The rates and patterns at which nucleotide substitutions occur vary significantly across the genome sequence of vertebrates. A prominent example is the difference in the rate of evolution of functional sequences versus nonfunctional (neutrally evolving) sequences, which is explained by the influence of natural selection on functional sequences. However, even within neutrally evolving sequences there is striking variation in the rates and patterns of nucleotide substitutions. Unraveling the underlying processes that induce this variation is necessary to understand the basic principles of variation in neutral substitution profiles, which in turn is crucial for the identification of regions in the genome where natural selection acts. This research question builds the main focus of the present thesis. I have studied the causes and consequences of variation in different patterns of nucleotide substitutions. In particular, I have investigated substitutional strand asymmetries in mammalian genes and could show that they result from the asymmetric nature of DNA replication and transcription. Comparative analysis of substitutional asymmetries then suggested that the organization of DNA replication and the level of transcription are conserved among mammals. Further, I have examined the variation in CpG mutation rate among human genes and could show that beside DNA methylation also GC content plays a decisive role in CpG mutability. In addition, I have studied the signatures of GC-biased gene conversion and its impact on the evolution of the GC isochore structure in chicken. By comparison of the results in chicken to previous results in human I found evidence that karyotype stability is critical for the evolution of GC isochores. Finally, beside the empirical studies, I have performed theoretical investigations of substitution rates in functional sequences. More precisely, I have explored the temporal dynamics of estimates of the ratio of non-synonymous to synonymous substitution rates dN/dS in a phylogentic-population genetic framework.

Seven Poems was written for a text in combination with live and recorded sounds. The sound sources used for the recorded portion of the composition came from Bb clarinet and male voice. The taped sound was processed with filters, amplifiers, modulators, reverberators, and various other signal modifiers to expand the timbral possibilities.The text that was used for this piece is a poem by Mark Strand written in 1970. This poem is divided into seven sections. Each of the sections has its own character, although all are related to the same idea - the separation of man from himself. / School of Music

Cell fate decisions incorporate distinct and overlapping mechanisms. The activity of caspase 3 was initially understood to be a cell death restricted event, however numerous studies have implicated this enzyme in the regulation of both differentiation and proliferation. How the activity of caspase 3 promotes a non-death cell fate remains unclear. Here we examine the role caspase 3 activity plays during skeletal muscle differentiation; in particular we explore the hypothesis that the mechanism of inducing DNA strand breaks during cell death is also a key feature of differentiation, albeit with a distinctly different outcome. We delineate the transient formation of Caspase 3/Caspase activated DNase (CAD) dependent DNA strand breaks during differentiation. The formation of these breaks is essential for differentiation and the regulation of specific genes. In particular expression of the cell cycle inhibitor p21 is related to the formation of a DNA strand break within the gene’s promoter element. Further, we explored the genome wide association of CAD using Chromatin Immunoprecipitation coupled to high through put sequencing (ChIP-seq). This approach identified a potential role for Caspase3/CAD in regulating the expression of Pax7. Here, a CAD directed DNA strand break in the Pax7 gene is correlated with decreased Pax7 expression, an outcome that has been shown to be critical for progress of the myogenic differentiation program. The regulation of Pax7 expression through a CAD induced DNA strand break raises an intriguing connection between this regulation and oncogenic transformation observed in alveolar rhabdomyosarcoma. The putative site of CAD induced DNA strand breaks that promote decreased Pax7 expression during differentiation corresponds to site of chromosomal translocations responsible for Pax7 fusion events in alveolar rhabdomyosarcoma.

Thesis (Ph. D.)--Utrecht University, 2007. / Afterword and vita in both English and Dutch. Includes bibliographical references (p. 115-120).

Thesis (M.A.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 85-90.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Partial embargo. Vita. Bibliography: 135-163.

The work presented in this thesis focusses primarily on the determination of protein structure at atomic resolution, with NMR spectroscopy as the principle investigative tool. The thesis is divided into four parts. Part I consists of Chapter 1 which provides an introduction to protein structure, folding and NMR spectroscopy. Part II, consisting of Chapters 2 and 3, describes the effects of aromatic interactions on nucleating structure in disordered regions of proteins, using variants of apo-cytochrome b5 as a model system. Part III consists of Chapter 4, which describes structural effects of introducing cross-strand disulfide bonds using variants of Thioredoxin. Part IV of this thesis consists of the Appendices A, B and C. Appendix A describes the purification and characterization of ilvM, the regulatory subunit of the E.coli enzyme AHAS II. Appendices B and C contain chemical shift information corresponding to Chapter 3 and Chapter 4 respectively. Part I : Introduction to protein structure, folding and solution structure studies Chapter 1 first gives a brief overview of protein structure followed by an introduction to protein folding, focussing on the forces involved in determining the final three-dimensional shape of the protein as well as the experimental and computational techniques involved in studying or predicting the fold of a given protein. The second section of this chapter details the methodology followed to obtain solution structures of proteins using NMR spectroscopy. Part II : Engineering aromatic interactions to nucleate folding in intrinsically disordered regions of proteins Chapter 2 describes site-specific mutagenesis, recombinant over-expression, purifica-tion and preliminary biophysical characterization of two aromatic mutants of the molten globule apo-cytochrome b5 (apocytb5) : H43F H67F cytochrome b5 (FFcytb5) and H43W H67F cytochrome b5 (WFcytb5). Analysis of the structure of wild-type apo - cytochrome b5 was done to introduce surface mutations and avoid perturbation of the interior pack-ing of the protein. The bacterial host E.coli BL21(DE3) was used for recombinant over-expression, and both mutant proteins were purified by anion-exchange chromatography followed by size-exclusion chromatography. Biophysical studies show a decrease in the hydrodynamic radii and surface hydropho-bicity of FFcytb5 and WFcytb5 compared to wt -apo cytb5. An increase in protein stability was also seen from the wt apocytb5 to WFcytb5 and FFcytb5 in the presence of the chemical denaturant Urea. Proton 1D NMR spectra exhibited sharp lines and good spectral dispersion in the amide region, indicating that both mutant proteins are well folded. In addition, conservation of two distinctive up field and downfield shifted resonances present in apocytb5 indicated that structural changes upon mutation accrued on the upon the scaffold of apocytb5. Chapter 3 describes solution structure studies to determine secondary and tertiary structure of FFcytb5 and WFcytb5. Structural studies were carried out using homonu-clear and heteronuclear NMR methods, for which isotopically enriched 15N- and 13C, 15N samples were prepared for each protein. Additionally a 2H, 13C, 15N ILV methyl labeled sample was prepared for FFcytb5 to obtain unambiguous NOE correlation data. The hydrogen bond network for WFcytb5 was determined using hydrogen/deuterium exchange data. The restraints required to define the orientations and interactions of the aromatic groups were obtained from 15N-edited NOESY HSQC, 13C -edited NOESY HSQC and 2D 1H - 1H NOE spectra. These correlations were crucial in determining the aromatic interactions present within each protein. The structure of FFcytb5 was calculated using 1163 NOE distance restraints, 179 φ and ψ dihedral angle restraints, along with 40 hydrogen bond restraints. Similarly the structure of WFcytb5 was calculated using 1282 NOE distance restraints, 177 φ and ψ dihedral angle restraints and 40 hydrogen bond restraints. The ensemble of structures obtained for FFcytb5 showed a root mean square deviation of 1.01±0.21 Å . The ensemble of structures obtained for WFcytb5 showed a root mean square deviation of 0.58±0.09 Å . In both cases, ≈ 80% of backbone dihedral angles were found to be in the allowed regions and ≈ 20% in the additionally allowed regions of the Ramachandran map. The final tertiary structure of both FFcytb5 and WFcytb5 consisted of a mixed four strand β -sheet with a four helix bundle resting on top and were seen to align well, with an RMSD of 0.6 Å. A comparison of the solution structures of apocytb5 with FFcytb5 and WFcytb5 convincingly showed the nucleation secondary and tertiary structure well beyond the site of mutation. The presence of aromatic trimers, non-canonical in context of the wt apoc-ytb5, was confirmed upon analysis of the structures of FFcytb5 and WFcytb5, with NOE correlations assigned to verify these interactions. The reduction in the hydrodynamic radii of FFcytb5 and WFcytb5 in relation to apocytb5 was also verified from tsuperscript15N-NMR relaxometry studies. The nucleation of long-range structure using aromatic interactions has been demonstrated in proteins for the first time, and can in principle be used to incorporate aromatic residues and interactions in protein design. Structural data, chemical shift data and restraints lists used for structure calculation of WFcytb5 and FFcytb5 were deposited with the PDB (accession numbers 5XE4 and 5XEE) and BMRB(accession numbers 36070, 36071) respectively1. Part III : Structural consequences of introducing disulfide bonds into β - sheets Chapter 3 describes the solution structure studies on two mutants of E.coli Thiore-doxin which were designed to incorporate a disulfide bond between two anti-parallel β-strands at the edge of the β-sheet. One mutant was designed with a disulfide bond at the hydrogen bonding position (HB, 78c90cTrx) and the other with the disulfide bond at the non-hydrogen bonding position (NHB, 77c91cTrx). Here we study the structural changes that accompany the introduction of a cross-strand disulfide and whether such structural changes could be correlated with the previously seen thermodynamic and catalytic changes. Solution structure studies were conducted using a suite of multidimensional heteronu-clear NMR experiments, for which isotopically enriched 15N and 13C, 15N labelled samples were used. The solution structure for 77c91cTrx was calculated using 1190 NOE distance restraints, 199 φ and ψ dihedral angle restraints and 48 hydrogen bond restraints. The solution structure for 78c90cTrx was calculated using 1123 NOE distance restraints, 197 φ and ψ dihedral angle restraints and 50 hydrogen bond restraints. The ensemble of structures for 77c91cTrx showed an RMSD of 0.78± 0.13 Å while the RMSD for the ensemble of structures of 78c90cTrx was seen to be 0.76±0.09 Å . In both cases, ≈ 80% of backbone dihedral angles were seen to be in the allowed regions and ≈ 20% in the additionally allowed regions of the Ramachandran map. The tertiary structures of both proteins were seen to have a 5-strand mixed β-sheet and 4 helices surrounding it. . A comparison of the solution structures of mutant and wt -Trx showed significant changes in secondary and tertiary structure. For example, an α helix was reduced from 3 turns to a single turn, and of the β-strands containing the mutation was elongated by 3 residues. A ≈ 50% loss of hydrogen bonds, primarily from the β -sheet, was seen for both mutants. The secondary and tertiary structure for both 77c91cTrx and 78c90cTrx was seen to be near identical, despite the greater strain of the disulfide bond at the hydrogen bonding position. In addition to this, the Ile75-Pro76 peptide bond is now seen to be in the trans conformation in 78c90cTrx, while in wt -Trx the Ile75-Pro76 peptide bond is in the cis conformation. This cis peptide bond is known to play a role in both folding and catalysis, and the solution structures were analyzed in the context of observed changes in folding and catalysis. The study shows that introducing disulfide bonds even at the edge of β sheets have long-range structural effects, and the structural effects cannot be directly correlated with the changes in stability. Part III: Appendix Appendix A describes the expression, purification and preliminary characterization of ilvM, the regulatory subunit of E.coliAHAS II, one of three enzyme isomers that catal-yse the first step in the synthesis of all branched chain amino acids. AHAS II is known to be insensitive to feedback regulation, but our studies showed that the presence of Ile, Leu and Val causes structural changes and increases the stability of ilvM. However we were not able to purify ilvM in sufficient quantities to proceed with solution structure studies. Appendices B and C contain chemical shift information for the structural studies carried out on FFcytb5, WFcytb5, 77c91cTrx and 78c90cTrx.

Rayons ionisants, dessiccation, ou encore métabolites secondaires exogènes sont autant de facteurs qui peuvent engendrer des dommages à l’ADN chez les bactéries du sol, notamment en provoquant la formation de cassures double-brin (DSB), préjudice majeur pour une cellule. Chez les procaryotes, l’évolution a sélectionné deux principaux mécanismes de réparation des DSB, à savoir la recombinaison homologue (RH) et le non-homologous end joining (NHEJ). La RH est un mécanisme quasi-ubiquiste dans le monde bactérien qui repose sur l’utilisation d’une copie intacte de la molécule endommagée comme matrice pour la réparation de la DSB. Contrairement à la RH, le NHEJ n’est présent que chez 20 à 25% des bactéries et est considéré comme un mécanisme mutagène puisque la réparation de la DSB se fait sans matrice homologue et peut entrainer l’ajout ou la délétion de nucléotides au site de cassure. Chez la bactérie modèle Mycobacterium, seuls deux acteurs sont nécessaires pour la réparation par NHEJ. Ainsi, un dimère de protéine Ku se fixe sur la cassure puis recrute la protéine multifonctionnelle LigD, qui catalyse le traitement puis la ligation des extrémités grâce à ses domaines polymérase, nucléase et ligase. Les mécanismes de réparation des DSB chez les Streptomyces étaient peu connus à l’initiation de ce travail. Cette bactérie présente des caractéristiques génomiques remarquables avec notamment un chromosome linéaire de grande taille (6 à 12 Mb). En ce qui concerne la RH, nous avons focalisé nos recherches sur les étapes tardives (post-synaptiques) et étudié le rôle du complexe RuvABC et de RecG impliqués chez Escherichia coli dans la migration de la croix de Holliday et de sa résolution. La construction de mutants simples et multiples a montré que bien que les gènes codant ces protéines soient très conservés chez les Streptomyces, leur déficience ne se traduit chez Streptomyces ambofaciens que par une faible baisse de la recombinaison suite à un événement de conjugaison. Aucune baisse de l’efficacité de recombinaison intrachromosomique n’a en revanche été observée. Ces résultats suggèrent que des acteurs alternatifs majeurs sont encore à découvrir chez les Streptomyces. Le décryptage du mécanisme de NHEJ chez S. ambofaciens constitue une première dans ce genre bactérien. Une étude génomique exhaustive a permis de révéler la très grande diversité du nombre d’acteurs potentiels de ce mécanisme (Ku, LigDom, PolDom, NucDom) et de l’organisation des gènes qui les codent.. L’analyse fonctionnelle a révélé que l’ensemble des acteurs étaient impliqués dans la réponse à l’exposition à un faisceau d’électrons accélérés, connus pour induire, entre autre, la formation de DSB. La génération de DSB, par coupure endonucléasique I-SceI, a par ailleurs permis de mettre en évidence au niveau moléculaire des réparations de type NHEJ (délétions ou insertions de quelques nucléotides, intégration de fragments d’ADN). Les cassures dans les régions terminales du chromosome sont accompagnées de grandes délétions (jusqu’à 2,1 Mb) et de réarrangements de grande ampleur incluant circularisations du chromosome et amplifications d’ADN. Les conséquences de la réparation de DSB chez S. ambofaciens sont en tous points similaires aux réarrangements observés spontanément ou par comparaison des génomes des espèces types. Ainsi, il est possible de lier la plasticité du génome à la réparation de DSB. En outre, l’intégration de matériel génétique exogène serait favorisée au cours de la réparation NHEJ ce qui donnerait à ce système de réparation une place importante dans le processus de transfert horizontal, mécanisme d’évolution majeur chez les bactéries / Ionizing radiation, desiccation or exogenous secondary metabolites are all factors that can cause DNA damage in soil bacteria, especially by triggering double strand breaks (DSB), the most detrimental harm for the cell. In prokaryotes, evolution selected two main DSB repair pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ). HR is almost ubiquitous in bacteria and relies on an intact copy of the damaged DNA molecule as a template for DSB repair. In contrast to HR, NHEJ is only present in 20 to 25% of bacteria and is considered as a mutagenic pathway since DSB repair is performed without the need of any template and can lead to nucleotide addition or deletion at DSB site. In the bacterial model Mycobacterium, two partners are sufficient for a functional NHEJ pathway. Thus, Ku protein dimer recognizes and binds the DSB and then recruits the multifunctional LigD protein for extremities treatment and ligation thanks to its polymerase, nuclease and ligase domains. At the beginning of this work, few informations on DSB repair in Streptomyces were available. This bacteria exhibits remarkable genomic features including a large linear chromosome (6 to 12 Mb). Regarding HR, we focused on the late stage (post-synaptic step) in studying the role of RuvABC complex and RecG, involved in branch migration and Holliday junction resolution in E. coli. Construction of single and multiple mutants showed that although the genes encoding these proteins are highly conserved in Streptomyces, their deficiency in Streptomyces ambofaciens only results in a mild decrease of recombination after conjugation events. Besides, no decrease of intrachromosomal recombination efficiency could be observed. These results suggest that major alternative factors are still to be discovered in Streptomyces. This work was also the first occasion to decipher a NHEJ pathway in Streptomyces. An exhaustive genomic study revealed a great diversity in the number of factors potentially implicated in this pathway (Ku, LigDom, PolDom, NucDom) and in the organization of their encoding genes. Functional analyses revealed that all the factors, whatever they are conserved or not between species, were involved in the response to electron beam exposure, known to induce, amongst other things, DSB formation. Generation of DSB by I-SceI endonuclease cleavage was also used to evidence at a molecular level NHEJ type DSB repair (deletions or insertions of several nucleotides, integration of DNA fragments). Targeted breaks in the terminal regions of the chromosome were accompanied by large deletions (up to 2.1 Mb) and major rearrangements including chromosome circularizations and DNA amplifications. Consequences of DSB repair in S. ambofaciens are in all points similar to chromosome rearrangements observed spontaneously or by comparing genomes of different species. Thus, it is possible to link the genome plasticity to DSB repair. In addition, the integration of exogenous genetic material would be favoured during NHEJ repair which would give this repair system a major role in the horizontal transfer process, known to be a main evolution mechanism in bacteria

Made available in DSpace on 2017-07-10T17:51:41Z (GMT). No. of bitstreams: 1 2015Micheli_Danzer.pdf: 7746878 bytes, checksum: 46102ff9d9387c68a2b535406f1fc888 (MD5) Previous issue date: 2015-04-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The understanding of the soil and of the reliev resulting from the concern with the environmental consequences of historical related to the use, coverage and interference in the dynamic evolution of systems and pedological. Describe the relations betwen soil and relief allows understand their spatial distribution and the action of the factors triggering or control their dynamics and evolution. The identification and description of the forms of reliev, the strands and of classes of soil associated constitute the basis for the interpretation and conservation of environments. This way, the objective of the research was to charaterize and understand the between the soils and the morphology of the strands by means of techniques and tools of geotecnologies. Hese, added to the comments and field inspections helped with the identification of types and forms of prominence and strands. As well as the delimitation and description of soil systems that integrate the morfoescultural subunit of New Santa Rosa, located in the west region of Parana. The abovementioned subunit, defined by Bade (2014) from the compartmentalisation of the Paraná Basin morfoescultural III, was selected and studied based on the proposal of taxonomy and classification of the embossment of Ross (1992). The data of steepness and hipsometria, forms of prominence and of the strands, the soil and geological structure, obtained by means of the thematic maps, were the main results. The study allowed, also, gather important information on the spatial organization of soil systems along the strands and thus, understanding their evolution in different scales of landscape units / A compreensão do solo e do relevo resulta da preocupação com as consequências ambientais históricas, relacionadas ao uso, cobertura e interferências na dinâmica e evolução dos sistemas pedológicos. Descrever as relações entre o solo e o relevo permite compreender a sua distribuição espacial e a ação dos fatores que desencadeiam e/ou controlam sua dinâmica e evolução. Por isso, a identificação e a descrição das formas de relevo, das vertentes e das classes de solos associadas constituem a base para a interpretação e conservação dos ambientes. Dessa forma, o objetivo da pesquisa foi caracterizar e entender as relações entre os solos e a morfologia das vertentes, por meio da utilização de técnicas e ferramentas de geotecnologias. Estas, somadas às observações e inspeções em campo, auxiliaram na identificação dos tipos e formas de relevo e das vertentes, bem como na delimitação e descrição dos sistemas pedológicos que integram a subunidade morfoescultural de Nova Santa Rosa, localizada na região Oeste do Paraná. A referida subunidade, definida por Bade (2014) a partir da compartimentação morfoescultural da Bacia do Paraná III, foi selecionada e estudada com base na proposta de taxonomia e classificação do relevo de Ross (1992). Os dados de declividade e hipsometria, das formas de relevo e das vertentes, dos solos e da estrutura geológica, obtidos por meio das cartas temáticas, foram os principais resultados. O estudo permitiu, também, reunir importantes informações sobre a organização espacial dos sistemas pedológicos ao longo das vertentes e assim, entender sua evolução nas diferentes escalas das unidades de paisagem

Ionising radiation potentially produces a broad spectrum of damage in DNA including single and double strand breaks (ssb and dsb) and base damages. It has been hypothesised that sites of complex damage within cellular DNA have particular biological significance due to an associated decreased efficiency in repair. The aim of this study is to gain further understanding of the formation of complex DNA damage. Irradiations of plasmid DNA illustrate that an increase in ionising density of the radiation results in a decrease in ssb yields/Gy but an increase in dsb per ssb, indicative of an increase in the number of complex damage sites per simple isolated damage site. As the mechanism for damage formation shifts from purely indirect at low scavenging capacities to a significant proportion of direct at higher scavenging capacities the proportion of complex damage increases. Comparisons with the yields of ssb and dsb simulated by Monte-Carlo calculations for AIK USX and a-particles also indicate this correspondence. The ionisation density of low energy, secondary electrons produced by photons was assessed experimentally from the dependence of the yield of OH radicals escaping intra-track recombination on photon energy. As energy decreases the OH radical yield initially decreases reflecting an increased ionisation density. However, with further decrease in photon energy the yield of OH radicals increases in line with theoretical calculations. Base damage yields were determined for low and high ionising density radiation over a range of scavenging capacities. As scavenging capacity increases the base damage:ssb ratios increases implying a contribution from electrons to base damage. It is proposed that base damage contributes to DNA damage complexity. Complex damage analysis reveals that at cell mimetic scavenging capacities, 23% and 72% of ssb have an additional spatially close damage site following y-ray and a-particle irradiation respectively.