Scrub typhus, caused by the obligate intracellular bacterium Orientia tsutsugamushi, afflicts one million people annually. Despite being a global health threat, little is known about O. tsutsugamushi pathogenesis. Here, we demonstrate that O. tsutsugamushi modulates the ER and ER-associated processes as mechanisms of nutritional virulence and immune evasion. To obtain amino acids to fuel replication, O. tsutsugamushi simultaneously induces ER stress and the unfolded protein response (UPR) while inhibiting ER-associated degradation (ERAD) during early infection time points. During exponential growth, the bacterium releases the ER bottleneck, resulting in generation of ERAD-derived amino acids that it parasitized for replication. The O. tsutsugamushi effector, Ank4, is linked to this process, as it impedes ERAD when ectopically expressed. O. tsutsugamushi expression of ank4 peaks during the ERAD inhibition window, but is absent when the pathway is restored. These data reveal a novel mechanism of nutritional virulence, whereby an obligate intracellular pathogen coordinates the modulation of multiple ER-associated processes. Like other intracellular pathogens, O. tsutsugamushi inhibits expression of MHC-I, but it does so in a novel manner by degrading the master regulator of MHC-I, NLRC5. This impedes production of the MHC-I components, human leukocyte antigen A and Beta-2 microglobulin. The NLRC5-reduction mechanism recapitulates across diverse cell types, but the degree and duration of inhibition is cell type-specific. NLRC5 modulation and MHC-I inhibition are linked to another O. tsutsugamushi Ank, Ank5. NLRC5 is a putative interacting partner of Ank5. Moreover, NLRC5 and MHC-I levels are reduced in cells ectopically expressing Ank5. To our knowledge, these are the first examples of a pathogen modulating NLRC5 to negatively regulate MHC-I expression and of a bacterial effector interacting with NLRC5. As we learn more about the bacterium’s ability to regulate its host cell, a unifying theme has emerged: modulation of the ER and ER-associated pathways. These projects reveal two novel mechanisms of O. tsutsugamushi pathogenesis, strategies to acquire the amino acids needed for replication and to decrease MHC-I antigen presentation by the host cell. These insights help in understanding how O. tsutsugamushi and potentially other related pathogens co-opt host cell processes to cause disease.

Classic Galactosemia (CG) is an autosomal recessive disorder caused by deleterious mutations of galactose-1-phosphate uridyltransferase (E.C. 2.7.7.12) (GALT) gene, which results in the inability to metabolize galactose and the accumulation of galactose-1-phosphate (gal-1-p) in patients' cells. Newborn screening has resulted in presymptomatic diagnosis and treatment. Although a galactose-restricted diet prevents the neonatal lethality of this disorder, many well-treated patients continue to develop debilitating complications such as premature ovarian insufficiency (POI), dyspraxic speech, ataxia and other neurological defects. The causes of these unsatisfactory outcomes remain unclear, but accumulation of gal-1-p is regarded as the major factor responsible for these chronic complications. In previous studies we found that gal-1-p was a competitive inhibitor of the UTP dependent, glucose-1-phosphate pyrophosphorylase (EC 2.7.7.9) and inositol monophosphatase (EC 3.1.3.25). As a result there were deficiencies in cellular UDP-glucose and UDP-galactose as well as impaired G-protein-stimulated inositol responses of Ca++ release, respectively. In this study, we found that when we challenged GALT-deficient yeast and GALT-deficient human diploid fibroblasts with galactose, these cells exhibited environmental stress and endoplasmic reticulum (ER) stress, which was characterized by the up-regulation of the gene encoding the master regulator of ER stress, GRP78/BiP. In separate studies using GALT-deficient diploid fibroblasts and comparative expression arrays, we found that the expression level of a tumor suppressor gene called aplysia ras homolog I (ARHI) was significantly higher in patient cells under galactose challenge. This ARHI gene was lost in rodents during evolution and GALT knockout mice did not express the human phenotype for galactosemia whereas over-expression of this gene in transgenic mice resulted in phenotypes characteristic of those seen in patients with galactosemia. We therefore propose here that ARHI could be an important target of galactose toxicity in Classic Galactosemia, and also explain the absence of patient phenotypes in GALT knock-out mice. In order to prevent accumulation of gal-1-p caused by GALT-deficiency, we experimentally screened over 300,000 chemical compounds against human galactokinase (GALK) in vitro. To date, we obtained from two high-throughput screenings (HTS), 200 GALK inhibitors with IC50s ranging from 700nM to 35μM. We subsequently established selectivity and toxicity profiles of 34 selected GALK inhibitors. Based on these results, we selected four compounds for further characterization, which included kinetic studies, site-directed mutagenesis and molecular docking experiments. From these experiments, we learned how these compounds interacted with the GALK enzyme and built detailed binding models for each of them. We demonstrated that three of the in vitro inhibitors of GALK could lower intracellular gal-1-p accumulation in GALT-deficient cells. Of considerable interest to us was that one of the compounds, cluster 25-1, not only reduced gal-1-p accumulation, but also corrected the level of GRP78/BiP back to background in the galactose-challenged GALT-deficient cells. These results were the first to demonstrate a direct link between GALT-deficiency and ER stress and provided proof of concept that we could prevent both gal-1-p accumulation and ER stress with GALK inhibitors in GALT deficient human cells. Lastly, we developed a new, virtual method of identifying novel GALK inhibitors by combining software-based, high-throughput virtual screening (HTVS) and fragment-based linkage using docking software. The initial HTVS validation experiments with compounds from the "ZINC" database identified four active GALK inhibitors with IC50s ranging from 70μM to 400μM. We then used HTVS to identify chemical fragments which bind to the active site of the human GALK enzyme. Using fragment-linking software, we identified chemical fragments which could potentially result in high-affinity inhibitors when chemically joined.

The role that each of the Notch receptors play in controlling alveolar development and cell fate determination in the mouse mammary gland has remained unclear. By utilizing a cre-conditional constitutively active intracellular Notch1 knock-in I define, in vivo, that ectopic Notch1 activation is sufficient to inhibit ductal outgrowth, cause the formation of alveolar-like cell accumulations, and promote Elf5+/ER- cell fate, at the expense of ER+ cell fate, in the mammary gland of pubescent mice. Furthermore, ectopic Notch1 in the pregnant mammary gland is sufficient to promote the formation of pregnancy/lactation-dependent lactating adenomas. These lactating adenomas consist of differentiated secretory cells and normally regress during involution but progress into non-regressing tumours after multiple pregnancies. These lactating adenomas exhibit decapitation secretions characteristic of apocrine differentiation. Together these results suggest that Notch1 may function to promote Elf5+/ER- cell fate and may be misregulated in pregnancy-associated masses and apocrine-carcinoma of the breast in humans.

The role that each of the Notch receptors play in controlling alveolar development and cell fate determination in the mouse mammary gland has remained unclear. By utilizing a cre-conditional constitutively active intracellular Notch1 knock-in I define, in vivo, that ectopic Notch1 activation is sufficient to inhibit ductal outgrowth, cause the formation of alveolar-like cell accumulations, and promote Elf5+/ER- cell fate, at the expense of ER+ cell fate, in the mammary gland of pubescent mice. Furthermore, ectopic Notch1 in the pregnant mammary gland is sufficient to promote the formation of pregnancy/lactation-dependent lactating adenomas. These lactating adenomas consist of differentiated secretory cells and normally regress during involution but progress into non-regressing tumours after multiple pregnancies. These lactating adenomas exhibit decapitation secretions characteristic of apocrine differentiation. Together these results suggest that Notch1 may function to promote Elf5+/ER- cell fate and may be misregulated in pregnancy-associated masses and apocrine-carcinoma of the breast in humans.

Androgen receptor (AR) is a member of the steroid hormone receptor family of molecules, and expansion of a CAG repeat encoding polyglutamine (poly-Q) in AR gene are associated with a progressive neuromuscular disease known as spinal bulbar muscular atrophy (SBMA) or Kennedy disease. The hallmark of SBMA diseases is formation of juxtanuclear AR inclusions that have been termed ¡¥AR aggregates¡¦.Previous studies showed that transgenic mice overexpressing wild-type AR exclusively in the skeletal muscle fibers display similar abnormalities to those observed in models of SBMA disease. To elucidate the mechanisms underlying toxicity conferred by wild-type protein aggregation within normal cells, a mouse embryonic stem cell (ESC) model with non-genetic modified settings in AR overexpression was used to display the common features of polyglutamine disease in this experiment. It was found that wild-type AR proteins are highly expressed and form nuclear aggregate inclusions in response to androgen treatment in ES cells, the formation of AR aggregates inhibit the differentiation of embryonic bodys and enhanced caspase-3 activity in androgens -induced apoptosis. In addition, it was also investigated that relation between chaperones¡BAR and the endoplasmic reticulum (ER) stress-induced pathways in ES cells in this study, and it was found that chaperones could colocalize with AR aggregates, these findings may help us to better understand the roles of the chaperones on AR aggregates.

No description available.

Database design methods and tools have been investigated in the paper. The action diagram construction process has been investigated and three data design methodologies IDE1X, IE and Chen‘ have been considered. The main definitions have been described and classified in detail. The problems of database design have been stated and table normal forms have been presented. The company‘s employee income tax database model has been designed on the basis of the theory. Four step Chen‘s diagram has been used to design the data model. The company‘s employee income tax database has been designed and implemented in MS Access environment. The data import program from MS Excel has been developed.

Viena iš problemų, su kuria susiduria informacinių sistemų kūrėjai, tai veiklos taisyklių atradimas. Tai procesas, kurio metu identifikuojamos veiklos taisyklės, įtakojančios kuriamą informacinę sistemą. Veiklos taisyklių atradimas gali trukti visą IS kūrimo laiką: daugelis jų atrandama specifikuojant vartotojų poreikius, taip pat projektuojant IS modelius. Derėtų nepamiršti, kad IS kūrimas – tai procesas, išsidėstęs laike, todėl tuo metu pati veikla, savaime aišku, nestovi vietoje ir gali generuoti naujas taisykles ar senų pakeitimus. Taigi taisyklės atrandamos viso proceso metu ir netgi po jo. Kurgi dėti, kaip dokumentuoti naujai atrastą taisyklę? Daugelis mokslininkų siūlo susikurti tam tikros formos blanką ir jame užrašyti veiklos taisyklės specifikaciją. Be abejo tai gali būti ir kompiuterinis failas. Šio darbo objektas veiklos taisyklėmis grindžiamas vartotojo reikalavimų kuriamai informacinei sistemai specifikavimas. Darbo tikslas yra pasiūlyti veiklos taisyklėmis grindžiamo vartotojo poreikių specifikavimo metodą ir CASE priemonę jam palengvinti. Iškelti uždaviniai yra tokie: • Apžvelgti veiklos taisyklių koncepciją; • Aptarti veiklos taisyklių struktūrizavimo metodus; • Apžvelgti veiklos taisyklių struktūrizavimo proceso eigą; • Realizuoti pasirinktą veiklos taisyklėmis grindžiamos vartotojo reikalavimų saugyklos modelį; • Pateikti išvadas ir pasiūlymus tolesniam sistemos vystymui. Laukiamas rezultatas veikianti vartotojo reikalavimų saugykla. / The success of requirements analysis often decides the quality of the finished information system (IS). Application of ideas behind the business rules approach for requirements analysis could help narrow the gap between business representatives and analysts thus paving the way to a better requirements specification. In this paper a framework for business rules driven IS requirements specification is presented. The proposed approach is based on discovery of functions, business decisions and underlying business rules. The management of captured requirements as well as the generation of relevant documentation is performed using the repository system, major implementation guidelines of which are also discussed. The prototype of the repository system has been implemented. The problems of the proposed approach that were discovered during the experimental implementation are also discussed.

The unfolded protein response (UPR) is a well conserved mechanism in eukaryotes that protects organisms from the damaging effects of endoplasmic reticulum (ER) stresses. Activation of the UPR will lead to two outcomes. It first attempts to restore cellular functions by enhancing protein folding capacity, inhibiting protein synthesis and promoting degradation of harmful proteins (the pro-survival pathway). However, if the stressful conditions are prolonged or severe, apoptosis will be induced (the pro-apoptotic pathway). The present study suggests that an isoform of the cellular protein Zhangfei (ZF, CREBZF) is linked to the pro-apoptotic pathway in the UPR by using DNA, protein and cell viability analyses. This isoform is known as the short-tail ZF (stZF). We demonstrated that stZF can be induced by prolonged ER stress. The protein of stZF is stable under ER stress and it has the ability to promote programmed cell death in the early stage of apoptosis through the induction of CHOP, a protein that plays a key role in the pro-apoptotic pathway of the UPR.

Notre laboratoire s'intéresse aux mécanismes impliqués dans la régulation de l'expression génique et plus particulièrement au rôle de la chromatine dans cette régulation. En effet, chez les eucaryotes l'ADN est compactée autour de protéines appelées histones créant ainsi des nucléosomes lesquels forment une structure plus complexe, la chromatine. Cette dernière est une barrière aux processus cellulaires touchant l'ADN dont la transcription. La compréhension de la régulation de la structure de la chromatine est essentielle pour saisir les variations de l'expression génique. Mon projet de doctorat a porté sur l'étude de la régulation des gènes cibles du récepteur aryl hydrocarbone (AhR), CYP1A1 et CYP1B1, et plus particulièrement sur le rôle du variant d'histone H2A.Z dans l'expression de ces gènes. AhR est un senseur moléculaire auquel va [i.e. vont] se lier de nombreux polluants appartenant principalement à ces deux grandes familles : les hydrocarbones aromatiques halogènes (HAH) et les hydrocarbones aromatiques polycycliques (PAH). En réponse à la liaison de ces polluants, AhR va induire l'expression de ses gènes cibles. CYP1A1 et CYP1B1 sont impliquées dans le métabolisme de l'estradiol (E2) en 2hydroxyestradiol et 4-hydroxyestradiol respectivement. Il a été proposé qu'une diminution du ratio CYP1A1/CYP1B1 soit importante pour l'initiation du cancer du sein. Au cours de mon doctorat, j'ai pu mettre en évidence un rôle du variant H2A.Z dans la régulation de l'expression de CYP1A1 et CYP1B1. J'ai aussi pu montrer que le statut de ER[alpha] déterminait l'importance de H2A.Z lors de l'induction de CYP1A1. De plus, nous avons observé que la déplétion de H2A.Z induit une augmentation de la méthylation de l'ADN au promoteur de CYP1A1. En parallèle, nous avons confirmé que ER[alpha] réprime spécifiquement l'induction de CYP1A1 sans affecter celle de CYP1B1. Nos résultats montrent qu'en présence de TCDD et d'E2, ER[alpha] et DNMT3B sont recrutés au promoteur de CYP1A1, ce qui conduit à une augmentation de la méthylation du promoteur de CYP1A1 et conséquemment à une diminution de son induction. AhR possède de nombreux ligands d'origine très variée qui peuvent être aussi bien toxiques que bénéfiques. Nous avons choisi de comparer deux de ces ligands : le TCDD et le DIM. Au cours de ces travaux, nous avons montré que le DIM utilisé à forte concentration (>50[mu]M) induit les gènes cibles de AhR (CYP1A1 et CYP1B1) mais aussi un arrêt de la croissance et la mort des cellules. À l'opposé, le traitement avec des concentrations plus faible [i.e. faibles] de DIM (10[mu]M) induit principalement les gènes cibles de ER[alpha] (TFF1 et GREB1) et la prolifération des cellules. Nous avons aussi montré que l'activation de ER[alpha] par le DIM est due à l'action de la protéine kinase A (PKA). En effet, l'inhibition de la PKA ainsi que la déplétion de ER[alpha] abolissent les effets du DIM sur l'expression de GREB1 et CYPIA1 ainsi que sur la prolifération cellulaire. En conclusion, nous avons dans un premier temps mis en évidence le rôle de deux protéines, DNMT3B et H2A.Z, dans la régulation de CYP1A1 dans les cellules MCF7. Nous avons ainsi découvert un nouveau corépresseur partenaire de ER[alpha] en DNMT3B et nous avons proposé une nouvelle façon pour ER[alpha] de promouvoir la carcinogenèse en dérégulant le ratio CYP1A1/CYP1B1. Dans un deuxième temps, nous avons montré que la concentration de DIM utilisée dans les expériences peut conduire à des résultats diamétralement opposés sur la croissance cellulaire.