O estado de metilação é suscetível a mudanças quando os organismos são expostos a agentes ambientais tais como componentes dos alimentos e medicamentos. Uma dieta rica em metionina (Met) poderia modular a concentração de S-adenosilmetionina (SAM) e S-adenosilhomocisteína (SAH) e alterar o estado de metilação da região promotora de genes supressores de tumores. Tanto a hipometilação global quanto a hipermetilação de genes específicos estão envolvidas na instabilidade genômica e poderiam resultar em dano ao DNA. Este estudo avalia se a dieta suplementada com Met associada a doxorrubicina (DXR), um fármaco antitumoral que induz espécies reativas, resulta em alterações no estado de metilação da região promotora do gene TP53, na razão SAM/SAH, na concentração de glutationa (GSH) e em dano ao DNA. Quarenta ratos machos Wistar foram separados em dois grupos: dieta suplementada com Met (ração comercial acrescida de 2% Met) e dieta controle (ração comercial) por seis semanas. Cada grupo foi subdividido em dois subgrupos que receberam DXR (1mg/Kg) ou solução salina intraperitoneal na terceira e sexta semanas de tratamento. Os rins e fígado foram utilizados para isolamento do DNA, determinação da concentração de SAM, SAH e GSH, e análise da instabilidade genômica. Todos os grupos apresentaram o mesmo estado de metilação da região promotora do gene TP53, determinado pelo método de análise de restrição combinada com bissulfito (COBRA). Este fato poderia ser explicado pelo índice de metilação (razão SAM/SAH) que permaneceu inalterado, possivelmente devido a uma adaptação do ciclo da Met que manteve a concentração de SAM. A depleção de GSH não ocorreu quando DXR foi associada a dieta suplementada com Met. Portanto, a suplementação com Met manteve a concentração de GSH em ratos tratados com DXR. A dieta suplementada com Met não induziu instabilidade genômica e não alterou o dano ao DNA induzido pela DXR. Em conclusão, DXR induz depleção de GSH que é inibida pela suplementação com Met. Entretanto, a mesma suplementação não previne a instabilidade genômica induzida pela DXR. A dieta suplementada com Met aumenta a concentração de SAH renal sem alterar a concentração de SAM e GSH. Tanto a dieta suplementada quanto a DXR não induzem hipermetilação na região promotora do gene TP53. / The DNA methylation status is susceptible to changes when organisms are exposed to environmental agents such as food components and drugs. A methionine-rich (Met) diet may modulate S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) concentrations, which could change the DNA methylation status in the promoter region of tumor suppressor genes. Global hipomethylation and gene-specific hipermethylation are involved in genomic instability and it could result in DNA damage. This study intends to evaluate if a Met-rich diet associated with doxorubicin (DXR), an antitumoral drug that induces reactive species, result in changes in the methylation status of the TP53 gene promoter, in the SAM/SAH ratio, in glutathione levels (GSH) and in DNA damage. Forty male Wistar rats were separated into two groups: Met-rich diet (standard chow plus 2% Met), and control diet (standard chow) for six weeks. Each group was subdivided into another two groups that received DXR (1mg/kg) or saline intraperitoneally in the third and sixth weeks of the experiment. The kidneys and the liver were removed for DNA isolation, SAM, SAH and GSH determination, and genomic instability assay. All groups showed the same unmethylated status in the TP53 promoter according to the Combined Bisulfite Restriction Analysis (COBRA). This could be explained by the fact that the methylation index (SAM/SAH ratio) remained unchanged, possibly because of an adaptive Met pathway that maintains SAM levels. GSH depletion did not occur when DXR was associated with the Met-rich diet. As a matter of fact, the Met-rich diet improved GSH concentration in DXR-treated rats. Met-rich diet did not induce genomic instability, and it did not alter DNA damage induced by DXR. In conclusion, DXR induces GSH depletion which is inhibited by Met supplementation. However, Met-rich diet may not prevent genomic instability induced by DXR. A Met-rich diet increases SAH levels; however, it does not change GSH and SAM levels. Neither Met supplementation nor DXR induced DNA hypermethylation in the TP53 gene promoter.

Thesis advisor: Charles Hoffman / Faithful chromosome segregation is necessary for the successful completion of mitosis and meiosis. The centromere is the site of kinetochore and microtubule attachment during chromosome segregation, and it is critical that the centromere is properly formed and maintained. Many proteins contribute to centromere formation, and this process has been extensively studied during the mitotic cell cycle. However, the roles of the centromere and its associated proteins during meiosis and their contribution to the fidelity of chromosome segregation process are not as well understood. Here, I aim to elucidate a mechanism that may contribute to aneuploidy in gametes, which is a major contributing factor in human infertility. In this study, I investigate the role of Abp1, the most prominent member of the transposase-derived protein family homologous to mammalian CENP-B in the assembly of centromeric chromatin during meiosis in the fission yeast Schizosaccharomyces pombe. I reveal that in contrast to its known role as a major regulator of LTR retrotransposons during the mitotic and meiotic cell cycles, Abp1 has a specialized role at the centromere during meiosis. My results indicate that Abp1 displays dynamic localization to the centromeres during meiosis compared to the vegetative cell cycle. I show that loss of abp1 impairs pericentromeric heterochromatin and the localization of Cnp1, a CENP-A ortholog, to the centromere central cores during meiosis. Moreover, Abp1 appears to suppress formation of meiotic neocentromeres by restricting deposition of Cnp1 at certain heterochromatin loci. Loss of abp1 has a drastic effect on chromosome segregation, resulting in dramatic frequency of aneuploidy. Furthermore, the genome surveillance role for retrotransposons by Abp1 appears to encompass centromeres as the mere insertion of an LTR sequence within the centromere central cores further exacerbates incidence of meiotic aneuploidy in abp1 null cells. This study provides intriguing insights into factors controlling the assembly of centromeric chromatin and its impact on the fidelity of chromosome segregation process during meiosis with important implications for advancing our understanding of the evolutionary forces driving the evolution of eukaryotic centromeres. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Hugh P. Cam / Meiotic homologous recombination (HR) is not uniform across eukaryotic genomes, creating regions of strong recombination activity dubbed recombination hotspots, and regions of low recombination activity dubbed coldspots. Considerable attention has led to discoveries of a host of factors controlling the formation of hotspots. However, the determinants of coldspots are not as clearly defined. I have previously shown that CENP-B homologs of the fission yeast Schizosaccharomyces pombe have a genome surveillance role in regulating the nuclear organization and expression of Tf2 retrotransposons. Here, I reveal an additional role for CENP-Bs in suppressing meiotic recombination of Tf2s. I describe the development of a random sporulation assay to rapidly screen thousands of meiotic progeny for recombination across a locus in a variety of genetic backgrounds. Loss of any CENP-B family members (Abp1, Cbh1, Cbh2), results in increased HR at Tf2s. I show that Abp1, which acts as the primary determinant of HR suppression at Tf2s, is required to maintain proper recombination exchange of homologous alleles flanking a Tf2. In addition, Abp1-mediated suppression of HR at Tf2s requires all three of its domains with distinct functions in transcriptional repression and higher-order genome organization. I show that this suppression is likely mediated by Abp1 binding to specific motifs near the 3’end of flanking LTRs. I demonstrate that HR suppression of Tf2s can be robustly maintained despite disruption to chromatin factors essential for transcriptional repression and nuclear organization of Tf2s. Intriguingly, I uncover a surprising cooperation between the histone methyltransferase Set1 responsible for histone H3 lysine 4 methylation and the non-homologous end joining pathway in ensuring the suppression of HR at Tf2s. Furthermore, I identify a role for the architectural protein condensin involved in 3D chromatin organization and chromosome condensation in restricting HR at Tf2s. My study identifies a molecular pathway involving functional cooperation between a transcription factor with epigenetic regulators, DNA repair pathway, and chromosome organizers to regulate meiotic recombination at interspersed repeats. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

L'athérosclérose est une maladie chronique à évolution lente caractérisée par la formation de plaques d'athérome, consistant en l’accumulation de lipoprotéines de basse densité (LDL), de leucocytes, de cellules spumeuses, la migration des cellules musculaires lisses (CML) et l’altération des cellules endotheliales (ECs). Ces phénomènes conduisent à la formation d'un noyau nécrotique incluant des régions calcifiées. La genèse de l'athérosclérose et de l’instabilité de la plaque d’athérome sont le résultat d'une synergie entre inflammation et stress oxydant. Les données actuelles identifient plusieurs populations de macrophages dans la plaque d’athérome présentant différents phénotypes en lien avec l’inflammation (pro-inflammatoire: M1, anti-inflammatoire: M2) ou avec des modifications redox de l’environnement (Mox). Stress oxydant et inflammation sont liés et jouent un rôle important dans (i) la dysfonction endothéliale induisant une diminution de la biodisponibilité du monoxyde d’azote (NO), (ii) l'oxydation des LDL, (iii) le remodelage de la lésion (régulation de protéases et d’antiprotéases) et (iv) la prolifération des CML. Les CML sont le deuxième type cellulaire le plus abondant dans la lésion athérosclérotique après les macrophages, leur hyperprolifération est la conséquence d’une dédifférenciation cellulaire d’un phénotype contractile à sécrétoire, augmentant leur capacité proliférative et migratoire. Les donneurs de NO, comme les S-nitrosothiols, connus également pour protéger contre le stress oxydant grâce essentiellement à la S-nitrosation, peuvent contrer la carence en NO. Parmi eux, le S- nitrosoglutathion (GSNO), forme physiologique de stockage de NO dans les tissus, spécifiquement metabolisé par la gamma-glutamyl transférase (GGT) peut être envisagé. La corrélation entre l’augmentation des concentrations sériques de GGT et les facteurs de risque cardiovasculaire a récemment été démontrée. En particulier, seule la b-GGT s'accumule dans les plaques d'athérome, et concorde avec l’apparition d'autres marqueurs histologiques de vulnérabilité de la plaque. Étant donné que, les macrophages et les CML sont les principaux types cellulaires retrouvés dans les lésions athérosclérotiques et semblaient être colocalisés avec la GGT, l'attention de ce travail de thèse a été centrée sur la compréhension de la provenance de la GGT et son rôle dans le métabolisme du GSNO au sein de la plaque d’athérome. Une première partie de ce manuscrit vise à identifier l'origine de la GGT accumulée dans la plaque d’athérome, et à élucider entre le stimulus inflammatoire et oxydant, qui est responsable de l'accumulation de GGT dans la plaque d'athérome. La deuxième partie a été consacrée à la restauration de la biodisponibilité de NO dans les CML en condition de stress oxydant avec un intérêt particulier porté sur l'identification des protéines S-nitrosés / Atherosclerosis is a slowly progressing chronic disease characterized by the formation of atherosclerotic plaques consisting of accumulated low density lipoprotein (LDL), leukocytes, foam cells, migrated smooth muscle cells (SMCs) and altered endothelial cells (ECs), leading to the formation of necrotic cores with calcified regions. Atherosclerosis genesis and subsequent instability of atherosclerotic plaques result from a synergy between inflammation and oxidative stress. Current data identified several macrophage populations within the atherosclerotic plaque showing different inflammatory phenotypes (pro-inflammatory: M1, anti-inflammatory: M2) or functions in response to redox changes in the environment (Mox). The oxidative stress linked to inflammation plays an important role in (i) endothelial dysfunction, with reduced nitric oxide (NO) bioavailability, (ii) LDL oxidation, (iii) lesion remodeling (regulation of proteases and antiproteases) and (iv) SMCs proliferation. Indeed, SMCs are the second more abundant cell type, after macrophages, in the atherosclerotic lesion because their dedifferentiation from contractile to secreting phenotype increased their proliferation and migration capacity. NO donors, like S-nitrosothiols, also known to protect from oxidative stress by S-nitrosation, could counteract this NO deficiency. Among them, the S-nitrosoglutathione (GSNO), a physiological storage form of NO in tissues, specifically catabolized by the gamma-glutamyltransferase (GGT) is considered. Recently, it has been shown that the increased serum level of GGT is an independent risk factor for cardiovascular mortality related to atherosclerotic disease. In particular, only the big fraction (b-GGT) has been detected inside human atherosclerotic plaques associated to CD68+ macrophage-derived foam cells. As macrophages and SMCs are the main cell types found in atherosclerotic lesion and seemed to be colocalized with GGT, the attention of this thesis work was focused on the understanding of GGT provenance and its role in the GSNO metabolism within the atherosclerotic plaque. A first part of the thesis was to identify the origin of GGT accumulating inside atherosclerotic plaques, and to decipher between inflammation and oxidative stress stimuli, which one is responsible of GGT accumulation in atherosclerotic plaques. The second part was dedicated to the restoration of NO bioavailability within SMCs under oxidative stress with a focus on the identification of S-nitrosated proteins

Les technologies de l’information et de la communication (TIC) occupent une place importante dans les débats publics sénégalais. En outre, elles semblent être au centre des préoccupations des dirigeants de ce pays, car les espoirs qu’elles suscitent dans le domaine de l’éducation sont assez nombreux. Mais, l’introduction des TIC dans les lycées soulève également des interrogations de la part des chercheurs et des acteurs éducatifs quant à leurs usages. Cette thèse porte sur les pratiques numériques des lycéens. L’objectif que nous poursuivons est de comprendre dans quelle mesure les usages des TIC peuvent leur permettre de s’approprier des savoirs. Ici, comprendre c’est, s’intéresser aux représentations, aux connaissances et aux compétences qui impliquent l’usage des outils informatisés, mais aussi aux sens des usages. La question des usages des TIC par les élèves suscite un intérêt particulier auprès des chercheurs de certaines disciplines, particulièrement ceux de la sociologie des usages, des sciences cognitives et des sciences de l’éducation. Ainsi, nous avons privilégié une approche théorique pluridisciplinaire et une pluralité méthodologique (le questionnaire, l’observation directe et l’entretien d’explicitation) en ayant recours à des outils spécifiques pour recueillir les données (dictaphone, notes, captures d’écrans). L’analyse a permis de voir que ces usagers-apprenants s’approprient les technologies numériques à des fins d’apprentissage et a mis en relief le « versant sombre » de leurs pratiques. Partant des données obtenues nous proposons quelques pistes de réflexion pour une intégration optimale des TIC dans les pratiques d’enseignement et d’apprentissage. / Information and communication technologies (ICTs) are central to Senegalese public debates. Moreover, the leaders of this country view them as very advantageous to education. However, their introduction in high schools raises a lot of questions from both researchers and educators as regards their usage. This study is about digital practices of high school students Our aim is to understand the extent to which the use of ICTs can enable them to acquire knowledge. Understanding in this regard implies looking at the perceptions, knowledge and skills related to the use of computerized tools, but also the meanings of this usage. The use of ICTs by pupils is also of particular interest to researchers in the fields of humanities and social sciences, especially those in the sociology, cognitive sciences and education areas. Thus, in order to better define the subject, this study used a multidisciplinary theoretical approach and a plural methodology (questionnaires, direct observation and interviews) as well as specific tools (voice-recorder, notes and screenshots) to collect data during two field investigations that were conducted in Dakar secondary schools (from March to April 2013 and November to December 2015). The analysis revealed that these learner-users use digital technologies for learning purposes but also highlighted the "dark side" of their practices. On the basis of the data obtained, we also suggest ways in which ICTs can be optimally integrated in teaching and learning practices.

S-acylation, commonly known as S-palmitoylation, is a reversible posttranslational lipid modification in which fatty acid, usually palmitic acid, covalently attaches to specific cysteine residues of proteins via thioester bonds. Palmitoylation enhances the hydrophobicity of proteins and contributes to their membrane association. It plays roles in protein trafficking, signalling, protein-protein interaction, protein stability and other important cellular functions. A family of Protein S-acyl Transferases (PATs) is responsible for this reaction. PATs are multi-pass transmembrane proteins that possess a catalytic Asp- His-His-Cys cysteine rich domain (DHHC-CRD) of ~50 amino acids. In Arabidopsis there are at least 24 such DHHC-CRD containing PAT proteins and they are named as AtPAT01 to AtPAT24. The function of only 2 AtPATs, AtPAT10 and AtPAT24 were studied in some detail, and a recent survey showed the ubiquitous expression pattern and different membrane localization habit of all 24 AtPATs. However, the biological function of the remaining 22 AtPATs in Arabidopsis was not reported when I started my project. Therefore, we carried out an initial screen of all the available T-DNA insertion lines of the 22 Arabidopsis PATs and identified transcriptional null mutants of 18 of the AtPATs. Among them, the k/o mutant plants of only 3 genes showed significantly altered phenotypes compared to wild-type Arabidopsis, and the mutants are named as atpat14, atpat21 and plp1(PAT-like Protein 1). This project aims to characterize these three putative PATs in details in terms of their PAT activities, catalytic domains, expression patterns, subcellular localizations and biological functions. AtPAT14 was proved as a PAT by yeast complementary and in vitro auto-acylation assays. Mutagenesis studies clearly demonstrated that the cysteine residue in the DHHCmotif is essential for the enzyme activity of AtPAT14. Transgenic Arabidopsis plants expressing AtPAT14-GFP were observed and it was shown that AtPAT14 is predominantly localized at the Trans-Golgi. The phenotype was observed in both atpat14-1 and atpat14-2 mutant lines and this showed that the leaves of both lines were aging much faster than the WT. Analysis of the levels of different phytohormones revealed that the mutant leaves contained much higher salicylic acid (SA) than the WT. This coincided with the increased transcript levels of genes involved in SA biosynthesis and signalling. Therefore, AtPAT14 mediated protein S-acylation plays important roles in leaf senescence via the regulation of SA biosynthesis and signalling pathways. AtPAT21 was also confirmed as a PAT and the DHHC its functional domain by similar approaches as for AtPAT14. The plasma membrane (PM) localized AtPAT21 plays essential roles in both male and female gametogenesis. As such, loss-of-function by TDNA insertion in AtPAT21 leads to the plant being completely sterile. Therefore, AtPAT21-mediated S-acylation of proteins(s) plays important roles in the reproduction of Arabidopsis. AtPLP1 (PAT-like Protein 1) contains the signature DHHC-CRD. However, it does not rescue the growth defects of akr1, pfa3 and swf1, the 3 yeast PAT mutants used in enzyme activity assays of other known PATs from plant and animals. Further, the cysteine residue in the DHHC motif was not essential for the function of AtPLP1 as mutated variant containing serine in place of cysteine of the DHHC motif can still rescue the growth defects of atplp1-1. Seedling establishment of atplp1-1 was impaired without external carbon source. This is because the efficiency in converting the seed storage lipid to sugar in the mutant is much lower than WT due to the defective β-oxidation process involved in the degradation of free fatty acids released from lipid during post-germinative growth. In addition, atplp1-1 seedlings are also de-etiolated in the dark, and this was coincided with more cytokinin (CK) and less active gibberellin (GA) related pathway in the mutant. Other defects were also found in atplp1-1, such as hypersensitive to abscisic acid (ABA) and sugar during seed germination and abnormal shoot apical meristem (SAM) in older plants. Therefore, protein S-acyltransferases play distinct and diverse roles throughout the life cycle, from seed germination, seedling growth to seed production in Arabidopsis. This is most likely through the palmitoylation of an array of proteins they modify. Hence, our results provide vital clues for future studies on the molecular mechanism as to how AtPATs operate in plant.

The Agricultural Development Fund (ADF) is the Government agency responsible for financing farmers in the Saudi Arabian agricultural sector. The ADF provides short-term loans and medium-term loans and funds specialised projects, as well as providing subsidies for some types of loans. From its establishment in 1962 until 2012, the ADF has lent more than £7.7 billion and the total number of loans exceeds 439,000. However, in spite of the facilities provided by the ADF, its annual reports show that the outstanding debt increased from £579 million in 1990 to £840 million in 2012; moreover, the reports reveal that some project areas have been receiving more funding than others. This thesis aims to assess the efficiency of the collection of loan repayments by the ADF. This aim will be achieved through four objectives: 1) analyse the current status of the regional distribution of agricultural loans to determine the optimal distribution of loans among regions to achieve development objectives, 2) examine the determinants of the efficiency of loan collection to identify and quantify factors that affect loan repayment, 3) explain the variation in collection rates among the ADF's branches and 4) provide recommendations to improve the probability of repayment and achieve the optimal distribution of loans. Variation in the current distribution of agricultural loans among regions has been observed. Using Multiple-Attribute Analysis, this variation has been identified as sub-optimal with respect to achieving development objectives. The highest priority regions for agricultural loans are the Northern, Jazan and Baha regions. Logistic modelling approaches have been used to determine factors affecting the repayment of short-term loans, medium-term loans and specialised projects; the empirical results of the overall model for short-term loans show that the age of the borrower and four regions have a positive significant effect on loan repayments, while the grace period, the loan value, the financial guarantee and three regions each have a significant negative effect. The results of the analysis of medium-term loans reveal that five variables have a significant effect on loan repayments: age, the grace period and loan value have a negative effect. Financial guarantee and three regions have a positive effect. The results for the specialised projects showed that the Hail region, feed and wheat project have a positive effect, while the Eastern region and greenhouses, livestock and vegetables projects have a negative effect on loan repayments, whereas the age of the borrower, the grace period, financial guarantee and loan value have no significant effect.

La presente investigación constituye un acercamiento crítico a la noción de inconmensurabilidad propuesta por Kuhn en 1962. Consideramos que es una noción insostenible tanto en su versión radical como en la moderada. Así, en primera instancia, realizaremos una revisión histórica del concepto en sus versiones ontológicas, epistemológicas y, sobre todo, semánticas. Nuestras críticas iniciales reposan sobre los alcances que Davidson, Putnam y Coseriu han hecho sobre el tema: el primero con su crítica a la idea de esquema conceptual, el segundo sobre la teoría de la referencia y, el tercero, sobre las posibilidades y condiciones de la traducción. Asumimos que el elemento vinculante entre estas críticas es el realismo que subyace a sus propuestas. Una vez realizada la revisión histórica y la recopilación de argumentos en contra de la noción en cuestión presentaremos nuestra propuesta. Esta defiende que la experiencia es el eje para poner en cuestión nuestro lenguaje y nuestro paradigma. Para llevar a cabo este trabajo, primero presentaremos los alcances centrales de Kuhn sobre esta noción. Luego presentaremos las perspectivas críticas a este concepto desde los tres autores mencionados. En un tercer momento reformularemos las críticas planteadas y presentaremos nuestra hipótesis sobre el tema: esta establece que la experiencia está por encima de las palabras y por encima de los paradigmas. Asimismo, propondremos que hay una salida práctica para los casos de inconmensurabilidad parcial: el uso de ―enunciados básicos‖ ya descritos por Popper y que equiparamos con la idea de ―teorías al paso‖ de Davidson. Cerraremos el trabajo con la revisión de las conclusiones alcanzadas en el desarrollo. / Tesis

Orientadora: Ilse Maria Beuren. / Dissertação (mestrado) - Universidade Regional de Blumenau, Centro de Ciências Sociais Aplicadas, Programa de Pós-Graduação em Ciências Contábeis.

The person of the counsellor, or what is sometimes referred to as the counsellor�s self, is the focus of this thesis. How the counsellor�s self is described and manifested during therapy constitute the two main research questions. Various perspectives are presented from psychoanalysis, behaviour therapy, cognitive behaviour therapy, person centered therapy and systems therapy. While issues pertaining to the counsellor�s self have been explored predominately by systems therapists, this study extends previous research by involving interviews with counsellors from a range of orientations. The study is conducted within an interpretative research paradigm, and data are collected and interpreted according to a qualitative approach. Semi-structured interviews with 16 counsellors, from a range of theoretical orientations, constitute the primary method of data collection. Other data sources include a short questionnaire sent to the same counsellors, the researcher�s reflective journal as well as recorded meetings between a peer debriefer and the researcher. Study participants describe the counsellor�s self as a multifaceted, positive and integrated entity. The counsellor�s self includes participant�s professional knowledge and skills as well as their beliefs, values, thoughts, feelings, personal style and an unknown aspect of self that some participants referred to as their unconscious. While somewhat influenced by past relationships and the client, the counsellor�s self is primarily autonomous and defined by the individual counsellor. Although the counsellor�s self has the capacity to change over time, in different environments and with different clients, the self also includes notions of stability and consistency. The counsellor�s self is involved in therapy as an inevitable presence, a deliberate tool and a stance. Participants highlighted the importance of self-awareness and various professional and personal constraints on the involvement of self. A central function of the self in therapy is in the therapeutic alliance. The study has implications for the training and supervision of counsellors and future psychotherapeutic research.