Orientador: Sueli Aparecida Calvi / Banca: Cilmery Suemi Kurokawa / Banca: Lisiane de Almeida Martins / Resumo: Não disponível / Abstract: Toll-like receptors (TLRs) present in innate immune cells recognize pathogen molecules and influence on immunity to control the host-parasite interaction. Our objective was to evaluate the mRNA expression of TLR-2 and 4, expression and production of IL-12, IFN-γ, TNF-α, IL-17, IL-10 and TGF-β and NO production during infection with Leishmania chagasi and correlate TLR2 and 4 expressions with cytokines production and NO. Infection resulted in increased TLR2, TLR4, IL-17, TNF-α and TGF-β expression at the beginning of infection, with a decrease at the final phase in according the parasitic load; IFN- γ and IL-12 decreased at the peak of parasitemia and increased at the final phase; IL-10 increased during the whole period under analysis. With respect to cytokines and NO production, TGF-β, TNF-α and IL-17 showed high rates at the initial phase, and IFN-γ and IL-12 showed high rates at the final phase; IL-10 and NO showed increasing production during the infection period evaluated. There was a positive correlation of TLR2 and 4 with TNF-α, IL-17, NO, IL-10 and TGF-β at the beginning of infection, and with TNF-α, IL-17 and TGF-β at the end. Our data suggest that L. chagasi was in contact with host's cells via TLR2 and 4, which resulted in cytokine modulation. This interaction could be considered as pathogenic mechanism in visceral leishmaniasis / Mestre

Orientador: Marcos Roberto de Mattos Fontes / Coorientador: Agnes Alessandra Sekijima Takeda / Banca: Maria Célia Bertolini / Banca: Rafael Lemos Miguez Counãgo / Banca: Valber de Albuquerque Pedrosa / Banca: Lucilene Delazari dos Santos / Resumo: Danos no DNA, podem ocorrer tanto por agentes genotóxicos endógenos quanto agentes exógenos, que podem promover a instabilidade do genoma e levar diretamente a doenças, como por exemplo, o câncer, alterações neurológicas, imunodeficiências e envelhecimento prematuro. Auxiliando na manutenção da estabilidade, as células apresentam uma série de vias de reparo de DNA, as quais realizam o processo em múltiplas etapas para resolver lesões específicas no DNA e manter a integridade do genoma. A importação nuclear é um pré-requisito para as funções das proteínas de reparo do DNA e dentre os mecanismos responsáveis pela regulação da importação nuclear, a via clássica constituída pelo heterodímero Importina-α/β é um dos principais mecanismos de deslocamento. A Importina-β (Impβ) atua como o transportador enquanto a Importina-α (Impα) atua como adaptador, reconhecendo as sequências de localização nuclear (NLS) presentes nas proteínas que possuem função no núcleo. Esse trabalho trata especificamente dos estudos estruturais de complexos da Impα peptídeos NLSs de proteínas relacionadas ao reparo de DNA utilizando técnicas de cristalografia e ensaios de afinidade pela técnica de calorimetria de titulação isotérmica (ITC). A expressão e purificação da Impα de Mus musculus truncada em sua porção N-terminal foi realizada, bem como a co-cristalização da Impα peptídeos NLSs de proteínas relacionadas ao reparo de DNA, correspondentes as sequências MLH1, PMS2, XPG1 e XPG2. Peptídeos mutados em regiões importantes de reconhecimento nuclear para os peptídeos MLH1 e PMS2 também foram selecionados para o desenvolvimento deste projeto. Dados de difração de raios-X foram coletados dos cristais obtidos e processados no intervalo de 2,0-2,8 Å de resolução. Com esses resultados, as estruturas contendo cNLSs MLH1, PMS2, XPG1 e XPG2 foram elucidadas. As proteínas do complexo MutLα, a MLH1 e... / Abstract: DNA damage can occur by endogenous and exogenous genotoxic agents, which may promote instability of the genome and directly lead to diseases such as cancer, neurological disorders, immunodeficiencies and even premature aging. Helping in the maintenance of the stability, the cells display a number of DNA repair pathways, which carry out the process in multiple steps to resolve specific DNA damage, and maintain the integrity of the genome. The nuclear import is a pre-requisite for the functions of DNA repair proteins and, among the mechanisms responsible for regulation of nuclear import, the classical pathway constituted by the heterodimer importin-α / β is a major shift mechanisms. Importin-β (Impβ) acts as the carrier while the importin α-(Impα) acts as an adapter recognizing the nuclear localization sequence (NLS) present in proteins that have function into the nucleus.This work concerns specifically the structural studies of complexes with Imp and NLSs peptides from proteins related to DNA repair using crystallographic techniques and binding assays by isotermal titration calorimetry technique (ITC). The expression and purification of Mus musculus Impα truncated at its N-terminal portion was performed, as well as co-crystallization with Impα NLSs peptides of proteins related to DNA repair, the corresponding sequences MLH1, PMS2, XPG1 and XPG2. Peptides mutated in key regions of nuclear recognition for MLH1 and PMS2 peptides were also selected for this project. X-ray diffraction data collected from crystals were obtained and processed in the range of 2.0-2.8 Å resolution. With these results, the structures containing cNLSs MLH1, PMS2, XPG1 and XPG2 were elucidated. The MutLα complex proteins, MLH1 and PMS2, related to the mismatch repair (MMR), bound to the Impα similarly to the T antigen NLS of SV40 in the major binding site. ITC experiments corroborate the crystallographic results, which suggest that both NLSs are classic... / Doutor

Parmi les différents phénomènes responsables de la déformation à court et long terme des roches poreuses, on s’intéresse dans ce travail à l’endommagement d’un calcaire oolitique en régime semi-fragile et sous différentes conditions hydriques. Dans ce but, nous avons élaboré un dispositif expérimental permettant de mesurer les vitesses des ondes ultrasonores et les déformations ainsi que d’écouter les émissions acoustiques sur le même échantillon, sous chargement mécanique et sous différentes conditions hydriques. En particulier, le dispositif expérimental permet de mesurer simultanément, les vitesses des ondes ultrasonores selon 3 directions de propagation (axiale, latérale et hors axe) et 3 polarisations différentes (P et 2 S perpendiculaires), ceci quasi-instantanément, ce qui in fine permet d’enregistrer les évolutions des vitesses des ondes ultrasonores en continu pendant le chargement. Ces mesures de vitesses permettent d’évaluer le tenseur acoustique à tout moment et donc de suivre en continu l’évolution de l’état d’endommagement de la roche. Le comportement endommageable à court et long terme du calcaire est modélisé à l’aide d’un modèle phénoménologique qui est basé sur une généralisation macroscopique des mécanismes microscopiques mis en évidence par les études expérimentaux (glissement-ouverture des fissures existantes, nucléation de nouvelles fissures, propagation et coalescence de fissures). Le modèle à court terme reproduit assez bien le comportement expérimental instantané de la roche (courbes contrainte-déformations et évolution des modules élastiques). Le modèle de comportement à long terme, découplé dans sa formulation du modèle à court terme, permet de reproduire qualitativement les courbes de fluage expérimentales / Among the different phenomena responsible for the short and long term deformation of porous rocks, we have studied in this work the damage of an oolithic limestone in the semi-brittle regime and under different hydrous conditions. For this purpose, we have developed an experimental device allowing the simultaneous and continuous measurement of strains and elastic wave velocities, as well as acoustic emissions, on the same sample under mechanical loading and under different hydrous conditions. Particularly, the experimental setup allows simultaneous and continuous measurement of the five elastic wave velocities in 3 different directions of propagation (axial, lateral and off-axis) and 3 different directions of polarization (P and 2 perpendicular S), this almost instantaneously. These velocity measurements allow to assess the acoustic tensor at any time and thus to continuously monitor the evolution of the damage of the rock. The short and long term damage behavior of the limestone is modelled thanks to a phenomenological model which is based on a macroscopic generalization of the microscopic mechanisms highlighted by the experimental study (sliding-opening of existing cracks, nucleation of new cracks, propagation and coalescence of cracks). The short-term model reproduces very well the instantaneous behavior (stress-strain curves and evolution of elastic moduli). The long-term model, whose formulation is uncoupled from the short-term model, allows reproducing qualitatively the experimental creep curves

Dans le contexte du développement durable, la fissuration des structures en béton armé est devenue un sujet d'étude central. Deux niveaux d'analyse de la durabilité d'un ouvrage apparaissent : l'échelle de la structure et l'échelle de la fissure. A l'échelle de la structure, les modèles à éléments finis basés sur la mécanique de l'endommagement peuvent être utilisés. Cependant, l'utilisation de ces modèles nécessite l'introduction d'une longueur caractéristique afin de prévenir la dépendance au maillage. Or l'introduction de cette longueur caractéristique empêche l'étude fine de la fissuration. A l'échelle de la fissure, la discontinuité peut être décrite de manière explicite à l'aide d'un modèle à éléments discrets. Grâce à ces modèles, les principaux mécanismes de dégradation du béton sont reproduits. Mais, l'étude d'ouvrages industriels en béton n'est pas possible à cause de la finesse de maillage nécessaire à l'étude de la fissuration. Dans ce travail, une méthode de couplage non-intrusive est proposée. A l'échelle globale, un modèle à éléments finis basé sur la mécanique de l'endommagement est utilisé. Les zones d'intérêt -- c'est-à-dire les zones de concentration d'endommagement -- sont analysées à l'échelle locale à l'aide d'un modèle à éléments discrets. Les résultats numériques obtenus à l'aide de la stratégie proposée sont comparés à des résultats expérimentaux afin de valider la méthode. Plusieurs cas tests sont étudiés afin de montrer les capacités de l'approche à modéliser la propagation des fissures pour une structure en béton sous chargement. Des problèmes spécifiques au béton comme l'effet d'échelle, l'impact des armatures ou l'effet 3D sont adressés. / In the context of sustainable development, the study of cracking of large concrete structures has become of primary importance. Two levels of analysis appear: the structure scale and the crack scale. At the structural level, non-linear finite element analyses based on continuum damage mechanics can be carried out. Nevertheless, the use of such models requires the introduction of a characteristic length to prevent the occurrence of spurious mesh dependency in case of strain softening. This characteristic length tends to smooth the discontinuity and thus makes the study of the cracks harder. At the crack level, an explicit description of the crack can be achieved using discrete element methods. This way, the main failure mechanisms of quasi-brittle materials are recovered. But the mesh density required for such modeling is nevertheless prohibitive to treat a whole industrial structure. A non-intrusive technique is proposed, allowing the use of finite element models at a global scale and a decoupled local analysis of some interesting areas, i.e. around cracks, for which a discrete element model is used. Numerical results obtained from the proposed strategy are compared with experimental results and show the applicability of the approach. Several test cases are studied proving the capability of the approach to model the cracks propagation of loaded concrete structures. Specific concrete problems such as scale effect, 3D effect or rebars impact are addressed.

DNA damage from both normal metabolic activities and environmental factors such as UV and radiation can cause as many as 1 million individual lesions to the DNA per cell per day (Lodish et al 2004). Cells respond to this continuous damage by employing many, highly efficient DNA repair mechanisms and undergo apoptosis when normal DNA repair fails. Of the many types of DNA damage that can occur, double strand breaks (DSBs) are the most toxic (Featherstone & Jackson 1999). A single unrepaired DSB is enough to induce cellular apoptosis and several mechanisms have developed to repair DSBs. The recognition, signalling and repair of DSBs involve large multi-­‐subunit complexes that bind to both the DNA and modified histone tails, which require modification of the chromatin in order to access their bind sites and function effectively (Allard et al 2004). Consequently several chromatin-­‐remodelling proteins have been implicated in DSB repair (van Attikum et al 2004, Chai et al 2005). LSH (Lymphoid specific helicase) is a putative chromatin-­‐remodelling enzyme that interacts with DNA methyltransferases and has been connected to DNA methylation (Myant & Stancheva, 2008). Knockouts of LSH or its homologues in A. thaliana and M. musculus show a reduction in DNA methylation of 60-­‐70% (Jeddeloh et al 1999, Dennis et al 2001). However in addition to this phenotype, knockout A. thaliana also have an increased sensitivity to DNA damage (Shaked et al 2006). A homologue of LSH has also been identified in S. cerevisiae, which interacts with known repair proteins (Collins et al 2007) and may be involved in DSB repair. Although the majority of Lsh-­‐/-­‐ mice die shortly after birth, 40% of the line produced by Sun et al survive and show unexplained premature aging (Sun et al 2004). As premature aging is a hallmark of increased acquisition of DNA damage there is the possibility of a conserved role for LSH in mammalian DNA damage repair. Here I show that LSH depleted mammalian cells have an increased sensitivity specifically to DSB inducing agents and show increased levels of apoptosis. Further analysis shows that cells lacking LSH repair DSBs slower, indicating a novel role for LSH in mammalian repair of DSB. I performed an in depth analysis of the DSB defects in LSH depleted cells in an attempt to elucidate the function of LSH in DSB repair. I found that LSH depleted cells can correctly recognise DSBs but recruit downstream signalling and repair factors, such as γH2AX, less efficiently. I show that reduced recruitment of downstream DSB repair factors is not accompanied by extended cell cycle checkpoint signalling. This suggests that LSH depleted cells continue through the mitosis with unrepaired DSBs, which most likely leads to apoptosis and the increased sensitivity to DSB inducing agents. These experiments also showed that recruitment of DSB signalling and repair factors is not impaired equally at all breaks, and I present a model system created to quantitatively compare individually breaks between WT and LSH depleted cells to identify DSB that require LSH for efficient repair. I also preformed an analysis of Lsh-­/-­ MEFs containing WT or catalytic null mutant LSH rescue constructs and I show that WT but not catalytic null LSH can restore efficient DSB repair. These studies identify a novel role for LSH in mammalian DSB repair and demonstrate the importance of its catalytic activity.

Budding yeast Slx4 binds to the structure-specific DNA repair nucleases Slx1 and Rad1XPF-Rad10ERCC1, and it was reported that Slx4 is essential for DNA flap cleavage by Rad1XPF-Rad10ERCC1 during certain types of DNA repair in yeast. At the outset of this thesis, bioinformatic analyses identified the uncharacterised protein BTBD12 in higher eukaryotes as a putative orthologue of yeast Slx4. In the first results chapter of this thesis, I describe the identification of BTBD12-interacting proteins, including XPF-ERCC1 and SLX1. These findings led me to refer to BTBD12 as human SLX4. I found that SLX4 binds to another structure-specific nuclease MUS81-EME1, and other proteins involved in telomere maintenance and cell cycle progression. The remainder of this chapter describes detailed biochemical analysis of the nuclease activities associated with the SLX4 complex isolated from human cells. Work from this lab and others revealed that depletion of SLX4 from human cells using siRNAs causes defects in the repair of DNA interstrand crosslinks (ICLs). Inherited mutations in humans that reduce the efficiency of ICL repair cause Fanconi anaemia (FA). The cellular sensitivity of SLX4 depleted cells to ICLs prompted me to investigate SLX4 as a candidate FA gene. Dr. Johan de Winter (VU University Medical Center, Amsterdam) and Dr. Detlev Schindler (University of Wurzburg) had identified several patients with unclassified FA that was not caused by mutations in the FA genes known at the time. In the second results I describe characterisation of SLX4, and the SLX4 holo-complex, in cells from some of these FA patients who had bi-allelic SLX4 mutations. In three of the patients SLX4 was expressed at normal levels but was missing part of the first, and all of the second, UBZ-type putative ubiquitin-binding domain. This prompted me to investigate the function of the SLX4 UBZ domains. I found that the first, but not the second, UBZ domain of SLX4 binds to ubiquitin in vitro and targets SLX4 to sites of DNA damage in vivo. Furthermore, the first but not the second SLX4 UBZ domain appears to be required for ICL repair, demonstrating the important of correctly localising SLX4 for DNA repair. In the final chapter, I present preliminary data which suggests that SLX4 is regulated in an unusual manner in during S-phase of the cell cycle, and that SLX4 interacts with the PLK1 kinase in a phosphorylation-dependent manner.

DNA damage response (DDR) pathways are essential in maintaining genomic integrity in cells, but many DDR proteins have other important functions such as in the innate immune sensing of cytoplasmic DNA. Some DDR proteins are known to be beneficial or restrictive to viral infection, but most remain uncharacterised in this respect. Non-homologous end joining (NHEJ) is a mechanism of double stranded DNA (dsDNA) repair that functions to rapidly mend broken DNA ends. The NHEJ machinery is well characterised in the context of DDR but recent studies have linked the same proteins to innate immune DNA sensing and, hence, anti-viral responses. The aim of this thesis is to further investigate the interplay between herpes simplex virus 1 (HSV-1), a dsDNA virus, and two NHEJ proteins, DNA protein kinase catalytic subunit (DNA-PKcs) and paralogue of XRCC4 and XLF (PAXX). PAXX was first described in the literature as a NHEJ protein in 2015, but whether it has any role in the regulation of virus infection has not been established. Here we show that PAXX acts as a restriction factor for HSV-1 because PAXX-/- (KO) cells produce a consistently higher titre of HSV-1 than the respective wild type (WT) cells. We hypothesised that this could be due to a role of PAXX binding viral DNA and directly inhibiting HSV-1 replication or activating an anti-viral innate immune response. We have been able to, at least partially, rule out both of these initial hypotheses by showing that there was a reduced number of viral genomes present in KO cells during active lytic infection, and that an identical level of type I interferons are produced from WT and KO cells during HSV-1 infection. Although further characterisation of HSV-1 infection in WT and KO cells has not defined the molecular mechanism of restriction of HSV-1 by PAXX, we have uncovered a potential role for PAXX in mitogen-activated protein kinase (MAPK) signalling. In addition, and consistent with its function in restriction of HSV-1 infection, we show that infection with this virus in WT cells induces a loss of nuclear PAXX protein. Preliminary data suggest that these changes in localisation may occur as a result of stimulation of the cells with DNA, but not the RNA analogue poly(I:C). The role of PAXX in the regulation of HSV-1 infection in vivo was investigated by studying KO mice. Despite previous observations that mice lacking NHEJ proteins have brain defects related to autoinflammatory pathology, there were no obvious defects in the development of Paxx-/- mice, and they had brains of normal weight. No significant difference in viral spread or viral protein expression was observed between WT and KO HSV-1 infected mice, and KO mice did not exhibit abnormal pathology. There were, however, small but significant differences in the cellular immune response to infection which might be explained by reduced MAPK signalling in KO cells. DNA-PKcs is another component of the NHEJ machinery that acts to assist in dsDNA break repair in the nucleus and as an innate sensor of cytoplasmic viral DNA, but the effect of DNA-PKcs on HSV-1 infection has not been fully explored. Murine skin fibroblasts (MSFs) derived from wild type and PRKDC-/- (DNA-PKcs deficient) mice were cultured ex vivo and used for innate immune studies. Although HSV-1 was able to infect and stimulate these cells, no differences in the stimulation of innate immune gene expression between the two genotypes was observed, suggesting that DNA-PKcs does not contribute to HSV-1 sensing in MSFs. It has previously been reported that the HSV-1 protein ICP0 targets DNA-PKcs for degradation, although the reason for this is unknown. We confirmed these data, although found it to be cell-type specific and explored this interaction further using PRKDC-/- RPE-1 cells created using CRISPR/Cas9. HSV-1 infection in these cells followed unusual dynamics, and the development of cytopathic effect was accelerated as compared to WT cells. Together these observations confirm that DNA-PKcs regulates HSV-1 infection, but more work is required to fully understand the mechanisms involved.

Obstacles such as DNA damage can block the progression of DNA replication forks. This is a major source of genome instability that can lead to cell transformation or death. The budding yeast MMS1 and MMS22 genes were identified in a screen for mutants that were hypersensitive to DNA alkylation that blocks replisome progression. I set out to investigate the cellular roles of these genes and found that cells lacking MMS1 or MMS22 are hypersensitive to a wide variety of genotoxins that stall or block replication forks, and are severely defective in their ability to recover from DNA alkylation damage. Homologous recombination (HR) is an important mechanism for the rescue of stalled or blocked replication forks and for the repair of double-strand breaks (DSBs). Strikingly, MMS1 and MMS22 are required for HR induced by replication stress but not by DSBs, and the underlying mechanisms were explored.I next identified the uncharacterized protein C6ORF167 (MMS22L) as a putative human Mms22 orthologue. MMS22L interacts with NF?BIL2/TONSL, the histone chaperone ASF1 and subunits of the MCM replicative helicase. MMS22L colocalizes with TONSL at perturbed replication forks and at sites of DNA damage. MMS22L and TONSL are important for the repair of collapsed replication forks as depletion of MMS22L or TONSL from human cells causes DNA damage during S–phase and hypersensitivity to agents that cause fork collapse. These defects are consistent with the observations that MMS22L and TONSL are required for the efficient loading of the RAD51 recombinase onto resected DNA ends and for efficient HR. These data indicate that MMS22L and TONSL are novel regulators of genome stability that enable efficient HR.

Magister Scientiae Dentium - MSc(Dent) / Aim To analyze the demographic profile, clinical data and radiographs of patients who had third molar surgery under general anaesthesia at the Faculty of Dentistry at the University of the Western Cape over a 10 year period. Introduction Minor oral surgical procedures are carried out by Maxillofacial and Oral Surgeons daily. The surgical removal of third molars is a large part of Minor Oral Surgery which is common throughout the world. The general impression of third molar surgery performed by experienced professionals is the ease of the operation, however no-matter how experienced one may be, a simple procedure should never be underestimated (Carvalho and Do Egito Vasconselos, 2011). New surgical techniques, as well as extensive training, skill and experience have led to the evolution of oral surgery and allowed this procedure to be carried out in a less traumatic manner. Certain factors precipitate third molar surgery to be performed in theatre as opposed to the dental clinic setting. These factors will be discussed in this research report.

No trabalho estudam-se alguns aspectos relativos à formulação teórica e à simulação numérica de modelos constitutivos para o concreto fundamentados na Mecânica do Dano Contínuo, incluindo-se os chamados métodos simplificados de análise estrutural. Inicialmente apresenta-se uma discussão sobre deformações permanentes e anisotropia induzidas pela evolução do dano. A resposta unilateral do concreto submetido a solicitações com inversão de sinal, também é comentada. Cada um dos fenômenos é ilustrado por respostas observadas experimentalmente. O modelo de dano proposto por Mazars para o concreto submetido a carregamento proporcionalmente crescente é então analisado. Em seguida, apresenta-se uma extensão do modelo considerando-se o aspecto unilateral no comportamento do concreto. Na sequência, analisa-se o modelo constitutivo proposto por La Borderie em seus aspectos de formulação e resposta numérica. O modelo é mais completo permitindo levar em conta todos os fenômenos discutidos anteriormente. Um outro aspecto considerado no trabalho é relativo à aplicação dos modelos estudados à análise de estruturas aporticadas. Em termos de discretização destacam-se a técnica de divisão dos elementos estruturais em estratos e os chamados modelos simplificados. Neste último caso, o modelo de Flórez-López é analisado e a simplificação consiste na definição prévia, sobre a estrutura discretizada através de elementos de viga e de coluna, de zonas de localização da plastificação e do dano; no limite com a evolução do processo de carregamento, aquelas zonas passam a se constituir em rótulas. Por último uma generalização do modelo anterior proposta por Álvares é estudada. Os resultados numéricos fornecidos pelos modelos são confrontados com os experimentais de vigas em concreto armado (biapoiadas e com diferentes taxas de armadura) e de um pórtico em concreto armado. / In this work some aspects related to the theoretical formulation and numerical simulation of constitutive models for the concrete based on the Continuum Damage Mechanics are studied, including simplified methods of structural analysis. Initially a discussion about permanent strains and anisotropy induced by the evolution of the damage is presented. The unilateral response of the concrete submitted to loadings with signal inversion is commented as well. Each one of the phenomena is illustrated by experimental tests. The damage model proposed by Mazars for the concrete under proportional increasing load is then analysed. Afterwards, it is shown an extension of the model considering the unilateral aspect ofthe concrete behaviour. In the sequence the constitutive model proposed by La Borderie is presented in its aspects of formulation and numerical response. This model is more complete, allowing to take in to account all the phenomena discussed previously. Another aspect considered in the work is related to the application of the models studied to the analysis of framed structures. Regarding the discretization, the technique of division of the structural elements in layers and the so called simplified models are enhanced. In this last case, the model proposed by Flórez-López is analysed and the simplification consists on previous definition of yielding and damage zones over the assembly of beam and colunm elements, which compose the discretized structure; in the limit evolution of the loading process, these zones become hinges. Finally, a generalization of the previous model proposed by Álvares is studied. The numerical results supplied by the models are compared with the experimental results of reinforced concrete beams (simply supported and with different reinforced rates) and of a reinforced concrete frame.