Proteomics of Poly(ADP-ribose) Polymerases during DNA Replication and Repair

En 2017, Statistique Canada a rapporté qu'un Canadien sur quatre mourra d’un cancer. Chaque jour, nous sommes confrontés à des facteurs environnementaux qui imposent à notre ADN un stress génotoxique. Ce stress peut avoir de graves conséquences au point de menacer notre intégrité génomique, comme les cassures d'ADN double-brin (DSBs). Heureusement, nos cellules ont deux voies principales pour combattre ce type de lésions : la recombinaison homologue (HR) et la Classical Non-Homologous End-Joining (CNHEJ). La voie HR, un type de réparation sans erreur utilisé dans la phase-S du cycle cellulaire, assure une réparation fidèle de la zone endommagée et conserve l'intégrité de l'information génétique. Les individus porteurs de mutations dans les protéines de cette voie, telles que BRCA1 et BCRA2, sont plus susceptibles de développer des cancers du sein et de l'ovaire. Récemment, la clinique a connu une percée majeure dans le traitement du cancer de l'ovaire. Une nouvelle classe de médicaments a été autorisée par la US Food and Drug Administration (FDA) pour traiter les cancers de l'ovaire récurrents qui présentent une HR défective. Ces médicaments inhibent un des acteurs les plus précoces dans la réponse aux dommages à l'ADN (DDR): la PARP-1 (Poly(ADP-ribose) polymerase-1). Lors de l'induction de dommages à l'ADN, la PARP-1 devient fortement activée, conduisant à la production massive de polymères de poly(ADP-ribose) (PAR) générés à partir de l'hydrolyse du nicotinamide adénine dinucléotide. Ce polymère agira comme une plateforme pour recruter des facteurs de réparation de l'ADN au site de réparation. L'application clinique réussie des inhibiteurs de la PARP (PARPi) vient des observations où les mutations ou l'extinction de BRCA1/2 entraînent une diminution de l'activité HR. L'inhibition de la PARP-1 combinée à cette déficience en HR favorise la mort cellulaire, un phénomène appelé létalité synthétique. Trois PARPi sont actuellement autorisés par la FDA et sont utilisés pour le traitement du cancer gynécologique. Malgré l'efficacité thérapeutique de ces inhibiteurs, les mécanismes induisant une régression tumorale ne sont pas complètement compris. Ainsi, il devient extrêmement important de déchiffrer davantage ces mécanismes pour atteindre le plein potentiel des PARPi. Pour ce faire, une recherche fondamentale sur les fonctions des PARPs, et de leurs partenaires dans la DDR, est essentielle et constitue l'objectif général de cette thèse. Durant mon doctorat, nous avons étudié l'influence de la PARP-1 dans la voie HR au moment de l'étape initiale de la résection, qui est essentielle pour l'élimination de l'ADN endommagé. Certaines études ont montré l'implication de la PARP-1 dans le recrutement de la protéine de résection MRE11. Ici, nous démontrons que la PARP-1 a une nouvelle fonction dans la résection des DSBs et nous proposons un nouveau modèle pour expliquer la létalité synthétique observée dans les tumeurs avec une HR défective. Pour compléter l'objectif de ce doctorat, nous avons étudié les rôles régulateurs de la PARP-1 au cours du processus HR, mais plus tard dans la résolution des lésions, c'est-à-dire au maximum de la formation des foyers RAD51, une étape cruciale pour la réparation efficace des DSBs via la HR. Nous avons observé que le PAR-interactome (PARylome) est, à ce moment, fortement enrichi en protéines impliquées dans le métabolisme de l'ARN. Plusieurs des protéines les plus abondantes étaient constituées d’hélicases d’ADN et d’ARN, et de facteurs de transcription. Puisque certains de ces gènes sont mutés dans les tumeurs, ils pourraient théoriquement être des cibles prioritaires pour une utilisation conjointe avec des PARPi. Nous avons également étendu notre étude de la PARylation à la chromatine, au niveau des histones. Nous avons constaté que les queues d'histones ne sont pas les seules cibles de la PARP-1 et que les domaines globulaires centraux sont également PARylés. Finalement, le grand intérêt clinique de la PARP-1 méritait une analyse approfondie de son expression systémique. Ainsi, j'ai terminé mes études en décrivant la distribution et l'abondance tissulaire de la PARP-1 dans les organes simiens, avec l'objectif principal de fournir des informations précieuses quant à l'efficacité potentielle des PARPi ou sa résistance, dans un tissu donné et maladies apparentées. En résumé, cette thèse fournit de nouvelles informations importantes sur les mécanismes orchestrés par la PARP-1 lors de la réponse aux DSBs, y compris les réseaux protéiques complexes engagés dans le remodelage des fonctions cellulaires nécessaire au maintien de l'intégrité génomique.In 2017, Statistics Canada reported that one out of four Canadians will die of cancer. Every day, we face environmental factors that burden our DNA with genotoxic stress. This stress can lead to severe types of DNA damage that can threaten our genomic integrity, namely double-strand breaks (DSBs). Fortunately, our cells have evolved with different repair mechanisms to deal with such lesions. There are two primary types of repair against DSBs: Homologous Recombination (HR) and Classical Non-Homologous End-Joining (CNHEJ). The HR pathway is an error-free repair mechanism used in the S-phase of the cell cycle to ensure faithful repair of the damaged area and thus preserve our genetic information. Individuals that bear mutations in proteins involved in this pathway, such as BRCA1 and BCRA2, have been associated with the development of breast and ovarian cancers. Almost 4 years ago, the field went through a major breakthrough in ovarian cancer care. A new class of drugs was accepted by the US Food and Drug Administration (FDA) to manage recurrent ovarian cancers that display HR-deficiencies. These drugs consist of inhibitor molecules against one of the earliest sensors of DNA damage in the cell: PARP-1 (poly(ADP-ribose) polymerase-1). Upon DNA damage induction, PARP-1 becomes highly activated, leading to the massive production of poly(ADP-ribose) (PAR) polymers, from the hydrolysis of nicotinamide adenine dinucleotide, which in turn modify several proteins posttranslationally and act as a scaffold to recruit DNA repair factors to the repair site. The successful application of PARP inhibitors (PARPi) arose from the observations that mutations or silencing of BRCA1/2, resulted in diminished HR activity. In the context of HR deficiency, the concomitant inhibition of PARP resulted in cell-death, an effect called synthetic lethality. Three PARPi are currently accepted by the FDA and are being clinically used for the treatment of gynaecological cancers. Notwithstanding the great promise of these inhibitors for other types of cancers, the mechanism by which these are inducing cancer lethality is not fully understood. Thus, it becomes of extreme importance to further decipher its mechanistic ways, to achieve full potential of PARPi in the clinic. To achieve this, fundamental research on the functions of PARPs and their protein partners in the DNA damage response is indispensable and constitutes the general aim of this thesis. During my doctoral work, we investigated the influence of PARP-1 during the HR pathway, primarily during the initial step of resection, which is essential for the removal of damaged DNA. Early reports of PARP-1 involvement in resection described the recruitment of the resection protein MRE11 to sites of damage in a PARP-1 dependent manner. Here, we demonstrate that PARP-1 has a novel function in DSB resection and we propose a new model for the synthetic lethality observed in HR-deficient tumors. To further complement the general aim of this doctorate, we investigated the regulatory roles of PARP-1 during the HR pathway, however in a later stage of HR resolution, at the peak formation of RAD51 foci, which is a crucial step for the efficient repair of DSBs through HR. We observed that the PAR-interactome (PARylome) at this stage was abundantly enriched with RNA-processing factors. Several of the most abundant proteins consisted of DNA and RNA helicases, as well as transcription factors, some of which were found to be mutated in tumors, and thus can be seen as potentially druggable targets to be used in combination with PARPi. We also extended our PARylome study to the chromatin proteome and investigated the histone PARylome upon DNA damage. Interestingly, we found that histone tails are not the only targets of PARP-1 and that globular domains are also targets of PARylation. Lastly, the high clinical interest of PARP-1 warrants studies addressing PARP-1 organ distribution. Thus, I finalized my studies by extensively describing and reporting PARP-1 tissular and cellular distribution and abundance in monkey organs, with the main objective of providing valuable information to any study assessing PARP inhibition efficacy and resistance in any given tissue and related diseases. In summary, this thesis provides important new information on the mechanisms PARP-1 is regulating during the response to DSBs, including the networks PARP-1 is orchestrating to potentially help reshape the cell environment, to efficiently repair the most lethal lesion our genome faces

Predicting fraud behaviour in online betting

Project Work presented as the partial requirement for obtaining a Master's degree in Statistics and Information Management, specialization in Information Analysis and ManagementFraud isn’t a new issue, there are discussions about this matter since the beginning of commerce. With the advance of the Internet this technique gained strain and became a billion-dollar business. There are many different types of online financial fraud: account takeover; identity theft; chargeback; credit card transactions; etc. Online betting is one of the markets where fraud is increasing every day. In Portugal, the regulation of gambling and online betting was approved in April 2015. In one hand, this legislation made possible the exploration of this business in a controlled and regulated environment, but on the other hand it encouraged customers to develop new ways of fraud. Traditional data analysis used to detect fraud involved different domains such as economics, finance and law. The complexity of these investigations soon became obsolete. Being fraud an adaptive crime, different areas such as Data Mining and Machine Learning were developed to identify and prevent fraud. The main goal of this Project is to develop a predicting model, using a data mining approach and machine learning methods, able to identify and prevent online financial fraud on the Portuguese Betting Market, a new but already strong business

The 1755 earthquake in the Algarve (South of Portugal): what would happen nowadays?

The 1755 Lisbon earthquake, which reached a magnitude of 8.5, remains the most powerful and destructive to hit Europe so far. Within minutes, many lives were lost, populations displaced, livelihoods, homes and infrastructures were destroyed. Although frequently associated to the city of Lisbon, one of the most important European cities at the time, this earthquake caused similar damage and casualties, if not greater, in the southwest of the Algarve, where the seismic intensity was estimated at IX-X Mercalli Intensity Scale. Some time later a tsunami increased the number of victims and the amount of damage. In some locations the tsunami caused greater destruction than the earthquake itself. The tsunami hit both coasts of the North Atlantic; however, the more destructive damage occurred in the Portuguese coast, south from Lisbon, in the Gulf of Cadiz and in the Moroccan coast. The downtown of Lisbon was flooded by waves that reached a height of 6 m. The water flooded an area with an extension of around 250 m from the coast. In the Southwest part of Algarve the waves reached a height between 10 and 15 m and the flooded area was much larger. Through the analysis of recent research works on the assessment of the 1755 tsunami parameters and the interpretation of the more reliable historical documents, it is our intention to analyse the destructive power of the tsunami in the Algarve and delimit the flooded area. Using simple techniques of simulation it is our intention to assess the impacts nowadays of the occurrence of a tsunami similar to the one that hit the Algarve in 1755, which would probably affect a greater number of people, buildings and infrastructures. This assessment is an important instrument not only in terms of disaster preparedness but also for the integration of risk mitigation measures in land use planning

The dilemma of wildfire definition: what it reveals and what it implies

This paper presents the results of an explorative survey, based on a questionnaire sent by email, about how wildfire experts, operating in different countries, perceive wildfire and express their mindset by defining "wildfire" from a list of 14 terms and how they justify their preference for the term selected as the most important. Using a five-point Likert Scale, results from 221 valid replies indicate a general convergence toward a reduced number of terms. Six of them exhibit a mean >3.20 (Disturbance, Natural hazard, Climate-sensitive hazard, Socio-ecological hazard, Socio-ecological disturbance, and Social-ecological hazard). The three most preferred terms (i.e., Disturbance, Natural hazard, and Climate-sensitive hazard) reflect wildfire as a natural process or phenomenon (about 59% of the replies). The three terms characterized by both the social and ecological adjectives (i.e., Socio-ecological hazard, Socio-ecological disturbance, Social-ecological hazard) occupy relatively less favorable positions in the ranking. For each term, a synthesis of the explanations given by the respondents is provided, together with a critical comment. Our findings show very different perceptions of wildfires inclusively within the same disciplinary field. In addition, for the same term selected, different definitions are often presented. This reflects sectorial, disciplinary, and personal perspectives of the wildfire phenomenon and the lack of a common understanding of wildfire "nature" (i.e., its own identity). The different perceptions on wildfire concept influence the knowledge that can be used by decision makers to improve wildfire management policies. This work puts into perspective one of the most widespread problems in science: the lack of appropriate and similar terminology across different scientific fields dealing with the same problem. A common conceptualization of the nature of wildfires and the creation of a common language across different scientific fields related to wildfires is of paramount importance to address the complexity of the existing problems, and enhance an interactive communication not only among scientific community but also with stakeholders and citizens

The rising temperature trend and elongation of the warm period in summer in the Algerian South-West, 1951-2010

Several studies indicate that the hot season is lengthening at the expense of the intermediate seasons, and extreme events are becoming more intense and spread out over time. What is the situation in a climate described as very hot? This work aims to examine the evolution of minimum and maximum temperatures in the hot period from June to September during 1951-2010 in the Bechar region, which is classified among the hottest in the world and located in the south-west of Algeria. This study will also focus on the extending of the warm period (the period between the first and the last recording of an extremely hot day). The results showed an evolution of maximum and minimum temperatures, leading to an upward trend in the mid-1990s. There was an average increase of 1°C at the maximum and 0.9°C at the minimum. The warm period experienced a sig-nificant expansion attributed to both the earliest and the latest extremely hot day events

The characterization of the possible interaction between the Chlamydia effector tarp and its outative chaperone CT043

A Chlamydia trachomatis é responsável por uma das infecções bacterianas sexualmente transmissíveis mais comuns na Europa e nos EUA. Esta bactéria Gram negativa é um patogénico intracelular obrigatório, com um ciclo biológico bifásico, no qual se observam duas morfologias bacterianas diferentes, o Corpo Elementar (CE) e o Corpo Reticular (CR). As formas infectantes desta bactéria são os CEs que têm capacidade de se agregar e invadir as células dos mamíferos. Este processo parece ser dependente do Sistema de Secreção Tipo III (T3SS), permitindo a secreção de efectores de Chlamydia para as células do hospedeiro, os quais modulam o esqueleto de actina das células eucariótas promovendo a invasão. Um dos primeiros efectores secretados pela Chlamydia designa-se TarP (Translocated Actin Recruiting Protein). Efectores ortólogos ao TarP estão presentes em todas as espécies do género Chlamydia. Estes ortólogos apresentam polimorfismos associados com alguns subdomínios, como é o caso o domínio rico em tirosina, que apenas se encontra presente no TarP descrito para C. trachomatis. Apesar das diferenças verificadas nos ortólogos das diferentes espécies de Chlamydia, todos têm a capacidade de recrutar e agregar actina permitindo a internalização da bactéria por parte da célula. Uma vez que, numa fase inicial da invasão, a secreção do TarP parece ser indispensável para a mesma, poderia pensar-se que, para que esta secreção seja eficiente, deve ser facilitada por uma chaperona do T3SS. Estas chaperonas são, normalmente, proteínas de baixo peso molecular (13-16 kDa), com um ponto isoeléctrico acídico e uma estrutura secundária com um motivo α-β-β-β-α-β-β. Através do estudo da estrutura secundária previram-se três chaperonas do T3SS, CT043 (Slc1), CT663 (Slc2) CT088 (Scc1), presentes em CEs de Chlamydia. A interacção destas chaperonas com o domínio N-terminal do TarP (aa 1-200) testou-se através de ensaios de co-imunoprecipitação. Observou-se que, das três chaperonas testadas, o TarP unicamente interage de forma específica com a chaperona Slc1. Tendo como base estes resultados, utilizou-se um ensaio de duplo híbrido para avaliar quais os efeitos que mutações a nível do N-ternimal do TarP (1-100) têm na sua interacção com Slc1. Este estudo teve como objectivo identificar os aminoácidos que estão envolvidos na ligação do TarP à chaperona Slc1. Através da utilização de diversos mutantes no N-terminal do TarP, concluiu-se que a interacção entre o TarP e Slc1 parece depender tanto de aminoácidos específicos como de interacções hidrofóbicas entre TarP e Slc1.Chlamydia trachomatis is the most common cause of sexually transmitted bacterial infection in Europe and in the United States. It is a Gram negative obligate intracellular pathogen with a unique biphasic development cycle, possessing two distinct morphological forms, the elementary body (EB) and the reticulate body (RB). The EBs are the infectious form and are able to attach and invade mammalian cells. This process is likely dependent on the Type III Secretion System, which allows secretion of Chlamydia effectors that modulate the actin skeleton of the host cell and promote invasion. One of the first effectors to be secreted is TarP (Translocated Actin Recruiting Protein). Orthologs of this effector are present in all Chlamydia with some polymorphisms associated with some subdomains. An example is the tyrosine rich domain, which is only present in the TarP homolog of the C. trachomatis species. Despite this difference, all chlamydial TarP are able to recruit and nucleate actin to promote the internalisation of Chlamydia. Since early secretion of TarP is crucial for invasion it was hypothesized that this secretion, in order to be efficient must be facilitated by a Type III Secretion chaperone. These usually are small proteins (13-16 kDa) with an acidic pI and a secondary structure motif of α-β-β-β-α-β-β. Three putative Chlamydia trachomatis Type III secretion chaperones, CT043 (Slc1), CT663 (Slc2), CT088 (Scc1), which are present in EBs have been predicted by their secondary structures. The interaction of these chaperones with the N-terminal domain of TarP (amino acids 1-200) was tested using pull-down assays. A specific interaction between TarP and Slc1 was observed, but not with Slc2 or Scc1. Using a bacterial two-hybrid assay, we evaluated the effects of mutating TarP(1-100) on its interaction with Slc1. The objective was to identify critical amino acid residues involved in chaperone binding. Using several mutants TarP derivatives, we were able to conclude that the interaction between TarP and Slc1 may depend of both specific amino acids and hydrophobic interactions

Comparative analysis of multisensor burned area products for the Brazilian Amazon - Region of the APA Triunfo do Xingu

Wildfires are not natural phenomena of the Amazon rainforest; therefore, they occur due to human activities, and their occurrences have increased in recent years. This situation requires continuous monitoring of this vast region, especially in areas where agricultural, livestock, mining, and infrastructure activities are located near protected areas (indigenous lands and nature conservation units). One of the conservation units that has recorded the highest increases in deforestation and fire rates is the Triunfo do Xingu Environmental Protection Area (APA Triunfo do Xingu), which since 2018 has registered the highest rates among other conservation units in the Amazon. The present study aims to develop two databases of burned areas from optical and microwave images for the years 2018, 2019, and 2020 for the APA Triunfo do Xingu using Google Earth Engine; then the results are compared with the DETER, MAPBIOMAS and MCD64A1 burned area bases to estimate existing similarities and divergences. This research uses the images of the Sentinel-2/S-2 and Landsat-8/L-8 optical satellites for the month of August of the years 2018, 2019, and 2020, a period of increased occurrences of active fires in the APA Triunfo do Xingu, to generate the burned area database that will serve as a reference for comparison with other burned area databases. Thus, images with spatial resolution, S-2 with 10 meters and L-8 with 30 meters and spectral (red, near, and medium infrared bands), are suitable for generating information with geometric and thematic quality, as the GEE allows the production of pixel mosaics excluding pixels with cloud and cloud shadows. The Sentinel-1/S-1 images used correspond to the VH cross-polarization, which is the most suitable polarization to map burned area than the VV polarization (Prasasti et al., 2020), having 10 meters of spatial resolution, being with speckle noise filter and with backscatter (DB) values. It is noteworthy that the S-1 images correspond to Band C, the wavelength of 5 cm, which reduces the interference of clouds when imaging the surface. In general, the BA-S1, BA-DETER, BA-MAPBIOMAS, and BA-MCD64A1 datasets obtained discrepant values concerning the reference dataset, which reveals that a consolidated base with values consistent with the reality of the fire regime of the study area is still to be achieved, as the discrepancies found reveal that the data and methodologies followed are not generating consistent quantitative information. Regarding the overlaps between the datasets, the spatial similarity between the datasets have a higher rate with polygons above 20 hectares, which allowed an average overlap of 63%, 69%, and 78% for the BA-S1, BA-MAPBIOMAS, and BA-MCD64A1. The BA-DETER dataset was below 20%, as it presents the lowest values of the number of polygons for the three years

O conceito de "fire smart territory": contributo para a mudança de perspetiva na gestão dos incêndios florestais em Portugal

A perspetiva sintomática centrada no controlo do fogo através daextinção rápida das ignições, não resolveu nem controlou o problemados incêndios florestais. Este tem uma natureza estruturalcuja solução só pode ser encontrada no âmbito do Sistema Social eEcológico Acoplado em que se origina e se desenvolve. Neste trabalhointroduzimos o conceito de Fire Smart Territory1 que assenta numanova perspetiva de gestão dos incêndios florestais que promove ummaior equilíbrio e inter-relação entre a prevenção e a extinção.Palavras -chave: Controlo do fogo, prevenção, risco.The symptomatic approach to fire management focused on "fire control"through the suppression of all fires did not solve or control theproblem of wildfires which has a structural root. The resolution ofthe problem can only be found in the Coupled Human and NaturalSystem where it is originated and developed. In this paper the conceptof "Fire Smart Territory" is introduced. It is based on a new perspectiveon the management of wildfires that promotes a better balance andinterrelationship between prevention and suppression