Com o objetivo de analisar a influência do fenômeno de congelamento e o tempo de criopreservação sobre as propriedades biomecânicas de tendões, os autores estudaram 40 tendões de Aquiles obtidos de 20 cadáveres humanos. De cada cadáver foram retirados dois tendões, sendo que um foi testado a fresco e o contralateral congelado a - 85o C, durante um período de seis ou 12 semanas. Os corpos de prova foram submetidos a ensaios de tração, com análise de suas propriedades biomecânicas. Os resultados foram comparados estatisticamente pelo método de "t-student", com índice de significância de 0,05, não havendo diferença significativa entre os grupos "congelado" e "a fresco" / For the purpose of analyzing the influence of the freezing phenomenon and cryopreservation time over the biomechanical properties of tendons, the authors studied 40 Achilles tendons from 20 human cadavers. Each cadaver had two tendons removed, one of which was tested whilst fresh and its contralateral whilst frozen at - 85o C, for a period of six or 12 weeks. The trial items were submitted to tensile testing in order to analyze their biomechanical properties. The results were compared statistically using the T-Student method, with a significance ratio of 0.05, there being no significant difference between the 'frozen' group and the 'fresh' group

Buscando alternativas ao enxerto ósseo autógeno para reconstruções craniofaciais, propõe-se a utilização de aloenxerto ósseo de crânio criopreservado, irradiado e enriquecido com medula óssea autógena.Vinte e um coelhos foram sacrificados e tiveram seus ossos parietais retirados, criopreservados e irradiados com irradiação gama em dose de 50kGy. Os aloenxertos foram implantados em defeitos nos crânios de vinte e um receptores. À esquerda implantava-se o aloenxerto ósseo puro e à direita o aloenxerto adicionado de medula óssea autógena. Os coelhos foram sacrificados em duas, cinco e dez semanas e os enxertos submetidos à análise macroscópica e histomorfométrica. Todos os enxertos apresentavam-se fixos ao leito. Os parâmetros histomorfométricos não mostraram diferenças estatisticamente significante. / Searching for alternatives to autogenous bone graft in craniofacial reconstructions, it is proposed deep-frozen, irradiated calvarial bone allograft added with autogenous bone marrow utilization. Twenty and one rabbits were sacrificed and their parietal bones harvest, deep-frozen and gamma irradiated with 50kGy. The allografts were implanted in twenty one other rabbits. At left side only allograft were placed, at right side were put allografts added with autogenous bone marrow. The animals were sacrificed in two, five and ten weeks. Macroscopical examination showed all grafts fixed at the host bone, the histomorphometric analysis did not showed significant statistical differences between the left and right sides.

Over the course of an organism's life, its genome is exposed to endogenous and exogenous chemical, physical and biological agents - genotoxins. These genotoxins alter its basic structural components - sugar residues, phosphodiester bonds, and nitrogenous bases. Organisms have therefore evolved a plethora of different strategies to both repair DNA lesions and maintain genomic stability. These DNA repair pathways are linked with several other cell pathways, including chromatin remodelling, DNA replication, transcription, cell cycle control, apoptosis - programmed cell death (PCD), thereby providing a coordinated cellular response to DNA damage. Biochemical mechanisms of DNA repair are relatively well understood in yeast and mammals, however, far less so in plants. While these repair mechanisms are evolutionary conserved, significant differences still remain. Therefore, further investigation is required. This thesis summarises the introduction of a novel plant model - the moss, Physcomitrella patens (Physcomitrella). As a haploid gametophyte with unique characteristics of high frequency of homologous recombination (HR), and apical growth of filaments, it is an ideal organism to study DNA repair in plants. Previous research on Physcomitrella regarding mechanisms of DNA lesion repair induced by...

O projeto de obtenção de huAcMos (Anticorpos Monoclonais Humanizados) tinha como escopo a humanização de anticorpos murinos com potencial terapêutico, inserção das sequências em vetores de expressão e transfecção em células CHO (do inglês, Chinese Hamster Ovary). A expressão do huAcMo Anti-CD18 resultou em baixos níveis da proteína recombinante e inciamos o processo de expressão de isoformas do huAcMo Anti-CD3. As células foram transfectadas com seqüências codificadoras do fragmento FvFc Anti-CD3 e clonadas pelo equipamento ClonePix FL. O fragmento foi caracterizado e demonstrou uma menor afinidade quando comparada com a molécula murina original. Ulizamos o sistema de recombinação homóloga (CHO Flp-In, Invitrogen) para expressão da molécula inteira do huAcMo Anti-CD3. Os clones foram caracterizados e demonstrou, assim como o fragmento FvFc, uma menor afinidade pelo alvo. As diferenças nas propriedades de ligação são freqüentemente encontradas após processos de humanização; dependendo da função efetora esta diminuição de afinidade não é negativa para a molécula. / The humanized antibodies (huMab) project intent to use murine antibodies with therapeutic potencial to obtain more human sequences with maintened specificity. The sequences were inserted in expression vectors and transfected in CHO (Chinese Hamster Ovary) cells. Anti-CD18 huMab expression results in low levels of recombinant protein and lead us to try the expression of Anti-CD3 isoforms. The cells were transfected for the expression of a FvFc antibody fragment and cloned using ClonePix FL equipment. The fragment characterization demonstrate a lower affinity when compared with the murine molecule. We use the homologous recombination system (CHO Flp-In) for the expression of the whole molecule of huMab Anti-CD3; like the FvFc fragment, the whole molecule demonstrate a lower affinity for the target. The differences in the affinity properties are frequently found after humanization process and depending on the expected efector functions is not negatively characterized.

This diploma thesis is focused on development of a fast method for site-directed gene mutagenesis in Streptococcus zooepidemicus based on the mechanism of natural competence. Several genes were selected based on experimental data which highly probably influence hyaluronic acid synthesis. The deletion of the selected genes from genomic DNA was performed as proof of concept, and the resulting recombinant strains were characterized regarding changes of hyaluronic acid precursor concentrations (glucuronic acid and N-acetylglucosamin) in time of cultivation and the end production of hyaluronic acid.

Les modifications post-traductionnelles des protéines par des polymères d’ADP-ribose (PAR) ou par phosphorylation permet l’assemblage des complexes de la réparation de l’ADN à la chromatine endommagée dont les fonctions sont essentielles pour assurer le maintien de la stabilité du génome. En réponse aux lésions de l’ADN, l’activité de synthèse de PAR des protéines PARP1 et PARP2 est fortement stimulée. Les PAR servent de signalisation pour le recrutement de multiples protéines, dont la protéine plateforme XRCC1.Les études menées au cours de cette thèse ont porté sur l’étude de la régulation des fonctions des protéines PARP1, PARP2 dans la réparation des cassures double brins (CDB) et l’étude des modifications de XRCC1 par phosphorylation en réponse à des dommages de l’ADN. En utilisant des substrats permettant de mesurer l’efficacité des différentes voies de réparation des CDB, nous avons démontré que PARP2, et non PARP1, est impliqué dans la régulation du choix des voies de la réparation des CDB. Plus spécifiquement, nous avons montré que PARP2 stimule l’initiation de la résection des extrémités des CDB dépendante de CtIP, indépendamment de son activité catalytique. Par des approches de vidéo-microscopie, nous avons pu déterminer que PARP2 limite l’accumulation de 53BP1 aux sites de dommages induits par micro-irradiation laser. Nous proposons que la protéine PARP2, en limitant le recrutement de la protéine 53BP1 aux sites de dommages, favorise la réparation des CDB dépendante de la résection des extrémités d’ADN, au détriment de la voie canonique de jonction des extrémités. Ces résultats sont les premiers démontrant un rôle de PARP2 dans le choix des voies de réparation des CDB.En parallèle, nous avons analysé comment la phosphorylation régule les fonctions de la protéine XRCC1. Par des approches in vitro et in vivo, nous avons pu déterminer que l’interdomaine 1 de XRCC1 est phosphorylé par la kinase CDK5. En réponse aux dommages induits par un agent alkylant, XRCC1 est activement déphosphorylé in vivo. De plus, nous avons observé que lorsque l’interdomaine 1 ne peut pas être phosphorylé in vitro, l’interaction de XRCC1 avec les PAR synthétisés par PARP1 et PARP2 augmente, et le recrutement de XRCC1 aux sites de dommages de l’ADN est accru. Ces résultats indiquent pour la première fois que la déphosphorylation de XRCC1 en réponse à un stress génotoxique participe activement à son recrutement aux sites de dommages.Dans leur ensemble, ces travaux ont contribué à améliorer nos connaissances fondamentales des réseaux de protéines impliquées dans la prise en charge des dommages de l’ADN. La compréhension de ces mécanismes est essentielle non seulement car ils participent au maintien de la stabilité du génome mais aussi du fait du développement exponentiel de nouvelles stratégies anti-tumorales qui visent à inhiber les voies de la réparation dans la but de cibler spécifiquement les cellules cancéreuses. / Post-translational modifications of proteins by polymers of ADP-ribose (PAR) or by phosphorylation allow the assembly of DNA repair protein complexes at damaged chromatin and are crucial to ensure genome stability. In response to DNA insults, the synthesis of PAR by the PARP1 and PARP2 proteins is strongly induced. PAR act as a signaling platform for the recruitment of multiples proteins at the sites of DNA damages, including the scaffold protein XRCC1. Research conducted during this PhD have been focused on studying the regulation of PARP1 and PARP2 functions in double-strands break repair (DSBR), and in investigating the role of XRCC1 modifications by phosphorylation in response to DNA damage.Using DNA repair assay allowing us to assess the accuracy of the different DSBR pathways, we demonstrated that PARP2, and not PARP1, is involved in the regulation of DNA double-strands break repair pathway choice. More precisely, we showed that PARP2 stimulates CtIP dependent initiation of end-resection at DSB, independently of its catalytic activity. By live cell imaging, we were able to determine that PARP2 limit 53BP1 accumulation at DNA damage sites induced by laser-microirradiation. We propose that by limiting 53BP1 accumulation at DNA damage sites, PARP2 stimulate DSB repair pathway that depend on DNA end-resection, thus counteracting the canonical end-joining pathway. These results are the first demonstrating a role for PARP2 in DNA DBSR pathway choice.In addition, we analyzed how the functions of XRCC1 are regulated by phosphorylation. Using in vitro and in vivo approaches, we were able to demonstrate that the linker 1 region of XRCC1 is phosphorylated by the CDK5 kinase. XRCC1 is actively dephosphorylated in response to DNA damage induced by an alkylating agent in vivo. We also observed that when the linker 1 cannot be phosphorylated, the XRCC1 interaction between the PAR synthetized by PARP1 and PARP2 is stimulated, and XRCC1 recruitement at the sites of DNA damage is far more efficient. These evidences indicate for the first time that the dephosphorylation of XRCC1 actively participate in its recruitment at the site of DNA damage. Put together, this work contributed to strengthen our fundamental knowledge of the protein network involved in the DNA damage response. Knowledge of those mechanisms is crucial since they participate in maintaining genome stability, and because new antitumoral drugs targeting DNA repair pathways in the attempt to specifically killed tumor cells are exponentially released.

BACKGROUND: The inactivation of the tumor suppressor gene BRCA1 is a predisposing factor for a breast/ovarian cancer development. Formation of cancer-specific alternative splicing variants with aberrant biological properties can represent additional mechanism decreasing the overall BRCA1 activity in DNA double strand break (DDSB) repair. In this study, we analyzed BRCA1 alternative splicing variants BRCA114-15 and 17-19 ascertained previously during the screening of high-risk breast cancer individuals. METHODS: We established a stable MCF-7 cell line-based model system for an in vitro analysis of BRCA1 variants. Using this system, we analyzed the impact of BRCA114-15 and 17-19 variants on DNA repair kinetics using comet assay and confocal immunomicroscopy. The capacity of DNA repair was assessed directly by an in vitro NHEJ assay and indirectly by a mitomycin C sensitivity test. The proliferation activities were determined by a clonogenic assay and growth curves. RESULTS: Overexpression of BRCA114-15 and 17-19 increases the endogenous level of DNA damage, slows down the DDSB repair, and decelerates the initial phase of radiation-induced foci formation and prolongs their persistence. Moreover, BRCA114-15 and 17-19 differentially influence the activity of HR and NHEJ and sensitivity of MCF-7 cells to ionizing...

Les réarrangements permettent une évolution rapide du génome par l’acquisition de séquences codantes exogènes, la perte de fonctions non-essentielles ou la création de nouvelles organisations génomiques. Différents mécanismes de réarrangements impliquant des éléments génétiques mobiles (EGM) ont été identifiés chez les archées, les bactéries et les eucaryotes. En revanche, on ignore l’origine des nombreuses inversions génomiques détectées pour les espèces du genre archéen Thermococcus. Mes travaux de thèse visent à améliorer la compréhension de l’évolution génomique chez les Thermococcales à travers l’étude de deux familles d’EGM : les familles de plasmides pTN3 et pT26-2. Plus précisément, je me suis intéressée aux recombinases à tyrosine (ou intégrases) que ces plasmides encodent et qui permettent leur intégration dans le chromosome de l’hôte. J’ai montré que l’intégrase plasmidique Intᵖᵀᴺ³ est responsable d’inversions dans le chromosome de son hôte Thermococcus nautili grâce à une activité catalytique inédite de recombinaison homologue. J’ai par la suite caractérisé deux autres intégrases de Thermococcales reliés phylogénétiquement à Intᵖᵀᴺ³ dont seulement une présente une activité de recombinaison homologue. La comparaison de leurs séquences primaires et la résolution de la structure de Intᵖᵀᴺ³ vont maintenant éclairer les déterminants génétiques responsables de la spécificité de site et de l’activité de recombinaison homologue. Les trois intégrases appartiennent à une classe de recombinases spécifique des archées qui catalyse une intégration suicidaire. Lors de l’intégration, le gène de l’intégrase est fragmenté et probablement désactivé. L’EGM intégré se retrouve piégé dans le chromosome. Les avantages évolutifs d’une telle activité suicidaire restent pour l’instant mystérieux. J’ai identifié 62 intégrases hyperthermophiles suicidaires et reconstruit leur histoire évolutive. Ces intégrases sont très prévalentes et recrutées par différents EGM. De plus, j’ai montré que l’une de ces intégrases présente in vitro une activité de recombinaison site-spécifique à des températures proches de l’ébullition de l’eau, représentant un avantage dans les environnements hyperthermophiles. / Genomes rapidly evolve through rearrangements that can generate new genome organizations or lead to the acquisition of foreign coding sequences or the loss of non-essential functions. Several mechanisms of rearrangement were uncovered for Archaea, Bacteria and Eukaryotes that involve mobile genetic elements (MGE). Species from the archaeal genera Thermococcus present numerous genomic inversions but none of the previously known inversion drivers. To better understand the genomic evolution of Thermococcales, I investigated two of their MGE families: the pTN3 and pT26-2 plasmid families. Specifically, I focused on the tyrosine recombinases (or integrase) that these plasmids encode and that catalyze their site-specific integration in the host chromosome. I demonstrated that the plasmidic integrase Intᵖᵀᴺ³ is responsible for chromosomal inversions in the host Thermococcus nautili through an unprecedented homologous recombination catalytic activity. I also characterized two other related Thermococcus integrases and only one catalyzes homologous recombination. The structure resolution of Intᵖᵀᴺ³ and primary sequence comparisons will now provide clues about the genetic determinants of site specificity and of the homologous recombination activity. The three integrases all belong to an archaeal-specific class of integrases that catalyzes a suicidal integration. The integrase gene is partitioned and presumably inactivated upon integration. The integrated MGE is then trapped into the chromosome. The evolutionary benefits of this suicide activity are puzzling. I identified 62 related suicidal hyperthermophilic integrases and reconstructed their evolutionary history. They are highly prevalent and recruited by diverse MGE. I also showed that one of these integrases can catalyze in vitro site-specific recombination at near boiling water temperature, representing an advantage in hyperthermophilic environments.

Ovarian cancer is serious and one of the most common gynecologic cancers. Carboplatin is the therapeutic agent of the first choice in the ovarian cancer therapy. However, after the primary therapeutic response to carboplatin, the relapse of the disease may occur with developed resistance to carboplatin. Chemoresistance and insufficient therapy response are considered to be the reason of the high mortality rate of ovarian cancer. The DNA damage response pathways play an important role in the therapeutic response and chemoresistance development. Restoration of homologous recombination function in cancers is the key mechanism of resistance development to platinum agents. Based on this knowledge, we formed our hypothesis, that the inhibition of homologous recombination could increase the sensibility to carboplatin. The main goal of this thesis was to define the role of double-strand breaks repair in response to chemotherapy of ovarian cancer. Protein MRE11 is part of the MRN complex, that participates in double-strand breaks repair. Using mirin as a pharmaceutic inhibitor of MRE11 we were aiming to determine the impact of homologous recombination on the effect of carboplatin and its role in resistant development to carboplatin. In the practical part of the thesis, we described the association between...

RAD18 is an E3 ubiquitin ligase that prevents the replication forks from collapsing caused by damaged DNA. As an important factor controlling replication, its dysregulation was shown to be associated with some human tumours. However, the clinical relevance of this finding is unknown. The aim of the thesis was evaluation of selected RAD18 variants that had been identified in breast and ovarian cancer patients. This work revealed functional defects of RAD18 variants not only in replication fork protection but also in repair of DNA double-strand breaks. This unconventional role of RAD18 is known to be dependent on upstream ubiquitination events, however, its contribution to the repair per se is not understood. This work aimed to elucidate the function of RAD18 in DNA double-strand break repair by homologous recombination focusing especially on its relationship with 53BP1. Data presented here show that RAD18 effectively disrupts 53BP1 accumulation in the repair foci by competition for the same binding partner and thus promotes resection of DNA ends. This antagonistic function of RAD18 is restricted both spatially (to the vicinity of the repair centre) and temporarily (to S phase). Moreover, it seems to be regulated by existence of RAD18 in two distinct complexes. Potential models for this regulation...