This diploma thesis critically examines traditional positivist interpretations concerning the birth of four core norms of the current legal regime governing activities of states in Outer Space: a) non-militarization, b) peaceful use, c) non- appropriation, and d) freedom of access. Traditional interpretations often explicate the emergence of the aforementioned principles in static terms, either as a result of universal aspirations of mankind to peaceful explore the final frontier, or alternatively as a product of rational calculations of self-interested states. Analysis of the first decade of the space age through the lens of post-structural genealogical method proposed by Richard Price reveals a much more complex picture. Application of critical genealogical approach indicates that the emergence of the four norms in question was contingent on the presence of particular historical circumstances and cognitive structures of the Cold War: a) fear of nuclear weapons, b) policy of containment, c) trauma of the Second World War, and d) highly politicized outer space discourse due to the presence of the space race. The findings also indicate that without the presence of the particular historical circumstances and cognitive structures that necessitated their emergence, the four principles in question are...

Yes / As an important economic insect, Bombyx mori is also a useful model organism for lepidopteran insect. Integrins are evolutionarily conserved fromsponges to humans, and play vital roles inmany physiological and pathological processes. To explore their diverse functions of integrins in insect, eleven integrins including sixα and five β subunitswere cloned and characterized fromsilkworm. Our results showed that integrins fromsilkwormown more family members compared to other invertebrates. Among those α subunits, integrins α1, α2, and the other four subunits belong to PS1, PS2, and PS3 groups, respectively. The β subunits mainly gather in the insect βν group except the β1 subunit which belongs to the insect β group. Expression profiles demonstrated that the integrins exhibited distinct patterns, but were mainly expressed in hemocytes. α1 and β2 subunits are the predominant ones either in the embryogenesis or larva stages. Interestingly, integrins were significantly up-regulated after stimulated by 20-hydroxyecdysone (20-E) in vivo. These results indicate that integrins performdiverse functions in hemocytes of silkworm. Overall, our results provide a newinsight into the functional and evolutionary features of integrins. / National Basic Research Programof China (No. 2012cb114603), the Research Fund for the Doctoral Program of Higher Education of China (20130182110003), the Natural Science Foundation of Chongqing (cstc2013jcyjys0007), and the Fundamental Research Funds for the Central Universities (SWU111014).

To further facilitate mitochondrial DNA (mtDNA) sequence analysis for human identity testing, a better understanding of its mutation rate is needed. Prior to the middle 1990's the mutation rate applied to a forensic or evolutionary analysis was determined by phylogenetic means, This method involved calculating genetic distances as determined by amino acid or DNA sequence variability within or between species. The mutation rate as determined by this method ranged from 0.025-0.26 nucleotide substitutions/ site/ myr (million years). With the recent advent of mtDNA analysis as a tool in human identity testing an increased number of observations have recently come to light calling into question the mutation rate derived from the phylogenetic method. The mutation rate as observed from forensic analysis appears to be much higher than that calculated phylogenetically. This is an area that needs to be resolved in human identity testing. Mutations that occur within a maternal lineage can lead to a possible false exclusion of an individual as belonging to that lineage. A greater understanding of the actual rate of mutation within a given maternal lineage can assist in determining criteria for including or excluding individuals as belonging to that lineage. The method used to assess the mutation rate in this study was to compare mtDNA sequences derived from the HVI and HVII regions of the D-loop from several different maternal lineages. The sequence information was derived from five unrelated families consisting of thirty-five individuals. One intergenerational mutational event was found. This derives to approximately 1.9 nucleotide substitutions/ site/ myr. This mutation rate was very consistent with several other similar studies. This increased mutation rate needs to be considered by forensic testing laboratories performing mtDNA sequence analysis prior to formulating any conclusive results.

<p>Glutathione transferases (GSTs) constitute a family of multifarious enzymes that conjugate glutathione (GSH) with a wide range of electrophiles. GSTs are grouped into different classes based on protein sequence similarities. Despite high sequence identities between GSTs of the same class they often display different substrate specificites. Human GST M1-1 is efficiently catalyzing the conjugation of GSH and various epoxide substrates, whereas the 84% sequence-identical GST M2-2 has low activities with the same substrates.</p><p>Evolutionary rate analysis was used to identify hypervariable amino acid positions among GST Mu class sequences. A Thr to Ser conversion of the variable residue 210 in GST M2-2 elicited a drastic increase in catalytic activity with epoxides, which is the characteristic activity of GST M1-1. This provides support for the usefulness of evolutionary analysis in identifying functionally important residues, although the additional mutations of two other variable residues did not confer any noteworthy changes in activity.</p><p>To further investigate the functional importance of residue T210 in GST M2-2 it was replaced by all other commonly occurring amino acids. The replacements caused marked changes in substrate specificity, stability, and expressivity, indicating how functionalities of a duplicated Mu class GST may easily be altered by point mutations. </p><p>The stereo- and regioselectivity in epoxide-conjugation catalyzed by GSTs M1-1 and M2-2 was investigated. The results show that a serine in position 210 is beneficial for high enantioselectivity with trans-stilbene oxide. However, an alanine in position 210 is more favorable for stereo- and regioselectivity with the smaller epoxide substrate styrene-7,8-oxide. </p><p>The low enantioselectivity of GST M1-1 was improved 10- and 9- fold with styrene-7,8-oxide and 1-phenylpropylene oxide, respectively, through different combination of site-specific mutations and posttranslational chemical modifications. The approach can be employed in more extensive screening experiments where a large variety of modifications easily can be tested.</p>

Glutathione transferases (GSTs) constitute a family of multifarious enzymes that conjugate glutathione (GSH) with a wide range of electrophiles. GSTs are grouped into different classes based on protein sequence similarities. Despite high sequence identities between GSTs of the same class they often display different substrate specificites. Human GST M1-1 is efficiently catalyzing the conjugation of GSH and various epoxide substrates, whereas the 84% sequence-identical GST M2-2 has low activities with the same substrates. Evolutionary rate analysis was used to identify hypervariable amino acid positions among GST Mu class sequences. A Thr to Ser conversion of the variable residue 210 in GST M2-2 elicited a drastic increase in catalytic activity with epoxides, which is the characteristic activity of GST M1-1. This provides support for the usefulness of evolutionary analysis in identifying functionally important residues, although the additional mutations of two other variable residues did not confer any noteworthy changes in activity. To further investigate the functional importance of residue T210 in GST M2-2 it was replaced by all other commonly occurring amino acids. The replacements caused marked changes in substrate specificity, stability, and expressivity, indicating how functionalities of a duplicated Mu class GST may easily be altered by point mutations. The stereo- and regioselectivity in epoxide-conjugation catalyzed by GSTs M1-1 and M2-2 was investigated. The results show that a serine in position 210 is beneficial for high enantioselectivity with trans-stilbene oxide. However, an alanine in position 210 is more favorable for stereo- and regioselectivity with the smaller epoxide substrate styrene-7,8-oxide. The low enantioselectivity of GST M1-1 was improved 10- and 9- fold with styrene-7,8-oxide and 1-phenylpropylene oxide, respectively, through different combination of site-specific mutations and posttranslational chemical modifications. The approach can be employed in more extensive screening experiments where a large variety of modifications easily can be tested.

abstract: Here I document the breadth of the CAP (Cysteine-RIch Secretory Proteins (CRISP), Antigen 5 (Ag5), and the Pathogenesis-Related 1 (PR)) protein superfamily and trace some of the major events in the evolution of this family with particular focus on vertebrate CRISP proteins. Specifically, I sought to study the origin of these CAP subfamilies using both amino acid sequence data and gene structure data, more precisely the positions of exon/intron borders within their genes. Counter to current scientific understanding, I find that the wide variety of CAP subfamilies present in mammals, where they were originally discovered and characterized, have distinct homologues in the invertebrate phyla contrary to the common assumption that these are vertebrate protein subfamilies. In addition, I document the fact that primitive eukaryotic CAP genes contained only one exon, likely inherited from prokaryotic SCP-domain containing genes which were, by nature, free of introns. As evolution progressed, an increasing number of introns were inserted into CAP genes, reaching 2 to 5 in the invertebrate world, and 5 to 15 in the vertebrate world. Lastly, phylogenetic relationships between these proteins appear to be traceable not only by amino acid sequence homology but also by preservation of exon number and exon borders within their genes. / Dissertation/Thesis / Masters Thesis Biology 2016

Cancer is a disease of the genome, requiring mutation or epimutation of specific genes to develop. The subsequent progression of cancer and response to therapies is also dictated to some degree by new mutation and clonal selection on that novel variation. However, it is thought that the majority of somatic mutations that occur in cancer are inconsequential passengers, and only a subset of functionally important driver mutations are of importance for cancer biology. This project set out to adapt and apply well-established methods from the field of molecular evolution to measure the selective forces driving the development of cancers. The ultimate objective being an improved understanding of which mutations help or hinder the progression of a cancer. Somatic cancer mutations were identified through the analysis of paired tumour and non-tumour whole-exome sequence data from the same individual. Primary data from 1005 patients was processed and complemented with additional publicly available pre-processed somatic variant calls from 4728 patients. Tumours were classified by tissue of origin and also their spectrum of substitution mutations. An advanced evolutionary analysis framework was established, allowing somatic single nucleotide variant data to be analysed as traditional organismal DNA sequence. Estimates of amino acid changing (non-synonymous) and synonymous mutation rates were derived and maximum likelihood tests of selection applied to identify genes and regions of genes subject to selective pressure during oncogenesis. While the meta-analysis of all patients provided unprecedented power for such a study, more refined analyses based on the stratification of patients gave insights into the pathways of importance for specific tissues of origin. Additionally, stratification of patients by the relative frequencies of different mutation types in a tumour also provided insights into how mutation profile influences the sites, genes and pathways that are perturbed in the development of cancer. Of particular interest here, was to test the hypothesis that both (1.) mutation spectrum and (2.) tissue of origin, set the evolutionary trajectory of a cancer. Building on this I sought to estimate their relative contributions. During this work an unexpected, localised mutation pattern was discovered and subsequent analysis demonstrated some loci to be highly susceptible to small segmental deletions in a subset of cancers. In the absence of a justifiable model of neutral segmental deletion it was not possible to infer whether these major mutations could be considered passengers or drivers of cancer progression. In contrast, an advantage of the evolutionary approach applied to nucleotide substitutions in protein coding sequences is that there is a justified model of neutral evolution (synonymous changes). Using this approach, I have not only been able to detect genes harbouring putative cancer driver mutations, but have also found evidence for genes subject to purifying selection in cancers where potentially disruptive mutations appear to be deleterious to cancer progression. Such genes, if they are non-essential in the adult organism, could provide a novel type of target for anti-cancer therapeutics.

Globally disseminated bacterial pathogens frequently cause epidemics that are of major importance in public health. Of particular significance is the capacity for some of these bacteria to switch into a new environment leading to the emergence of pathogenic clones. Understanding the evolution and epidemiology of such pathogens is essential for designing rational ways for prevention, diagnosis and treatment of the diseases they cause. Whole-genome sequencing of multiple isolates facilitating comparative genomics and phylogenomic analyses provides high-resolution insights, which are revolutionizing our understanding of infectious diseases. In this thesis, a range of population genomic analyses are employed to study the molecular mechanisms and the evolutionary dynamics of bacterial pathogen niche adaptation, specifically between humans, animals and the environment. A large-scale population genomic approach was used to provide a global perspective of the host-switching events that have defined the evolution of Staphylococcus aureus in the context of its host-species. To investigate the genetic basis of host-adaptation, we performed genome-wide association analysis, revealing an array of accessory genes linked to S. aureus host-specificity. In addition, positive selection analysis identified biological pathways encoded in the core genome that are under diversifying selection in different host-species, suggesting a role in host-adaptation. These findings provide a high-resolution view of the evolutionary landscape of a model multi-host pathogen and its capacity to undergo changes in host ecology by genetic adaptation. To further explore S. aureus host-adaptive evolution, we examined the population dynamics of this pathogen after a simulated host-switch event. S. aureus strains of human origin were used to infect the mammary glands of sheep, and bacteria were passaged in multiple animals to simulate onward transmission events. Comparative genomics of passaged isolates allowed us to characterize the genetic changes acquired during the early stages of evolution in a novel host-species. Co-infection experiments using progenitor and passaged strains indicated that accumulated mutations contributed to enhanced fitness, indicating adaptation. Within-host population genomic analysis revealed the existence of population bottlenecks associated with transmission and establishment of infection in new hosts. Computational simulations of evolving genomes under regular bottlenecks supported that the fitness gain of beneficial mutations is high enough to overcome genetic drift and sweep through the population. Overall, these data provide new information relating to the critical early events associated with adaptation to novel host-species. Finally, population genomics was used to study the total diversity of Legionella longbeachae from patient and environmental sources and to investigate the epidemiology of a L. longbeachae outbreak in Scotland. We analysed the genomes of isolates from a cluster of legionellosis cases linked to commercial growing media in Scotland and of non-outbreak-associated strains from this and other countries. Extensive genetic diversity across the L. longbeachae species was identified, associated with intraspecies and interspecies gene flow, and a wide geographic distribution of closely related genotypes. Of note, a highly diverse pool of L. longbeachae genotypes within compost samples that precluded the genetic establishment of an infection source was observed. These data represent a view of the genomic diversity of this pathogen that will inform strategies for investigating future outbreaks. Overall, our findings demonstrate the application of population genomics to understand the molecular mechanisms and the evolutionary dynamics of bacterial adaptation to different ecological niches, and provide new insights relevant to other major bacterial pathogens with the capacity to spread between environments.

Resumo: A composição de quasiespécies do vírus da Hepatite C (HCV) pode ter implicações importantes com relação à persistência viral e à resposta a terapia baseada em Interferon. A região NS5A completa foi analisada para avaliar se a composição de quasiespécies do HCV 1a/1b está relacionada à resposta ao tratamento combinado de interferon peguilado (PEGIFN) e ribavirina. Seiscentos e noventa seqüências correspondentes a região não estrutural 5A (NS5A) completa foram geradas a partir de amostras coletadas antes, durante a após a administração da terapia de pacientes respondedores, não respondedores e respondedores ao final do tratamento. Este estudo apresenta evidências de que a homogeneidade da composição de quasiespécies, e a baixa complexidade e diversidade da região NS5A em amostras préterapia estão associados à resposta virológica sustentada. Portanto, a alta diversidade e complexidade de quasiespécies podem fornecer ao vírus melhores oportunidades de evadir a terapia antiviral. Análises filogenéticas não demonstraram o agrupamento das seqüências de acordo os padrões específicos de resposta ao tratamento. Contudo, o agrupamento distinto de seqüências pré e pós-terapia foi observado, sugerindo que um processo adaptativo ocorreu durante o período analisado. Adicionalmente, a dinâmica evolutiva da composição de quasiespécies demonstrou estar sob pressão seletiva purificadora ou purificadora relaxada, o que é condizente com a população de quasiespécies diversificada no pré-terapia, seguida de um aumento em freqüência de quasiespécies predominantes nas amostras pós-tratamento, provavelmente devido a conferirem alguma vantagem ao vírus. Estes resultados sugerem que a diversidade de quasiespécies da região NS5A pode ser importante para o entendimento dos mecanismos de baixa resposta virológica sustentada em pacientes com Hepatite C crônica / Abstract: The quasispecies composition of Hepatitis C virus (HCV) could have important implications with regard to viral persistence and response to interferon-based therapy. The complete NS5A was analyzed to evaluate whether the composition of NS5A quasispecies of HCV 1a/1b is related to responsiveness to combined interferon pegylated (PEG-IFN) and ribavirin therapy. Six hundred and ninety full-length NS5A sequences were generated from samples collected before, during and after treatment from virological sustained responder, non-responder and the end-of-treatment responder patients. This study provides evidence that homogeneity of quasispecies composition, low diversity and less complexity of the NS5A region pre-therapy are associated with viral clearance. Therefore, higher diversity and complexity of quasispecies could offer the virus a better opportunity of evading anti-viral therapy. Phylogenetic relationships concerning complete NS5A sequences obtained from patients did not demonstrate clustering associated with specific response patterns. However, distinctive clustering of pre/post-therapy sequences was observed, suggesting that an evolutionary process occurred during the time course examined. In addition, the evolution of quasispecies over time was subjected to purifying or relaxed purifying selection. This could explain the initial diversified composition of quasispecies at baseline, followed by an increase in the frequency of a predominant quasispecies in 'after treatment' samples of non-responders and end-of-treatment responders, probably because it offers some advantage for the virus. These results suggest that quasispecies diversity of the NS5A region could be important for elucidating the mechanism underlying treatment failure in patients infected with chronic hepatitis C / Orientador: Paula Rahal / Coorientador: Isabel Maria Vicente Guedes Carvalho-Mello / Banca: Camila Malta Romano / Banca: Jonny Yokosawa / Banca: Maurício Lacerda Nogueira / Banca: Fátima Pereira de Souza / Doutor

Made available in DSpace on 2014-06-11T19:32:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-25Bitstream added on 2014-06-13T20:03:27Z : No. of bitstreams: 1 jardim_acg_dr_sjrp.pdf: 1650863 bytes, checksum: 6ecdc00802358d3e90fac9c3024108d4 (MD5) / A composição de quasiespécies do vírus da Hepatite C (HCV) pode ter implicações importantes com relação à persistência viral e à resposta a terapia baseada em Interferon. A região NS5A completa foi analisada para avaliar se a composição de quasiespécies do HCV 1a/1b está relacionada à resposta ao tratamento combinado de interferon peguilado (PEGIFN) e ribavirina. Seiscentos e noventa seqüências correspondentes a região não estrutural 5A (NS5A) completa foram geradas a partir de amostras coletadas antes, durante a após a administração da terapia de pacientes respondedores, não respondedores e respondedores ao final do tratamento. Este estudo apresenta evidências de que a homogeneidade da composição de quasiespécies, e a baixa complexidade e diversidade da região NS5A em amostras préterapia estão associados à resposta virológica sustentada. Portanto, a alta diversidade e complexidade de quasiespécies podem fornecer ao vírus melhores oportunidades de evadir a terapia antiviral. Análises filogenéticas não demonstraram o agrupamento das seqüências de acordo os padrões específicos de resposta ao tratamento. Contudo, o agrupamento distinto de seqüências pré e pós-terapia foi observado, sugerindo que um processo adaptativo ocorreu durante o período analisado. Adicionalmente, a dinâmica evolutiva da composição de quasiespécies demonstrou estar sob pressão seletiva purificadora ou purificadora relaxada, o que é condizente com a população de quasiespécies diversificada no pré-terapia, seguida de um aumento em freqüência de quasiespécies predominantes nas amostras pós-tratamento, provavelmente devido a conferirem alguma vantagem ao vírus. Estes resultados sugerem que a diversidade de quasiespécies da região NS5A pode ser importante para o entendimento dos mecanismos de baixa resposta virológica sustentada em pacientes com Hepatite C crônica / The quasispecies composition of Hepatitis C virus (HCV) could have important implications with regard to viral persistence and response to interferon-based therapy. The complete NS5A was analyzed to evaluate whether the composition of NS5A quasispecies of HCV 1a/1b is related to responsiveness to combined interferon pegylated (PEG-IFN) and ribavirin therapy. Six hundred and ninety full-length NS5A sequences were generated from samples collected before, during and after treatment from virological sustained responder, non-responder and the end-of-treatment responder patients. This study provides evidence that homogeneity of quasispecies composition, low diversity and less complexity of the NS5A region pre-therapy are associated with viral clearance. Therefore, higher diversity and complexity of quasispecies could offer the virus a better opportunity of evading anti-viral therapy. Phylogenetic relationships concerning complete NS5A sequences obtained from patients did not demonstrate clustering associated with specific response patterns. However, distinctive clustering of pre/post-therapy sequences was observed, suggesting that an evolutionary process occurred during the time course examined. In addition, the evolution of quasispecies over time was subjected to purifying or relaxed purifying selection. This could explain the initial diversified composition of quasispecies at baseline, followed by an increase in the frequency of a predominant quasispecies in ‘after treatment’ samples of non-responders and end-of-treatment responders, probably because it offers some advantage for the virus. These results suggest that quasispecies diversity of the NS5A region could be important for elucidating the mechanism underlying treatment failure in patients infected with chronic hepatitis C