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Xylella fastidiosa é uma bactéria gram-negativa, não flagelada, agente causal de doenças de importância econômica como a doença de Pierce nas videiras e a clorose variegada dos citros (CVC) nas laranjeiras. O objetivo do presente trabalho foi realizar análises in silico das sequências promotoras dos genes deste fitopatógeno em uma tentativa de arrecadar novas evidências para o melhor entendimento da dinâmica de regulação transcricional de seus genes, incluindo aqueles envolvidos em mecanismos de patogenicidade e virulência. Para tanto, duas estratégias foram utilizadas para predição de elementos cis-regulatórios em regiões promotoras do genoma da cepa referência 9a5c, comprovadamente associada à CVC. A primeira, conhecida como phylogenetic footprinting, foi empregada para identificação de elementos regulatórios conservados em promotores de unidades transcricionais ortólogas, levando em consideração o conjunto de genes de X. fastidiosa e 7 espécies comparativas. O critério para identificação de unidades transcricionais ortólogas, isto é, unidades trancricionais oriundas de espécies distintas e cujos promotores compartilham elementos cis-regulatórios, foi paralelamente estudado utilizando-se informações regulatórias das bactérias modelos: Pseudomonas aeruginosa, Bacillus subtilis e Escherichia coli. Os resultados obtidos com análise de phylogenetic footprinting nos permitiu acessar a rede regulatória transcricional da espécie de forma compreensiva (global). Foram estabelecidas 2990 interações regulatórias, compreendendo 80 motivos distribuídos nos promotores de 56.8% das unidades transcricionais do genoma de X. fastidiosa. Na segunda estratégia recuperamos informações regulatórias experimentalmente validadas em E. coli e complementamos o conhecimento de dez regulons de X. fastidiosa, através de uma metodologia de scanning (varredura), dos quais algumas interações regulatórias já haviam sido previamente descritas por outros trabalhos. Destacamos os regulons de Fur e CRP, reguladores transcricionais globais, que se mostraram responsáveis pela modulação de genes relacionados a mecanismos de invasão e colonização do hospedeiro vegetal entre outros. Por fim, análises comparativas em regiões regulatórias correspondentes entre cepas foram realizadas e diferenças possivelmente associadas a particularidades fenotípicas foram identificadas entre 9a5c e J1a12, um isolado de citros não virulento, e 9a5c e Temecula1, um isolado de videira causador da doença de Pierce. / Xylella fastidiosa is a gram-negative, non-flagellated bacterium responsible for causing economically important diseases such as Pierce\'s disease in grapevines and Citrus Variegated Clorosis (CVC) in sweet orange trees. In the present work we performed in silico analysis on promoter sequences of protein-coding genes from this phytopathogen, including those involved in virulence and pathogenic mechanisms, in an attempt to better understand the underlying transcriptional regulatory dynamics. Two strategies for cis-regulatory elements prediction were applied on promoter sequences from 9a5c strain genome, a proven causal agent of CVC. The first one, known as phylogenetic footprinting, involved the prediction of regulatory motifs conserved on promoter sequences of orthologous transcription units from X. fastidiosa and a set of 7 comparatives species. The criteria to identify orthologous transcription units, i. e., those from different species and whose promoter sequences share at least one common regulatory motif, was studied based on regulatory information available for model organisms: Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli. The results obtained with the phylogenetic footprinting analysis permitted us to access the underlying transcriptional regulatory network from the species in a comprehensive manner (genome-wide), with a total of 2990 regulatory interactions corresponding to 80 predicted motifs distributed on promoter sequences of 56.8% of all transcription units. In the second strategy regulatory information from E. coli was recovered and used to expand the knowledge of ten regulons in X. fastidiosa, through a scanning process, of which some regulatory interactions were previously described by independent studies. We emphasize some genes related to host invasion and colonization present in the Fur and CRP regulons, two global transcription regulators. Lastly, comparative analysis on corresponding regulatory regions among strains were performed and differences possibly associated to phenotypic variation were identified between 9a5c and J1a12, a non-virulent strain isolated from orange trees, and between 9a5c and Temecula1, a strain associated to Pierce\'s disease on grapevines.

Durch ihren Einfluß auf die Genexpression reguliert die zirkadiane Uhr physiologische Funktionen vieler Organe. Obwohl der zugrundeliegende allgemeine Uhrmechanismus gut untersucht ist, bestehen noch viele Unklarheiten über die gewebespezifische Regulation zirkadianer Gene. Neben ihrer gemeinsamen 24-h-Periode im Expressionsmuster unterscheiden diese sich darin, zu welcher Tageszeit sie am höchsten exprimiert sind und in welchem Gewebe sie oszillieren. Mittels Überrepräsentationsanalyse lassen sich Bindungsstellen von Transkriptionsfaktoren identifizieren, die an der Regulation ähnlich exprimierter Gene beteiligt sind. Um diese Methode auf zirkadiane Gene anzuwenden, ist es nötig, Untergruppen ähnlich exprimierter Gene genau zu definieren und Vergleichsgene passend auszuwählen. Eine hierarchische Methode zur Kontrolle der FDR hilft, aus der daraus entstehenden Menge vieler Untergruppenvergleiche signifikante Ergebnisse zu filtern. Basierend auf mit Microarrays gemessenen Zeitreihen wurde durch Promotoranalyse die gewebespezifische Regulation von zirkadianen Genen zweier Zelltypen in Mäusen untersucht. Bindungsstellen der Transkriptionsfaktoren CLOCK:BMAL1, NF-Y und CREB fanden sich in beiden überrepräsentiert. Diesen verwandte Transkriptionsfaktoren mit spezifischen Komplexierungsdomänen binden mit unterschiedlicher Stärke an Motivvarianten und arrangieren dabei Interaktionen mit gewebespezifischeren Regulatoren (z.B. HOX, GATA, FORKHEAD, REL, IRF, ETS Regulatoren und nukleare Rezeptoren). Vermutlich beeinflußt dies den Zeitablauf der Komplexbildung am Promotor zum Transkriptionsstart und daher auch gewebespezifische Transkriptionsmuster. In dieser Hinsicht sind der Gehalt an Guanin (G) und Cytosin (C) sowie deren CpG-Dinukleotiden wichtige Promotoreigenschaften, welche die Interaktionswahrscheinlichkeit von Transkriptionsfaktoren steuern. Grund ist, daß die Affinitäten, mit denen Regulatoren zu Promotoren hingezogen werden, von diesen Sequenzeigenschaften abhängen. / A circadian clock in peripheral tissues regulates physiological functions through gene expression timing. However, despite the common and well studied core clock mechanism, understanding of tissue-specific regulation of circadian genes is marginal. Overrepresentation analysis is a tool to detect transcription factor binding sites that might play a role in the regulation of co-expressed genes. To apply it to circadian genes that do share a period of about 24 hours, but differ otherwise in peak phase timing and tissue-specificity of their oscillation, clear definition of co-expressed gene subgroups as well as the appropriate choice of background genes are important prerequisites. In this setting of multiple subgroup comparisons, a hierarchical method for false discovery control reveals significant findings. Based on two microarray time series in mouse macrophages and liver cells, tissue-specific regulation of circadian genes in these cell types is investigated by promoter analysis. Binding sites for CLOCK:BMAL1, NF-Y and CREB transcription factors are among the common top candidates of overrepresented motifs. Related transcription factors of BHLH and BZIP families with specific complexation domains bind to motif variants with differing strengths, thereby arranging interactions with more tissue-specific regulators (e.g. HOX, GATA, FORKHEAD, REL, IRF, ETS regulators and nuclear receptors). Presumably, this influences the timing of pre-initiation complexes and hence tissue-specific transcription patterns. In this respect, the content of guanine (G) and cytosine (C) bases as well as CpG dinucleotides are important promoter properties directing the interaction probability of regulators, because affinities with which transcription factors are attracted to promoters depend on these sequence characteristics.

En el llevat Saccharomyces cerevisiae els canvis en les condicions osmòtiques del medi extracel.lular són sensades per la MAP cinasa Hog1, la qual permet dur a terme l'adaptació cel.lular mitjançant la modulació de l'expressió gènica, de la traducció i de la progressió del cicle cel.lular. A l'inici d'aquest projecte de tesi, els mecanismes pels quals Hog1 controla l'expressió gènica no eren del tot coneguts. El nostre objectiu va ser caracteritzar el mecanisme molecular pel qual Hog1 modula la transcripció en resposta a estrès osmòtic. Hem aconseguit demostrar que el reclutament de Hog1 als promotors sensibles a estrès osmòtic per part del factor de transcripció és essencial per al reclutament i activació de la RNA polimerasa II, mecanisme que podria estar conservat en les cèl.lules eucariotes. També hem identificat noves activitats remodeladores de cromatina implicades en la resposta gènica a osmoestrès mediada per Hog1. Vàrem realitzar un cribatge genètic per identificar mutacions que provoquessin osmosensibilitat i una reducció en l'expressió de gens de resposta a estrès osmòtic. Aquest cribatge ens va permetre identificar nous reguladors de la transcripció mediada per osmoestrès: la histona deacetilasa Rpd3 i els complexes SAGA i mediador. Els nostres resultats permeten, doncs, definir un important paper per a Rpd3, SAGA i mediador en la inducció gènica mediada per Hog1, i han estat importants per assolir una millor visió de com les cinases activades per estrès regulen la iniciació de la transcripció. / In Saccharomyces cerevisiae, changes in the extracellular osmotic conditions are sensed by the HOG MAPK pathway, which elicits the program for cell adaptation, including modulation of gene expression, translation and cell-cycle progression. At the beginning of this PhD Project, the mechanisms by which Hog1 was controlling gene transcription were not completely understood. Our main objective was to characterize the molecular mechanisms by which the Hog1 MAPK modulates transcription upon osmostress. We have shown that anchoring of Hog1 to osmoresponsive promoters by the transcription factor is essential for recruitment and activation of RNA polymerase II, a mechanism that might be conserved among eukaryotic cells. In addition, we identified novel chromatin modifying and remodelling activities involved in the Hog1-mediated osmostress gene expression. We performed a genome-wide genetic screening searching for mutations that render cells osmosensitive and displayed reduced expression of osmoresponsive genes. Rpd3 histone deacetylase, SAGA and Mediator complexes were identified as novel regulators of osmostress-mediated transcription. Thus, our results define a major role for Rpd3, SAGA and Mediator in the Hog1-mediated osmostress gene induction, and have been important to achieve a better view of how a SAPK regulates transcription initiation.

En el sector de la promoció construcció, i en especial, en el subsector de la promoció construcció d'habitatges, l'empresari ha de tenir un bon coneixement de les variables d'entorn ja que la consideració de les mateixes seran fonamentals a l'hora de prendre decisions sobre planificació estratègica. En l'actualitat vivim una fase de canvis socioeconòmics que dificulten la previsió del comportament futur de les variables d'entorn. Per tant, el subjecte decisor es troba en un ambient d'incertesa que s'aguditza per la majoritària presència de factors qualitatius difícils de quantificar. Llavors, l'empresari promotor constructor haurà de recórrer a tècniques operatives de gestió que tinguin present aquesta situació i això serà possible a partir de les eines que ens ofereix la lògica borrosa. Aquesta tesi s'ha estructurat en tres parts: En la primera part, exposem les característiques específiques i l'evolució del sector. En la segona part, expliquem la metodologia i, en la tercera part, exposem diverses aplicacions de la metodologia borrosa per l'establiment de noves estratègies de gestió aplicades al sector objecte d'estudi. / In the management of promoters builders business area, specially in the subarea of house promotion building, to gather a large information of the variables around becomes essential. Their being taken into consideration is fundamental when considering decisions upon strategic foresight. Today, socioeconomic changes make difficult to forecast the future behaviour of the variables around and the uncertain atmosphere intensifies due to the majority presence of qualitative factors hard to quantify. In the management of promoters builders business it turns as necessary to apply to operative techniques that take into account the present situation. This is possible by using the tools fuzzy logics grant us. The first part of this thesis gives specific characteristics of the area and its evolution. Methodology is explained in the second part and in the third part different practical examples of fuzzy logic methodology are presented to establish new management strategies applied to this field.

All living organisms are made of cells and they store their hereditary information in the form of double stranded DNA. In all organisms DNA replication and repair is essential for cell division and cell survival. These processes require deoxyribonucleotides (dNTPs), the building blocks of DNA. Ribonucleotide reductase (RNR) is catalyzing the rate limiting step in the de novo synthesis of dNTPs. Active RNR is a heterodimeric protein complex. In S phase cells, the mouse RNR consists of the R1 and the R2 proteins. The R1/R2 RNR-complex supplies the cell with dNTPs required for DNA replication. Outside S-phase or in non-proliferating cells RNR is composed of R1 and p53R2 proteins. The R1/p53R2 RNR-complex supplies cells with dNTPs required for mitochondrial DNA replication and for DNA repair. An undisturbed dNTP regulation is important since unbalanced dNTP pools results in DNA mutations and cell death. Since unbalanced pools are harmful to the cell, RNR activity is regulated at many levels. The aim of this thesis is to study how the mouse RNR genes are regulated at a transcriptional level. We have focused on the promoter regions of all three mouse RNR genes. Primer extension experiments show that the transcription start of the TATA-less p53R2 promoter colocalizes with an earlier unidentified initiator element (Inr-element). This element is similar to the known Inr-element in the mouse R1 promoter. Furthermore, functional studies of the R1 promoter revealed a putative E2F binding element. This result suggests that the S phase specific transcription of the R1 gene is regulated by a similar mechanism as the R2 promoter which contains an E2F binding site. Finally we have established a method to partially purify the transcription factor(s) binding the upstream activating region in the mouse R2 promoter by phosphocellulose chromatography and affinity purification using oligonucleotides immobilized on magnetic beads. This method will allow us to further study the transcription factors responsible for activating expression of the R2 protein. This method has a potential to be utilized as a general method when purifying unknown transcription factors.

Xylella fastidiosa é uma bactéria gram-negativa, não flagelada, agente causal de doenças de importância econômica como a doença de Pierce nas videiras e a clorose variegada dos citros (CVC) nas laranjeiras. O objetivo do presente trabalho foi realizar análises in silico das sequências promotoras dos genes deste fitopatógeno em uma tentativa de arrecadar novas evidências para o melhor entendimento da dinâmica de regulação transcricional de seus genes, incluindo aqueles envolvidos em mecanismos de patogenicidade e virulência. Para tanto, duas estratégias foram utilizadas para predição de elementos cis-regulatórios em regiões promotoras do genoma da cepa referência 9a5c, comprovadamente associada à CVC. A primeira, conhecida como phylogenetic footprinting, foi empregada para identificação de elementos regulatórios conservados em promotores de unidades transcricionais ortólogas, levando em consideração o conjunto de genes de X. fastidiosa e 7 espécies comparativas. O critério para identificação de unidades transcricionais ortólogas, isto é, unidades trancricionais oriundas de espécies distintas e cujos promotores compartilham elementos cis-regulatórios, foi paralelamente estudado utilizando-se informações regulatórias das bactérias modelos: Pseudomonas aeruginosa, Bacillus subtilis e Escherichia coli. Os resultados obtidos com análise de phylogenetic footprinting nos permitiu acessar a rede regulatória transcricional da espécie de forma compreensiva (global). Foram estabelecidas 2990 interações regulatórias, compreendendo 80 motivos distribuídos nos promotores de 56.8% das unidades transcricionais do genoma de X. fastidiosa. Na segunda estratégia recuperamos informações regulatórias experimentalmente validadas em E. coli e complementamos o conhecimento de dez regulons de X. fastidiosa, através de uma metodologia de scanning (varredura), dos quais algumas interações regulatórias já haviam sido previamente descritas por outros trabalhos. Destacamos os regulons de Fur e CRP, reguladores transcricionais globais, que se mostraram responsáveis pela modulação de genes relacionados a mecanismos de invasão e colonização do hospedeiro vegetal entre outros. Por fim, análises comparativas em regiões regulatórias correspondentes entre cepas foram realizadas e diferenças possivelmente associadas a particularidades fenotípicas foram identificadas entre 9a5c e J1a12, um isolado de citros não virulento, e 9a5c e Temecula1, um isolado de videira causador da doença de Pierce. / Xylella fastidiosa is a gram-negative, non-flagellated bacterium responsible for causing economically important diseases such as Pierce\'s disease in grapevines and Citrus Variegated Clorosis (CVC) in sweet orange trees. In the present work we performed in silico analysis on promoter sequences of protein-coding genes from this phytopathogen, including those involved in virulence and pathogenic mechanisms, in an attempt to better understand the underlying transcriptional regulatory dynamics. Two strategies for cis-regulatory elements prediction were applied on promoter sequences from 9a5c strain genome, a proven causal agent of CVC. The first one, known as phylogenetic footprinting, involved the prediction of regulatory motifs conserved on promoter sequences of orthologous transcription units from X. fastidiosa and a set of 7 comparatives species. The criteria to identify orthologous transcription units, i. e., those from different species and whose promoter sequences share at least one common regulatory motif, was studied based on regulatory information available for model organisms: Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli. The results obtained with the phylogenetic footprinting analysis permitted us to access the underlying transcriptional regulatory network from the species in a comprehensive manner (genome-wide), with a total of 2990 regulatory interactions corresponding to 80 predicted motifs distributed on promoter sequences of 56.8% of all transcription units. In the second strategy regulatory information from E. coli was recovered and used to expand the knowledge of ten regulons in X. fastidiosa, through a scanning process, of which some regulatory interactions were previously described by independent studies. We emphasize some genes related to host invasion and colonization present in the Fur and CRP regulons, two global transcription regulators. Lastly, comparative analysis on corresponding regulatory regions among strains were performed and differences possibly associated to phenotypic variation were identified between 9a5c and J1a12, a non-virulent strain isolated from orange trees, and between 9a5c and Temecula1, a strain associated to Pierce\'s disease on grapevines.

This work presents the results ofour research of cell-free nucleic acids (cfNA). The first part shows changes in methylation patterns of immune response genes promoters that are detectable in plasma during the hemodialysis sessions and also differences in methylation between patients and healthy subjects. Alterations include genes that play their role in the regulation of hematopoiesis and these changes are in close relation with the need of anemia therapy. In the other plasma cfNA study we detected miRNA signatures in patients with acute myeloid leukemia at diagnosis (6 highly abundant miRNAs found) and in remission achieved after standard chemotherapy (trend to n01malization, lower levels ofthese miRNAs). Another part of work presents data from the study of potential non-invasive biomarker of bladder cancer. The amounts of cfDNA in urine are higher in patients than in healthy subjects and there were found 5 down-regulated miRNAs. Simultaneously it was established set of 30 miRNAs that are constantly present in urine supematants independently on sex, age and healthy status of subjects. The last part presents analysis ofcell-free fetal DNA. We analyzed differences between a new quantification method - droplet digital PCR and real-time PCR which is used routinely nowadays. Slightly more precise was...

“Why do immigrants and refugees need community health workers/lay health workers (CHWs) if Canada already has a universal health care system?” Abundant evidence demonstrates that despite the universality of our health care system marginalized populations, including immigrants and refugees, experience barriers to accessing the health system. Evidence on the role of CHWs facilitating access is both lacking and urgently needed. This dissertation contributes to this evidence by providing a thick description and thorough analytical exploration of a CHW model, in Edmonton, Canada. Specifically, I examine the activities of the Multicultural Health Brokers Co-operative (MCHB Co-op) and its Multicultural Health Brokers from 1992 to 2011 as well as the relationship they have with Alberta Health Services (AHS) Edmonton Zone Public Health. The research for this study is based on an instrumental and embedded qualitative case study design. The case is the MCHB Co-op, an independently-run multicultural health worker co-operative, which contracts with health and social services providers in Edmonton to offer linguistically- and culturally-appropriate services to marginalized immigrant and refugee women and their families. The two embedded mini-cases are two programs of the MCHB Co-op: Perinatal Outreach and Health for Two, which are the raison d’être for a sustained partnership between the MCHB Co-op and AHS. The phenomenon under study is the Multicultural Health Brokers’ practice. I triangulate multiple methods (research strategies and data sources), including 46 days of participant and direct observation, 44 in-depth interviews (with Multicultural Health Brokers, mentors, women using the programs, health professionals and outsiders who knew of the work of the MCHB Co-op and Multicultural Health Brokers), and document review and analysis of policy documents, yearly reports, training manuals, educational materials as well as quantitative analysis of the Health Brokers’ 3,442 client caseload database. In addition, data include my field notes of both descriptive and analytical reflections taken throughout the onsite research. I also triangulate various theoretical frameworks to explore how historically specific social structures, economic relationships, and ideological assumptions serve to create and reinforce the conditions that give rise to the need for CHWs, and the factors that aid or hinder their ability to facilitate marginalized populations’ access to health and social services. Findings reveal that Multicultural Health Brokers facilitate access to health and social services as well as foster community capacity building in order to address settlement, adaptation, and integration of immigrant and refugee women and their families into Canadian society. Findings also demonstrate that the Multicultural Health Broker model is an example of collaboration between community-based organizations and local systems in targeting health equity for marginalized populations; in particular, in perinatal health and violence against women. A major problem these workers face is they provide important services as part of Canada’s health human resources workforce, but their contributions are often not recognized as such. The triangulation of methods and theory provides empirical and theoretical understanding of the Multicultural Health Brokers’ contribution to immigrant and refugee women and their families’ feminist urban citizenship.

“Why do immigrants and refugees need community health workers/lay health workers (CHWs) if Canada already has a universal health care system?” Abundant evidence demonstrates that despite the universality of our health care system marginalized populations, including immigrants and refugees, experience barriers to accessing the health system. Evidence on the role of CHWs facilitating access is both lacking and urgently needed. This dissertation contributes to this evidence by providing a thick description and thorough analytical exploration of a CHW model, in Edmonton, Canada. Specifically, I examine the activities of the Multicultural Health Brokers Co-operative (MCHB Co-op) and its Multicultural Health Brokers from 1992 to 2011 as well as the relationship they have with Alberta Health Services (AHS) Edmonton Zone Public Health. The research for this study is based on an instrumental and embedded qualitative case study design. The case is the MCHB Co-op, an independently-run multicultural health worker co-operative, which contracts with health and social services providers in Edmonton to offer linguistically- and culturally-appropriate services to marginalized immigrant and refugee women and their families. The two embedded mini-cases are two programs of the MCHB Co-op: Perinatal Outreach and Health for Two, which are the raison d’être for a sustained partnership between the MCHB Co-op and AHS. The phenomenon under study is the Multicultural Health Brokers’ practice. I triangulate multiple methods (research strategies and data sources), including 46 days of participant and direct observation, 44 in-depth interviews (with Multicultural Health Brokers, mentors, women using the programs, health professionals and outsiders who knew of the work of the MCHB Co-op and Multicultural Health Brokers), and document review and analysis of policy documents, yearly reports, training manuals, educational materials as well as quantitative analysis of the Health Brokers’ 3,442 client caseload database. In addition, data include my field notes of both descriptive and analytical reflections taken throughout the onsite research. I also triangulate various theoretical frameworks to explore how historically specific social structures, economic relationships, and ideological assumptions serve to create and reinforce the conditions that give rise to the need for CHWs, and the factors that aid or hinder their ability to facilitate marginalized populations’ access to health and social services. Findings reveal that Multicultural Health Brokers facilitate access to health and social services as well as foster community capacity building in order to address settlement, adaptation, and integration of immigrant and refugee women and their families into Canadian society. Findings also demonstrate that the Multicultural Health Broker model is an example of collaboration between community-based organizations and local systems in targeting health equity for marginalized populations; in particular, in perinatal health and violence against women. A major problem these workers face is they provide important services as part of Canada’s health human resources workforce, but their contributions are often not recognized as such. The triangulation of methods and theory provides empirical and theoretical understanding of the Multicultural Health Brokers’ contribution to immigrant and refugee women and their families’ feminist urban citizenship.