The aim of this Ph.D. thesis was to analyze the morphine-induced changes of frontal brain cortex protein composition in rats exposed to increasing doses of morphine (10-50 mg/kg) for prolonged period of time (10 days). The first part of this work was oriented to the analysis of the phenomenon of hypersensitization/superactivation of adenylyl cyclase (AC), which is regarded as one of the crucial molecular mechanisms causing drastic pathological consequences of drug addiction. The increase of AC activity represents a "compensatory" response and is functionally related to the desensitization of G protein response to prolonged morphine exposure of target cells. The clear desensitization of µ-OR- and δ-OR-stimulated G protein response by morphine was demonstrated in our laboratory by analysis of the dose-response curves of DAMGO and DADLE-stimulated, high-affinity [35 S] GTPγS binding in plasma membranes isolated from frontal brain cortex of rats exposed to morphine according to the same protocol as that used in my Ph.D. thesis (10-50 mg/kg, 10 days). The κ-OR-stimulated [35 S] GTPγS binding was unchanged. It has been determined the amount of all AC isoforms (AC I-IX) in plasma membranes (PM) isolated from control and morphine-treated rats which were sacrificed 24 hours since the last dose of morphine....

La vaccination muqueuse est un moyen efficace de lutter contre les pathogènes qui utilisent les muqueuses comme porte d’entrée. Cependant, la vaccination muqueuse avec des antigènes non réplicatifs nécessite l’utilisation d’adjuvants. Les molécules de la famille de la toxine du choléra, l’entérotoxine thermolabile d’E.coli (LT), la toxine du choléra (CT) ainsi que le mutant LT-R192G et les sous-unités B non toxiques de ces toxines (LTB et CTB) ont été montrées augmenter les réponses immunitaires contre des antigènes coadministrés par voie muqueuse. Cependant leur mécanisme d’action est complexe et reste encore mal connu et des différences entre molécules entières et sous-unités B ont été rapportées ainsi que, pour une même molécule, des différences selon le modèle utilisé. Dans ce travail, nous avons étudié les effets de ces cinq molécules sur les réponses anticorps ainsi que sur les lymphocytes T CD4 dans un modèle murin d’immunisation intrarectale avec des pseudoparticules virales de rotavirus (VLP-2/6). Chez les souris non immunisées, nous avons montré que ces molécules, à l’exception de la CTB, diminuent in vitro les lymphocytes T régulateurs naturels CD4+CD25+Foxp3+, probablement par un mécanisme d’apoptose. Chez les souris immunisées, toutes les molécules étudiées induisent une même réponse anticorps sérique et fécale spécifique des VLP-2/6, qu’il s’agisse des molécules entières connues pour leur fort pouvoir adjuvant ou des sous-unités B qui, elles, ont été rapportées avoir un plus faible effet adjuvant voire un effet tolérogène dans certaines études. Concernant la réponse T CD4, les réponses spécifiques de l’antigène et de l’adjuvant ont été analysées. Des différences importantes ont été mises en évidence entre ces molécules. Notamment, seules les molécules entières (LT, LT-R192G et CT) induisent la production d’IL-2 et l’activation de lymphocytes T CD4+CD25+Foxp3- mémoires spécifiques de l’antigène tout en permettant la mise en place d’une régulation médiée par des lymphocytes T régulateurs CD4+CD25+Foxp3+ (boucle d’autorégulation), qui pourraient jouer un rôle majeur lors d’une réponse secondaire, afin d’éviter les réactions inflammatoires délétères. Malgré ces différences, toutes les molécules étudiées induisent la production d’IL-17, suggérant le rôle majeur de cette cytokine dans l’effet adjuvant.L’influence de la voie d’administration sur ces effets est en cours d’étude grâce à la comparaison avec la voie intranasale / Mucosal immunization is an important goal of vaccine development to protect against pathogens that use mucosa as portals of entry. However, the use of non-replicating antigens requires the addition of adjuvants.Cholera-like enterotoxins, cholera toxin (CT) from Vibrio cholerae and the heat-labile enterotoxin (LT) from toxinogenic strains of E. coli, as well as the mutant LR-192G and their B subunits (CTB and LTB) have been shown to increase immune responses against unrelated co-administered antigens by mucosal routes. However, their mechanism of action is very complex and not completely understood and differences exist between holotoxins and B subunits and within molecules, differences exist between the models used.In this work, we have studied the effects of these five molecules on antibody responses and on CD4+ T cell responses in a murine model of intrarectal immunization using rotavirus-like particles (2/6-VLP). In non-immunized mice, we have shown that all molecules, except CTB, decreased CD4+CD25+Foxp3+ natural regulatory T cells, probably by induction of apoptosis.In immunized mice, all molecules induced similar VLP-2/6 specific systemic and fecal antibody responses, teither he holotoxins, which are well known for their strong adjuvanticity or their B subunits with a less strong adjuvanticity but with also a tolerogenic effect in some studies.Regarding the CD4+ T cell response, antigen- and adjuvant- specific responses have been analysed. Important differences have been highlighted between the molecules. Among others things, only whole toxins (LT, LT-R192G and CT) trigger IL-2 production and activation of antigen specific memory CD4+CD25+Foxp3- T cells and at the same time antigen specific CD4+CD25+Foxp3+ regulatory T cells are activated which may control the effector response (Feedback loop regulation) and avoid deleterious inflammation. In spite of these differences, all studied molecules triggered IL-17 production, suggesting the major role of this cytokine in adjuvanticity. We are currently comparing the intrarectal and intranasl routes in order to evaluate the role played by the route of immunisation in different effects of these molecules

Introdução: A grande variabilidade individual em resposta a fármacos antitabagismo sugere que tratamentos específicos podem ser mais efetivos em determinados subgrupos de fumantes. No contexto de medicina personalizada, o principal objetivo do presente estudo foi avaliar se polimorfismos nos genes CHRNA4, CHRNB2, CYP2B6 e ANKK1 estão associados com a resposta às terapias de cessação tabágica em pacientes provenientes de um programa de assistência ao fumante. Métodos: Estudo de coorte com 483 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona em monoterapia ou coadministrada com terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. Os polimorfismos CHRNA4 (rs1044396 e rs2236196), CHRNB2 (rs2072660 e rs2072661) e ANKK1 (rs1800497) foram genotipados pela análise da curva de melting e os polimorfismos CYP2B6 *9 (rs3745274), *4 (rs2279343), *5 (rs3211371) foram genotipados por restrição enzimática. Resultados: Os pacientes com o genótipo CC para o polimorfismo CHRNA4 (rs10443196) obtiveram menor taxa de sucesso no tratamento com vareniclina (29,5%) em comparação com os portadores dos genótipos CT ou TT (50,9%) (P=0,007; n=167). Os genótipos CT ou TT foram associados com maior odds ratio para o sucesso (OR=1,67; IC 95%=1,10-2,53; P=0,02), em um modelo multivariado. Os pacientes com o genótipo AA para o polimorfismo CYP2B6 (rs2279343) obtiveram maior taxa de sucesso no tratamento com bupropiona (48,0%) em comparação com portadores dos genótipos AG ou GG (35,5%) (P=0,05; n=237). O genótipo AA foi associado com maior odds ratio para o sucesso no tratamento (OR=1,92; IC 95%=1,08-3,42; P=0,03), em um modelo multivariado. Não foram observadas diferenças significativas nos escores FTND e Issa com relação aos polimorfismos estudados. Conclusão: Os polimorfismos CHRNA4 (rs1044396) e CYP2B6 (rs2279343) estão associados com a cessação tabágica em indivíduos tratados com vareniclina e bupropiona, respectivamente. Sugere-se que estes polimorfismos influenciam a resposta farmacológica e podem ser importantes para o desenho de uma farmacoterapia individualizada / Background: The large individual variability in response to drugs for smoking cessation suggests that specific treatments can be more effective in particular subgroups of smokers. In the context of personalized medicine, the main aim of the present study was to evaluate whether the CHRNA4, CHRNB2, CYP2B6 and ANKK1 polymorphisms are associated with response to smoking cessation therapies in patients from a smoker assistance program. Methods: This cohort study enrolled 483 smoking patients patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion in monoterapy or plus nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. The CHRNA4 (rs1044396 and rs2236196), CHRNB2 (rs2072660 and rs2072661) and ANKK1 rs1800497 polymorphisms were genotyped by high resolution melting analysis and the CYP2B6 *9 (rs3745274), *4 (rs2279343) and *5 (rs3211371) were genotyped by restriction fragment lenght polymorphisms. Results: Patients with CHRNA4 rs1044396 CC genotype had lower success rate in treatment with varenicline (29.5%) compared with carriers of CT or TT genotypes (50.9%) (P=0.007, n=167). The CT or TT genotypes were associated with higher odds ratio for success (OR=1.67, 95%CI=1.10-2.53, P=0.02), in a multivariate model. Patients with CYP2B6 rs2279343 AA genotype had higher success rate in treatment with bupropion (48.0%) compared with carriers of AG or GG genotypes (35.5%) (P=0.05, n=237). The AA genotype was associated with higher odds ratio for success (OR=1.92, 95%CI=1.08-3.42, P=0.03), in a multivariate model. We did not observe significant differences in the FTND and Issa scores according to the studied polymorphisms. Conclusion: The CHRNA4 rs1044396 and CYP2B6 rs2279343 are associated with smoking cessation in individuals on varenicline and bupropion terapies, respectively. We suggest that these polymorphisms influence the pharmacological response of these drugs and it might be important in the design of individualized pharmacotherapy

Introdução: A grande variabilidade individual em resposta a fármacos antitabagismo sugere que tratamentos específicos podem ser mais efetivos em determinados subgrupos de fumantes. No contexto de medicina personalizada, o principal objetivo do presente estudo foi avaliar se polimorfismos nos genes CHRNA4, CHRNB2, CYP2B6 e ANKK1 estão associados com a resposta às terapias de cessação tabágica em pacientes provenientes de um programa de assistência ao fumante. Métodos: Estudo de coorte com 483 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona em monoterapia ou coadministrada com terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. Os polimorfismos CHRNA4 (rs1044396 e rs2236196), CHRNB2 (rs2072660 e rs2072661) e ANKK1 (rs1800497) foram genotipados pela análise da curva de melting e os polimorfismos CYP2B6 *9 (rs3745274), *4 (rs2279343), *5 (rs3211371) foram genotipados por restrição enzimática. Resultados: Os pacientes com o genótipo CC para o polimorfismo CHRNA4 (rs10443196) obtiveram menor taxa de sucesso no tratamento com vareniclina (29,5%) em comparação com os portadores dos genótipos CT ou TT (50,9%) (P=0,007; n=167). Os genótipos CT ou TT foram associados com maior odds ratio para o sucesso (OR=1,67; IC 95%=1,10-2,53; P=0,02), em um modelo multivariado. Os pacientes com o genótipo AA para o polimorfismo CYP2B6 (rs2279343) obtiveram maior taxa de sucesso no tratamento com bupropiona (48,0%) em comparação com portadores dos genótipos AG ou GG (35,5%) (P=0,05; n=237). O genótipo AA foi associado com maior odds ratio para o sucesso no tratamento (OR=1,92; IC 95%=1,08-3,42; P=0,03), em um modelo multivariado. Não foram observadas diferenças significativas nos escores FTND e Issa com relação aos polimorfismos estudados. Conclusão: Os polimorfismos CHRNA4 (rs1044396) e CYP2B6 (rs2279343) estão associados com a cessação tabágica em indivíduos tratados com vareniclina e bupropiona, respectivamente. Sugere-se que estes polimorfismos influenciam a resposta farmacológica e podem ser importantes para o desenho de uma farmacoterapia individualizada / Background: The large individual variability in response to drugs for smoking cessation suggests that specific treatments can be more effective in particular subgroups of smokers. In the context of personalized medicine, the main aim of the present study was to evaluate whether the CHRNA4, CHRNB2, CYP2B6 and ANKK1 polymorphisms are associated with response to smoking cessation therapies in patients from a smoker assistance program. Methods: This cohort study enrolled 483 smoking patients patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion in monoterapy or plus nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. The CHRNA4 (rs1044396 and rs2236196), CHRNB2 (rs2072660 and rs2072661) and ANKK1 rs1800497 polymorphisms were genotyped by high resolution melting analysis and the CYP2B6 *9 (rs3745274), *4 (rs2279343) and *5 (rs3211371) were genotyped by restriction fragment lenght polymorphisms. Results: Patients with CHRNA4 rs1044396 CC genotype had lower success rate in treatment with varenicline (29.5%) compared with carriers of CT or TT genotypes (50.9%) (P=0.007, n=167). The CT or TT genotypes were associated with higher odds ratio for success (OR=1.67, 95%CI=1.10-2.53, P=0.02), in a multivariate model. Patients with CYP2B6 rs2279343 AA genotype had higher success rate in treatment with bupropion (48.0%) compared with carriers of AG or GG genotypes (35.5%) (P=0.05, n=237). The AA genotype was associated with higher odds ratio for success (OR=1.92, 95%CI=1.08-3.42, P=0.03), in a multivariate model. We did not observe significant differences in the FTND and Issa scores according to the studied polymorphisms. Conclusion: The CHRNA4 rs1044396 and CYP2B6 rs2279343 are associated with smoking cessation in individuals on varenicline and bupropion terapies, respectively. We suggest that these polymorphisms influence the pharmacological response of these drugs and it might be important in the design of individualized pharmacotherapy

This thesis investigates the molecular evolutionary and macroevolutionary consequences of flight loss in insects. Chapter 2 tests the hypothesis that flightless groups have smaller effective population sizes than related flighted groups, expected to result in a consistent pattern of increased non-synonymous to synonymous ratios in flightless lineages due to the greater effect of genetic drift in smaller populations. Chapter 3 tests the hypothesis that reduced dispersal and species-level traits such as range size associated with flightlessness increase extinction rates, which over the long term will counteract increased speciation rates in flightless lineages, leading to lower net diversification. The wide-spread loss of flight in insects has led to increased molecular evolutionary rates and is associated with decreased long-term net diversification. I demonstrate that the fundamental trait of dispersal ability has shaped two forms of diversity—molecular and species—in the largest group of animals, and that microevolutionary and macroevolutionary patterns do not necessarily mirror each other. / Generously funded by NSERC with a Canada Graduate Scholarship and the Government of Ontario with an Ontario Graduate Scholarship to T. Fatima Mitterboeck; NSERC with a Discovery Grant to Dr. Sarah J. Adamowicz

La présente thèse par articles aborde différentes facettes du dépistage prénatal de certaines aneuploïdies au premier trimestre de la grossesse. L’introduction retrace l’historique du dépistage prénatal et énonce les différents marqueurs biochimiques et échographiques associés aux aneuploïdies. La première publication démontre que le tabagisme maternel abaisse significativement les niveaux sanguins maternels de PAPP-A et de la fraction libre de la β-hCG et augmente significativement la clarté nucale, confirmant la nécessité de contrôler cette co-variable dans le calcul de risque final, du moins pour la trisomie 18. Le deuxième article identifie des seuils de clarté nucale au-delà desquels la biochimie génétique n’apporte aucune valeur additionnelle au dépistage prénatal de la trisomie 21 et de la trisomie 18. Pour les fœtus avec clarté nucale supérieure aux seuils établis, un diagnostic prénatal intrusif devrait être offert sans délai. Le troisième et dernier article porte sur la première détermination des niveaux plasmatiques maternels de la protéine FLRG (follistatin-related gene) au premier trimestre de grossesse et sur son rôle potentiel à titre de marqueur biochimique dans le dépistage prénatal de la trisomie 21. Bien que détectables, les niveaux plasmatiques maternels de FLRG ne sont pas significativement altérés en présence de fœtus avec syndrome de Down. Dans la discussion générale, les trois articles sont abordés sous un angle plus spécifique au Québec. Des données complémentaires et originales y sont présentées. Une discussion sur l’évolution future du dépistage prénatal est entamée et des axes de recherche sont proposés. / In this thesis by articles, we explore different facets of first trimester prenatal screening of aneuploidy. Introduction retraces the origin of prenatal screening and enunciates current biochemical and ultrasound markers associated with aneuploidy. In the first article, impact of maternal smoking on first-trimester prenatal screening results is assessed for Down syndrome and trisomy 18. Both maternal blood levels of PAPP-A and free β-hCG are significantly decreased by maternal smoking while fetal nuchal translucency (NT) thickness is significantly increased. Without adjustment, this results in an increase of false positives, at least for trisomy 18. Based on these results, adjustment for smoking should be mandatory in first-trimester prenatal screening. In the second article, we identify NT threshold values above which biochemical screening provides no additional benefit. In pregnancies in which NT is above the proposed upper cut-offs, invasive prenatal screening should be offered without undue delay. In the third and last article, maternal plasma levels of follistatin- related gene protein (FLRG) are determined for the first time in first trimester of pregnancy. Its potential role as a new marker for Down syndrome is assessed. Although FLRG can be successfully detected in maternal plasma, its levels are not significantly altered by the presence of Down syndrome fetuses. In the general discussion, articles are mainly addressed under a Quebec standpoint. Additional and complementary original data are presented and different clinical research avenues are proposed.

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

Made available in DSpace on 2014-12-17T15:36:35Z (GMT). No. of bitstreams: 1 SebastiaoFS.pdf: 2551917 bytes, checksum: 3538250a323b39c32585d41d316bcb6a (MD5) Previous issue date: 2009-09-08 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The primary and accessory optic systems comprise two set of retinorecipient neural clusters. In this study, these visual related centers in the rock cavy were evaluated by using the retinal innervations pattern and Nissl staining cytoarchigtecture. After unilateral intraocular injection of cholera toxin B subunit and immunohistochemical reaction of coronal and sagittal sections from the diencephalon and midbrain region of rock cavy. Three subcortical centres of primary visual system were identified, superior colliculus, lateral geniculate complex and pretectal complex. The lateral geniculate complex is formed by a series of nuclei receiving direct visual information from the retina, dorsal lateral geniculate nucleus, intergeniculate leaflet and ventral lateral geniculate nucleus. The pretectal complex is formed by series of pretectal nuclei, medial pretectal nucleus, olivary pretectal nucleus, posterior pretectal nucleus, nucleus of the optic tract and anterior pretectal nucleus. In the accessory optic system, retinal terminals were observed in the dorsal terminal, lateral terminal and medial terminal nuclei as well as in the interstitial nucleus of the superior fasciculus, posterior fibres. All retinorecipient nuclei received bilateral input, with a contralateral predominance. This is the first study of this nature in the rock cavy and the results are compared with the data obtained for other species. The investigation represents a contribution to the knowledge regarding the organization of visual optic systems in relation to the biology of species. / Os sistemas visual prim?rio e ?ptico acess?rio compreendem dois conjuntos de grupamentos neurais, que recebem proje??o direta da retina. Nesse estudo, estes dois sistemas foram avaliados com rela??o a citoarquitetura e padr?o de inerva??o retiniana, usando como tra?ador neural anter?grado, a subunidade B da toxina col?rica revelada por imunoistoqu?mica para detectar este tra?ador em terminais sobre grupamentos neuronais (alvos) no enc?falo do moc? (Kerodon rupestris), um roedor nativo do Nordeste Brasileiro. Os resultados permitiram identificar os componentes do sistema visual prim?rio o complexo geniculado lateral, o complexo pr?-tectal e o col?culo superior. O complexo geniculado lateral cont?m o n?cleo geniculado lateral dorsal, o n?cleo geniculado lateral ventral e o folheto intergeniculado. Todos recebem fibras da retina com predomin?ncia contralateral, menos intensa para o folheto intergeniculado. O complexo pr?-tectal cont?m os n?cleos pr?-tectal anterior, pr?-tectal medial, pr?-tectal posterior, olivar pr?-tectal e n?cleo do trato ?ptico, os quais recebem proje??o retiniana predominantemente contralateral. Do mesmo modo, o col?culo superior recebe fibras da retina contralateral nas camadas superficiais. Tamb?m foi identificado o sistema ?ptico acess?rio completo no moc?, constitu?do pelos n?cleos terminal medial, terminal lateral, terminal dorsal e intersticial do fasc?culo superior posterior. Esses n?cleos recebem inerva??o retiniana com forte predomin?ncia contralateral, sendo que o n?cleo terminal medial, embora preserve a predomin?ncia contralateral, exibe uma evidente inerva??o ipsolateral.

The guanosine monophosphate synthetase (GMPS) is a class I glutamine amidotransferase, involved in the de-novo purine nucleotide biosynthesis. The enzyme catalyzes the biochemical transformation of xantosine (XMP) into guanosine monophosphate (GMP) in presence of ATP, Mg2+ and glutamine. All GMPSs consist of two catalytic sites 1) for GATase activity 2) for the ATPPase activity. The two catalytic sites may be housed in the same polypeptide (two-domain-type) or in separate polypeptides (two-subunit-type). Most of the studies have been performed on two-domain-type GMPSs, while only one study has been reported from two-subunit-type GMPS (Maruoka et al. 2009). The two-subunit-type GMPS presents an example where the component reactions of a single enzymatic reaction are carried out by two distinct subunits. In order to get better understanding of structural aspects and mechanistic principle that governs the GMPS activity in two-subunit-type GMPSs, we initiated the study by taking GMPS of Methanocaldococcus jannaschii as a model system. The GMPS of M. jannaschii (Mj) is a two-subunit-type protein. The GATase subunit catalyzes the hydrolysis of glutamine to produce glutamate and ammonia. The ATPPase subunit catalyses the amination of XMP to produce GMP using the ammonia generated in GATase subunit. Since the two component reactions are catalysed by two separate subunits and are coupled in the way that product of one reaction (ammonia) acts as a nucleophile in the second reaction. The cross-talk between these two subunits in order to maximise the efficiency of overall GMPS warrants investigation. The GATase activity is tightly regulated by the interaction with ATPPase domain/subunit, in all GMPS except in the case of P. falciparum. This interaction is facilitated by substrate binding to the ATPPase domain/subunit. Though, the conditions for the interaction between two subunits is known in a two-subunit-type GMP synthetase from P. horikoshii, the structural basis of substrate dependent interaction is not known. As a first step to understand the structural basis of interaction between the Mj GATase and Mj ATPPase subunits, we have determined the structure of Mj GATase (21 kDa) subunit using high resolution, multinuclear, multidimensional NMR spectroscopy. Sequence specific resonance assignments were obtained through analysis of various 2D and 3D hetero-nuclear multidimensional NMR experiments. NMR based distance restraints were obtained from assignment of correlations observed in NOE based experiments. Data were acquired on isotopically enriched samples of Mj GATase. The structure of Mj GATase (2lxn) was solved by using cyana-3.0 using NMR based restraints as input for the structure calculation. The ensemble of 20 lowest-energy structures showed root-mean-square deviations of 0.35±0.06 Å for backbone atoms and 0.8±0.06 Å for all heavy atoms. Attempts were also made to obtain assignments for the 69.6 kDa dimeric ATPPase subunit. Partial assignments have been obtained for this subunit. The GATase subunit is catalytically inactive. So far, there has been only one published report on a two-subunit-type GMPS from P. horikashii. The study has shown that the catalytic activity of GATase is regulated by the GATase-ATPPase interaction which is facilitated by the substrate binding to the ATPPase subunit. For the first time, we have provided the structural basis of interaction between GATase-ATPPase (112 kDa) in a two-subunit-type GMPS. Observed line width changes were used to identify residues in GATase residues that are involved in the Mj GATase-ATPPase interaction. Our data provides a possible explanation for conformational changes observed in the Mj GATase subunit upon GATase-ATPPase interaction that lead to GATase activation. Ammonia is generated in GATase subunit and is very reactive and labile. Thus, the faithful transportation of ammonia from GATase to ATPPase subunit is very crucial for optimal GMPS activity. Till date, a PDB query for GMPS retrieves only one structure which belongs to two-subunit-type GMPS, where authors have determined the structures of GATase and ATPPase subunits separately. However, the structure of holo-GMPS is not determined yet. Using interface information from experimental data and HADDOCK, we have constructed a model for the holo-GMPS from M. jannaschii. A possible ammonia channel has been deduced using the programs MOLE 2.0 and CAVER 2.0. This ammonia channel has a length of 46 Å, which is well within the range of the lengths calculated for similar channels in other glutamine amidotransferase. It had been suggested earlier that in addition to the magnesium required for charge stabilization of ATP, additional binding sites were present on GMPS. The effect of excess Mg2+ requirement on the GMPS activity has been studied in two-domain-type GMPS. However, the interaction between GATase and Mg2+ has been not investigated in any GMPS. This prompted us to investigate the effect of MgCl2 on Mj GATase subunit. For the first time, using chemical shift perturbation, we have established interaction between Mj GATase and Mg2+. The dissociation constant (Kd) of the Mj GATase-Mg2+ interaction was determined. The Kd value was found to be 1 mM, which indicates a very weak interaction. The substrate of the GATase subunit is glutamine. The condition of the hydrolysis of the glutamine is known in GMPS. However, the binding of the glutamine and associated conformational changes in GATase have been not studied in GMPS. Furthermore, till date there is no structure available for the glutamine bound GMPS/GATase. Using isotope edited one dimensional and two-dimensional NMR spectroscopy; we have shown that the Mj GATase catalytic residues are not in a compatible conformation to bind with glutamine. Thus, a conformational change in Mj GATase subunit is a pre-requisite condition for the binding of glutamine. These conformational changes are brought by the Mj GATase-ATPPase interaction.

In animals, some of the most critical regulators of gene expression are nuclear hormone receptors (NRs) and their coregulators, specifically the Mediator complex. Of particular interest are the NRs implicated in metabolic and developmental regulation and in carcinogenesis: thyroid hormone receptors (TRs) and retinoid X receptors (RXRs). In this work, I venture to elucidate some aspects of gene expression regulation by these NRs: the degree of evolutionary conservation of signalling based on NRs and their coregulators; the mechanisms of negative regulation by NRs; and possible implications of these findings for clinical medicine. State-of-the-art bioinformatical, genome editing and microscopic techniques are applied at three levels of animal evolution to study NRs and Mediator. Reverse genomics in human patients suffering from the syndrome of resistance to thyroid hormones β are used to infer the structure and function of TRβ subdomains. Alignments, binding studies and in vivo experiments in Trichoplax adhaerens allow identification of a close orthologue of human RXR at the basis of metazoan evolution. Employing database queries, genome editing and microscopy, we describe a correct orthologue of the Mediator subunit 28 in Caenorhabditis elegans, indicating a complete homology of the Mediator complex...