Indiana University-Purdue University Indianapolis (IUPUI) / RNA Polymerase II (RNAPII) is responsible for transcription of messenger RNA (mRNA) and many small non-coding RNAs. Progression through the RNAPII transcription cycle is orchestrated by combinatorial posttranslational modifications of the C-terminal domain (CTD) of the largest subunit of RNAPII, Rpb1, consisting of the repetitive sequence (Y1S2P3T4S5P6S7)n. Disruptions of proteins that control CTD phosphorylation, including the phosphatase Rtr1, cause defects in gene expression and transcription termination. There are two described RNAPII termination mechanisms. Most mRNAs are terminated by the polyadenylation-dependent cleavage and polyadenylation complex. Most short noncoding RNAs are terminated by the Nrd1 complex. Nrd1-dependent termination is coupled to RNA 3' end processing and/or degradation by Rrp6, a nuclear specific subunit of the exosome. The Rrp6-containing form a 3'-5' exonuclease complex that regulates diverse aspects of nuclear RNA biology including 3' end processing and degradation of a variety of noncoding RNAs (ncRNAs). It remains unclear whether Rrp6 is directly involved in termination. We discovered that deletion of RRP6 promotes extension of multiple Nrd1-dependent transcripts resulting from improperly processed 3' RNA ends and faulty transcript termination at specific target genes. Defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of Nrd1 depletion from the nucleus. Our data indicate Rrp6 acts with Nrd1 globally to promote transcription termination in addition to RNA processing and/or degradation. Furthermore, we found that deletion of the CTD phosphatase Rtr1 shortens the distance of transcription before Nrd1-dependent termination of specific regulatory antisense transcripts (ASTs), increases Nrd1 occupancy at these sites, and increases the interaction between Nrd1 and RNAPII. The RTR1/RRP6 double deletion phenocopies an RRP6 deletion, indicating that the regulation of ASTs by Rtr1 requires Rrp6 activity and the Nrd1 termination pathway.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The root system architecture of higher plants plays an essential role in the uptake of water and nutrients as well as the production of hormones. These root systems are highly branched with the formation of post-embryonic organs such as lateral roots. The initiation and development of lateral roots has been well defined. WAG1 and WAG2 are protein-serine/threonine kinases from Arabidopsis that are closely related to PINOID and suppress root waving. The wag1;wag2 double mutants exhibit a strong root waving phenotype on vertical hard agar plates only seen in wild-type roots when the seedlings are grown on inclined plates. Here an additional root phenotype in the wag1;wag2 mutant is reported. The wag1;wag2 double mutant displays both an increased total number and density of emerged lateral roots (approximately 1.5-fold). An increased LRP density of 1.5-fold over wild-type is observed. To ascertain the role of WAG1 and WAG2 in lateral root development we examined promoter activity in the WAG1::GUS and WAG2::GUS lines. The WAG1 promoter showed no detectable activity at any stage of development. The WAG2 promoter was active in stage IV onward, however there was no detectable activity in the cell types associated with initiation events. The lateral root density and spatial patterning in wild-type, when grown on inclined hard agar plates, was similar to wag1;wag2 on vertical plates. Seedlings of both genotypes were treated with hormones such as auxin and MeJA, and inhibitors. Auxin response in wag1;wag2 was normal with a similar number of LR as the wild-type after treatment. Treatment with MeJA resulted in a similar induction of LRP in both genotypes, however the percent lateral root emergence in wag1;wag2 was reduced while Col-0 was increased compared to controls. Treatment with the calcium blocker tetracaine resulted in wag1;wag2 displaying a wild-type level of LR but had no significant effect on wild-type. Genetic analysis of the wag1;wag2 LR pathway revealed that WAG1 and WAG2 are acting in the same pathway as AUX1, AXR1and PGM1. pgm1-1 was not previously reported to have a LR defect but showed decreased LR formation here, while pgm1;wag1;wag2 had a similar LR density to wag1;wag2. TIR7 and ARG1 were both deduced to operate in separate pathways from WAG1 and WAG2. The data presented here shows that the wag1;wag2 double mutant has an increased number of LR compared to Col-0. This defect appears to be caused by increased pre-initiation events and seems to be tied to the root waving phenotype. However, the treatment with MeJA revealed a possible role for WAG1 or WAG2 in LRP development, potentially under stress conditions. Calcium also seems to play a significant role in the wag1;wag2 LR phenotype, possibly independent of the root waving phenotype.

Ce travail porte sur l’étude des mécanismes de phosphorylation des protéines par les sérine/thréonine kinases chez Staphylococcus aureus. Nous avons, tout d’abord, mis en évidence et caractérisé une seconde Ser/Thr-kinase, nommée Stk2. Cette kinase présente peu d’homologies avec les autres Ser/Thr-kinases bactériennes décrites à ce jour, en particulier avec la première Ser/Thr-kinase mise en évidence précédemment chez S. aureus, Stk1. Nous avons ensuite caractérisé dix sites d’autophosphorylation de Stk2 et nous avons montré que trois sites sont nécessaires à son activité. Enfin, nous avons montré que le régulateur global de virulence, SarA, est phosphorylé à la fois par Stk1 et Stk2. La phosphorylation de SarA influence sa capacité de liaison à l’ADN. Cette étude contribue à mieux appréhender, au niveau moléculaire, le rôle des Ser/Thr-kinases dans le métabolisme des bactéries et, plus particulièrement, dans la régulation de leur virulence / We report that protein phosphorylation on serine and threonine is required for controlling staphylococcal virulence. We identified and characterized a second serine/threonine kinase, Stk2, in S. aureus. Biochemical analyses revealed that this enzyme displays autokinase activity on both threonine and serine residues. Stk2 is atypical in the sense that it exhibits a weak similarity with the first Ser/Thr-kinase previously detected, Stk1, and its undergoes a different mechanism of activation compared to the other bacterial Ser/Thr-kinases described so far. We also showed that SarA, a major transcription factor that regulates more than a hundred virulence genes, is phosphorylated by both Stk1 and Stk2. Phosphorylation of SarA leads to strong effects on its ability to bind DNA. The study of Stk1 and Stk2, at the molecular level, provides a better understanding of the role of these staphylococcal Ser/Thr-kinases in bacterial metabolism and, in particular, in the regulation of virulence

A Doença de Alzheimer (DA) é um transtorno progressivo que acomete o Sistema Nervoso Central, causando demência em idosos. A doença leva a uma diminuição da memória, dificuldade no raciocínio e pensamento e alterações comportamentais. A fisiopatologia da doença corresponde ao aumento na concentração do peptídeo -amilóide com consequente deposição e formação da placa amiloide; e também ao aparecimento dos emaranhados neurofibrilares, que são agregados de proteína tau hiperfosforilada. A enzima glicogênio sintase cinase 3 beta (GSK-3) está diretamente envolvida nos dois processos e, por isso, a busca por novos inibidores dessa enzima é uma importante estratégia terapêutica para o tratamento da doença. Neste trabalho utilizou-se a triagem virtual em 7 bancos de dados de moléculas aplicando cinco diferentes estratégias in silico, através de planejamento de fármacos baseado em ligantes e baseado em estrutura, combinada com estudos in silico de farmacocinética, toxicidade e atividade biológica, seguido de posteriores ensaios de inibição enzimática in vitro. Obteve-se três compostos pela metodologia de farmacóforo, (Estratégia 3) dos quais dois deles demonstraram atividade inibitória interessante para GSK-3, na faixa de micromolar. A partir das outras quatro estratégias foram selecionados 16 compostos que futuramente serão também testados utilizando o mesmo protocolo de ensaio in vitro aqui utilizado. / Alzheimer\'s disease (AD) is a progressive disorder that affects the Central Nervous System, causing dementia in the elderly. The disease leads to decreased memory, difficulty in reasoning and thinking, and behavioral changes. The pathophysiology of the disease corresponds to the increase in -amyloid peptide concentration with consequent deposition and formation of the amyloid plaque and to the appearance of neurofibrillary tangles, which are aggregates of hyperphosphorylated tau protein. The enzyme glycogen synthase kinase-3 beta (GSK-3) is directly involved in both processes and, therefore, the search for new inhibitors of this enzyme is an important therapeutic strategy for the treatment of the disease. In this work, we used virtual screening in 7 molecule databases applying five different in silico strategies, using the ligand-based and structure-based drug design methodologies, combined with in silico studies of pharmacokinetics, toxicity and biological activity, followed by subsequent assays enzymatic inhibition in vitro. We obtained three compounds by the pharmacophore methodology (Strategy 3) of which two of them demonstrated interesting inhibitory activity for GSK-3 in the micromolar range. From the other four strategies, 16 compounds were selected which in future will also be tested using the same in vitro assay protocol used here.

Dans le cadre d’un projet de drug design, l’amélioration de la prédiction de l’affinité représente toujours un défi malgré les nombreux efforts déployés dans ce sens. De plus, les constantes cinétiques d’association et de dissociation sont d'un intérêt majeur pour la découverte de nouveaux médicaments, notamment au stade précoce de l'optimisation des molécules afin de mieux évaluer leurs tolérances et efficacités. De par la récente émergence des études de constantes cinétiques, il existe peu de méthodes de prédiction de ces dernières et aucune approche efficace n'a encore été développée pour estimer correctement ces paramètres cinétiques.En relevant ces deux défis, le premier volet de cette thèse consiste au développement de nouvelles méthodes qui permettent dans un premier temps d’améliorer la prédiction de l’affinité par docking flexible et dans un deuxième temps la prédiction des constantes cinétiques d’association et de dissociations (kon et koff) grâce à des simulations de dynamique moléculaire accélérées.Dans le second volet de cette thèse, nous avons conçu de nouveaux inhibiteurs des LIM kinases, cibles émergentes impliquées dans plusieurs physiopathologies incluant la neurofibromatose et le cancer. Nos composés ont de bonnes affinités et sélectivités in vitro, et d’excellentes activités et tolérances évaluées sur des tests cellulaires. / In a drug design project, improving the prediction of affinity is still an issue despite the considerable efforts made in this direction. In addition, binding kinetic constants are of major interest for the discovery of new drugs, in particular at the early stage of molecules optimization to better evaluate their tolerance and efficacy. Due to the recent emergence of the importance of binding kinetics, methods of kinetic rates prediction remain scarce and no efficient computational approach has still been developed to correctly estimate kinetic parameters.In order to challenge these two problematics, the first part of this thesis consists in the development of new methods that allow, first, to improve the prediction of affinity by a flexible docking and, second, to predict the ligand binding/unbinding pathways and binding kinetic rates (kon and koff) by enhanced molecular dynamics simulations.In the second part of this thesis, we have designed novel inhibitors of LIM kinases, emerging targets involved in several pathophysiologies including neurofibromatosis and cancer. Our compounds have good affinities and selectivities in vitro, and excellent activities and tolerances evaluated on cellular tests.

PTP69D is a receptor protein tyrosine phosphatase (RPTP) with two intracellular catalytic domains (Cat1 and Cat2), which has been shown to play a role in axon outgrowth and guidance of embryonic motorneurons, as well as targeting of photoreceptor neurons in the visual system of Drosophila melanogaster. Here, we characterized the developmental role of PTP69D in the giant fiber (GF) neurons; two interneurons in the central nervous system (CNS) that control the escape response of the fly. In addition to guidance and targeting functions, our studies reveal an additional role for PTP69D in synaptic terminal growth in the CNS. We found that inhibition of phosphatase activity in catalytic domain (Cat1) proximal to the transmembrane domain did not affect axon guidance or targeting but resulted in stunted terminal growth of the GFs. Cell autonomous rescue and knockdown experiments demonstrated a function for PTP69D in the GFs, but not its postsynaptic target neurons. In addition,complementation studies and structure-function analyses revealed that for GF terminal growth, Cat1 function of PTP69D requires the immunoglobulin and the Cat2 domain but not the fibronectin type III repeats nor the membrane proximal region. In contrast, the fibronectin type III repeats, but not the immunoglobulin domains, were previously shown to be essential for axon targeting of photoreceptor neurons. Thus, our studies uncover a novel role for PTP69D in synaptic terminal growth in the CNS that is mechanistically distinct from its function during earlier developmental processes. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Estudos recentes demonstram que o Hormônio Tiroideano (HT) é capaz de modular rapidamente o estado de fosforilação de proteínas quinases relacionadas ao processo de hipertrofia cardíaca. Evidências experimentais indicam que a proteína quinase ativada por AMP (AMPK) seja um alvo importante no controle do crescimento hipertrófico, uma vez que a ativação desta enzima determina ampla variedade de efeitos siológicos, incluindo o controle de proteínas relacionadas à síntese protéica. Dessa forma, os objetivos deste estudo foram os de avaliar os efeitos do HT sobre a modulação da via de sinalização da AMPK, além de verificar o possível envolvimento desta quinase no modelo de hipertrofia in vitro induzida pelo HT. Os resultados obtidos mostraram que rapidamente este hormônio ativa a AMPK e proteínas relacionadas a esta sinalização. Além disso, a estimulação farmacológica da AMPK atenua a hipertrofia de cardiomiócitos induzida pelo HT. Estes dados sugerem que a AMPK seja uma possível ferramenta terapêutica em doenças cardiovasculares como a hipertrofia cardíaca. / Some studies have shown that Thyroid Hormones (TH) are also able to rapidly modulate the phosphorylation state of protein kinases related to cardiac hypertrophy process by non-genomic actions. In this sense, experimental evidences indicate that AMP-activated protein kinase (AMPK) is an important target in the control of hypertrophic growth, since the activation of this enzyme determines wide variety of physiological effects, including the control of enzymes related to protein synthesis. Thus, the objective of this study was to evaluate the effects of TH on the modulation of AMPK signaling pathway and to check the possible involvement of this kinase in vitro model of hypertrophy induced by TH. The results showed that this hormone rapidly activates AMPK and related proteins to this signaling. Furthermore, pharmacological stimulation of AMPK attenuated cardiomyocyte hypertrophy induced by TH. These data suggest that AMPK may correspond to a possible therapeutic tool in cardiovascular disease.

Ip Wai-Ki. / "July 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 261-290). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Estudos recentes demonstram que o Hormônio Tiroideano (HT) é capaz de modular rapidamente o estado de fosforilação de proteínas quinases relacionadas ao processo de hipertrofia cardíaca. Evidências experimentais indicam que a proteína quinase ativada por AMP (AMPK) seja um alvo importante no controle do crescimento hipertrófico, uma vez que a ativação desta enzima determina ampla variedade de efeitos siológicos, incluindo o controle de proteínas relacionadas à síntese protéica. Dessa forma, os objetivos deste estudo foram os de avaliar os efeitos do HT sobre a modulação da via de sinalização da AMPK, além de verificar o possível envolvimento desta quinase no modelo de hipertrofia in vitro induzida pelo HT. Os resultados obtidos mostraram que rapidamente este hormônio ativa a AMPK e proteínas relacionadas a esta sinalização. Além disso, a estimulação farmacológica da AMPK atenua a hipertrofia de cardiomiócitos induzida pelo HT. Estes dados sugerem que a AMPK seja uma possível ferramenta terapêutica em doenças cardiovasculares como a hipertrofia cardíaca. / Some studies have shown that Thyroid Hormones (TH) are also able to rapidly modulate the phosphorylation state of protein kinases related to cardiac hypertrophy process by non-genomic actions. In this sense, experimental evidences indicate that AMP-activated protein kinase (AMPK) is an important target in the control of hypertrophic growth, since the activation of this enzyme determines wide variety of physiological effects, including the control of enzymes related to protein synthesis. Thus, the objective of this study was to evaluate the effects of TH on the modulation of AMPK signaling pathway and to check the possible involvement of this kinase in vitro model of hypertrophy induced by TH. The results showed that this hormone rapidly activates AMPK and related proteins to this signaling. Furthermore, pharmacological stimulation of AMPK attenuated cardiomyocyte hypertrophy induced by TH. These data suggest that AMPK may correspond to a possible therapeutic tool in cardiovascular disease.