Salmonella enterica is an enteric pathogen with a broad host tropism that can cause disease ranging from self-limited gastroenteritis to enteric fever. The evolution of S. enterica as a pathogen is driven by the horizontal acquisition of genes that promote virulence and survival within host immune cells, as well as the coordinated regulation of these and ancestral genes by two-component systems (TCS). TCS integrate environmental cues with the transcriptional reprogramming of bacteria, and in the case of Salmonella, result in niche-specific gene expression in response to anti-bacterial cues produced by the host. The TCS SsrA-SsrB in S. enterica is considered the master regulator for intracellular virulence, where SsrA is a sensor kinase that triggers the activation of the DNA binding protein SsrB. The full suite of genes regulated by SsrB in S. enterica, as well as the cues that activate this TCS, have not been fully characterized. Here, we demonstrated that horizontally acquired and ancestral genes in the S. enterica genome have evolved to be regulated by SsrB, and the repression of a set of ancestral genes involved in flagellar motility promotes evasion of the host immune system. Additionally, we identified the production of reactive oxygen species (ROS) by host immune cells as a signal that can activate a cluster of genes regulated by the SsrA-SsrB TCS, likely mediated by SsrA sensing of these ROS. Together, these results expand our understanding of the complex interplay between the pathogen S. enterica and the host that results in bacterial infections. / Thesis / Doctor of Philosophy (PhD) / Salmonella enterica (S. enterica) is a species of bacteria that can cause food poisoning in various animals, including humans, through consumption of contaminated food and water. During an infection, host cells activate numerous defense mechanisms to prevent disease. S. enterica has evolved to turn specific genes on or off in response, resulting in modifications to bacterial and host cell behaviour that promote infection. The timing of these genetic changes is controlled by proteins that can sense specific environmental signals and adjust gene expression accordingly. The specific signals sensed by S. enterica that allow for adaptive gene expression within the host, and the types of genes that are regulated to promote survival, have not been fully identified. Here, we show that S. enterica evolved to repress genes involved in flagellar motility to hide from the host immune response. We further demonstrate that S. enterica can sense anti-bacterial molecules produced by the host, called reactive oxygen species, to trigger specific changes in gene expression. Together, this work reveals novel aspects for the molecular basis of Salmonella enterica pathogenesis.

Salmonella enterica is an intracellular bacterial pathogen of humans and the causative agent of the acute gastrointestinal disease, salmonellosis, and the chronic systemic infection, typhoid fever. Sensor proteins convert environmental signals, including signals detected within the host environment, into biochemical signals to control cellular responses. It has been previously established that the two component regulatory system SsrA-SsrB, consisting of the integral membrane sensor kinase protein SsrA and the cytoplasmic DNA-binding response regulator SsrB are essential for regulation of bacterial factors during systemic intracellular infection. The first chapter of this thesis describes characterization of the sensor kinase SsrA. The structure of the periplasmic sensor domain is modeled and evidence is presented that it is involved in enhancing signaling activity in response to environmental acidification encountered within the intracellular environment. A mechanism whereby protonation of histidine residues within this region in response to acidification drives conformational strain and thereby signaling is proposed. The second chapter describes identification of the DNA-binding motif of SsrB within regulated promoters as well as its regulon. Integration of experimental data with comparative genomics data resulted in identification of the palindromic heptameric DNA recognition motif of SsrB as well as identification of novel SsrB-regulated promoters. In addition, a DNA microarray analysis is described wherein the complete SsrB regulon is identified. Finally, the third chapter describes regulatory input of SsrB to the S. enterica type VI secretion system. This chapter also describes the contribution of this system to systemic dissemination of S. enterica during host infection. Altogether, these data advance understanding of how Salmonella controls factors essential for disease in response to the host environment during infection. / Thesis / Doctor of Philosophy (PhD)

The thesis elaborates on the development of the rating at capital markets and its regulation in the context of the global financial crisis. The first part focuses on the definition of the rating, the kinds and types of grant, explanation of the symbols in the rating scale, the rating process and activities of credit ratings agencies. The second part compares the different development of credit rating industry in the United States of America and in the European Union. It also deals with the participation of credit rating agencies in the financial crisis. The last part is concerned with the Dodd -- Frank Act and Regulation 1060/2009 on credit rating agencies as two different measures granted after crises. The thesis analyzed also their impact on the credit rating industry.

Changes in cis- and trans-regulatory elements are among the prime sources of genetic and phenotypical variation at species level. The introduction of cis- and trans- regulatory variation has played important roles in driving diversity, phenotypical differentiation, and evolution of humans. Therefore, variation that occurs on cis- and trans- regulatory elements becomes imperative to better understanding of human genetic diversity and its evolution. In this research, around 3360 gene regulatory factors (GRF) from the human genome were catalogued. This catalog includes genes that code for proteins that perform gene regulatory activities such DNA-depending transcription, RNA polymerase II transcription cofactor and co-repressor activity, chromatin binding and remodeling, among other 218 regulatory functions. This GRF catalog allowed us to initially explore how some GRF genes have evolved in humans, archaic humans (Neandertal and Denisovan) and non-human primate species. We discussed the likely phenotypical and medical effects that evolutionary changes in GRF genes may have introduced into the human genome; for instance, traits associated to speech and language capabilities, genomic recombination hotspots, diseases, among others. By using genome-wide datasets, we additionally looked for GRFs likely to be candidates for positive selection in three human populations: Utah Residents with Northern and Western Ancestry (CEU), Han Chinese in Beijing (CHB), and Yoruba in Ibadan (YRI). As result, we produced a set of candidates that gathers genes that may have contributed in shaping the phenotypical diversity currently observed in these populations; for instance, by introducing regulatory diversity at population-specific level. We additionally identified six GRF classes enriched for genes located in regions that are likely candidates for positive selection at population specific level. We found that out of the 41 DNA-binding GRF classes classified so far, six groups exhibited enrichment for genes located on regions that may have been under positive selection: C2H2 zinc finger, KRAB-ZNF zinc finger, Homeo domain, Tryptophan cluster, Fork head/winged helix and, and High-mobility HMG domain. We additionally identified three KRAB-ZNF gene clusters, in the chromosomes one, three, and 16, for the Asian population that exhibit regions with extended haplotype homozygosity EHH (larger than 100 kb). This EHH suggests that these regions have undergone positive selection in CHB population. Finally, considering that a representative fraction of the phenotypic diversity observed between humans and its closely related species are likely explained by changes in cis-regulatory elements (CREs), we investigated putative binding sites for the transcription factor GABPa. Using ChIP-Seq data generated from a human cell line (HEK293T), 11,619 putative GABPa CREs were found, Out of which 224 are putative human-specific. To experimentally validate the transcriptional activity of these human-specific CREs, reporter gene essays and knock-down experiments were performed. Our results supported the functionality of these human-specific GABPa CREs and suggest that at least 1,215 genes are primary targets of GABPa. Finally, further analyses depict scenarios that put together transcriptional regulation by GABPa and the evolution of particular human traits; for instance, cognitive abilities, breast morphology, lipids and glucose metabolic pathways, among others.

Changes in cis- and trans-regulatory elements are among the prime sources of genetic and phenotypical variation at species level. The introduction of cis- and trans- regulatory variation has played important roles in driving diversity, phenotypical differentiation, and evolution of humans. Therefore, variation that occurs on cis- and trans- regulatory elements becomes imperative to better understanding of human genetic diversity and its evolution. In this research, around 3360 gene regulatory factors (GRF) from the human genome were catalogued. This catalog includes genes that code for proteins that perform gene regulatory activities such DNA-depending transcription, RNA polymerase II transcription cofactor and co-repressor activity, chromatin binding and remodeling, among other 218 regulatory functions. This GRF catalog allowed us to initially explore how some GRF genes have evolved in humans, archaic humans (Neandertal and Denisovan) and non-human primate species. We discussed the likely phenotypical and medical effects that evolutionary changes in GRF genes may have introduced into the human genome; for instance, traits associated to speech and language capabilities, genomic recombination hotspots, diseases, among others. By using genome-wide datasets, we additionally looked for GRFs likely to be candidates for positive selection in three human populations: Utah Residents with Northern and Western Ancestry (CEU), Han Chinese in Beijing (CHB), and Yoruba in Ibadan (YRI). As result, we produced a set of candidates that gathers genes that may have contributed in shaping the phenotypical diversity currently observed in these populations; for instance, by introducing regulatory diversity at population-specific level. We additionally identified six GRF classes enriched for genes located in regions that are likely candidates for positive selection at population specific level. We found that out of the 41 DNA-binding GRF classes classified so far, six groups exhibited enrichment for genes located on regions that may have been under positive selection: C2H2 zinc finger, KRAB-ZNF zinc finger, Homeo domain, Tryptophan cluster, Fork head/winged helix and, and High-mobility HMG domain. We additionally identified three KRAB-ZNF gene clusters, in the chromosomes one, three, and 16, for the Asian population that exhibit regions with extended haplotype homozygosity EHH (larger than 100 kb). This EHH suggests that these regions have undergone positive selection in CHB population. Finally, considering that a representative fraction of the phenotypic diversity observed between humans and its closely related species are likely explained by changes in cis-regulatory elements (CREs), we investigated putative binding sites for the transcription factor GABPa. Using ChIP-Seq data generated from a human cell line (HEK293T), 11,619 putative GABPa CREs were found, Out of which 224 are putative human-specific. To experimentally validate the transcriptional activity of these human-specific CREs, reporter gene essays and knock-down experiments were performed. Our results supported the functionality of these human-specific GABPa CREs and suggest that at least 1,215 genes are primary targets of GABPa. Finally, further analyses depict scenarios that put together transcriptional regulation by GABPa and the evolution of particular human traits; for instance, cognitive abilities, breast morphology, lipids and glucose metabolic pathways, among others.