GHRP-6) were synthesized, with dimethylhydroxy silane linkers placed at various positions.

The human papillomavirus (HPV) has been a “companion” of humanity for as long as humanity has existed. The migration of peoples around the globe has given rise to more than 170 different types of the virus, which cause a variety of conditions. All five genera of HPV infect epithelial cells in the body, but only the Alphapapillomaviruses infect the genital mucosa. Most infections are benign and typically regress to subclinical within two years, but persistent infections can cause precancerous lesions. HPV types 16 and 18 are among the highest risk and account for the majority of cervical cancer, and more than 90% of all other HPV-related cancers. While the two vaccines, Gardasil and Cervarix, have been successfully implemented in the US market and some European and Asian countries, complete world penetrance has been burdened by multiple factors, including financial constraints and social norms. Treatments for established papillomas are invasive (cryosurgery, conization, etc.) and advanced malignant HPV-related tumors have been targeted with chemo- and radiotherapy with varied success. The high morbidity and long-term effects of current treatment options make clear the need for easy-to-administer, low-cost therapies, which can specifically treat both early and advanced HPV-associated cancers.<div><br></div><div>The hallmark of HPV tumors is the inactivation of p53, an evasion strategy key to the progression of HPV- derived cancers. Through an interaction between the viral protein E6 and the E3 ubiquitin ligase E6AP, p53 is polyubiquitinated and targeted for proteasomal degradation, allowing infected cells to bypass their own defense mechanisms. This work explores interruption of the association between E6 and E6AP as an opportunity to combat the infection and resulting malignancies.<br></div><div><br></div><div>In the first part of this project, disruption of the E6-E6AP interactions is pursued through the development of helical stabilized peptidomimetics of the LxxLL motif, which E6AP uses for E6 recognition and binding. Several reports have indicated that targeting the E6 binding groove is a viable means for disrupting the interaction. However, reported peptides were not cell permeable or optimized for α-helicity and proteolytic resistance (for reference, the LxxLL motif is an α-helix when bound). To address this challenge a peptide stabilization strategy was applied, which uses an all-hydrocarbon chain to connect two non-adjacent residues and enforce α-helicity. Results from in silico simulations and biochemical assay with these new stapled peptides showed that affinity for E6, α-helicity, and cell permeability can all be improved with the installment of the proper staple.<br></div><div><br></div><div>The second question examined by this work is whether fragment-based drug design can be successfully employed to derive new small-molecule inhibitors of the formation of the E6-E6AP complex. From a design perspective, the significant challenge was to define discreet binding hot-spots capable of accommodating fragments with reasonable affinity, which can then be linked together into a complete ligand. Using existing structural knowledge of the E6 protein and computational hot-spot searching tools, three previously-unidentified regions (sub-pockets) on E6 were discovered, which are near but not directly engaged by either the E6AP motif or p53. Using high-throughput in silico and biochemical screening, three sets of sub-pocket specific fragments were defined and elaborated into larger molecules with two different scaffolds. As a result, the work herein presents a stepwise approach to targeting the E6-E6AP protein-protein interaction – the discovery of new binding hot spots, the identification of site-specific fragments, and the design of complete molecules with versatile scaffold.<br></div>

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / SÃndrome de DefecaÃÃo ObstruÃda (SDO) pode ser produzida por alteraÃÃo funcional (anismus) ou por alteraÃÃes anatÃmicas, especialmente retocele e prolapso mucoso retal. VÃrias tÃcnicas cirÃrgicas vÃm sendo utilizadas no tratamento da retocele, com vias de acesso transvaginal, perineal ou transanal, aquelas mais utilizadas por ginecologistas e a transanal por coloproctologistas, isto em funÃÃo das pacientes recorrerem a especialistas diferentes, dependendo da predominÃncia dos sintomas, sendo apresentados resultados semelhantes. O objetivo deste estudo foi avaliar os resultados do tratamento cirÃrgico de pacientes portadoras de SDO por retocele e prolapso mucoso retal, submetidas à ressecÃÃo transanal da retocele e mucosectomia com um grampeador (procedimento âTRREMSâ. Foram avaliadas 35 pacientes, sendo uma nulÃpara e as demais multÃparas, com idade mÃdia de 47,5 Â10,83 anos (31 â 67), portadoras de retocele e prolapso mucoso retal interno, sendo 13 (37,10%) com grau II e 22 (62,90%) com grau III. Os parÃmetros avaliados foram os escores de defecaÃÃo obstruÃda e de constipaÃÃo, o Ãndice funcional para continÃncia assim como a avaliaÃÃo da dor, satisfaÃÃo com o resultado e com a funÃÃo sexual e realizaÃÃo de defecografia no prà e no pÃs-operatÃrio. O escore mÃdio de SDO de 10,63 no prÃ-operatÃrio reduziu significativamente para 2,91 no pÃs-operatÃrio (p = 0,000). O escore mÃdio de constipaÃÃo de 15,23 no prÃ-operatÃrio reduziu significativamente para 4,46 no pÃs-operatÃrio (p = 0,000). O Ãndice funcional mÃdio para continÃncia, de 2,77 no prÃ-operatÃrio reduziu significativamente para 1,71 no pÃs-operatÃrio (p = 0,000). A dor no primeiro dia pÃs-operatÃrio, avaliada atravÃs da escala visual analÃgica (EVA) apresentou valor mÃdio de 5,23 reduzindo para 1,20 no oitavo dia (p = 0,000). A satisfaÃÃo com o resultado do tratamento, avaliada tambÃm atravÃs da EVA, ao final do primeiro mÃs foi 79,97, no terceiro 86,54, no sexto 87,65 e no dÃcimo segundo 88,06. TambÃm se obteve elevaÃÃo significativa, entre os valores mÃdios de 42,91 no prÃ-operatÃrio e 70,41 no sexto mÃs de pÃs-operatÃrio, para a satisfaÃÃo sexual avaliada atravÃs da EVA (p = 0,000). A defecografia demonstrou reduÃÃo significativa do tamanho mÃdio da retocele de 19,23 mm  8,84 (3 â 42) para 6,68 mm  3,65 (0 â 17) na fase de repouso e de 34,89 mm  12,30 (20 â 70) para 10,94 mm  5,97 (0 â 25) na fase evacuatÃria quando comparado o prà com o pÃs-operatÃrio (p = 0,000) (P=0,000) respectivamente. Procedimento âTRREMSâ à uma tÃcnica segura, eficiente e produziu resultados anatÃmicos e funcionais satisfatÃrios e nÃveis reduzidos de complicaÃÃes pÃs-operatÃrias / Obstructed defecation syndrome (ODS) can be induced by functional changes (anismus) or anatomical abnormalities, especially rectocele and rectal mucosal prolapse (RMP). Several surgical techniques with transvaginal, perineal or transanal access have been used in the treatment of rectocele. The first two are more commonly used by gynecologists, the last one is favored by proctologists. Depending on the prevalence of symptoms, patients may go to either specialist with the same result. The objective of the present study was to make a clinical and functional evaluation of patients submitted to the TRREMS procedure (transanal repair of rectocele and rectal mucosectomy with a single circular stapler) as treatment for ODS caused by rectocele and RMP. The study included 35 female patients (34 of whom multiparous) aged 47.5Â10.83 years (31â67) diagnosed with ODS caused by RMP-associated rectocele grade II (n=13; 37.1%) or grade III (n=22; 62.9%). The study parameters included SDO and constipation scores, functional continence index, sexual function and treatment outcome satisfaction and pre- and postoperative defecographic measures. The average preoperative ODS score (10.63) was significantly reduced after surgery (2.91) (p=0.000). The average constipation score fell from 15.23 to 4.46 (p=0.000). The average functional continence score decreased from 2.77 to 1.71 (p=0.000). Between the first and the eighth postoperative day, the average visual analog scale pain score fell from 5.23 to 1.20 (p=0.000). Using the same scale, satisfaction with the treatment outcome was 79.97, 86.54, 87.65 and 88.06 at 1, 3, 6 and 12 months, respectively, and the average sexual function satisfaction was 42.91 (19â70) and 70.41 (39â97) before and after surgery, respectively (p=0.000). On defecography, average reductions in rectocele size were from 19.23Â8.84 mm (3â42) to 6.68Â3.65 mm (0â17) at rest and from 34.89Â12.30 mm (20â70) to 10.94Â5.97 mm (0â25) during evacuation (both p=0.000). The TRREMS procedure is a safe, efficient technique associated with satisfactory anatomical and functional results and reduced levels of postoperative pain and complications.

Le but de cette thèse a concerné le développement de deux types de structures oligomériques contraintes, basées soit sur des dérivés non naturels d’acides aminés cycliques, soit sur des dimères cycliques de Peptide Nucleic Acids (PNA), en vue d’analyser leur tendance à adopter des conformations pré-organisées, imitant les conformations actives des protéines ou des acides nucléiques. La première partie de cette thèse a trait à l’élaboration de synthons clefs dérivant d’acides α-aminés cycliques non naturels (acides 4-oxopipécoliques 6-substitués), à leur utilisation potentielle dans la synthèse de peptidomimétiques contraints, homogènes et hétérogènes, ainsi qu’à leurs limites dans cette utilisation. Dans le cadre de ce travail, une nouvelle voie de synthèse permettant d’accéder à des résidus N-protégés d'acides 4-oxo-pipécoliques 6substitués a été mise au point. La deuxième partie de cette thèse concerne l’élaboration de dimères d’α-PNA (di-α-PNA) cycliques, dans lesquels les chaînes latérales de deux monomères d’α-PNA consécutifs sont «agrafées» via un pont lactame. Une stratégie de synthèse a tout d'abord été développée en phase liquide, puis appliquée à la synthèse en phase solide de di-α-PNA «agrafés» modèles, incorporant des bases nucléiques thymine. / The purpose of this thesis concerned the development of two kinds of constrained oligomeric structures, based either on unnatural cyclic α-amino acids derivatives or on cyclic Peptide Nucleic Acid (PNA) dimers, in view of analyzing their propensity to adopt pre-organized conformations mimicking the active conformations of proteins or nucleic acids. The first part of this thesis reports on the elaboration of new cyclic αamino-acids (6-substituted 4-oxopipecolic acids) building blocks, their potential use in the synthesis of constrained homogenous and heterogeneous peptidomimetics as well as their limitations in this use. As part of this work, a new methodology has been developed for the synthesis of N-protected 6-substituted 4-oxo-pipecolic acids residues. The second part of this thesis reports on the elaboration of constrained α-PNA dimers (di-α-PNA), in which the side-chains of two consecutive α-PNA monomers are “stapled” via a lactam bridge. A synthetic orthogonal strategy has been first developed in liquid-phase then applied to the solid-phase synthesis of models “stapled” di-α PNA incorporating thymine nucleobases.

Ce mémoire présente les travaux de thèse visant à étudier le comportement parasismique des éléments de murs à ossature en bois aux travers d’approches expérimentales et du développement d’une méthodologie d’application de méthodes basées sur la performance sismique. Les approches expérimentales sont constituées de trois campagnes d’essais : (1) une série d’essais monotones et cycliques à l’échelle d’assemblages agrafés et pointés, (2) une série d’essais monotones et cycliques à l’échelle de l’élément de mur à ossature en bois et (3) une série d’essais dynamiques sur table vibrante. La base de données constituée par les résultats d’essais permet alors l’examen des propriétés de résistance et de raideur des éléments de murs suivant les conditions de sollicitation (vitesse d’essai, chargement vertical). Le développement d’une modélisation à l’échelle macro du comportement cyclique et dynamique de tels éléments est également proposé à l’aide de modèles de loi de comportement hystérétiques. Une méthodologie d’application aux structures à ossature bois de méthodes basées sur la performance sismique (méthode N2 et MPA) ainsi qu’une analyse de vulnérabilité - construction de courbes de fragilité - à l’aide de la méthode N2 sont proposées. / This thesis presents a study of the seismic behavior of light timber frame walls with stapled and nailed sheathings through experimental approaches and the development of a methodology for the application of seismic performance-based methods. The experimental approaches consist of three test campaigns: (1) a series of static tests on stapled and nailed connections, (2) a series of static tests performed on light timber frame walls and (3) a series of dynamic tests performed on light timber frame walls on a vibrating table. The database consists of these test results then allows the examination of strength and stiffness properties of the wall elements according to the stress conditions (strain rate, vertical load). The development of a macro-scale modeling of the cyclic and dynamic behavior of such elements is also proposed using constitutive law models. A framework of the application to light timber frame structures of seismic performance-based methods based (N2 method and MPA method) and a vulnerability analysis - fragility curves - using the N2 method are proposed.