Le gène ALK code pour un récepteur à activité tyrosine kinase exprimé principalement dans le système nerveux central et périphérique au cours du développement chez le mammifère. Ces informations font suspecter un rôle développemental du récepteur ALK bien que ses fonctions précises ne sont pas connues. De la même manière, ses ligands et les voies de signalisation qui lui sont associés demeurent mal caractérisés. En 2008, des mutations ponctuelles du gène ALK ont été identifiées dans des formes sporadiques et familiales de neuroblastome. Deux hotspots de mutation sont situés dans le domaine tyrosine kinase. Ces travaux de thèse ont permis d’étudier l’implication du gène ALK dans l’oncogenèse du neuroblastome sous des aspects cellulaires, génomiques et fonctionnels. L’étude de la régulation de l’activation et de l’adressage de la protéine ALK a révélé qu’à la différence des récepteurs sauvages, l’activation constitutive conduit à une rétention intracellulaire associée à une maturation incomplète des récepteurs mutés. Par ailleurs, la caractérisation complète du locus ALK dans le neuroblastome a mis en évidence une fréquence élevée de réarrangements chromosomiques. Ce travail a notamment identifié un réarrangement chromosomique conduisant à l’expression d’un variant du récepteur ALK tronqué pour une partie du domaine extracellulaire. Ce variant, associé à l’agressivité tumorale, est activé de façon constitutive et présente des propriétés oncogéniques. Enfin, l’analyse de souris knock-in exprimant les mutations AlkF1178L et AlkR1279Q a révélé un rôle majeur du gène Alk dans le développement du système nerveux sympathique. En effet, ces souris présentent une hyperplasie des ganglions sympathiques mise en place chez l’embryon et associée à une augmentation de la prolifération cellulaire à la naissance. Ce travail a aussi mis en évidence la coopération oncogénique in vivo entre les mutations du gène Alk et la surexpression du gène MYCN dans le neuroblastome. Les tumeurs générées chez les souris partageant ces deux altérations se développent après une courte période de latence et avec une pénétrance complète. L’analyse des tumeurs a mis en évidence des propriétés conférées aux tumeurs par les mutations du gène Alk. En effet, ces tumeurs expriment des marqueurs cholinergiques et montrent des signes de différenciation. Ces animaux permettent de mieux comprendre le rôle du gène ALK dans l’oncogenèse du neuroblastome et constituent de bons modèles d’étude préclinique des neuroblastomes dépendants de ALK. / The ALK protein is a receptor tyrosine kinase mainly expressed in the central and peripheral nervous system during the development in mammals. These informations suggest that the ALK receptor might have a developmental role even though its functions remain unknown. Ligands and signaling pathways associated to this receptor are also poorly characterized. In 2008, point mutations of the ALK gene have been identified in sporadic and familial cases of neuroblastoma with two main hotspots of mutation in the kinase domain. This thesis project allowed to study the involvement of the ALK gene in neuroblastoma oncogenesis under cellular, genomic and functional aspects.The study of ALK protein activation and subcellular localization revealed that the constitutive activation of the kinase domain lead to an intracellular retention and to a lack of glycosylation. The genomic characterization of the ALK locus pointed out the high frequency of chromosomal rearrangements in neuroblastoma cell lines. This work notably identified the expression of a chromosomal rearrangement leading to a truncated protein variant of ALK lacking a part of the extracellular domain. This variant, associated with the tumoral aggressiveness, is constitutively activated and harbor oncogenic properties. Finally, the analysis of the knock-in mice expressing the two AlkF1178L and AlkR1279Q mutations demonstrated that the ALK gene has a key role in the regulation of the developing sympathetic nervous system. Indeed, these mice have an hyperplasia of sympathetic ganglia set up during embryogenesis and associated with an increased neuroblast proliferation at birth. This work also described the oncogenic cooperation between ALK mutations and MYCN overexpression in neuroblastoma. Tumors, generated in mice sharing those two alterations, arise with a full penetrance and a short latency. Histological and transcriptomic analysis of tumors identified specific properties provided by ALK mutations. Indeed, these tumors express cholinergic markers and harbor signs of differentiation. Those animals allow to better understand the role of the ALK gene in neuroblastoma oncogenesis and constitute good preclinical models for ALK dependant neuroblastoma.

Orientador: Maria de Lourdes Morales Horiguela / Banca: Beatriz Bojikian Matsubara / Banca: Ivan de Melo Araújo / Banca: Paulo Sérgio Teixeira do Prado / Banca: Tânia Moron Saes Braga / Resumo: Este trabalho avalia e compara os alunos do sexto ano de medicina da Faculdade de Medicina de Marília em relação ao estágio de terapia intensiva. Para execução do trabalho, os alunos foram divididos em dois grupos, controle e experimental, e a análise dos ganhos em relação ao estágio foram feitas intra e intergrupos. Os objetivos deste trabalho são avaliar e comparar os ganhos teóricos e práticos de cada grupo antes e após o estágio de terapia intensiva, e comparar os dois grupos entre si após a inserção de treinamento em atividades práticas programadas e inseridas no grupo experimental. Usou-se um questionário fechado, aplicado aos alunos antes e apos o estágio de terapia intensiva, em ambos os grupos, para avaliar os ganhos teóricos e práticos em relação ao estágio. No grupo experimental foram inseridas atividades práticas para treinamento dessas habilidades durante o estágio de UTI. Essas atividades práticas foram escolhidas de acordo com a literatura que apontava as necessidades de formação prática para os graduandos em medicina, elaboradas e aplicadas com base no rearranjo de contingências, de acordo com a análise do comportamento, que foi o método didático usado. O treinamento dos alunos foi realizado pela pesquisadora docente especialista na área de terapia intensiva, que supervisionou o trabalho diretamente. A análise estatística recebeu tratamento especial para cada comparação e cada grupo de questões. Os dados foram descritos utilizando-se medidas de freqüência absoluta (n) quando a comparação era feita no mesmo grupo entre fases (Pré x Pós- estágio - intra-grupo) utilizando o teste de Wilcoxon para escala ordinal e o teste de McNemar quando escala nominal, e utilizou medidas relativas (%) quando a comparação foi feita entre grupos independentes (Controle x Experimental - intergrupos) usando o teste de Mann-Whitney para... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study evaluates and compares the students of the sixth year of medicine, School of Marília Medicine, in relation to the stage of intensive care. To undertake the work, students were divides into two groups, control and experimental, and analysis of earnings in relation to the stage were made within and intergroup. The objectives of this study are assessed and compared the theoretical and practical gains of each group before and after the stage of intensive care, and compare the two groups among themselves, after the insertion of training in practical activities planned and included in the experimental group. The study was divided into two phases, first with the control group and the second phase with the experimental group. A closed questionnaire was applied to students before and after the stage of intensive care, in both groups, to assess the theoretical and practical gains on the stage. In the experimental group were included practical activities for training of these skills during the stage of ICU. These practical activities were chosen according to literature that emphasized the need for practical training for graduate students in medicine, developed and implemented based on the rearrangement of contingencies, according to the analysis of behavior, which was the teaching method used. The training of students was conducted by the researcher teacher expert in the field of intensive care, which oversaw the work directly. Statistical analysis has received special treatment compared to each and every group of issues. The data were described using the measures of absolute frequency (n) when the comparison was made between the same group stages (Pre x Post-stage - intra-group) using the Wilcoxon test for ordinal scale and McNemar test when nominal scale was used, and used measures on percent (%) when the comparison was made between independent groups... (Complete abstract click electronic access below) / Doutor

The present work deals with m-th order compact Sobolev embeddings on a do- main Ω ⊆ Rn endowed with a probability measure ν and satisfying certain isoperi- metric inequality. We derive a condition on a pair of rearrangement-invariant spaces X(Ω, ν) and Y (Ω, ν) which suffices to guarantee a compact embedding of the Sobolev space V m X(Ω, ν) into Y (Ω, ν). The condition is given in terms of compactness of certain operator on representation spaces. This result is then applied to characterize higher-order compact Sobolev embeddings on concrete measure spaces, including John domains, Maz'ya classes of Euclidean domains and product probability spaces, among them the Gauss space is the most stan- dard example. 1

Asymmetric synthesis and catalysis is one of the leading research areas in chemistry society, for its versatility and efficiency in obtaining chiral molecules that found the vast majority in natural active compounds and synthetic drugs. Developing asymmetric catalytic methodology is at the frontier in both industrial and academic research laboratories. Enantioselective organocatalysis has emerged as a powerful synthetic tool that is complementary to metal-catalyzed transformations. The development of chiral phosphoric acid and metal phosphate as catalysts has been a breakthrough in recent years. Chiral phosphoric acids have been shown to be powerful catalysts in many organic transformations. Moreover, chiral metal phosphates, which formed by simply replacing the proton in phosphoric acid with metals, have introduced new catalytic activations and broaden the scope of phosphoric acids. This thesis details new highly enantioselective chiral phosphoric acid-catalyzed Pinacol rearrangement and robust alkaline phosphates catalytic system, which utilizes novel carbonyl activation. The Pinacol rearrangement has long been known to be difficult to control in terms of regioselectivity and stereoselectivity. The initial studies found that indolyl-diol compounds can be treated with chiral phosphoric acids to afford the Pinacol rearrangement with high regio- and enantioselectivity. Over 16 chiral phosphoric acids were screened, and it was found an H8-BINOL-phosphoric acid variant with 1-naphthyl groups at 3 and 3' position was the excellent catalyst. This asymmetric transformation is tolerant toward variety of substituents both on the indole ring and migrating groups. During the study, it was found that different ways to generate the catalyst had critical effect on this catalytic transformation. Only those phosphoric acids washed with HCl after column chromatography afforded the rearrangement products with high enantioselectivity. And those without treating with HCl were found contaminated by alkaline metals. These "contamination" catalysts were also found active with carbonyl activations. A highly enantioselective catalytic hetero-Diels-Alder reaction of alpha-keto esters has been developed with chiral alkaline metal phosphates. A calcium 1-naphthyl-BINOL phosphate was found to be the optimum catalyst. A large range of alpha-keto esters as well as isatins can be applied in this alkaline phosphates catalytic system with high efficiency and selectivity. The structure of the catalyst is detailed for the first time by X-ray crystal structure analysis. A proposed Transition state model is provided based on the catalyst crystal structure and Raman spectroscopy analysis. This methodology was further developed with an asymmetric Mukaiyama-Michael addition of beta,gamma-unsaturated alpha-keto ester. The best catalyst was found to be a magnesium chiral phosphate. And the transformation was found capable of tolerating a wide variety of beta,gamma-unsaturated alpha-keto esters.

Dans un premier chapitre, nous nous intéressons à l'utilisation des Carbènes N-Hétérocycliques comme catalyseurs organiques nucléophiles pour la polymérisation par ouverture de cycle de l'octamethylcyclosiloxane et pour des réactions de polycondensation.<br />Dans un deuxième chapitre, nous effectuons une revue bibiliographique sur des composés phosphorés présentant deux fonctions ylures cumulées : les carbodiphosphoranes. <br />Dans le troisième chapitre, nous présentons la synthèse et la coordination de nouveaux carbodiphosphoranes cycliques. Nous montrons que ces ligands ont un caractère fortement donneur.<br />Dans le quatrième chapitre, nous étudions la thermolyse d'un de ces composés qui conduit par réarrangement à la formation deun 1.2 5-azaphosphète. Une étude théorique met en évidence une inter-conversion entre les carbodiphosphoranes et les disphosphinocarbènes.

<p>Malt lymphoma is a malignant disease that can arise in a variety of extra nodal sites. Previous studies indicate that tumour arise from more mature B-cells.</p><p>Our purpose was to examine the presence of clonality and somatic hypermutation of immunoglobulin (IgVн) of MALT lymphomas.</p><p>Paraffin-embedded tumour samples from13 MALT lymphoma were subjected to rearrangement analysis, by using PCR, heteroduplex gels and sequence analysis.</p><p>Successful amplification was seen in 10/13 cases and sequences of IgVн genes were obtained in 6/13, all of them were mutated. The percentage of mutation compared to germline sequences was 1,1% to 8,6% monoclonal rearrangemang. It was demonstrated that 5 of 7 clones were derived from the Vн3 family, 2 from Vн1 and 1 from the Vн 4 family.</p>

The process of evolution is of both scientific and medical interest. This thesis presents several studies using complete genomic reference sequences, comparative genomic data, and intraspecific diversity data to study the two key processes of evolution: mutation and selection. Large duplications, deletions, inversions, and translocations of DNA contribute to genomic variation both between and within species. Human chromosomes 15 and 17 contain a high percentage of dispersed, recently duplicated sequences. Examination of the relationships between these sequences showed that the majority of all duplications within each chromosome could be linked through core sequences that are prone to duplication. Comparison to orthologous sequences in other mammals allowed a reconstruction of the ancestral state of the human chromosomes, revealing that regions of rearrangement specific to the human lineage are highly enriched in chromosome-specific duplications. Comparison to copy number variation data from other studies also shows that these regions are enriched in current human structural variation. One specific region, the MAPT locus at 17q21.31, known to contain an inversion polymorphism in Europeans, was resequenced completely across both human orientation haplotypes and in chimpanzee and orangutan, revealing complex duplication structures at the inversion breakpoints, with the human region being more complex than chimpanzee or orangutan. Fluorescent in-situ hybridization analysis of human, chimpanzee, and orangutan chromosomes showed inversion polymorphisms of independent origin in all three species, demonstrating that this region has been a hotspot of genomic rearrangement for at least twelve million years. These results reveal a mechanistic relationship between sequence duplication and rearrangement in the great apes. We also generated a draft sequence of the chimpanzee genome and compared it to that of the human. Among other findings, this showed that CpG dinucleotides contribute 25% of all single base mutations, with a rate of mutation ~10-fold that of other bases, and that the male mutation rate in great apes is ~5-6 times the female rate, a higher ratio than had been observed in comparisons of primates and rodents. We detected six regions of probable recent positive selection in humans with a statistical method relying on chimpanzee sequence to control for regional variation in mutation rates. Finally, resequencing of several lines of domestic chicken and comparison to the reference chicken genome identified a number of gene deletions fixed in domestic lines and also several potential selective sweeps. Of particular interest are a missense mutation in TSHR nearly fixed in all domestic chickens and a partial deletion of SH3RF2 fixed in a high growth line. The TSHR mutation may play a role in relaxation of seasonal reproduction. A high-resolution QTL mapping experiment showed that the SH3RF2 deletion is significantly associated with increased growth. This work provides important new insights into the mechanics of evolutionary change at both the single nucleotide and structural level and identifies potential targets of natural and artificial selection in humans and chickens.

Despite major advances in our understanding of the biology of childhood acute myeloid leukemia (AML) and the development of new cytotoxic drugs, the prognosis of long-term survival is still only 60-65 %. In the present research, we studied the pharmacokinetics of drugs used in the induction therapy of childhood AML and performed in vitro drug sensitivity testing of leukemic cells from children with AML. The aims of the studies were to correlate the results of the analysis to biological and clinical parameters and to identify subgroups of AML with specific drug sensitivity profiles in order to better understand why treatment fails in some patients and how therapy may be improved. Blood samples were analysed to study the pharmacokinetics of doxorubicin (n=41), etoposide (n=45) and 6-thioguanine (n=50). Doxorubicin plasma concentration and total body clearance were correlated to the effect of induction therapy, and doxorubicin plasma concentration was an independent factor for complete remission, both in univariate and multivariate analysis including sex, age, and white blood cell count at diagnosis. For etoposide and 6-thioguanine no correlation was found between pharmacokinetics and clinical effect. Children with Down syndrome (DS) tended to reach higher blood concentrations of etoposide and thioguanine nucleotides, indicating that dose reduction may be reasonable to reach the same drug exposure as in children without DS. Leukemic cells from 201 children with newly diagnosed AML, 15 of whom had DS, were successfully analysed for in vitro drug sensitivity by the fluorometric microculture cytotoxicity assay (FMCA). We found that samples from children with DS were highly sensitive to most drugs used in AML treatment. In non-DS children, the t(9;11) samples were significantly more sensitive to cytarabine (p=0.03) and doxorubicin (p=0.035) than other samples. The findings might explain the very favorable outcome reported in children with DS and t(9;11)-positive AML. A specific drug resistance profile was found for several other genetic subgroups as well. A detailed study of MLL-rearranged leukemia showed that cellular drug sensitivity is correlated both to partner genes and cell lineage, findings that support the strategy of contemporary protocols to include high-dose cytarabine in the treatment of patients with MLL-rearrangement, both in AML and acute lymphoblastic leukemia (ALL). Our results indicate that drug resistance and pharmacokinetic studies may yield important information regarding drug response in different sub-groups of childhood AML, helping us to optimize future chemotherapy in childhood AML.

Microwave-Assisted Thermolysis of ortho-Substituted Aroylsilanes Marc Tremblay Master of Science Department of Chemistry University of Toronto 2008 The microwave-assisted thermolysis of ortho-substituted aroylsilanes has been investigated. When irradiated at 250ºC in DMSO or o‑dichlorobenzene for 10 minutes, aroylsilanes form siloxycarbenes that react following different pathways depending on the solvent and the structure of the starting material. It is shown that in the case of substrates having an O‑allyl or an O‑propargyl chain ortho to the acylsilane, cycloaddition occurs followed by a cascade ring opening to give respectively chroman‑4-one and chromen‑4-one derivatives in up to 66% yield. Among the major competitive pathways were the insertion of the siloxycarbene into allylic C–H bonds and decomposition of the acylsilane group to the corresponding aldehyde, followed by Claisen rearrangement.

Microwave-Assisted Thermolysis of ortho-Substituted Aroylsilanes Marc Tremblay Master of Science Department of Chemistry University of Toronto 2008 The microwave-assisted thermolysis of ortho-substituted aroylsilanes has been investigated. When irradiated at 250ºC in DMSO or o‑dichlorobenzene for 10 minutes, aroylsilanes form siloxycarbenes that react following different pathways depending on the solvent and the structure of the starting material. It is shown that in the case of substrates having an O‑allyl or an O‑propargyl chain ortho to the acylsilane, cycloaddition occurs followed by a cascade ring opening to give respectively chroman‑4-one and chromen‑4-one derivatives in up to 66% yield. Among the major competitive pathways were the insertion of the siloxycarbene into allylic C–H bonds and decomposition of the acylsilane group to the corresponding aldehyde, followed by Claisen rearrangement.