Dissertationen zum Thema „Insertion elements“

Thesis (Ph.D.) -- University of Adelaide, Dept. of Molecular Biosciences, 2001?
Copy of author's previously published article inserted. Errata sheet attached to front cover. Includes bibliographical references (14 p.).

Transposable elements (TEs) are mobile DNA sequences that are a source of mutations and can target specific sites in host genome. Understanding the molecular mechanisms of TE target site preferences is a fundamental challenge in functional and evolutionary genomics. Here we used accurately mapped TE insertions in the Drosophila melanogaster genome, from large-scale gene disruption and resequencing projects, to better understand TE insertion site mechanisms. First we test predictions of the palindromic target site model for DNA transposon insertion using artificially generated P-element insertions. We provide evidence that the P-element targets a 14 bp palindromic motif that can be identified at the primary sequence level that differs significantly from random base composition in the D. melanogaster genome. This sequence also predicts local spacing, hotspots and strand orientation of P-element insertions. Next, we combine artificial P-element insertions with data from genome- wide studies on sequence properties of promoter regions, in an attempt to decode the genomic factors associated with P-element promoter targeting. Our results indicate that the P-element insertions are affected by nucleosome positioning and the presence of chromatin marks made by the Polycomb and trithorax protein groups. We provide the first genome-wide study which shows that core promoter architecture and chromatin structure impact P-element target preferences shedding light on the nuclear processes that influence its pattern of TE insertions across the D. melanogaster genome. In an effort to understand the natural insertion preferences of a wide range of TEs, we then used genome resequencing data to identify insertions sites not present in the reference strain. We found that both Illumina and 454 sequencing platforms showed consistent results in terms of target site duplication (TSD) and target site motif (TSM) discovery. We found that TSMs typically extend the TSD and are palindromic for both DNA and LTR elements with a variable center that depends on the length of the TSD. Additionally, we found that TEs from the same subclass present similar TSDs and TSMs. Finally, by correlating results on P-element insertion sites from natural strains with gene disruption experiments, we show that there is an overlap in target site preferences between artificial and natural insertion events and that P-element targeting of promoter regions of genes is a natural characteristic of this element that is influenced by the same features has the artificially generated insertions. Together, the results presented in this thesis provide important new findings about the target preferences of TEs in one of the best-studied and most important model organisms, and provide a platform for understanding target site preferences of TEs in other species using genomic data.

Le moustique Chironomus riparius est présent dans toute la région Paléarctique, et il est bien caracterisé au niveau morphologique et cytogénétique. Les connaissances sur la variabilité génétique de cette éspèce sont cependant très reduites. Ici nous décrivons le polymorphisme, chez six populations naturelles, des sites d'insertion de quatre éléments transposables (CTRT, MEC, NLRCth1, TFB1) au moyen d'une technique TID (Transposon Insertion Display) derivée de la technique S-SAP (Sequence -Specific Amplification Polymorphism) et aussi de l'AFLP (Amplified Fragment Length Polymorphism). La seule étude sur le polymorphisme alloenzymatique faite sur cette espèce a decelé une differenciation très rèduite entre des populations de Russie. Nos resultats indiquent que tous les amplicons identifiés sont polymorphes. Le degré de différenciation entre individus au sein de chaque population est d'un ordre de grandeur plus élevé que la différenciation parmi les populations. Néanmoins, la valeur de st est significative (P< 0. 001): cela suggère que les populations sont génétiquement plus differenciées entre-elles qu'un échantillon aléatoire d'individus

Composite materials are increasingly being used in advanced structural ap- plications. Debonding of adjacent laminate layers, also known as delamination, is considered to be one of the most dominant damage mechanisms affecting the behavior of composite laminates. Various numerical methods for simulating delamination in composite materials do exist, but they are generally limited to small-scale structures due to their complexity and high numerical cost. In this thesis, a novel technique aimed to allow efficient simulation of delamination in large-scale laminated composite structures is presented. During the transient analysis, continuum elements within regions where delamination has the potential to initiate are adaptively split through their thickness into two shell elements sandwiching a cohesive element. By elimi- nating the a priori requirement to implant cohesive elements at all possible spatial locations, the computational efforts are reduced, thus lending the method suitable for treatment of practical size structures. The methodol- ogy, called the local cohesive zone method (LCZ), is verified here through its application to Mode-I, Mode-II and Mixed-Mode loading conditions, and is validated using a dynamic tube-crushing loading case and plate impact events. Good agreement between the numerical results and the available experimental data is obtained. The results obtained using the LCZ method are compared favourably with the numerical results obtained using the con- ventional cohesive zone method (CZM). The numerical performance of the method and its efficiency is investi- gated. The efficiency of the method was found to be superior compared to that of the conventional CZM, and was found to increase with increasing model size. The LCZ method is shown to have a lower effect on reducing the structural stiffness of the structure, compared to the conventional CZM. The results obtained from the application of the LCZ method to the various cases tested are encouraging, and prove that the local and adaptive insertion of cohesive zones into a finite element mesh can effectively capture the delamination crack propagation in laminated composite structures. It is expected that further improvements in speed and accuracy will be attained once the algorithm is embedded within commercial finite element solvers as a built-in feature.
Applied Science, Faculty of
Graduate

The regulation and evolution of the inverted repeat transposable element Tel, found in the nematode Caenorhabditis elegans, was studied. The stability of Tel elements in the N2 strain genome was investigated by cloning seventeen N2 Tel elements. To examine their structural integrity, sixteen cloned N2 Tel elements were restriction mapped and, in the case of some variants, their DNA was partially sequenced. Two restriction site variants, Tcl(Eco).12 and Tcl(Hpa-).9, were found. Tel(1.5).10b had lost 89 bp from one end, while Tcl(1.7).28 contained a 55 bp insertion. Two additional elements, Tcl(0.9).2 and Tcl(0.9).14, had different internal deletions. Each element was about 900 bp in length. The majority of Tel elements cloned from the N2 strain were found to have identical restriction maps. Somatic excision of Tel elements in the N2 genome was demonstrated. Tel elements in N2 are apparently both structurally and functionally intact. Nevertheless, mobilization of Tel elements in the N2 germline is restricted. Two new transposable element families, Barney (also known as TCbl) and TCb2, were discovered in a closely related nematode, Caenorhabditis briggsae due to Tel identity. These two families, distinguished through differential inter-element hybridization, showed multiple banding differences between strains. The open reading frames (ORFs) of Tel and Barney share 71% DNA sequence and 74% amino acid sequence identity. The putative terminus of Barney exhibits 68% identity with the 54 bp terminal repeat of Tel. Partial sequencing of TCb2 revealed that its ORF is equally diverged from Barney and Tel. The basis of the sequence heterogeneity observed in the C. briggsae transposons and not in the C. elegans transposons could be due to either horizontal transfer or alternate paths of divergence. Significant sequence identity was found between Tel, Barney, and HB1 (a transposable element from Drosophila melanogaster) within their coding regions and terminal repeats. These sequence similarities define a subclass of inverted repeat transposable elements inhabiting two different phylla, Arthropoda and Nematoda.
Medicine, Faculty of
Medical Genetics, Department of
Graduate

Five transposon Tn5 mutants of bean rhizobia (Rhizobium leguminosarum b.v. phaseoli) and the wild type strain were used in ecological studies to evaluate the efficacy of transposon Tn5 as a phenotypic marker in rhizobia for ecological studies in two Sonoran desert soils. All mutants possessed chromosomal insertions of the transposable element. Survival of each mutant strain was compared to that of the wild type strain under non stress, moisture stress and temperature stress conditions in Pima silty clay loam and Brazil to sandy loam. The genetic stability of Tn5 in terms of transposition of the element within the chromosome and the Tn5 coded antibiotic resistant phenotype was determined in cells recovered throughout the survival period. Under non stress conditions, the viable Tn5 mutant population decreased in size. Two mutants showed significantly (p < 0.01) lower populations than the wild type at the end of 30 days in the silty clay loam. In the sandy loam, four of the five mutant populations were significantly lower than the wild type. Tn5 was genetically stable in both soils. Under moisture stress conditions, the decline of the Tn5 mutant and wild type populations corresponded to a decline in soil moisture content. The finer textured soil afforded more protection to the cells than the coarse textured soil. There were no indications of Tn5 instability under moisture stress. In both soils under temperature stress, sizes of all populations declined rapidly and after 12 days, the mutant cells when screened using the Tn5 coded markers were significantly less in numbers than the wild type indicating a loss of Tn5 coded antibiotic resistance phenotype. There were no significant differences in numbers between wild type and mutant cells when screened using only the intrinsic markers. DNA:DNA hybridizations confirmed that the lack of Tn5 coded antibiotic resistance phenotype was probably not due to a deletion or transposition of the element. Under non stress conditions Tn5 is a useful ecological marker, but each Tn5 mutant has to be evaluated independently under specific environmental conditions to determine the efficacy of Tn5 as an ecological marker.

A major challenge of modern Biology is elucidating the genetic basis of adaptation. While there are many SNP-based studies trying to elucidate the genetic basis of genotype-phenotype relationships, the role of transposable element (TE)-induced mutations is understudied. Recent evidences demonstrate that TEs are a powerful tool to identify the genetic basis of adaptive phenotypic traits. Drosophila melanogaster is a good model to study adaptation because it is original from subtropical Africa and only recently colonized out-of-Africa environments. To identify and characterize the role of several candidate TEs in D. melanogaster adaptation, we have followed two different strategies: locus-specific and trait-specific. In the first chapter, we have characterized both at the molecular and phenotypic level FBti0019386, a previously identified candidate adaptive TE. We first elucidated the evolutionary history of this natural insertion and provided evidences of genomic signatures of positive selection. We then explored several phenotypes related to known phenotypic effects of nearby genes, and having plausible connections to fitness variation in nature. We found that flies with FBti0019386 insertion had a shorter developmental time and were more sensitive to stress, which are likely to be the adaptive effect and the cost of selection of this mutation, respectively. Interestingly, these phenotypic effects are not consistent with a role of FBti0019386 in temperate adaptation as has been previously suggested. Indeed, a global analysis of the population frequency of FBti0019386 showed that climatic variables explain well the FBti0019386 frequency patterns only in Australia. These results suggest that further functional validation should be gathered before concluding that a candidate loci is under spatially varying selection. Finally, although FBti0019386 insertion could be inducing the formation of heterochromatin by recruiting HP1a (Heterochromatin Protein 1a) protein, the insertion is associated with up-regulation of sra in adult females. In the second chapter of this thesis, we have studied the impact of several TE insertions in a highly conserved and ecologically relevant trait: the immune response. To do that, we first performed a new genome-wide screening in order to identify a dataset of candidate TEs involved in adaptation. By increasing the number of populations and the number of TEs analyzed compared to similar studies, we were able to increase the number of identified candidate TEs: a total of 121 TEs. Interestingly, we found that genes associated with those TEs are enriched for stress-related functions, specifically we detected a significant enrichment for immune response functions. We combined allele-specific expression (ASE), enhancer assays, and TSS detection experiments to characterize the impact of these TEs in oral immune response to the gram-negative bacteria Pseudomonas entomophila. We were also able to associate the 12 candidate TEs with gene expression changes, and determine some of the molecular mechanisms behind these expression changes. We showed that the allele with the TE was differently expressed in 13 out of the 16 analyzed genes under non-infected and/or infected conditions in at least one of the two genetic backgrounds analyzed. We also show that different TEs alter gene expression by adding promoters and enhancer regulatory sequences to their nearby genes. Although we found evidences pointing to a possible role of TEs in immune response regulation, more experiments should be performed in order to link the identified TEs with a fitness effect in this trait. Overall, our two integrative approaches allowed us to shed light on the role of TEs in generating genomic natural variation potentially underlying adaptation. The results obtained in this work illustrate that TEs are a good tool to bridge the gap between genotypic and phenotypic evolution.
Un dels reptes actuals en biologia és explicar la base genètica de l’adaptació. Mentre que hi ha molts estudis basats en SNPs intentant trobar les bases genètiques de les relacions genotip-fenotip, no s’ha estudiat tant bé el paper de les mutacions generades pels elements mòbils (TEs, de l’anglès Transposable Elements). Evidències recents demostren que els TEs són una eina potent per identificar les bases genètiques dels fenotips adaptatius. Drosophila melanogaster és un bon model per estudiar l’adaptació, ja que és originària de l’Àfrica subtropical i recentment ha colonitzat altres ambients. Per identificar i caracteritzar el paper de diversos TEs candidats per l’adaptació a D. melanogaster, hem seguit dues estratègies diferents: locus-específica i tret-específica. En el primer capítol hem caracteritzat a nivell molecular i fenotípic el TE FBti0019386, identificat prèviament com a candidat per l’adaptació. Primer hem estudiat la història evolutiva d’aquesta inserció i hem demostrat que està associada a senyals genòmiques de selecció positiva. Després hem explorat diferents fenotips relacionats amb els efectes fenotípics coneguts dels gens del costat, que poguessin tenir connexions plausibles amb la variació de la fitness a la natura. En el segon capítol, hem estudiat l’impacte de diferents TEs en la resposta immune. Per això, hem mostrejat el genoma buscant TEs candidats per l’adaptació en quatre poblacions naturals. Després, hem combinat anàlisis d’expressió específica d’al·lel, assajos d’enhancer i detecció de TSS per caracteritzar l’impacte d’aquests TEs durant la resposta a infecció amb el bacteri gram-negatiu Pseudomonas entomophila. Hem trobat que l’al·lel amb el TE s’expressa de manera diferent en 13 dels 16 gens analitzats en condicions control i/o d’infecció en almenys un dels dos fons genètics analitzats. Hem demostrat que alguns d’aquests TEs alteren l’expressió afegint promotors i enhancers als gens del costat. Tot i que les evidències assenyalen cap a un possible paper dels TEs en la regulació de la resposta immune, es requereixen més experiments per associar els TEs identificats amb un efecte en la fitness. En resum, les dues estratègies integratives seguides ens han permès mostrar el paper dels TEs en la generació de variació natural genòmica potencialment implicada en l’adaptació.

La réfrigération magnétique, basée sur l'effet magnétocalorique (EMC), est une alternative intéressante aux méthodes de réfrigération traditionnelles, basées sur des cycles de compression/détente, car elle présente des rendements énergétiques nettement plus élevés et permet d'éviter l'utilisation de gaz nocifs contribuant à l'effet de serre et problématiques pour l'environnement. Cette technologie s'appuie sur l’EMC géant de certains matériaux magnétiques autour de la température ambiante. Cet effet permet d'augmenter ou de diminuer la température du matériau lors de son aimantation ou désaimantation adiabatique autour de sa température de transition magnétique.La majeure partie des travaux de thèse se focalise sur la famille des matériaux de type La(Fe,Si)13 dans lesquels un effet magnétocalorique géant a été mis en évidence et pour lesquels la faisabilité industrielle semble la plus favorable. Dans un premier temps, les propriétés structurales et magnétiques de ces alliages sont explorées et optimisées, en remplaçant aussi bien la terre rare que le métal de transition par d'autres éléments. Les méthodes d’élaboration, des traitements thermiques, ainsi que le contrôle de la stœchiométrie sont guidées par les caractérisations structurales, microstructurales, physiques (thermiques et magnétiques).D’autre part, l'effet de l'insertion d'éléments interstitiels légers est également étudié et une grande partie du travail porte sur la détermination des conditions de stabilité de ces interstitiels dans les matériaux. Grâce à l'extension des distances Fe-Fe, la température de Curie de la phase magnétocalorique peut être augmentée jusqu'à des plages proches de latempérature ambiante. L'influence d’une faible concentration en carbone sur les propriétés magnétiques des échantillons est examinée avant hydrogénation et la teneur en carbone est optimisée.Afin d'étudier la diffusion des éléments interstitiels, la cinétique de sorption d'hydrogène est étudiée par la méthode de Sieverts ainsi que par diffraction neutronique. La diffraction neutronique in situ et à haute résolution permet une localisation des atomes interstitiels et donne accès au schéma d’insertion. Cette étude permet de préciser l’effet de l’insertion d’interstitiels légers et des substitutions d’éléments de terre rare sur la structure des alliages métalliques complexes de type La-Fe-Si. Nous montrons que la dépression ou l’accélération de la cinétique d'hydrogénation peut être liée à la variation hétérogène particulière de la maille et des liaisons dans la structure de type NaZn13. Un mécanisme pour le chemin de diffusion est suggéré.Le mécanisme d'insertion d'atomes légers est non seulement fortement lié à l'espace disponible, mais aussi associés à la facilité du chemin de diffusion dans le réseau. Nous démontrons avec des résultats expérimentaux qu'une addition modérée de carbone dans la phase La(Fe,Si)13 avant l'hydrogénation peut effectivement ralentir la cinétique d'insertion de l'hydrogène. Dans les phases La-Ce-Fe-Si, une insertion de carbone peut aider à retenir les atomes d'hydrogène lors de la désorption, par conséquent, offre une possibilité d'avoir une meilleure stabilité des matériaux hydrogénés pour des applications à long terme. La stabilité des matériaux hydrogénés est mesurée par DSC et une amélioration de la stabilité thermique du matériau est réalisée par un dopage au carbone.Un volet exploratoire est consacré aux alliages Fe-Cr-Ni et Fe-Cr-Mn qui pourraient potentiellement avoir un effet magnétocalorique exploitable. Les transitions magnétiques et structurales de ces alliages de compositions différentes sont étudiées et leur potentiel d'application magnétocalorique est discuté
The magnetocaloric effect (MCE) is characterized by a magnetic entropy change and an adiabatic temperature change. The NaZn13-type La(Fe,Si)13 system has attracted wide interest because of its first-order ferromagnetic phase transition with a large magnetocaloric effect. The transition temperature can be flexibly adjusted through substitution or interstitial insertion. Particularly, hydrogen interstitials can adapt the temperature range to room-temperature applications. Precise adjustment can be achieved by full hydrogen absorption then partial desorption. However, fully hydrogenated alloys are unstable upon heating. It is important to have a better understanding of its hydrogen stability to optimize its application potential.In the first part, the structural, magnetic, and magnetocaloric properties of La(Fe,Si)13 phases are studied. In particular, we have investigated the effect of substitution of Ce on the La site and Mn on the Fe sites. The partial substitution of Ce results in the decrease of TC with decreasing lattice constant. At the same time, Ce substitution for La results in a reduced volume of the octahedral interstitial site due to steric effect. The interstitial insertion is impeded by Ce partial substitution.Secondly, the effects of interstitial atoms such as hydrogen and carbon are examined. These elements are able to enter the interstitial voids in the La(Fe,Si)13 phase, expanding the lattice. Through the extension of Fe-Fe distances, the Curie temperature of the magnetocaloric phase can be raised up to room temperature range. The influence of small concentration of carbon on the magnetic properties of samples is examined prior to hydrogenation and carbon content is optimized. In order to investigate the interstitial dynamics, the hydrogen sorption kinetics is studied by the means of Sieverts’ volumetric method and neutron diffraction. Particular attention has been given to the adjustment of the structure in the course of hydrogen/deuterium interstitial absorption and desorption.Steady-state and in-situ neutron diffractions provide precise information of the interstitial atom location of the sequential filling of the accommodating sites. The structural investigation allows specifying the deformations undergone in the complex metallic alloys La-Fe-Si when subjected to light interstitial insertion or rare earth substitution at the cation site. We show that the depression or enhancement of the hydrogenation kinetics may be related to the particular inhomogeneous cell variation of bonding in the structure. A mechanism for the diffusion path is suggested.The mechanism is light atom insertion into the interstitial sites is not only strongly related to the available space for accommodation, but also associated with the facility of the diffusion path in the lattice. We demonstrate with experimental results that a modest addition of carbon in the La-Fe-Si phase prior to hydrogenation can effectively slow down the hydrogen insertion kinetics. In Ce-substituted La-Ce-Fe-Si phases, carbon insertion can help retain hydrogen atoms during desorption, therefore, offering a prospect to have improved stability of hydrogenated materials for long-term applications. The hydrogen stability of the material is examined by means of thermal desorption in DSC and an enhancement of the thermal stability of the material is achieved with carbon-doping.Lastly, in the search of new rare-earth-free materials for magnetocaloric applications, we have explored the capacity of alloys of types FeCrNi and FeCrMn. The magnetic and structural transitions of these alloys of different compositions are studied and their potential for magnetocaloric application is examined in this thesis

Mobile genetic elements are occupied by this project. It is aimed at their features, which can be used for their detection. It also deals with issue of conversion of symbolic sequence into numerical form. Classifications of mobile genetic elements are explained, basic types of mobile genetic sequences are described, and principles of numerical maps and detection in symbolic represetation are also clarified. Conversion of symbolic genetical sequences by chosen numerical map and calculation of normalized correlation values for set of mobile genetic elements are compiled. Analysis of the mobile genetic elements properties is performed for design of detector. The library of themes is created at the end for usage by designed detector.

Os elementos genéticos móveis (mobile genetic elements MGEs), são elementos extremamente importantes para plasticidade e evolução dos genomas. Uma das classes mais importantes de MGEs são as sequências de inserção (insertion sequences - IS). Estes elementos são encontrados em bactérias e archaea e apresentam grande diversidade de famílias e mecanismos de movimentação. Uma família interessante de IS é IS200/605, esta família encontra-se distribuída em bactérias, archaea e vírus e utiliza substratos de DNA de fita simples para seu processo de transposição. Neste trabalho, através da análise de dados públicos de transcritoma identificamos RNAs sobrepostos ao 3\' de genes tnpB de IS200/605 em archaea e bactérias, estes transcritos foram chamados de sense overlapping transcripts (sotRNAs). As extremidades 5\' e 3\' dos sotRNAs foram mapeadas através de dados de RNA-seq de pequenos RNAs (sRNA-seq) e validadas através da técnica de C-RACE. Análises de sequência e estrutura secundária demonstraram que estes RNAs apresentam um motivo conservado chamado de RE-like. Utilizando a sequência consenso deste motivo pudemos identificar RNAs intergênicos derivados de IS200/605 em H. salinarum NRC-1. Para caracterização funcional, construímos linhagens superexpressando os RNAs VNG_sot0042 e VNG_R0052, ambos contendo o motivo RE-like. Curvas de crescimento, utilizando as linhagens construídas demonstraram que a superexpressão destes RNAs aumenta o crescimento de H. salinarum demonstrando sua funcionalidade. Devido a presença do motivo RE-like, extremidades 5\' e 3\' determinadas e fenótipo visualizado em curva de crescimento padrão, o sotRNA VNG_sot0042 foi estudado mais a fundo. Realizamos experimentos de RNA-seq para avaliar o impacto desta superexpressão no transcritoma de H. salinarum NRC-1, assim como experimentos de SILAC para identificação de proteínas parceiras em larga escala. Nestes ensaios identificamos proteínas e genes associados ao processo de adesão, geração de células persistentes e resistência a metais pesados. Ensaios de adesão e sobrevivência a metais pesados demonstraram que a linhagem de superexpressão apresenta maior capacidade de aderir a vidro e maior sobrevivência em diversas condições de estresse. Deste modo, podemos sugerir que ncRNAs derivados de IS200/605 são importantes moléculas regulatórias em H. salinarum NRC-1 e nos ajudam a compreender a manutenção de IS200/605 e seus derivados nos genomas de procariotos.
Mobile genetic elements (MGEs) are extremely important for plasticity and evolution of genomes. Their impacts are diverse and could be related to antibiotic resistence or symbiosis. One of the most important class of MGEs are insertion sequences (ISs). These elements are found widespread throughout bacteria and archaea, presenting a great diversity of families. An interesting family is the IS200/605, this family is found widespread in bacteria, archaea and viruses and is divided in three subgroups according to it\'s genetic composition: IS200 with tnpA gene alone, IS605 with tnpA and tnpB and IS1341 with tnpB only. Another interesting aspect is the utilization of single-stranded DNA as a substrate during the transposition process. In this work, through the analysis of public available transcriptomic data we identified transcripts that overlaps the 3\' end of tnpB in IS200/605 in both bacteria and archaea. These transcripts were named sense overlapping transcripts (sotRNAs). Sequence and secondary structure analysis showed a conserved motif present in sotRNAs, the RE-like motif. Using the consensus sequence of this motif we identified novel intergenic ncRNAs containing this motif that are derived from IS200/605. For functional characterization we overexpressed a sotRNA (VNG_sot0042) and a intergenic (VNG_R0052), both containing the RE-like motif. Standard growth curves demonstrated that the overexpression of these ncRNAs improve H. salinarum growth showing that this RNA is functional. To further evaluate the impact of the overexpressions we prepared RNA-seq libraries of the strain overexpressing VNG_sot0042 and in parallel performed SILAC experiments to identify potential protein-RNA interaction partners. Differentially regulated genes and interacting proteins associated with adhesion and persistent cells generation were found. Adhesion and survival assays showed that the lineage overexpressing VNG_sot0042 has a better capability to adhere in glass surfaces and survive more in diverse stressful conditions.

One hundred and thirty-six Salmonella enterica strains, isolated from humans, animals, environmental and plant sources in Australia from 23 serovars, were examined for the streptomycin resistance gene strA and strB, the sulfonamide resistance gene sul2, and the tetracycline resistance gene tetA(A) and tetA(B). Thirteen strains were identified as containing the strA-strB genes located on the transposon Tn5393. S. enterica serovar Hadar accounted for 11 of these strains, 6 of which were isolated from humans and 5 were from ducks. This investigation is therefore the first report of the Tn5393 transposon being detected in bacterial strains from a human source in Australia.RSF1010 plasmids were identified and extracted from 4 S. enterica strains, and were further confirmed by restriction enzyme profiling using PstI, SspI and EcoRV. Small non-conjugative plasmid p9123 was extracted and characterised from 3 of the S. enterica strains and also confirmed by restriction enzyme digestion. An RSF1010-like plasmid was also identified in 3 of the strains. This plasmid was found to be approximately 2.6 kb larger than RSF1010, and possibly derived from the RSF1010 plasmid via insertion of the tetracycline resistance gene tetA(A) between strB and mobC genes.An IS26-strB-strA-sul2-repC-repA-IS26 antibiotic resistance region was identified in 33 S. enterica strains, among these were 23 serovar Typhimurium isolates, 8 serovar Bovismorbificans, 1 serovar Senftenberg and 1 isolate where the serovar could not be conclusively identified. The 23 Typhimurium strains were further characterised by PCR and Southern hybridisation analysis using a blaTEM gene probe. The analysis identified two classes of antibiotic resistance gene clusters. Eleven S. enterica serovar Typhimurium strains harboured an IS26-strB-strA-sul2-repC-repA-IS26-blaTEM-1-IS26 antibiotic resistance gene cluster and another 10 S. enterica serovar Typhimurium strains contained an IS26-strB-strA-sul2-repC-repA-IS26-blaTEM-1 gene cluster, without the IS26 element downstream of the blaTEM-1 gene. Two strains contain elements of these gene clusters but further investigation is needed to fully identify these.Further linkage PCR amplifications revealed that the IS26-strB-strA-sul2-repC-repA-IS26-blaTEM-1-IS26 antibiotic resistance gene cluster was possibly inserted into the 3P-CS of a class 1 integron (In4 type) and truncated the 3P-CS region. Three derivatives were identified, of which the dfrA5-intI1 type was most commonly found downstream of the blaTEM-1-IS26 region. Southern hybridisation analysis using an IS200 gene probe revealed that strains which contain different antibiotic resistance gene clusters also display different but related IS200 profiles.The antibiotic resistance gene clusters of 19 S. enterica serovar Typhimurium strains were transferred to an E. coli 294 Rifr recipient either by direct mating or triparental mating methods. These experiments confirmed that the antibiotic resistance gene clusters were located on conjugative or mobilisable plasmids. The antibiotic resistance gene clusters of 4 S. enterica serovar Typhimurium strains could not be transferred to the E. coli 294 Rifr recipient. These experimental results suggest that the antibiotic resistance gene cluster of IS26-strB-strA-sul2-repC-repA-IS26-blaTEM-1-IS26 might move as one genetic element between distinct plasmid backbones.

PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO
O método de elementos finitos é uma ferramenta amplamente utilizada na análise e simulação de fenômenos físicos. Uma etapa crucial desse método consiste na discretização de um domínio contínuo em uma malha de elementos finitos. A precisão da solução obtida na análise está diretamente relacionada á qualidade da malha utilizada, a qual é determinada pela forma e consistência de seus elementos. Grande parte dos trabalhos existentes na literatura é baseada em métodos clássicos, como Avanço de Fronteira, Triangulação de Delaunay com Restrição e Decomposição de Domínios. Utilizando uma abordagem diferente, esta dissertação propõe um método de geração a posteriori, em que restrições do domínio são inseridas iterativamente na malha, a qual é deformada localmente a fim de satisfazer cada nova restrição. Para validar o método proposto, os resultados obtidos são analisados através de critérios de qualidade comumente utilizados na literatura.
The finite element method (FEM) is a widely used tool in the analysis and simulation of physical phenomena. The discretization of a continuous domain into a finite element mesh is an essential FEM step. The accuracy of the solution is highly dependent on the quality of the input mesh, which is mainly evaluated through the shape of the elements. Most of the related works is based on traditional methods, such as Advancing Front, Constrained Delaunay Triangulation and Domain Decomposition. Unlike these methods, this work proposes an iterative mesh generation method with deferred constraint insertion, in which an initially regular mesh is locally deformed in order to satisfy each new domain constraint. In addition, in order to validate the proposed method, this work evaluates each output mesh according to quality criteria commonly used in the literature.

Mycoplasma agalactiae est responsable de l'agalactie contagieuse, maladie des petits ruminants difficilement contrôlée et figurant sur la liste de l’OIE. Afin d’évaluer la diversité génétique de ce pathogène, 101 isolats ont été comparés par trois techniques (VNTR, RFLP, répertoire vpma). Les résultats révèlent une grande homogénéité génétique dont la souche type PG2 est représentative. Quelques isolats font exception telle la souche 5632 que nous avons séquencée et analysée ici. La comparaison des génomes et des protéomes entre 5632 et PG2 indiquent que la plasticité de ces génomes est liée à d’importants échanges d'ADN et à la présence de nombreux éléments génétiques mobiles (10% du génome). Ces analyses révèlent également une forte dynamique au sein de répertoires de gènes codant des protéines de surfaces. Pour les mycoplasmes, bactéries minimales dépourvues de paroi, ces évènements ont certainement joués un rôle dans leur survie et leur adaptation à des hôtes complexes
Mycoplasma agalactiae is responsible of contagious agalactia, a disease of small ruminants that is still difficult to control and is listed by the OIE. In order to evaluate the genetic diversity of this pathogen, 101 isolates were compared using three techniques (VNTR, RFLP, vpma repertoire). Results revealed a high genetic homogeneity with the PG2 type strain as representative. Some isolates however diverged such as the 5632 which was sequenced and analysed here. Whole comparative genomic and proteomic analyses of the 5632 and PG2 strains indicate that their genomic plasticity resides in important genes flux and in the presence of several mobile genetic elements (10% of the genome). These analyses also revealed that specific loci encoding repertoire of surface proteins are highly dynamic. For these minimal bacteria that lack a cell-wall, these events have most likely played a major role in their survival and adaptation to complex hosts

Les techniques d'insertion d'aiguille font partie des interventions chirurgicales les plus courantes. L'efficacité de ces interventions dépend fortement de la précision du positionnement des aiguilles dans un emplacement cible à l'intérieur du corps du patient. L'objectif principal dans cette thèse est de développer un système robotique autonome, capable d'insérer une aiguille flexible dans une structure déformable le long d'une trajectoire prédéfinie. L’originalité de ce travail se trouve dans l’utilisation de simulations inverses par éléments finis (EF) dans la boucle de contrôle du robot pour prédire la déformation des structures. La particularité de ce travail est que pendant l’insertion, les modèles EF sont continuellement recalés (étape corrective) grâce à l’information extraite d’un système d’imagerie peropératoire. Cette étape permet de contrôler l’erreur des modèles par rapport aux structures réelles et ainsi éviter qu'ils divergent. Une seconde étape (étape de prédiction) permet, à partir de la position corrigée, d’anticiper le comportement de structures déformables, en se reposant uniquement sur les prédictions des modèles biomécaniques. Ceci permet ainsi d’anticiper la commande du robot pour compenser les déplacements des tissus avant même le déplacement de l’aiguille. Expérimentalement, nous avions utilisé notre approche pour contrôler un robot réel afin d'insérer une aiguille flexible dans une mousse déformable le long d'une trajectoire (virtuelle) prédéfinie. Nous avons proposé une formulation basée sur des contraintes permettant le calcul d'étapes prédictives dans l'espace de contraintes offrant ainsi un temps d'insertion total compatible avec les applications cliniques. Nous avons également proposé un système de réalité augmentée pour la chirurgie du foie ouverte. La méthode est basée sur un recalage initial semi-automatique et un algorithme de suivi peropératoire basé sur des marqueurs (3D) optiques. Nous avons démontré l'applicabilité de cette approche en salle d'opération lors d'une chirurgie de résection hépatique. Les résultats obtenus au cours de ce travail de thèse ont conduit à trois publications (deux IROS et un ICRA) dans les conférences internationales puis à un journal (Transactions on Robotics) en cours de révision
Needle-based interventions are among the least invasive surgical approaches to access deep internal structures into organs' volumes without damaging surrounding tissues. Unlike traditional open surgery, needle-based approaches only affect a localized area around the needle, reducing this way the occurrence of traumas and risks of complications \cite{Cowan2011}. Many surgical procedures rely on needles in nowadays clinical routines (biopsies, local anesthesia, blood sampling, prostate brachytherapy, vertebroplasty ...). Radiofrequency ablation (RFA) is an example of percutaneous procedure that uses heat at the tip of a needle to destroy cancer cells. Such alternative treatments may open new solutions for unrespectable tumors or metastasis (concerns about the age of the patient, the extent or localization of the disease). However, contrary to what one may think, needle-based approaches can be an exceedingly complex intervention. Indeed, the effectiveness of the treatment is highly dependent on the accuracy of the needle positioning (about a few millimeters) which can be particularly challenging when needles are manipulated from outside the patient with intra-operative images (X-ray, fluoroscopy or ultrasound ...) offering poor visibility of internal structures. Human factors, organs' deformations, needle deflection and intraoperative imaging modalities limitations can be causes of needle misplacement and rise significantly the technical level necessary to master these surgical acts. The use of surgical robots has revolutionized the way surgeons approach minimally invasive surgery. Robots have the potential to overcome several limitations coming from the human factor: for instance by filtering operator tremors, scaling the motion of the user or adding new degrees of freedom at the tip of instruments. A rapidly growing number of surgical robots has been developed and applied to a large panel of surgical applications \cite{Troccaz2012}. Yet, an important difficulty for needle-based procedures lies in the fact that both soft tissues and needles tend to deform as the insertion proceeds in a way that cannot be described with geometrical approaches. Standard solutions address the problem of the deformation extracting a set of features from per-operative images (also called \textit{visual servoing)} and locally adjust the pose/motion of the robot to compensate for deformations \cite{Hutchinson1996}. [...]To overcome these limitations, we introduce a numerical method allowing performing inverse Finite Element simulations in real-time. We show that it can be used to control an articulated robot while considering deformations of structures during needle insertion. Our approach relies on a forward FE simulation of a needle insertion (involving complex non-linear phenomena such as friction, puncture and needle constraints).[...]

This thesis develops a numerical technique in which the Particle Finite Element method is applied to simulate solid intrusion problems in Geotechnical Engineering. The thesis describes the numerical development work that made the method functional and showcases its potential with various application problems. An explicit stress integration scheme is developed for elasto-plastic, large-strain constitutive models using a multiplicative split of the deformation gradient. This scheme uses adaptive substepping and a yield violation drift correction technique. Low-order (linear) elements are employed to speed computation. These elements may suffer volumetric locking in quasi-incompressible conditions. For soils, such conditions appear under undrained loading or at critical state. To alleviate this problem, mixed formulations are developed and stabilization techniques are applied. Two different three-field mixed formulations for the coupled hydro-mechanical problem are presented, adding either the effective pressure or the Jacobian as nodal variables to the solid skeleton displacement and water pressure. Stabilization terms are used in the mass conservation equation of the biphasic medium and in the rest of scalar equations. Several mixed formulations are also implemented for the simplified single-phase problem, which approximates saturated soil behavior under undrained conditions. A total stress approach is used in a parametric study of tube sampling in clay. The parameters explored include sampler geometry (round-tipped or beveled cutting shoes; outer diameter to wall thickness ratios); constitutive parameters, roughness factor and boundary conditions are studied. Outputs are analyzed in terms of classical sampling disturbance measures: the centerline strain path and the specific recovery ratio. The results show good agreement with experimental evidence and question the frequently accepted reference role of Strain Path method solutions. The set of simulations in which a rough interface behavior is considered has been used to assess the theory proposed by Paikowsky and Whitman (1990) to predict the occurrence of a plug inside of an open-ended pile. The numerical results obtained here corroborate this theory: a plug inside of the tube is formed once the mobilized forces are equal to those that would mobilize a smooth closed-ended pile. Indeed, the failure mechanism that prevails during the penetration of a plugged tube is coincident with that of a closed-ended pile. The last analysis of this work is the hydro-mechanical simulation of the cone penetration test in a Modified Cam Clay soil. A parametric analysis covers the effect of the permeability of the soil -from drained to undrained conditions- and the interface friction angle. The effect of these parameters on the cone resistance, sleeve friction and pore pressure at three potential measurement points is fully characterized. These numerical results are used to assess several techniques to estimate the permeability of soils from CPTu testing. Special attention is paid to on-the-fly techniques, in which permeability could be directly estimated from the CPTu data stream without the need for any stoppage.
Esta tesis desarrolla una técnica numérica en la que se aplica el método de Partículas y Elementos Finitos (Particle Finite Element Method) para simular problemas de intrusión de objetos sólidos en Ingeniería Geotécnica. La tesis describe los trabajos numéricos desarrollados para lograr tal objetivo y muestra el potencial del método mediante varios problemas de aplicación. Los nuevos desarrollos numéricos incluyen: esquemas de integración para modelos constitutivos elasto-plásticos en deformaciones finitas, nuevos métodos de integración de las restricciones de contacto con un cuerpo rígido y formulaciones mixtas estabilizadas para el problema hidromecánico. Se desarrolla un esquema explícito para la integración de tensiones para modelos constitutivos elasto-plásticos en deformaciones finitas usando una descomposición multiplicativa del gradiente de deformación. Este esquema utiliza técnicas adaptativas para la elección del paso así como esquemas para corregir la violación de la condición de consistencia. Se pueden modelar cuerpos rígidos con forma y movimiento preespecificados. Las restricciones de contacto se introducen mediante un método de penalización en el que el comportamiento tangencial se trata con una analogía elastoplástica. Se emplea una técnica de integratión implícita para este modelo de contacto elastoplástico; además, también se describe y evalúa una técnica alternativa utilizando un algoritmo Implex. Para acelerar el cálculo, se emplean elementos de bajo orden (lineales). Estos elementos pueden sufrir bloqueo volumétrico en condiciones casi incompresibles. En suelos, tales condiciones aparecen bajo carga no drenada o en estado crítico. Para aliviar este problema, se desarrollan formulaciones mixtas y se aplican técnicas de estabilización. Se presentan dos formulaciones mixtas de tres campos para el problema hidromecánico acoplado, que tienen la presión efectiva o el Jacobiano como variables nodales además del desplazamiento del esqueleto sólido y la presión de agua. Se usan técnicas de estabilización en la ecuación de conservación de masa del medio bifásico y en el resto de ecuaciones escalares. También se implementan varias formulaciones mixtas para el problema simplificado de una sola fase, ya que aproxima el comportamiento del suelo saturado en condiciones no drenadas. Los primeros ejemplos de aplicación son tres problemas de tensión total: la indentación de una cimentación rígida en faja, el movimiento de una barra en T (T-Bar) y un ensayo de penetración de cono rugoso. Todos son problemas frecuentemente utilizados para validar la implementación numérica; por lo que permiten comparar el rendimiento del esquema numérico desarrollado con otras técnicas. Parece que la estrategia numérica seguida por PFEM, obtiene resultados similares a los obtenidos con métodos numéricos alternativos con ahorros significativos en esfuerzos computacionales. También se usa un enfoque de tensión total para un estudio paramétrico de muestreo mediante tubo en arcilla. Los parámetros explorados incluyen la geometría del muestreador (zapatas de corte de punta redonda o biselada, relaciones de diámetro exterior a grosor de pared); además, se estudian los parámetros constitutivos, el factor de rugosidad y las condiciones de contorno. Los resultados se analizan en términos de medidas clásicas de alteración de la muestra: la deformación vertical en el eje de simetría y la relación de recuperación específica. Los resultados muestran un buen acuerdo con las evidencias experimentales y cuestionan la solución -frecuentemente aceptada- obtenida mediante el Strain Path method.
Aquesta tesi desenvolupa una tècnica numèrica en la qual s'aplica el mètode de Partícules i Elements Finits (Particle Finite Element Method) per simular problemes d’intrusió d'objectes sòlids en Enginyeria Geotècnica. La tesi descriu els treballs numèrics desenvolupats per aconseguir aquest objectiu i mostra el potencial del mètode mitjançant diversos problemes d’aplicació. Els nous desenvolupaments numèrics inclouen: esquemes d’integració per a models constitutius elasto-plàstics a deformacions finites, nous mètodes d’integració de les restriccions de contacte amb un cos rígid i formulacions mixtes estabilitzades pel problema hidromecànic. Es desenvolupa un esquema explícit per a la integració de tensions per a models constitutius elasto-plàstics en deformacions finites usant una descomposició multiplicativa del gradient de deformació. Aquest esquema utilitza tècniques adaptatives per a l’elecció del pas així com esquemes per corregir la violació de la condició de consistència. Es poden modelar cossos rígids la forma i moviment dels quals està predefinida. Les restriccions de contacte s'introdueixen mitjançant un mètode de penalització en el qual el comportament tangencial es tracta amb una analogia elastoplàstica. Es fa servir una tècnica d'integration implícita per a aquest model de contacte elastoplàstic; a més, també es descriu i avalua una tècnica alternativa utilitzant un algoritme Implex. Per accelerar el càlcul, s'empren elements de baix ordre (lineals). Aquests elements poden patir bloqueig volumètric en condicions gairebé incompressibles. En sòls, aquestes condicions apareixen sota càrrega no drenada o en estat crític. Per evitar aquest problema, es desenvolupen formulacions mixtes i s'apliquen tècniques d’estabilització. Es presenten dues formulacions mixtes de tres camps per al problema hidromecànic acoblat, que tenen la pressió efectiva o el Jacobià com a variables nodals a més del desplaçament de l'esquelet sòlid i la pressió d'aigua. S'usen tècniques d’estabilització en l’equació de conservació de massa del medi bifàsic i a la resta d'equacions escalars. També s'implementen diverses formulacions mixtes per al problema simplificat d'una sola fase, ja que aproxima el comportament del sòl saturat en condicions no drenades. Els primers exemples d’aplicació són tres problemes de tensió total: la indentació d'una fonamentació rígida en faixa, el moviment d'una barra en T (T-Bar) i un assaig de penetració de con rugós. Tots són problemes freqüentment utilitzats per validar implementacions numèriques; per la qual cosa permeten comparar el rendiment de l'esquema numèric desenvolupat amb altres tècniques. Sembla que l’estratègia numèrica seguida per PFEM, obté resultats similars als obtinguts amb mètodes numèrics alternatius amb estalvis significatius en esforços computacionals. També es fa servir un enfocament de tensió total per un estudi paramètric de mostreig mitjançant tub en argila. Els paràmetres explorats inclouen la geometria del mostrejador (sabates de tall de punta rodona o bisellada, relacions de diàmetre exterior a gruix de paret); a més, s'estudien els paràmetres constitutius, el factor de rugositat i les condicions de contorn. Els resultats s'analitzen en termes de mesures clàssiques d’alteració de la mostra: la deformació vertical en l'eix de simetria i la relació de recuperació especifica. Els resultats mostren un bon acord amb les evidències experimentals i qüestionen la solució -sovint acceptada- obtinguda mitjançant el Strain Path method. El conjunt de simulacions en el qual es considera un comportament d’interfície rugós s'ha utilitzat per avaluar la teoria proposada per Paikowsky and Whitman (1990) per predir el taponament de pilots oberts. Els resultats numèrics obtinguts aquí corroboren aquesta teoria: el tub es tapona una vegada que les forces mobilitzades són iguals a les que mobilitzaria un pilot de base tancada amb interfície és llisa. De fet, el mecanisme de fallada que preval durant la penetració d'un tub obstruït és coincident amb el d'un pilot de base tancada. L’última anàlisi d'aquest treball és la simulació hidromecànica de l'assaig de penetració de con en un sòl Cam Clay modificat. L’anàlisi paramètric abasta l'efecte de la permeabilitat del sòl -des de condicions drenades a no drenades- i l'angle de fricció de la interfície. Es caracteritza per complet l'efecte d'aquests paràmetres sobre la resistència del con, la fricció en el fust i la pressió de porus en tres possibles punts de mesura. Aquests resultats numèrics s'utilitzen per avaluar diverses tècniques per estimar la permeabilitat dels sòls a partir de l'assaig CPTu. Es presta especial atenció a les tècniques sobre-la-marxa (on-the-y), en què la permeabilitat es pot estimar directament a partir de les dades obtingudes durant la penetració del CPTu, sense necessitat de realitzar cap parada.

In introduction of work, I presented the state of the art of measuring insertion loss of the EMI filter and the description of individual methods of measuring is also included. After that I dealt with global optimization methods and some of them I theoretically described. Mainly PSO method was studied carefully, and the principle of optimization in details are presented in the thesis, because this method was used for optimization of the filters parameters in the thesis. Next part of work deals with theoretical and mathematical expression of insertion loss of the EMI filters. Due to the mathematical expression of the insertion loss, the values of parasitic elements of given filter have been optimized in different measuring systems. This optimization was applied on synthetic data. By this step the functionality of optimization was proved and also the fitness function has been tuned and optimised. The last part of thesis works with optimization on measured data of given filters. I found out inaccuracy in mathematical expression of given model of EMI filter, therefore it was necessary to come up with the new filter model. The accuracy increases by using the new filter model. In next step the new model was enlarged by new spurious components.

The development of algorithms that effectively modify all-hexahedral finite element mesh is currently an active area of research. Mesh modification can be used to improve mesh quality reduce the time required to mesh a model, and improve the finite element analysis results. However, general modification of all-hexahedral meshes has proven difficult because of the global effects of local modifications. This thesis explains the global constraints on modifying all-hexahedral meshes and then presents three mesh modification techniques that make it possible to do local modifications while accounting for the global effects. These techniques are sheet insertion, sheet extraction, and mesh cutting. Sheet insertion is used to refine a mesh by inserting sheets of hexahedral elements into existing meshes. Sheet extraction coarsens existing meshes by deleting sheets of elements from the mesh. Mesh cutting is used to modify a simple mesh to fit it to complex geometric feature. The mesh modification techniques are covered in detail with representative graphics. Examples are given that demonstrate the application of each technique to the mesh generation process.

La réfrigération magnétique à température ambiante, une alternative à la réfrigération classique, nécessite des matériaux magnétocaloriques performants. L'effet magnétocalorique se traduit par une variation d'entropie et une variation de température du matériau lorsqu'il est soumis à un champ magnétique. Les composées de type LaFe13-xSix présentent des atouts intéressants. Nous avons tout d'abord exploré cette famille de composé. Par substitutions chimiques et par insertion d'éléments interstitiels, nous avons étudié plusieurs compositions dont celles de type LaFe13-x-yCoySix et les hydrures LaFe13-xSixHy qui se sont révélées être les plus prometteuses. Les caractérisations structurales, microstructurales, thermiques et magnétiques entreprises nous ont guidés dans l'optimisation des méthodes d'élaboration : fusion à arc ou induction, des traitements thermiques, du contrôle de la stœchiométrie, etc. Dans un second temps nous avons développé la mise au point de ces matériaux à plus grande échelle en collaboration avec des partenaires industriels
Magnetic refrigeration at room temperature, an alternative to conventional refrigeration, requires efficient magnetocaloric materials. The magnetocaloric effect is reflected by an entropy variation or a temperature variation of the material undergoes a magnetic field. The LaFe13-xSix type compounds present interesting advantages. First step consisted in investigating this system. Through chemical substitutions and interstitial elements insertion, we studied several compositions, including those of LaFe13-x-yCoySix type and hydrides LaFe13-xSixHy which proved to be the most promising ones. The structural, microstructural, thermal and magnetic characterization have led us in the optimization of the elaboration method, arc melting or induction melting, heat treatment, stoichiometry control, and so on. In a second step we set up the synthesis of these materials at a larger commercial scale with our industrial partners

Thesis advisor: Gabor T. Marth
Structural variations (SVs), like single nucleotide polymorphisms (SNPs) and short insertion-deletion polymorphisms (INDELs), are a ubiquitous feature of genomic sequences and are major contributors to human genetic diversity and disease. Due to technical difficulties, i.e. the high data-acquisition cost and/or low detection resolution of previous genome-scanning technologies, this source of genetic variation has not been well studied until the completion of the Human Genome Project and the emergence of next-generation sequencing (NGS) technologies. The assembly of the human genome and economical high-throughput sequencing technologies enable the development of numerous new SV detection algorithms with unprecedented accuracy, sensitivity and precision. Although a number of SV detection programs have been developed for various SV types, such as copy number variations, deletions, tandem duplications, inversions and translocations, some types of SVs, e.g. copy number variations (CNVs) in capture sequencing data and mobile element insertions (MEIs) have undergone limited study. This is a result of the lack of suitable statistical models and computational approaches, e.g. efficient mapping method to handle multiple aligned reads from mobile element (ME) sequences. The focus of my dissertation was to identify and characterize CNVs in capture sequencing data and MEI from large-scale whole-genome sequencing data. This was achieved by building sophisticated statistical models and developing efficient algorithms and analysis methods for NGS data. In Chapter 2, I present a novel algorithm that uses the read depth (RD) signal to detect CNVs in deep-coverage exon capture sequencing data that are originally designed for SNPs discovery. We were one of the early pioneers to tackle this problem. In Chapter 3, I present a fast, convenient and memory-efficient program, Tangram, that integrates read-pair (RP) and split-read (SR) signals to detect and genotype MEI events. Based on the results from both simulated and experimental data, Tangram has superior sensitivity, specificity, breakpoint resolution and genotyping accuracy, when compared to other recently published MEI detection methods. Lastly, Chapter 4 summarizes my work for SV detection in human genomes during my PhD study and describes the future direction of genetic variant researches
Thesis (PhD) — Boston College, 2013
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

Transposable elements (TEs) are utilized for the creation of a wide range of transgenic organisms. However, in some systems, this technique is not very efficient due to low transposition frequencies and integration into unstable or transcriptionally inactive genomic regions. One approach to ameliorate this problem is to increase knowledge of how transposons move and where they integrate into target genomes. Most transposons do not insert randomly into their host genome, with class II TEs utilizing target sequences of between 2 – 8 bp in length, which are duplicated upon insertion. Furthermore, amongst insertion sites, certain sites are preferred for insertion and hence are classified as hot spots, while others not targeted by TEs are referred to as cold spots. The hypothesis tested in this analysis is that in addition to the primary consensus target sequence, secondary and tertiary DNA structures have a significant influence on TE target site preference. Bioinformatics was used to predict and analyze the structure of the flanking DNA around known insertion sites and cold spots for various TEs, to understand why insertion sites are used preferentially to cold spots for element integration. Hidden Markov Models were modeled and trained to analyze datasets of insertions of the P element in the Drosophila melanogaster genome, the Tc1 element in the Caenorhabditis elegans genome, and insertions of the Mos1, piggyBac and Hermes transposons into the target plasmid pGDV1. Analysis of the DNA structural profiles of the insertion sites for the P element and Hermes transposons revealed that both transposons targeted regions of DNA with a relatively high degree of bendability/flexibility at the insertion site. However, similar trends were not observed for the Tc1, Mos1 or piggyBac transposons. Hence, it is believed that the secondary structural features of DNA can contribute to target site preference for some, but not all transposable elements.

Este trabalho analisou o processo de moldagem por injeção de micro elementos ópticos difrativos usinados em insertos de cobre eletrolítico com ferramenta de diamante com ponta única. Quatro tipos de microestruturas características foram selecionados neste estudo, a saber: lente anesférica, lente de Fresnel, grade de difração (blaze grating) e sensor de frente de onda. A análise da fidelidade de replicação foi feita considerando aspectos dimensionais micrométricos e nanométricos para a microestrutura e acabamento. Um perfilometro óptico e microscópio eletrônico de varredura foram utilizados para avaliar os insertos usinados e as características dos replicados. Uma ferramenta de diamante com geometria especial, com meio raio, foi usada para usinar as características de difração. As superfícies usinadas apresentaram baixo acabamento superficial, na faixa de 16 nm Rms. As simulações numéricas foram realizadas para avaliar o desempenho do processo de moldagem por injeção com polimetilmetacrilato (PMMA), e os resultados foram utilizados para orientar a injeção do polímero. Com base na simulações numéricas as temperaturas do molde e pressões de injeção foram variadas entre 85ºC/130°C e 70 bar/130 bar, respectivamente. A influência destes parâmetros no desempenho do processo de replicação foi analisada. A análise quantitativa da replicação foi feita através de um parâmetro denominado grau de replicação que define a relação entre a altura nominal da microestrutura do inserto e à altura da microestrutura na réplica de polímero. A grade de difração e o sensor de frente de onda apresentaram os melhores níveis de replicação: 98% e 99%, respectivamente. Os resultados experimentais mostraram que o processo de moldagem por injeção é uma técnica viável para replicar com alta qualidade microcaracterísticas de elementos ópticos de difração gerados por torneamento com ferramenta de diamante com ponta única.
This work investigated the injection molding process of micro diffractive optical elements machined on electrolytic copper inserts by single point diamond turning. Four types of microstructure features were selected in this study, namely: aspherical lens, Fresnel lens, blaze grating and the wavefront sensor. The replication fidelity was evaluated in terms of dimensional micrometric features found in the microstructure and the surface finish. An optical profiler and scanning electron microscopy were used to assess the machined inserts and the replicated features. A special geometry diamond tool with half radius was used to machine the diffraction features. The machined surfaces presented very low surface finish in the range of 16 nm Rms. Numerical simulations were carried out to evaluate the performance of the injection molding process with polymethylmethacrylate (PMMA), and the results were used to guide the polymer injection. Based on numerical simulations mold temperatures and injection pressures were varied between 85°C/130°C and 70 bar/130 bar, respectively. The influence of these parameters on performance of the replication process was assessed. The quantitative assessement of the replication was made by using a parameter called degree of replication which defines the ratio between the nominal height of the microstructure in the insert and the height of the microstructure in the polymer replica. The blaze grating and the wavefront sensor presented the best degrees of replication: 98% and 99%, respectively. The experimental results showed that injection molding process is a viable technique to replicate high quality micro features of optical diffraction elements generated by single point diamond turning.

L'objectif de ce travail de these est d'isoler des elements transposables de petunia pour developper un outil d'etiquetage de genes vegetaux par la mutagenese insertionnelle. Le gene adh codant pour l'alcool deshydrogenase, enzyme conferant une sensibilite a l'alcool a ete utilise comme piege pour element instables. Les experiences realisees ont consiste a selectionner sur pollen des mutants adh instables, par insertion d'un element transposable a partir d'un genotype portant une instabilite de la pigmentation des fleurs (allele an214) et d'un genotype portant une instabilite de la pigmentation des tissus chlorophylliens (allele lu13). Des mutants stables ont par ailleurs ete selectionnes apres une mutagenese chimique a l'ems. Nous ne disposons pas a l'issue de ces travaux des mutants instables souhaites, il semble que les frequences de transposition des deux elements ne sont pas favorables a ce type de recherche. Les plantes que nous avons selectionnees semblent, comme dans le cas de la mutagenese a l'ems etre des mutants leaky. L'objectif initial de la recherche n'est pas atteint mais nous avons montre qu'il est possible d'augmenter la frequence de transposition par un stress au froid ce qui permet d'envisager de nouvelles experiences efficaces de selection de mutants par mutagenese insertionnelle

Transposable elements (TEs) are mutagenic mobile DNA sequences whose excision and insertion are powerful drivers of evolution. Some TE families are known to target specific genome features, and studying their insertion preferences can provide information about both TE biology and the state of the genome at these locations. To investigate this, collecting large numbers of insertion sites for TEs in natural populations is required. Genome resequencing data can potentially provide a rich source of such insertion sites. The field of detecting these "non-reference" TE insertions is an active area of research, with many methods being released and no comprehensive review performed. To drive forward knowledge of TE biology and the field of non-reference TE detection, we created McClintock, an integrated pipeline of six TE detection methods. McClintock lowers the barriers against use of these methods by automating the creation of the diverse range of input files required whilst also setting up all methods to run simultaneously and standardising the output. To test McClintock and its component methods, it was run on both simulated and real Saccharomyces cerevisiae data. Tests on simulated data reveal the general properties of component methods' predictions as well as the limitations of simulated data for testing software systems. Overlap between results from the McClintock component methods show many insertions detected by only one method, highlighting the need to run multiple TE detection methods to fully understand a resequenced sample. Utilising the well characterised properties of S. cerevisiae TE insertion preferences, real yeast population resequencing data can act as a biological validation for the predictions of McClintock. All component methods recreated previously known biological properties of S. cerevisiae TE insertions in natural population data. To demonstrate the versatility of McClintock, we applied the system to Drosophila melanogaster resequencing data. 27 Schneider's cell lines were sequenced and analysed with McClintock. In addition to demonstrating the scalability of McClintock to larger genomes with more TE families, this exposed ongoing transposition in S2 cell lines. Likewise, the use of non-reference TE insertions as variable sites allowed us to recreate the relationships between S2 sub-lines, confirming that S1, S2, and S3 were most likely established separately. The results also suggest that there are several S2 sub-lines in use and that these sub-lines can differ from each other in TE content by hundreds of non-reference TE copies. Overall this thesis demonstrates that the McClintock pipeline can highlight problems in TE detection from genome data as well as revealing that much can still be learned from this data source.

La présente thèse de doctorat a pour objectif d’améliorer la modélisation du phénomène de rupture dans les matériaux fragiles. Elle porte une attention particulière aux mécanismes de rupture des objets célestes. L’un des problèmes posant le plus de défis aux scientifiques spécialisés dans l’étude de la mécanique de la rupture est la propagation d’une fissure dans un maillage éléments finis, et ce pour des chemins arbitraires. Dans cette étude, ce problème est abordé en utilisant une technique de remaillage avancée utilisant des éléments finis cohésifs permettant la propagation de fissures suivant des directions arbitraires et indépendantes du maillage. La direction de la fissure est calculée suivant le critère du taux de restitution d’énergie maximal, implémentée à l’aide d’un modèle éléments finis et de la méthode Gθ. Les effets de différents paramètres numériques et physiques relatifs à la fissure ou à l’énergie libérée lors de la rupture sont investigués.Bien que différentes preuves de fissures et/ou fragments à la surface de corps célestes de notre système solaire induits par des variations cycliques de la température ont été détaillées, la compréhension de ces mécanismes de propagation dans des objets célestes reste très parcellaire. La fracturation thermique de roches en surface associée à l’impact de micro-météorites peutéventuellement conduire à la rupture complète de fragments de matière et à la production de régolithes. Cette dernière est définie comme la couche de matériau non consolidée qui recouvre la surface des planètes. Afin de comprendre ces mécanismes, l’étude s’attarde sur un exemple précis, celui de l’astéroïde (101955) Bennu. Pour ce faire, elle utilise un modèle thermoélastique couplé avec un modèle linéaire élastique de mécanique de la rupture permettant de considérer les variations cycliques de température liées aux alternances jour/nuit. En utilisant cette méthodologie, il a été observé que les fissures se propagent préférentiellement dans les directions : Nord vers Sud, Nord-Est vers Sud-Ouest et Nord-Ouest vers Sud-Est. Finalement, une analyse de fatigue est effectuée afin d’estimer la vitesse de croissance de la fissure.Les méthodes détaillées précédemment ont été implémentées dans Cimlib, une librairie C++ dévelopée au CEMEF. Au sein de cette librairie, une méthode permettant la propagation d’une ou plusieurs fissures, suivant des directions arbitraires, en 2D et au sein d’un environnement de calcul en parallèle est à présent disponible. Concernant l’extension de cette méthode à des problèmes 3D, une première approche a été mise au point. Elle permet de propager un front de fissure suivant une direction arbitaire. La structure développée permet d’ouvrir de nouvelles possibilités pour de nombreuses applications, telles que l’étude de la rupture de matériaux composites à l’échelle mesoscopique
The present PhD thesis aims at providing a better modeling of fracture phenomenon in brittle materials, with special attention focused on fracture processes taking place in astronomical bodies. One of the most challenging issues in computational fracture mechanics is the propagation of a crack through a finite element mesh for arbitrary crack paths. In this work, this problem is approached by means of an advanced remeshing technique that propagates a crack using cohesive elements through arbitrary directions (mesh-independent). The crack direction is computed using the maximal energy release rate criterion which is implemented using finite elements and the Gθ method. The effects of different numerical and physical parameters regarding the crack path and fracture energy have been investigated. Even though it has been shown that temperature cycles on airless bodies of our Solar System can cause damaging of surface materials (Thermal cracking), propagation mechanisms in the case of space objects are still poorly understood. Thermal cracking of surface rocks, in addition to the impact of micrometeorties, can eventually lead to rocks’ breakup and produce fresh regolith, the latter being the layer of unconsolidated material that covers planetary surfaces. For this reason, the present work combines a thermoelasticity model together with linear elastic fracture mechanics theory to predict fracture propagation in the presence of thermal gradients generated by diurnal temperature cycling and under conditions similar to those existing on asteroid (101955) Bennu. Using the implemented methodologies, it is found that in asteroid Bennu, cracks preferentially propagate in the North to South (N-S), in the North-East to South-West (NE-SW) and in the North-West to South-East (NW-SE) directions. Finally, thermal fatigue analysis was performed in order to estimate the crack growth rate.Aforementioned methodologies have been implemented in Cimlib, a C++ in-house finite element library developed at CEMEF. Inside Cimlib, a methodology allowing two-dimensional crack propagation through arbitrary directions with the option of handling multiple cracks in the domain and inside a parallel environment was developed. Regarding three-dimensional scenario, a first approach where a crack front was propagated through an arbitrary direction was achieved. Concerning numerical modeling of crack propagation, the developed framework opens new possibilities for various applications such as composites cracking at the meso-scale

Basic problems of the EMI mains filter is explained in the introduction of this thesis. These problems appear during the measurement of insertion loss of filters and by design of EMI mains filter. The important problem is uncertainly of the terminating impedances. Eventualities measurement insertion loss of EMI mains filter and motivation of creation filter models are shown. The following part of this project shows the electronic components, from which are realized the EMI mains filter. Parasitic properties of these real devices are also discussed. The circuitry of EMI mains filter is described by the Modified Nodal Voltage Method. The optimization method called Participle Swarm Optimization is introduced, too. Basic principle and terminology are mentioned. The third part of this project is focused on the own filter and spurious components. Influence of own filter and spurious filters components on insertion loss curve are showed. Magnetic factor coefficient k is searched by the optimization method PSO. The last part of this thesis deals with creation of the alternative circuits with spurious filters components. The performances of the alternative circuits were compared with the measured data for symmetrical and asymmetrical measurement setup. The breakages in the insertion loss curve were used for determining the spurious components.

Transmissibility is the most common metric used for isolator characterization. However, engineers are becoming increasingly concerned about energy transmission through an isolator at high frequencies and how the compliance of the machine and foundation factor into the performance. In this study, the transfer matrix approach for isolator characterization is first reviewed. Two methods are detailed for determining the transfer matrix of an isolator using finite element simulation. This is accomplished by determining either the mobility or impedance matrix for the isolator and then converting to a transfer matrix. One of the more useful metrics to characterize the high frequency performance of an isolator is insertion loss. Insertion loss is defined as the difference in transmitted vibration in decibels between the unisolated and isolated cases. Insertion loss takes into account the compliance on the source and receiver sides. Accordingly, it has some advantages over transmissibility which is a function of the damping and mounted resonant frequency. A static analysis is to preload the isolator so that stress stiffening is accounted for. This is followed by modal and forced response analyses to identify the transfer matrix of the isolator. In this paper, the insertion loss of spring isolators is examined as a function of several geometric parameters including the spring diameter, wire diameter, number of active coils, and height. Results demonstrate how modifications to these parameters affect the insertion loss and the first surge frequency.

Enclosures are a very common way to reduce noise emissions from machinery. However, enclosures display complex acoustic behavior that is difficult to predict. The research presented in this thesis uses the boundary element method in order to better understand the acoustic behavior of a partial enclosure. Insertion loss was used as the performance measure and the effect of several design factors on the overall insertion loss was documented. Results indicate that the most important factors affecting enclosure performance are the opening size, amount of absorption, and the source-to-opening distance.

Although sygma-aryl and sygma-vinylpalladium(II) complexes are commonly used as electrophiles in C—C bond forming reactions, recent research has demonstrated that the same palladium intermediates can also react with carbon-heteroatom multiple bonds in a nucleophilic manner. Continuing with the interest of our group on the palladium-catalysed intramolecular coupling of aryl halides whit carbonyl compounds, we have expanded our previous findings to substrates containing other carbon-heteroatom multiple bonds. In this context, the first objective of the PhD Thesis has been the study of the dual nature of the sygma-arylpalladium(II) species in aldehyde containing compounds. Thus, after some preliminary studies on the ambiphilic character of the sygma-arylpalladium(II) intermediates involved in the palladium-catalysed intramolecular reactions of (2-iodoanilino)-aldehydes, we have described an efficient new methodology for the synthesis of dibenzo[b,e]azepin-11-ones based on the intramolecular acylation of aryl iodides with aldehydes. Continuing whit our interest in these nucleophilic addition processes, we have developed an efficient methodology for the preparation of a small library of tetrahydroisoquinolin-4-ols based on the palladium-catalysed intramolecular nucleophilic addition of a-(2-iodobenzylamino)- aldehydes. Finally, we have expanded the influence of the heteroatom (nitrogen, oxygen, and sulphur) on the course of the palladium-catalysed intramolecular reactions of aryl iodides and aldehydes having heteroatom—containing tethers. Through an extensive experimental-computational (DFT) study we have concluded that the nature of the heteroatom in the tether is not decisive for the outcome of these reactions. Continuing our research for methodologies that help us to increase the synthetic potential of organopalladium chemistry, we decide to investigate the feasibility of the palladium-catalysed intramolecular coupling of aryl halides and diazoderivatives as a methodology for the synthesis of nitrogen heterocycles. So, the second objective was the study of the Pd-catalysed intramolecular coupling of aryl halides with N-tosylhydrazones and sygma-diazoesters in nitrogen containing substrates. We have found that palladium can be used to catalyse the C(sp3)-H insertion of metal carbenoids derived from sygma-diazoesters to form pyrrolidines through intramolecular assembly of C(sp3)-C(sp3) bonds. This reaction is the first example of palladium-catalyzed C(sp3)-C(sp3) bond assembly starting from diazocarbonyl compounds. The coupling reaction can be catalyzed by both Pd(0) and Pd(II), is regioselective, and shows a broad functional group tolerance.
Las especies de tipo sigma-aril y sigma-vinilpaladio(II) actúan generalmente como electrófilos en las reacciones de formación de enlaces C—C. Durante los últimos años se ha demostrado que en ciertos casos estas mismas especies pueden cambiar al carácter electrófilo usual por un carácter nucleófilo y adicionarse a enlaces múltiples carbono-heteroátomo. En este sentido, se ha estudiado el carácter ambifílico (electrófilo vs. nucleófilo) de los intermedios de 6-arilpaladio(11) derivados de 2-haloanilinas frente al grupo formílo. Hemos sido capaces de controlar la reactividad (sigma-arilación vs. acilación del haluro arílico) de esos intermedios mediante la modificación de las condiciones de reacción. Eso ha permitido desarrollar una metodología eficiente para la síntesis de una pequeña librería de dibenzoazepinas, compuestos importantes desde el punto de vista farmacéutico, mediante la reacción de acilación nucleófila intramolecular catalizada por Pd(0). A continuación hemos centrado nuestro interés en la reacción de adición nucleofílica intramolecular de yoduros arílicos a aldehídos catalizada por Pd(0) en sustratos de tipo sigma-(2- iodobenzylamino)-aldehído. Esta reacción ha permitido la síntesis de diversas tetrahydroisoquinolin-4-oles. Para finalizar el estudio de las reacciones de acoplamiento intramolecular de yoduros arílicos con aldehídos catalizadas por Pd(0) en sustratos de tipo 2-iodoanilino- y sigma-(2- iodobenzylamino)-aldehídos, hemos investigado la influencia que los heteroátomos de nitrógeno, oxígeno y azufre poseen en el curso de la reacción. El estudio combinado experimental-computacional (DFT) ha permitido concluir que la naturaleza del heteroátomo no juega un papel clave en ese tipo de transformaciones. La segunda parte de esta Tesis Doctoral se ha focalizado en el estudio de las reacciones de acoplamiento catalizadas por paladio entre yoduros de arilo y diazo derivados. El estudio ha permitido el descubrimiento de una nueva metodología para la síntesis de pirrolidinas basada en la reacción de inserción de carbenos de paladio derivados de sigma-diazoesteres sobre enlaces C(sp3)—H.

Partial enclosures are commonly used to reduce machinery noise. However, it is well known in industry that enclosures sometimes amplify the sound at low frequencies due to strong acoustic resonances compromising the performance. These noise issues are preventable if predicted prior to prototyping and production. Though boundary and finite element approaches can be used to accurately predict partial enclosure insertion loss, modifications to the model require time for remeshing and solving. In this work, partial enclosure performance at low frequencies is simulated using a plane wave transfer matrix approach. Models can be constructed and the effect of design modifications can be predicted rapidly. Results are compared to finite element analysis and measurement with good agreement. The approach is then used to design and place resonators into a sample enclosure. Improvements in enclosure performance are predicted using plane wave simulation, compared with acoustic finite element analysis, and then validated via measurement.

This project investigated the impact of femoral nail curvature on the ease of nail insertion, extraction and overall fit. Results from the cadaveric experiment showed that nails with curvature which closely match patient's anatomy are easier to insert and remove, and have a better overall fit after implantation. This finding supports the idea of further reducing the nail curvature for populations with strong bowed femora. The finite element study conducted in this project demonstrated that the interference fit method can be potentially used to quantitatively evaluate the performance of new nail designs in the future.

Among transition metals used in organic chemistry, palladium has greatly contributed to the development of modern organic synthesis. This thesis is focused on the development of novel and efficient methodologies for the synthesis of nitrogen heterocycles employing palladium catalysed reactions. In this work, DFT calculations complement the experimental work in order to gather mechanistic insights of the reported transformations. In the first part of the thesis an exhaustive study of the intramolecular palladium catalysed α-arylation of sulfones is described. This metal-mediated process was successfully combined with conjugated additions to generate domino processes of two, three and four steps that allowed the synthesis of functionalised indoles and tetrahydroisoquinolines. From a mechanistic point of view, DFT calculations suggest that a concertated metalation-deprotonation process followed by reductive elimination is involved in the indole ring formation. Next, the intramolecular palladium catalysed α-arylation reaction was explored using nucleophiles derived from sulfonates, sulfonamides and phosphonates. Although feasible, these nucleophiles are less efficient in the intramolecular cyclization leading to the tetrahydroisoquinoline ring. Domino processes based on α-arylation/Michael addition reactions starting from sulfonates and sulfonamides using diverse Michael acceptors were also reported. Attempts to pursue domino process starting from phosphonates were unsuccessful. In the second part of the thesis, a synthetic methodology for the synthesis of nitrogen heterocycles based on palladium catalysed decomposition of α- diazoacetamidas was studied. Decomposition of α-diazo-α-(methoxycarbonyl) acetanilides catalyzed by palladium leads to the chemoselective Csp2-H insertion and the synthesis of oxindole type products. Although Pd(0) and Pd(II) catalysts are able to promote this transformation, Pd(0) catalysts such as Pd2(dba)3 or [IMes)Pd(NQ)]2 yield the best results. DFT calculations suggest that the insertion reaction involves a novel stepwise mechanism that implies a palladium mediated 1,5-hidrogen migration followed by reductive elimination that yields two products: the insertion product and the Pd(0) catalyst. Finally, we studied the palladium catalysed decomposition of α-diazo-α- (methoxycarbonyl) acetamides to promote intramolecular Csp3-H insertion to form β-lactams. In these transformations, though both Pd (0) and Pd(II) catalysts can be used, Pd(II) complexes were found to be the most versatile and efficient.
De entre todos los metales de transición utilizados en síntesis orgánica, el paladio ha ocupado un lugar central durante décadas y ha sido uno de los metales que más ha contribuido al desarrollo de este campo. En la presente Tesis Doctoral se han desarrollado diversas metodologías para la síntesis de heterociclos nitrogenados basados en procesos dominó catalizados por paladio. Por otro lado, los estudios experimentales se han combinado con estudios computacionales DFT para desentrañar el mecanismo implicado en estas transformaciones. En la primera parte de la Tesis Doctoral se ha llevado a cabo un extenso estudio de la reacción de α-arilación intramolecular de sulfonas catalizado por paladio. La combinación de la reacción de α-arilación con la subsiguiente adición conjugada a diversos aceptores de Michael, ha permitido el desarrollo de procesos dominó en 2, 3 y 4 etapas para la síntesis de tetrahidroisoquinolinas e indoles diversamente funcionalizados. Los estudios computacionales DFT de este proceso para la formación de indoles sugieren que la reacción de α-arilación transcurre mediante un proceso de desprotonación-metalación concertado (CMD) seguido de una eliminación reductora. H SO2R' SO2R' Pd(0) I base SO2R' SO2R' PhO2S SO2R' n= 0,1 N n Aza-Michael N -arilación N n n adición N R conjugada n=1 SO2R' SO2R' adición conjugada n=0 R'O2S SO2R' N -eliminación N R R A continuación, la reacción de α-arilación intramolecular catalizada por paladio se ha extendido a la utilización de sulfonatos, sulfonamidas y fosfonatos. Aunque los tres tipos de nucleófilos pueden utilizarse en la reacción de α-arilación para la preparación de tetrahidroisoquinolinas, la reacción transcurre de manera menos eficiente en relación a las sulfonas estudiadas. Asimismo, la versatilidad de los procesos dominó desarrollados a partir de ésteres sulfónicos y sulfonamidas fue menor. Por otro lado, los fosfonatos han resultado ser completamente inactivos en los diferentes procesos domino estudiados. La reacción de α-arilación de sulfonatos, sulfonamidas y ésteres sulfónicos también se ha estudiado computacionalmente. Estos estudios han puesto de manifiesto que la reacción transcurre por mecanismos distintos en función del nucleófilo y de la base utilizada. En la segunda parte de la Tesis, se ha estudiado la viabilidad de los complejos de paladio en reacciones de inserción de carbenos en enlaces C-H generados a partir de α-diazoacetamidas. La descomposición de las α-diazo-α-(metoxicarbonil) acetanilidas catalizadas por complejos de paladio ha dado lugar a la formación de oxindoles como resultado de una inserción quimioselectiva en el enlace Csp2-H. Aunque tanto los catalizadores de Pd(0) como los de Pd(II) pueden promover esta reacción de inserción, los mejores resultados se han obtenido al utilizar complejos de Pd(0), en especial Pd2(dba)3 o [(IMes)Pd(NQ)]2. Los cálculos computacionales DFT de este proceso sugieren que la inserción tiene lugar mediante un mecanismo por etapas inédito que implica una migración 1,5 de hidrógeno promovida por el paladio seguido de un proceso de eliminación reductora. Finalmente, también se ha estudiado la descomposición de α-diazo-α- (metoxicarbonil) acetamidas catalizada por paladio para promover inserciones Csp3-H y la formación de β-lactamas. Aunque tanto los catalizadores de Pd(0) como de Pd(II) son capaces de promover esta reacción, los catalizadores de Pd(II) son más eficientes y versátiles.

La comparaison de la nature de 1674 mutants obtenus dans plusieurs contextes genetiques et conditions physiologiques, a montre que la mobilite des sequences is n'etait pas constante au cours de la croissance bacterienne. En milieu riche, la transposition des is est totalement absente en phase de croissance exponentielle, et ne se produit qu'en phase stationnaire. Ce phenomene semble etre independant du facteur de transcription rpos, specifique de la phase stationnaire. Cependant, la transposition peut se produire en phase de croissance exponentielle dans un milieu ou la croissance est ralentie par des carences nutritionnelles. Nous avons aussi identifie une region chromosomique impliquee dans la transposition. La deletion de cette region permet la mobilisation d'is1 et d'is5 des la phase de croissance exponentielle, mais il n'a pas ete possible d'affecter ce phenomene a un gene unique de cette region. L'hypothese formulee a l'issue de ce travail est que la transposition de certaines is est favorisee, lorsque le bon fonctionnement cellulaire est altere. Des donnees recentes montrent par exemple que le dysfonctionnement du ribosome en fonction des ressources nutritives augmente la transposition d'is1.

Understanding how organisms adapt to their environment remains an open question in Biology. So far, most projects focus on the study of single nucleotide polymorphism variants, while other types of mutations, likely to play a role in adaptation, are largely ignored. Far instance, the effects of transposable elements (TE), which are potent mutagens that introduce genomic variability in natural populations sometimes driving phenotypic adaptations to environmental conditions, are not so well studied. The aim of this work was to characterize the ecological adaptive effects of the Drosophila melanogaster roo insertion FBti0019985, which is located in the 5'UTR of the transcription factor CG18446. To that end, we studied its possible phenotypic effects and investigated the molecular mechanisms behind those functional changes. First, we discovered that besides FBti0019985, the CG18446 prometer region harbours 19 independent roo insertions. The presence of these recurrent roo insertions in the CG18446 promoter region is likely to be the result of several bursts of transposition. We suggest that chromatin accessibility could be one of the factors explaining the multiple insertions. We also investigated whether the identified insertions were functionally equivalent by performing S'RACE, gene expression, and cold-stress survival experiments. We found that only FBti00 19985 was associated with CG18446 up-regulation in embryos and with increased viability in nonstress and under cold-stress conditions. Second, we further studied the molecular and phenotypic effects of FBti00 19985 in different developmental stages and under different stress conditions. Performíng gene expression analysis and in vivo enhancer reporter assays we found that FBti0019985 drives the expression of its nearby gene CG18446 depending on both the developmental stage and the environmental conditions. We associated the presence of FBti0019985 with CG18446 down­ regulation. However, in embryos under nonstress conditions and in guts under immune-stress, FBti0019985 caused a CG18446 up-regulation. lndeed, we associated this up-regulation with an enhancer activity of the element under these specific contexts. Finally, with the generation of mutant strains with FBti0019985 deletions in O. melanogaster natural populations using the CRISPR/Cas9-mediated homology-directed repair technique, we could also associate the presence of FBti0019985 with tolerance to P. entomophila infection. Overall, this work gives more evidences of the role of TEs in relevant adaptive traits . Moreover, it also reflects the importance of considering the effect of a candidate adaptive insertion under different contexts to fully characterize its adaptive consequences.
Entender como los organismos se adaptan a su ambiente es aún una pregunta abierta en biología. Hasta ahora, muchos proyectos se han centrado en el estudio de polimorfismos de nucleótidos simples {SNPs), mientras que otros tipos de mutaciones, que probablemente tengan un rol importante en la adaptación, han sido ignorados. Por ejemplo, los efectos de los transposones, que son potentes mutágenos que introducen variabilidad genómica en poblaciones naturales y, a veces, provocan adaptaciones fenotípicas al ambiente, no están tan bien estudiados. El objetivo de este trabajo ha sido caracterizar los efectos adaptativos del transposón de la familia roo FBti0019985, presente en Drosophila melanogaster. Este transposón está insertado en la región 5'UTR del factor de transcripción CG18446. Hemos estudiado sus posibles efectos fenotípicos y hemos investigado los mecanismos moleculares detrás de estos cambios funcionales. Primero, hemos descubierto que, a parte del transposón FBti0019985, la región promotora del gen CG18446 contiene 19 inserciones independientes de la familia roo. La presencia de estas inserciones recurrentes en la región promotora de CG18446 puede ser el resultado de varios estallidos de transposición. Sugerimos que la accesibilidad de la cromatina puede ser uno de los factores que explicarían las múltiples inserciones encontradas. También hemos investigado si los transposones identificados eran funcionalmente equivalentes haciendo análisis de expresión y experimentos de supervivencia bajo estrés por frío. Hemos encontrado que solamente el transposón FBti00 19985 está asociado con una sobreexpresión del gen CG18446 en embriones y con un incremento de la viabilidad en condiciones normales y de estrés por frío. Segundo, hemos estudiado en detalle los efectos moleculares y fenotípicos del transposón FBtiO0 19985 en diferentes estadios del desarrollo y bajo diferentes condiciones de estrés. Haciendo análisis de expresión y ensayos de potenciación in vivo, hemos encontrado que FBti0019985 potencia la expresión del gen CG18446 dependiendo del estadio embrionario y de las condiciones ambientales. Hemos asociado la presencia de FBti0019985 con una reducción de la expresión de CG18446. En cambio, en embriones en condiciones normales y en intestinos bajo estrés por infección, FBti0019985 causa una sobreexpresión de CG18446. De hecho, hemos asociado esta sobreexpresión con una actividad potenciadora del transposón en estos mismos contextos. Finalmente, con la generación de cepas mutantes sin FBti0019985 usando la técnica CRISPR/Cas9, también hemos podido asociar la presencia de este transposón con tolerancia a la infección con s. En general, este trabajo aporta más evidencias sobre el rol de los transposones en rasgos adaptativos. Además, también refleja la importancia de considerar el efecto de las inserciones candidatas de tener un rol adaptativo bajo diferentes contextos para caracterizar completamente sus consecuencias adaptativas.

Thesis advisor: Gabor T. Marth
During the last few years, numerous new sequencing technologies have emerged that require tools that can process large amounts of read data quickly and accurately. Regardless of the downstream methods used, reference-guided aligners are at the heart of all next-generation analysis studies. I have developed a general reference-guided aligner, MOSAIK, to support all current sequencing technologies (Roche 454, Illumina, Applied Biosystems SOLiD, Helicos, and Sanger capillary). The calibrated alignment qualities calculated by MOSAIK allow the user to fine-tune the alignment accuracy for a given study. MOSAIK is a highly configurable and easy-to-use suite of alignment tools that is used in hundreds of labs worldwide. MOSAIK is an integral part of our genetic variant discovery pipeline. From SNP and short-INDEL discovery to structural variation discovery, alignment accuracy is an essential requirement and enables our downstream analyses to provide accurate calls. In this thesis, I present three major studies that were formative during the development of MOSAIK and our analysis pipeline. In addition, I present a novel algorithm that identifies mobile element insertions (non-LTR retrotransposons) in the human genome using split-read alignments in MOSAIK. This algorithm has a low false discovery rate (4.4 %) and enabled our group to be the first to determine the number of mobile elements that differentially occur between any two individuals
Thesis (PhD) — Boston College, 2010
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

The application of controlled potential electrolysis for the elemental analysis by the inductively coupled plasma for atomic emission spectrometry using a direct sample insertion device will be described. The purpose of the technique is to improve the ICP detection limits over those obtained by conventional nebulization of liquid samples and to separate trace metallic elements from a complex matrix. Preconcentration and separation are achieved by electrodeposition using different electrode types. A conventional graphite electrode, a hanging mercury drop electrode, a reticulated vitreous carbon electrode and a tubular graphite electrode are studied. Advantages and limitations of the different electrodes and techniques will be discussed.

This study concerns with acoustical analysis of exhaust mufflers for earth-moving machinery. The study arises from the fact that there is a need for further noise reduction emitted by earth-moving machinery produced by Hidromek Inc. in order to be on the safe side of the limits stated in European Noise Directive 2000/14/EC. The acoustical performance of the muffler is investigated both experimental and numerical means. A three-dimensional finite element method is performed to calculate the transmission loss. An experimental setup is also developed to measure the transmission loss. Chung-Blaser, two-source and two-load methods are applied to measure the transmission loss of single expansion chamber with extended inlet/outlet. The experimental setup is verified by comparing the test results obtained by two-load method with one-dimensional analytical solution obtained by transfer matrix method. Transmission loss of the muffler of interest, calculated by finite element method is compared with the experimental results. Sound power level of earth-moving machinery and insertion loss of the muffler is measured to investigate the acoustical performance of the muffler. These results can then be served as guidance for the acoustical design of a muffler.

La perforación de roca es un proceso que se encuentra presente en la minería, la construcción y la extracción de hidrocarburos. Normalmente, para la perforación de roca, se utilizan taladros percusivos. Los taladros percusivos tienen dos componentes importantes: el elemento que genera el impacto, llamado martillo o pistón de impacto y el elemento que se encuentra en contacto con la roca, llamado indentador o bit que aloja el elemento perforante llamado inserto. Se presenta el diseño de un equipo de laboratorio que permite al investigador estudiar el daño en una muestra de roca utilizando distintos tipos de insertos (ya sea cónicos o balísticos de distintos diámetros), distintas configuraciones geométricas de los insertos en un taladro percusivo, así como distintos ángulos de barrido. El equipo de laboratorio consiste en un cilindro neumático cuyo pistón (martillo) se acelera hasta una velocidad de 5 m/s. Dicho pistón impacta contra el indentador, transfiriendo su energía a través del indentador hasta una muestra de roca. En el extremo del indentador se encuentran los insertos de carburo de tungsteno, quienes se encargan de perforar la roca. El peso combinado del equipo es aproximadamente 50 kg. El equipo fue diseñado de tal manera que sea posible controlar el ángulo de barrido por medio de un sistema de giro que utiliza un motor DC con escobillas con un torque nominal de 1,6 Nm y que consume una potencia de 0,48 W. El equipo, además, cuenta con distintos tipos de sensores para registrar datos relevantes a la investigación. La velocidad del pistón de impacto se mide por medio de dos sensores inductivos separados por una distancia conocida de 24 mm. El indentador cuenta con sensores extensiométricos que miden la deformación del elemento. La roca cuenta con un acelerómetro para registrar la energía recibida. El ensayo se encuentra semi-automatizado, el control está a cargo de un PLC en comunicación con una PC, donde se registra la velocidad de impacto, las deformaciones del indentador y la roca. El PLC gobierna el funcionamiento secuencial del ensayo, activando y desactivando las electroválvulas correspondientes. El costo aproximado (sin considerar los costos de anclaje) es de S./ 25000. Los dos conceptos de mayor costo son la fabricación de los elementos y los sensores utilizados.
Tesis

The work and results presented in this dissertation concern two complimentary studies that are rooted in surface acoustic waves and transducer study. Surface acoustic wave devices are utilized in a variety of fields that span biomedical applications to radio wave transmitters and receivers. Of interest in this dissertation is the study of surface acoustic wave interaction with polydimethylsiloxane. This material, commonly known as PDMS, is widely used in the microfluidic field applications in order to create channels for fluid flow on the surface of a piezoelectric substrate. The size, and type of PDMS that is created and ultimately etched on the surface of the substrate, plays a significant role in its operation, chiefly in the insertion loss levels experienced. Here, through finite element analysis, via ANSYS® 15 Finite Element Modeling software, the insertion loss levels of varying PDMS sidewall channel dimensions, from two to eight millimeters is investigated. The simulation is modeled after previously published experimental data, and the results demonstrate a clear increase in insertion loss levels with an increase in channel sidewall dimensions. Analysis of the results further show that due to the viscoelastic nature of PDMS, there is a non -linear increase of insertion loss as the sidewall dimensions thicken. There is a calculated variation of 8.31 decibels between the insertion loss created in a microfluidic device with a PDMS channel sidewall thickness of eight millimeters verse a thickness of two millimeters. Finally, examination of the results show that insertion loss levels in a device are optimized when the PDMS sidewall channels are between two and four millimeters. The second portion of this dissertation concerns the calibration of an ultrasonic transducer. This work is inspired by the need to properly quantify the signal generated by an ultrasonic transducer, placed under a static loading condition, that will be used in measuring ultrasonic bone conducted frequency perception of human subjects. Ultrasonic perception, classified as perception beyond the typical hearing limit of approximately 20 kHz, is a subject of great interest in audiology. Among other reasons, ultrasonic signal perception in humans is of interest because the mechanism by which either the brain or the ear interprets these signals is not entirely understood. Previous studies have utilized ultrasonic transducers in order to study this ultrasonic perception; however, the calibration methods taken, were either incomplete or did not properly account for the operation conditions of the transducers. A novel transducer calibration method is detailed in this dissertation that resolves this issue and provides a reliable means by which the signal that is being created can be compared to the perception of human subjects.

Boundary-value problem for elliptical equation with conjugate conditions of non-ideal contact is considered. Computing algorithms of the increased accuracy order of sampling for the problem under consideration basing on the finite element methods are constructed. The solution results of the models examples are indicated.
Розглянуто задачу, що описується еліптичним рівнянням з умовами спряження неідеального контакту та різними крайовими умовами. На основі методу скінчених елементів (МСЕ) побудовані обчислювальні алгоритми підвищеного порядку точності дискретизації задачі. Отримано результати розв’язку модельного прикладу.

1S801 is a transposable element isolated from Pseudomonas syringae pathovar (pv.) phaseolicola, the causal agent of halo blight of bean. Fragments of the element are present in multiple copies on an indigenous plasmid, pMMC7105, of strain LR781, and have been implicated as sites of homologous recombination leading to imprecise excision of the chromosomally integrated form of the plasmid. The element, which has been completely sequenced, is 1512 base pairs in length and is unusual among transposable elements in that it does not have direct or inverted repeats in its termini. The terminal regions of the element were uncoupled from the two major open reading frames, and trans-acting activity of the putative transposase was demonstrated in Escherichia coli (recA). An alignment of the sequences of thirteen insertions defined the precise borders of the element, and demonstrated that it does not duplicate its targets upon insertion. The target specificity of IS801 is similar to, but more degenerate than, the target specificity of two transposable elements to which it is closely related, IS91 and IS1294. The consensus derived from the aligned target sequences is G/C-A/G-A-C/G, and the target tetramer is found immediately adjacent to the right terminus of the element upon transposition. IS91 was demonstrated to mobilize 1S801, but not with the specificity characteristic of 1S801. The structure of 1S801 and the characteristics of IS91-activated transposition of 1S801 are discussed in light of a proposed model for IS91 transposition, and it is suggested that 1S801 could have been derived from IS91 by a modification of its left end. Remnants of IS801 are present near avirulence genes of various P. syringae pathovars, suggesting that the element has been involved in genetic rearrangements in the vicinity of these loci.
Graduation date: 1996