Dissertations / Theses on the topic 'CASQ1'

Calsequestrin1 (CASQ1) is the main Ca2+ buffer protein localized in the lumen of the skeletal muscle sarcoplasmic reticulum. Two isoforms of CASQ, encoded by two different genes, have been identified: the skeletal isoform (CASQ1) expressed in fast-twich skeletal fibres and the cardiac isoform (CASQ2) expressed in cardiac muscle and in slow-twich skeletal fibres. Crystallographic studies have shown that the protein is made of three thioredoxin-like domains linked by short amino acidic sequences. The main characteristic of this molecule is its ability to polymerize in presence of Ca2+, binding more and more Ca2+ ions. Mutations in CASQ2 have been reported in an autosomal-recessive form of Catecholaminergic Polymorphic Ventricular Tachycardia, whereas recently a heterozygous missense mutation has been identified in the CASQ1 gene in patients with a vacuolar aggregate myopathy. This thesis is focused on the characterization of four missense variants in the CASQ1 gene, identified in patients with different myopathies. The identified mutations affect conserved amino acids of the CASQ1 protein. Turbidity measurements in presence of increasing Ca2+ concentrations showed that two of these mutations lead to a significant reduction in Ca2+-dependent aggregation. In agreement, limited trypsin proteolysis experiments showed that these amino acid substitutions enhanced the conformational flexibility of CASQ1, which become more susceptible to trypsin cleavage in comparison to wild type. On the contrary, the CASQ1 mutation associated with the vacuolar myopathy showed an increased Ca2+-dependent aggregation. These results support the hypothesis that distinct mutations in the CASQ1 gene may cause different phenotypes.

Rhabdomyolysis is an acute, and frequently severe, pathological event, characterized by rapid necrosis and destruction of striated muscle tissue. It is clinically characterized by muscle pain, weakness and emission of dark urine. The mechanisms that lead to rhabdomyolysis are various and different, and share common alterations such as dysfunction of the pumps Na + / K + and Ca2 + ATPase, and rupture of the sarcolemma, which in turn determine calcium homeostasis alterations, mitochondrial dysfunction, proteases activation, reduced availability of ATP and, overall, cell apoptosis and rhabdomyolysis. Several causes can initiate this vicious circle, both acquired and genetic. When facing an episode of rhabdomyolysis, the identification of the etiological cause can be extremely complex, long, and costly. Nevertheless, the correct identification of the underlying cause is of utmost importance for the correct information regarding prognosis (risk of recurrence) and for the family genetic counselling. Despite the application of extended diagnostic work up frequently it is not easy to distinguish if the rhabdomyolysis episode is due to a genetic disorder or whether it is the result of an abnormal effort, of an infectious episode or effect of a toxic. The main objective of this study is the evaluation of the diagnostic process of rhabdomyolysis, and to propose an updated, comprehensive, rational and cost effective protocol based on the latest information and techniques. We first studied the diagnostic process currently in use for rhabdomyolysis in a large retrospective study including 208 patients (aim 1). We characterized the clinical, molecular and radiological features of a new genetic cause of rhabdomyolysis, the CASQ1-related myopathy (aim 2). We determined the clinical spectrum of limb-girdle muscular dystrophy 2E (LGMD2E), investigating the risk of rhabdomyolysis of this disease (aim 3). We demostrated the role of EMG with provocative test (Long Exercise Test) as a sensitive and specific diagnostic test in the work up of rhabdomyolysis, especially in Glycogenosis type V (aim 4). We proposed a multi gene panel that should be studied by Next Generation Sequencing (NGS) in patients presenting rhabdomyolysis (aim 5) In conclusion, the results of this study suggest a new diagnostic algorithm for rhabdomyolysis. In this algorithm, the clinical features and few first-line tests (blood tests, EMG, acylcarnitines, grip test) exclude the most frequent causes or the treatable ones. The subsequent muscle biopsy will identify certain myopathies and guide toward the use of specific genetic panels for metabolic myopathies, muscular dystrophies or mitochondrial diseases that should be studied by NGS. The diagnostic algorithm that we propose will allow cost reduction and time optimization, and hopefully will increase the rate of etiological diagnosis of rhabdomyolysis, a serious event with major impact on patients' lives that, to date, remains poorly diagnosed.<br>La rabdomiolisi è un evento patologico acuto e talvolta grave, caratterizzato da rapida necrosi e distruzione del tessuto muscolare striato alla quale corrisponde clinicamente dolore e debolezza muscolare ed emissione di urine scure. I meccanismi che portano alla rabdomiolisi sono vari e differenti ma condividono alterazioni comuni quali la disfunzione delle pompe Na+/K+ e Ca2+ ATPAsi, e la rottura del sarcolemma, che a loro volta determinano alterazioni dell’omeostasi del calcio, disfunzione mitocondriale, attivazione di proteasi, riduzione della disponibilità di ATP e, complessivamente, apoptosi cellulare e rabdomiolisi. La corretta identificazione della causa sottostante è di estrema importanza per una corretta informazione al paziente a fini prognostici (rischio di ricorrenza) e per il counseling genetico familiare. Nonostante l’applicazione di estesi work up diagnostici, non è sempre facile determinare se alla base di un episodio di rabdomiolisi vi sia una patologia genetica o se sia esclusivamente conseguenza di uno sforzo, di un episodio infettivo o dell’effetto di un tossico. L’obiettivo principale del presente studio è stato, pertanto, definire l’iter diagnostico ottimale in caso di rabdomiolisi, al fine di proporne uno aggiornato, completo, razionale ed economico che includa le più recenti nozioni e tecniche. Abbiamo inizialmente valutato l'iter diagnostico utilizzato ad oggi nei pazienti che presentano rabdomiolisi, in un ampio studio retrospettivo che ha incluso 208 pazienti (aim 1). Abbiamo quindi studiato le caratteristiche cliniche, radiologiche e molecolari di una causa genetica di rabdiomiolisi di recente identificazione, la miopatia da mutazoine nel gene CASQ1 (aim 2). Abbiamo in seguito valutato lo spettro clinico della distrofia dei cingoli tipo 2 E (LGMD2E), valutando il rischio di rabdomiolisi in tale patologia (aim 3). Abbiamo poi dimostrato il ruolo di EMG con test provocativi (Long Exercise Test) come test sensibile e specifico utilizzabile in pazienti affetti da rabdomiolisi, in particolare nella glicogenosi tipo 5 (aim 4). Abbiamo infine creato un pannello di geni che dovrebbe essere studiato mediante Next Generation Sequencin (NGS) in pazienti affetti da rabdomiolisi (aim 5). In conclusione, con le evidenze raccolte nel presente studio, proponiamo un nuovo algoritmo diagnostico per le rabdomiolisi. In tale algoritmo le caratteristiche cliniche e pochi test di primo livello (esami ematochimici, EMG, profilo delle acilcarnitine, “grip test”) permettono di escludere le cause più frequenti o trattabili. Tale algoritmo permetterà di contenere i costi ed ottimizzare i tempi della diagnosi, e auspicabilmente aumenterà il rate di diagnosi etiologiche della rabdomiolisi, un evento grave e con un notevole impatto sulla vita dei pazienti ma che ad oggi resta scarsamente diagnosticato.

Das RNA-guided adaptive Immunsystem CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) immunisiert prokaryotische Zellen gegenüber mobilen genetischen Elementen (MGEs). Bei der Adaption wird eine kurze Nukleinsäurensequenz (prespacer) von den MGEs gewonnen, verarbeitet und schließlich als spacer in das CRISPR-Array integriert. Cas1 und Cas2, die Hauptbestandteile der Adaption, bilden einen Integrase-Komplex, welcher neue spacer in das CRISPR-Array integriert. Der molekulare Mechanismus für die Adaptiondes Typ II-A Systems, welches cas9, cas1, cas2, csn2 und tracrRNA codiert, ist bis heute nicht vollständig verstanden. Daher untersuchten wir die Anforderungen der verschiedenen Cas-Proteine für den Adaptionsprozess. Wir verifizierten die Adaptions-Aktivität von Typ II-A Systemen des Streptococcus thermophilus LMD-9 anhand von Adaptionsstudien nach Phagen-Infektion. Dabei beobachteten wir höhere Akquisitionsraten im CRISPR3-Lokus im Vergleich zum CRISPR1-Lokus. Unsere Plasmid-basierte Adaptionsstudie bestätigte die Notwendigkeit von Cas9, zusätzlich zu Cas1, Cas2 und Csn2 bei der Adaption. Der yeast two-hybrid und der pull-down Ansatz zeigten sowohl spezifische Interaktionen zwischen den Cas-Proteinen, als auch Interaktionen zwischen Cas-Proteinen sowie DNA-Reparatur Proteinen. Die Regionen der Cas1 und Cas9 Interaktion wurden durch SPOT peptide assay identifiziert. Zusammenfassend weist unsere Studie darauf hin, dass Cas-Proteine sowohl mit Proteinen innerhalb, als auch außerhalb des CRISPR-Cas Systems interagieren, und bietet somit eine Basis für die Erforschung der möglichen Funktionen von DNA-Reparatur Proteinen in CRISPR-Cas Systemen und vice versa.<br>The RNA guided adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats) Cas (CRISPR-associated) immunizes prokaryotic cells against mobile genetic elements (MGEs). During spacer acquisition stage, a short nucleic acid sequence (prespacer) is acquired from the MGEs, processed and finally integrated into the CRISPR array as a spacer, which serves as genetic memory to defend against the invasion of the cognate MGEs. The molecular mechanism for the spacer acquisition of the type II A systems, which encode cas9, cas1, cas2, csn2 and tracrRNA, is still not fully understood. Therefore, we investigated the requirement of the different Cas proteins for spacer acquisition. We verified the acquisition activity of the type II A systems of Streptococcus thermophilus LMD 9 via spacer acquisition studies by phage challenge. We observed higher acquisition rates in the CRISPR3 locus compared to the CRISPR1 locus. Our plasmid-based spacer acquisition study confirmed in addition to Cas1, Cas2 and Csn2 the requirement of Cas9 for spacer acquisition. Yeast two hybrid and pull down approaches revealed specific interactions among the Cas proteins, as well as interactions between Cas and DNA repair proteins. The interaction regions of Cas1 with Cas9 were identified by SPOT peptide assay. Altogether, our study suggests that Cas proteins interact with proteins within and beyond the CRISPR Cas systems, and it provides a basis for the investigation of the potential roles of DNA repair proteins in the CRISPR Cas systems and/or vice versa.

Causal explanations that individuals use to explain events in their lives are referred to as explanatory style. Three dimensions: internal-external, stable-unstable, and global-specific have most frequently been measured. Internal, stable, global explanations for negative events represent a pessimistic style, whereas these same explanations for positive events are considered optimistic. Explanations for negative events that are stable and global are considered to reflect hopelessness. The psychometric properties of the most commonly used measure of explanatory style for children, the Children Attributional Style Questionnaire (CASQ; Kaslow, Tanenbaum & Seligman, 1978) are poor. This is a limitation to research and theoretical advancement. Four studies were conducted in this project to investigate the measurement of explanatory style in 9-12 year old children. In Study 1, children (N = 173) completed the CASQ in a group to investigate the psychometric properties of the composite scales and subscales and the relationship between explanatory style and depressive symptoms. Internal consistency and inter-item correlations of the composite scales and subscales were poor. Regression analyses showed explanatory style for negative events (pessimism or hopelessness) made weak but significant unique contributions to the explanations of depressive symptoms. Study 2 (N = 72) investigated the stability of the CASQ scales longitudinally. The internal consistency and inter-item correlations for the CASQ scales were poor. The stability of explanatory style was low. The predicted relationship between depressive symptoms and explanatory style was found to be inconsistent, emerging at Time 1 but not at Time 2, 12 months later. Study 3 (N = 79) examined the forced-choice response scale of the CASQ using a fuzzy set approach. A fuzzy set scale which uses a Likert-type response that ranged from completely true to completely false was used to determine how well a child’s response of choice, their natural response, matched both the selected and non-selected response from the CASQ. Items on the CASQ that measure both pessimism and hopelessness were found to be a poor match to the natural responses of children. Little separation was found between the selected and non-selected responses for all items. The internal consistency of the CASQ was poor when the forced choice scoring approach was used. When Likert-type fuzzy values were used, good internal consistency was obtained. Providing a wider range of responses, obtained using fuzzy values, produced a more sensitive measure of the components of explanatory style. When the CASQ was scored according to the forced choice protocol weak, significant relationships were found between explanatory style and depressive symptoms, and explanatory style and neuroticism. There were no significant relationships found for either pessimism or hopelessness, with either depression or neuroticism using Likert-type fuzzy values. Study 4 elicited spontaneous causal explanations following success or failure on tasks that were familiar or unfamiliar. Task familiarity was manipulated. Using an interview format, children (N = 111) responded to questions, eliciting causal explanations, following task success or failures. Likert-type scales measured the internality, stability or globality of the explanation. Results showed that, following failure on two familiar tasks, acceptable levels of internal consistency were obtained on the subscales used to produce the measure of hopelessness and for the composite measure of hopelessness. This same pattern did not emerge following failure on combinations of familiar and unfamiliar events or on two tasks that were unfamiliar. Stable and global explanations and the composite measure of hopelessness, following failure on familiar tasks, were also positively related with depressive symptoms but not neuroticism. These results show that a reliable measure of hopelessness can be obtained from spontaneous explanations for failure at familiar events. Under these conditions the theoretically predicted relationship between explanatory style and depressive symptoms emerges. Conclusions were drawn about the theoretical conceptualisation of explanatory style and measurement recommendations were made that apply to 9- to 12-year-old children. Explanations for familiar events produced a consistent measure of explanatory style. The use of a Likert-type response scale to assess agreement with internal, stable, global components were shown to improve scale reliability. The findings are discussed in relation to theory and the measurement of explanatory style in children.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Phychology<br>Griffith Health<br>Full Text

This study explores the use of two tests of optimism: the Youth Life Orientation Test (YLOT) and the Children’s Attributional Style Questionnaire (CASQ) in six English primary schools with children aged between 9 years and 12 years. The study grew out of some problems I was confronted with as part of my professional practice regarding the outcomes for children in a school that was failing to meet Government Targets in attainment. In the study I worked with the staff and 9 – 11 year old children in six primary schools in rural, town, suburban and inner-city contexts. The total number of children was 305. The children were tested using the CASQ and YLOT and a range of other measures. Cronbach alpha internal consistency coefficients (coefficient alpha) and test-retest coefficients for the subscales and overall scale of the CASQ and YLOT were calculated. The validity of each measure was explored using evidence from: test content; internal structure; relations to other variables; and from the consequences of testing. Lastly the use of the YLOT as a proxy and nature of any associations between the measures used was explored looking at individual; school and community level data. The study found that the YLOT has good psychometric properties and could be used as a basis for further work both professionally and for research. The Cronbach alpha reliability coefficient for the whole scale was 0.81. The psychometric properties of the CASQ were poor in that the subscales had very low reliability coefficients and the aggregated total scale reliability coefficient was still too low at 0.53 to be able to recommend the use of the CASQ. Before the CASQ could be used there would need to be extensive work to increase its reliability and validity through lengthening the test or changing the format of the questions to reduce their specificity. The use of the YLOT as a proxy indicator of mental health and associations with school and community level data were discussed. The YLOT could provide an indication of well being particularly in relation to childhood depression. The community level data were not sensitive enough to discern hypothesised associations between communities and the children attending the schools sited in the communities.

Ces travaux ont porté sur la mise en place et le développement d’un pilote d’oxydation bactérienne de l’As(III) en As(V), pour la potabilisation et le traitement d’effluents contaminés en arsenic. Le procédé d’oxydation biologique de l’arsenic fait appel à consortium bactérien autotrophe apparenté au genre Thiomonas, le consortiumCAsO1. Deux techniques de dosages ont été mises au point : le dosage d’As(total) par Spectrométrie d’Absorption Atomique Four Graphite (GF-AAS), et celui d’As(III), par Spectrométrie d’Absorption Atomique avec Génération d’Hydrures (HG-AAS), pour des domaines de concentration de 0,2 à 20 µg. L-1. Le pilote de traitement et le matériau support (pouzzolane) ont été caractérisés (DTS, capacité d’adsorption…). Ainsi, les performances d’oxydation de CAsO1 ont été évaluées : pour des temps de séjours supérieurs à 2 heures, 95% de la concentration en As(III) est oxydé, quel que soit le type d’alimentation étudié (ascendante ou descendante) et plus de 80%, pour un temps de séjour de 1 heure<br>This work concerned the evaluation and the development of an arsenic(III)-oxidizing population in reactors, for drinking water production and waste water arsenic contaminated treatment. The process of the biological oxidation of arsenite was carried out with an autotrophic bacterial population named CAsO1. This population was phylogenetically related to Thiomonas. Two easy to handle analytical methods were developed: the determination of total arsenic was carried out by Graphite Furnace Atomic Absorption Spectrometry (GF-AAS), and that concerning As(III), by Hydride Generation Atomic Absorption Spectrometry (HG-AAS), for concentration range from 0,2 to 20 µg. L-1. The treatment pilot and the support material (pozzolana) were characterized (RTD, adsorption capacity. . . ). Thus, performances of oxidation of CAsO1 were evaluated: for residence times higher than 2 hours, 95% of As(III) was oxidized, whatever the type of circulation (up- or down-flow) and more than 80%, for a residence time of 1 hour

Les cardiomyocytes dérivés des cellules souches pluripotentes humaines induites (hiPSC-CMs) représentent des modèles in vitro prometteurs pour plusieurs applications scientifiques et thérapeutiques allant de la modélisation de pathologies à la découverte de médicaments et de la toxicologie prédictive à la médecine régénérative. Malgré les nombreux progrès dans ce domaine, les protocoles de différenciation actuels ne permettent pas d’atteindre le stade de maturité que l’on retrouve chez le myocarde adulte de l’Homme. En effet, certaines caractéristiques majeures des hiPSC-CMs demeurent similaires à celles de cardiomyocytes fœtaux telles que l’expression de plusieurs gènes cardiaques, l’électrophysiologie ou leur fonction contractile. En effet, des analyses transcriptomiques réalisés au sein de notre laboratoire à Sanofi ont révélé que les gènes KCNJ2 et CASQ2, impliqués respectivement dans l’électrophysiologie et la gestion du calcium, étaient sous-exprimés dans les hiPSC-CMs en comparaison aux cardiomyocytes adultes. Cette thèse avait pour objectif d’améliorer la maturation des hiPSC-CMs en utilisant des technologies d’édition du génome. Ainsi, nous avons généré des lignées stables de hiPSC-CMs qui expriment de manière inductible KCNJ2 ou CASQ2 ou les deux gènes simultanément puis nous avons examiné leurs phénotypes fonctionnels et électrophysiologiques par le biais de méthodes d’analyses complémentaires. A la suite à l’induction de l’expression de KCNJ2 et CASQ2 par la doxycycline, les hiPSC-CMs montraient des bénéfices phénotypiques tels que la diminution drastique de la fréquence des battements spontanés, une hyperpolarisation du potentiel de repos membranaire, la diminution de la durée du potentiel d’action et l’amélioration du flux de calcium transitoire. En plus de ces bénéfices attendus, l’expression concomitante de ces deux gènes a amélioré la pente de la pointe du potentiel de champ extracellulaire associée au courant sodique ainsi que la gestion du calcium. Nous avons ensuite évalué le bénéfice de l’expression de ces transgènes sur la toxicologie prédictive en testant des molécules agonistes ou antagonistes de canaux ioniques utilisées classiquement dans le cadre des essais précliniques de toxicité cardiaque. Nous avons notamment observé plus d’arythmies induites par l’E4031 avec les hiPSC-CMs exprimant conjointement KCNJ2 et CASQ2 par rapport aux cardiomyocytes contrôles. Ainsi, les hiPSC-CMs exprimant simultanément KCNJ2 et CASQ2 présentent un phénotype plus mature que les hiPSC-CMs natifs et de tels cardiomyocytes édités génétiquement peuvent être utiles pour l’évaluation de la toxicité cardiaque de nouveaux médicaments candidats<br>Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a very promising model for several scientific and therapeutic applications ranging from disease modeling to drug discovery, and from predictive toxicology to regenerative medicine. Despite numerous efforts, current protocols do not yet lead to a maturation phenotype equivalent to adult human myocardium. Indeed, key features of hiPSC-CMs remaining closer to fetal stages of development, such as gene expression, electrophysiology and function. Transcriptome analysis performed at Sanofi have confirmed these findings at the genome-wide level. Indeed, KCNJ2 and CASQ2 which are implicated in the two major physiological characteristics of cardiac cells, their electrophysiological behavior and calcium handling, respectively, were expressed at very low levels in hiPSC-CMs in comparison with adult cardiomyocytes. This thesis aimed to improve the maturation of hiPSC-CMs by using genome editing technologies. We generated stable hiPSC-CMs with inducible expression of KCNJ2, or CASQ2 or both genes (KCNJ2-CASQ2 hiPSC-CMs) and studied their functional and electrophysiological phenotype by several complementary methods. Upon doxycycline induction of KCNJ2 and CASQ2, KCNJ2-CASQ2 hiPSC-CMs displayed phenotypic benefits expected from previous studies of each maturation gene, including a drastic reduction of spontaneous beating, hyperpolarized resting membrane potential, shortened action potential duration and enhanced calcium transients. In addition, co-expression of the two genes enhanced Na+ spike slope of extracellular field potential and Ca2+ handling. We tested four reference drugs and observed signatures of known cardiac effects in KCNJ2-CASQ2 hiPSC-CMs, including arrhythmias induced by QT prolonging drug (E-4031), which were more easily detected than in control hiPSC-CMs. Therefore, KCNJ2-CASQ2 hiPSC-CMs exhibited a more mature phenotype than hiPSC-CMs and such genetically engineered hiPSC-CMs could be useful for testing cardiac toxicity of novel candidate drugs

Les objectifs essentiels de cette thèse portent (i) sur l'examen du cadre théorique sur lequel se fonde la théorie du " Mode explicatif " (e. G. , Abramson, Seligman, Teasdale, 1978), (ii) sur la validation d'un outil permettant une évaluation des modes explicatifs chez les enfants pratiquant des activités sportives diverses, et (iii) sur l'étude des effets des modes explicatifs sur des variables comportementales. La première partie présente les antécédents et les conséquences du mode explicatif chez des enfants âgés de 8 à 14 ans. La deuxième partie comporte une dizaine d'études ayant pour but de présenter la validation de l'outil de mesure, version française courte du CASQ (QEMEE-R), contextualisée à la fois pour les domaines scolaire et sportif. La troisième partie a pour but de présenter succinctement les bases théoriques d'un modèle inspiré des travaux d'Eccles et Wigfield (2002) incluant deux études visant à tester à la fois les effets du mode explicatif sur les résultats scolaires et sur les performances sportives. L'ensemble des études a permis de mettre en évidence son organisation hiérarchique, du plus général au plus contextualisé, et l'existence non pas de deux profils, optimiste et pessimiste, mais de quatre profils. Ces deux aspects constituent une évolution théorique dans le domaine de la recherche sur les modes explicatifs<br>The thesis aims to (i) valid, purify a short French version from “Children's Attributional Style Questionnaire, CASQ”, this one would be adapted in French culture and with sport children; (ii) examine the theoretical conceptual model of the attributional/explanatory style not only in sport but also in school context. It is based upon data stemming from the Attributional Reformulation of Helplessness Theory (e. G. , Abramson, Seligman, Teasdale, 1978). Despite its interest, we found a very little studies in school context for children have been realised, nothing of studies concerning the children in the sport context. The thesis consists of three parts: the first one presents the theoretical base of explanatory style theory, studies concerning the psychological effects of explanatory style for children and adolescents in many fields. In the second parts, using many methods and samples, we examine the psychometric proprieties of an experimental French version of CASQ (QEMEE), and purify a short version (QEMEE-R). Basing to this version and in order to examine many theoretical hypothesis, we contextualise a version in sport and in school context. In the third parts, we realised two studies in sport and school contexts. Basing to Eccles and Wigfield model (2002), we tested three hypotheses concerning the effect of the contextualised and/or general explanatory style to predict the result in sport and in school domains. These two studies confirm the existence of (i) hierarchical organisation of children's the explanatory style, (ii) not only two but four profiles of children's explanatory style. These result constitute an theatrical evaluation in explanatory style search

La polyarthrite rhumatoïde (PR) est unemaladie auto-immune complexe qui entraîne uneinflammation synoviale et une hyperplasie pouvantprovoquer une érosion osseuse et une destruction ducartilage dans les articulations. L'étiologie de la PR restepartiellement inconnue, mais elle implique de multiplescascades de signalisation croisées et l'expression demédiateurs pro-inflammatoires. Dans la première partie demon projet de doctorat, nous présentons un effortsystématique pour construire une base de connaissancessur la PR, entièrement annotée et validée par des experts.Cette carte de la PR illustre les voies moléculaires et designalisation importantes impliquées dans la maladie. Latransduction du signal est systématiquement représentéedes récepteurs au noyau en utilisant la représentationstandard de notation graphique en biologie des systèmes(SBGN). La curation manuelle est basée sur des critèresstricts et spécifique aux études sur l'homme, limitantl'apparition de faux positifs sur la carte. Cette carte peutservir de base de connaissances interactive pour la maladiemais aussi de tableau pour la visualisation des donnéesomiques. De plus, c’est une excellente base pour ledéveloppement d'un modèle informatique. La naturestatique de la carte PR pourrait fournir une compréhensionrelativement limitée du comportement émergeant dusystème dans différentes conditions. La modélisationinformatique pourra révéler les propriétés dynamiques duréseau par le biais de perturbations in silico et peut êtreutilisée pour tester et prédire des hypothèses.Dans la deuxième partie du projet, nous présentons unpipeline permettant la construction automatisée d'un grandmodèle booléen, à partir d'une carte d'interactionsmoléculaires. Pour cela, nous avons développé l'outilCaSQ (CellDesigner as SBML-qual), qui automatise laconversion des cartes moléculaires en modèles booléensexécutables basés sur la topologie et la sémantique descartes. Le modèle booléen résultant pourrait être utilisépour des simulations in silico afin de reproduire lecomportement biologique connu du système et de prédirede nouvelles cibles thérapeutiques. Pour l'analyse deperformance de l’outil, nous avons utilisé différentescartes et modèles de maladies en mettant l'accent sur lagrande carte moléculaire de la PR.Dans la troisième partie du projet, nous présentons nosefforts pour créer un modèle dynamique (booléen) àgrande échelle pour les synoviocytes de type fibroblastede polyarthrite rhumatoïde (RA-FLS). Parmi denombreuses cellules de l'articulation et du systèmeimmunitaire impliquées dans la pathogenèse de la PR, lesRA-FLS joue un rôle important dans l'initiation et laperpétuation de l'inflammation articulaire destructrice.Les RA-FLS expriment des cytokinesimmunomodulatrices, des molécules d'adhésion et desenzymes de modélisation matricielle. De plus, les RAFLSprésentent des taux de prolifération élevés et unphénotype résistant à l'apoptose. Les RA-FLS peuventégalement se comporter comme les principaux moteurs del'inflammation, et les thérapies dirigées contre les RA FLSpourraient devenir une approche complémentaire auximmunothérapies. Le défi est de prédire les conditionsoptimales qui favoriseraient l'apoptose des RA FLS,limiteraient l'inflammation, ralentiraient le taux deprolifération et minimiseraient l'érosion osseuse et ladestruction du cartilage<br>Rheumatoid arthritis (RA) is a complexautoimmune disease that results in synovial inflammationand hyperplasia leading to bone erosion and cartilagedestruction in the joints. The aetiology of RA remainspartially unknown, yet, it involves a variety of intertwinedsignalling cascades and the expression of pro-inflammatorymediators. In the first part of my PhD project, we present asystematic effort to construct a fully annotated, expertvalidated, state of the art knowledge-base for RA. The RAmap illustrates significant molecular and signallingpathways implicated in the disease. Signal transduction isdepicted from receptors to the nucleus systematically usingthe systems biology graphical notation (SBGN) standardrepresentation. Manual curation based on strict criteria andrestricted to only human-specific studies limits theoccurrence of false positives in the map. The RA map canserve as an interactive knowledge base for the disease butalso as a template for omic data visualization and as anexcellent base for the development of a computationalmodel. The static nature of the RA map could provide arelatively limited understanding of the emerging behaviorof the system under different conditions. Computationalmodeling can reveal dynamic network properties throughin silico perturbations and can be used to test and predictassumptions.In the second part of the project, we present a pipelineallowing the automated construction of a large Booleanmodel, starting from a molecular interaction map. For thispurpose, we developed the tool CaSQ (CellDesigner asSBML-qual), which automates the conversion ofmolecular maps to executable Boolean models based ontopology and map semantics. The resulting Booleanmodel could be used for in silico simulations to reproduceknown biological behavior of the system and to furtherpredict novel therapeutic targets. For benchmarking, weused different disease maps and models with a focus onthe large molecular map for RA.In the third part of the project we present our efforts tocreate a large scale dynamical (Boolean) model forrheumatoid arthritis fibroblast-like synoviocytes (RAFLS).Among many cells of the joint and of the immunesystem involved in the pathogenesis of RA, RA FLS playa significant role in the initiation and perpetuation ofdestructive joint inflammation. RA-FLS are shown toexpress immuno-modulating cytokines, adhesionmolecules, and matrix-modelling enzymes. Moreover,RA-FLS display high proliferative rates and an apoptosisresistantphenotype. RA-FLS can also behave as primarydrivers of inflammation, and RA FLS-directed therapiescould become a complementary approach to immunedirectedtherapies. The challenge is to predict the optimalconditions that would favour RA FLS apoptosis, limitinflammation, slow down the proliferation rate andminimize bone erosion and cartilage destruction

碩士<br>國立成功大學<br>臨床醫學研究所<br>102<br>Ovarian cancer is the most lethal gynecological malignancy. One of the main reasons for this highest fatality rate is that the molecular pathogenesis of ovarian cancer is poorly understood. To improve clinical outcomes, it would be desirable to identify of ovarian cancer-associated genes for uncovering the pathogenesis. Based on microarray analysis performed in our laboratory, a dysregulated gene CASZ1, which encodes a zinc finger transcription factor and functions as a tumor suppressor, was identified and found that expressed at higher levels in ovarian cancer. The apparently contrasting expression patterns and the association of CASZ1 with ovarian cancer are unknown. Here, the elevated expression of CASZ1 was confirmed by quantitative RT-PCR in ovarian cancer cell lines and tumor tissues. Moreover, higher CASZ1 expression was significantly associated with poor progression-free survival. To investigate the functional relevance of CASZ1 for malignant behavior in ovarian cancer, CASZ1 was stably knocked down using lentiviral-mediated shRNA constructs in MCAS and RMUG cell lines. Inhibition of CASZ1 expression significantly reduced the migration and invasion ability of ovarian cancer cells, though did not appear to affect cell proliferation. Using phalloidin staining, we observed a significant decrease in filopodia formation at leading edge in MCAS and RMUG cell lines silencing CASZ1. Besides, knockdown of CASZ1 showed a 50-60% decrease in soft agar clonogenicity. We also transfected CASZ1a and CASZ1b in TOV21G and A2780CP70 cell lines and demonstrated that both CASZ1a and CASZ1b promote cell migration and invasion. Furthermore, when injected CASZ1-depleted cells via the tail vein into NOD/SCID mice, lung tumor nodules were significantly decreased. These results suggested that CASZ1 may play a role in ovarian cancer cell migration and invasion, and that inhibition of CASZ1 expression could serve as a therapeutic strategy to overcome ovarian cancer.

Malaria is a global health concern responsible for thousands of deaths worldwide annually. Concerted efforts employing various preventive and treatment strategies have contributed a lot in controlling the disease, but we are still far from achieving complete eradication. Antimalarial drugs have been on the forefront of malaria control strategies, but their effectiveness is jeopardised due to increasing development of drug resistance in parasites. Interestingly, it has been noted that certain genetic mutations, especially those in red blood cells, confer natural resistance against malaria in endemic populations. Screening such mutations have promising translational potential as they may lead towards novel therapeutic targets or host-directed-therapies to overcome malaria resistance. In this study, N-Ethyl-N-Nitrosourea (ENU) mutagenesis, has been used as a tool to screen for novel erythrocyte variants in mouse model and assess their role in malaria susceptibility. A recent phenotypic screen for red cell abnormality identified a novel Casd1 gene mutation in mice that conferred a complete protective effect against P. chabaudi and P. berghei rodent malaria parasite. Casd1 is a eukaryotic 7(9)-O-acetyltransferase that resides in the lumen of Golgi complex as a membrane integrated protein. It functions by acetylating sialic acid residues that are subsequently decorated on the cell surface. The mutation was characterised to be a single base substitution at the splice site of Casd1 gene leading to skipping of the exon 14. An in silico analysis of the secondary structure of mutant Casd1 protein predicted a loss of connecting loop between two transmembrane helices affecting the topology of transmembrane domain. The initial screen identified a loss of Ter119 binding to erythrocytes derived from Casd1 mutated mice (ENU25^Casd1/Casd1). Ter119 is an erythroid lineage marker that recognises 7-9-di-O acetyl sialic acid epitope on the erythrocyte surface. Abrogation of Ter119 signal from the erythrocyte surface may indicate the loss of Casd1 function in ENU25^Casd1/Casd1 mice. The erythrocytes from ENU25^Casd1/Casd1 mice were further assessed for their susceptibility to invasion by malaria parasites. The relative parasitaemia was significantly reduced in the mutant erythrocytes as compared to the wild type, suggesting a novel role of Casd1 in merozoite invasion. Furthermore, the parasitised erythrocytes were tracked during infection assays to assess the role of Casd1 mutation in clearance of malaria parasites. These assays revealed a drastic clearance of exogenous blood injected into ENU25^Casd1/Casd1 mice as compared to wildtype blood. The clearance response in ENU25^Casd1/Casd1 mice was subsequently identified to be dose dependent and non-specific to malaria infection. Overall, these findings suggest that Casd1 plays a role in malaria susceptibility by modulating merozoite invasion. The results also revealed a novel role of Casd1 in self-nonself recognition which results in bystander clearance of the malaria infected foreign red blood cells in ENU25^Casd1/Casd1 mice.

Fragestellung: Um die Auswirkungen von genetischen Varianten für CASP1 in vivo analysieren zu können, sollte in dieser Arbeit ein Tiermodell generiert werden. Dadurch könnten die zugrundeliegenden Pathomechanismen der an „ICE-Fieber“ leidenden Patienten in einem gesamten Organismus aufgeklärt werden, da die Untersuchung von Patientenmaterial nur sehr eingeschränkt möglich ist. Ein Mausmodell stellt eine der besten Alternativen zur Analyse von Primärmaterial dar, da die Immunsysteme von Maus und Mensch sehr ähnlich sind. Ergebnisse: Um die natürliche Expression und Regulation der Procaspase-1 im Mausmodell zu gewährleisten, wurde für die Generierung ein BAC-Transgen verwendet. Mittels homologer Rekombination wurde die künstliche Variante C284A von Casp1 inseriert. Diese führt zu einer Zerstörung des enzymatischen Zentrums der Caspase-1 und zum vollständigen Verlust der enzymatischen Aktivität. Nach zwei Pronukleusinjektionen konnten lediglich drei Gründertiere mit mosaikartiger Expression des zufällig im Genom integrierten Transgens Casp1C284A und in der F1-Generation nur ein Tier mit stabil integriertem Transgen identifiziert werden. Die Nachkommen dieses transgenen Tieres zeigten keine basale Expression von Casp1C284A, jedoch konnte nach Stimulation von BMDCs mit LPS in vitro die Expression sowohl auf RNA- wie auch auf Proteinebene nachgewiesen werden. Ebenfalls eine erhöhte Sekretion der proinflammatorischen Zytokine TNF-α und IL-6 wurde in den Zellen der transgenen Tiere detektiert. Gleichfalls konnte nach Stimulation mit LPS in vivo eine gesteigerte Entzündungsreaktion in den Tieren mit Casp1C284A gezeigt werden, da ein stärkerer und länger anhaltender Abfall der peripheren Körpertemperatur und außerdem eine gesteigerte Sekretion von TNF-α und IL-6 zu verzeichnen war. Eine gesteigerte Inflammation des fetalen Gewebes, ausgelöst durch die integrierte künstliche Variante der Procaspase-1, könnte die Frage nach der geringen Anzahl der generierten Gründertiere beantworten. Hierfür wurde weiterführend eine Maus mit einem konditionalen Casp1C284A-Konstrukts generiert, welches zusätzlich eine zeit- als auch zelltyp-spezifische Expression der Procaspase-1 mit zentraler Mutation ermöglicht. Nach erfolgreichem Screening der ES-Zellen konnten diese in Blastozysten mikroinjiziert und Chimäre identifiziert werden. Eine embryonale Letalität des transgenen Konstrukts konnte durch die Verpaarung der ki-Tiere R26_Casp1C284A mit PGK-Cre-Tieren, die Cre-Rekombinase ubiquitär exprimieren, nahezu ausgeschlossen werden, da die Nachkommen alle lebensfähig waren und eine basale Expression des „knock-ins“ in mehreren Organen und auch in BMDCs nachgewiesen wurde. Ferner konnte nach Induktion einer Inflammation in vivo mit einer subletalen Dosis von LPS ein gesteigerter und länger anhaltender peripherer Temperaturabfall in den ki-Tieren ähnlich zu den transgenen Tieren detektiert werden. Desgleichen wurde eine Tendenz zu einer gesteigerten Sekretion der Zytokine TNF-α und IL-6 verzeichnet. Schlussfolgerungen: Mit dem transgenen Casp1C284A-Mausmodell als auch mit dem konditionalen R26_Casp1C284A-Modell konnte gezeigt weren, dass eine inaktive Variante der Procaspase-1 zur Entstehung einer gesteigerten Inflammation in einem gesamten Organismus führen kann. Somit können die im Rahmen dieser Arbeit generierten Tiermodelle zur Analyse der Pathomechanismen der an „ICE-Fieber“ leidenden Patienten herangezogen werden. In künftigen Studien kann ferner geklärt werden, ob die Entzündungsreaktionen durch eine verstärkte Interaktion von Casp1C284A mit der Kinase RIP2 verursacht werden und dadurch ähnlich wie im Patienten eine Aktivierung des proinflammatorischen Moleküls NFκB ausgelöst wird. Im Anschluss könnte eine spezifische Inhibierung des RIP2-Signalweges in diesen Mausmodellen getestet werden und schließlich im Patienten Anwendung finden. Die in dieser Arbeit generierten Mausmodelle könnten somit zur Erprobung zukünftiger therapeutischer Konzepte dienen.:Inhaltsverzeichnis Abkürzungsverzeichnis 1 Einleitung 1.1 Das angeborene und adaptive Immunsystem 1.2 Sensoren des angeborenen Immunsystems 1.2.1 Membran-gebundene Rezeptoren 1.2.2 Intrazelluläre PRRS 1.2.3 Inflammasome als Multiproteinkomplexe 1.2.4 Das NLRP3-Inflammasom 1.2.5 Metabolische Krankheiten, die mit Inflammasom-Aktivität assoziiert werden 1.2.6 Inflammasom-assoziierte autoinflammatorische Erkrankungen 1.2.6.1 Das Cryoporin-assoziierte periodische Syndrom (CAPS) 1.2.6.2 Familian Mediterranean Fever (FMF) 1.2.6.3 Pyogenic arthritis, pyoderma gangrenosum and acne syndrome (PAPA) 1.3 Caspase-1 1.3.1 Caspasen im Allgemeinen 1.3.2 Der Caspase-1 Gen-Lokus und das Caspase-1 Gen 1.3.3 Das Caspase-1 Protein 1.3.4 Funktionen von Caspase-1 1.3.4.1 Prozessierung der Zytokine pro-IL-1ß und pro-IL-18 1.3.4.2 Induktion von Pyroptose 1.3.4.3 Aktivierung der Caspase-1 durch ER-Stress 1.3.4.4 Aktivierung des Transkriptionsfaktors NFκB 1.3.4.5 weitere Funktionen 1.3.5 Caspase-1 Genvarianten und Entzündung 1.4 Mausmodelle 1.4.1 NLRP3 1.4.2 Pyrin 1.4.3 Caspase-1 2 Zielsetzung 3 Material und Methoden 3.1 Material 3.1.1 Antibiotika 3.1.2 Antikörper 3.1.3 Bakterienstämme 3.1.4 BAC-Klon 3.1.5 Plasmide 3.1.6 Zelllinie 3.1.7 Chemikalien und Substanzen 3.1.8 Enzyme 3.1.9 Größenstandards 3.1.10 Oligonukleotide 3.1.11 Kommerzielle Kits 3.1.12 Puffer und Lösungen 3.1.13 Mauslinien 3.1.14 Medien 3.1.15 Geräte 3.1.16 Software 3.2 Molekularbiologische Methoden 3.2.1 Agarosegelelektrophorese 3.2.2 Amplifikation von Nukleinsäuren mittels Polymerasekettenreaktion 3.2.3 Aufreinigung von DNA-Fragmenten aus Agarosegelen 3.2.4 Aufreinigung von BAC-DNA mittels Elektroelution 3.2.5 Aufreinigung von Subklon-Plasmid-DNA für die Elektroporation von ES-Zellen 3.2.6 Bestimmung der Konzentration und der Reinheit von DNA und RNA 3.2.7 c-DNA-Synthese 3.2.8 Dephosphorylierung von 5´-Phosphatresten 3.2.9 DNA-Isolierung 3.2.9.1 DNA-Isolierung aus Bakterienzellen 3.2.9.2 DNA-Isolierung aus eukaryotischen Zellen 3.2.10 DNA-Sequenzierung 3.2.11 Enzymatische Restriktionsspaltung von DNA-Fragmenten 3.2.12 Homologe Rekombination von DNA-Sequenzen 3.2.13 Ligation von DNA-Fragmenten mit linearisierten Vektoren 3.2.14 Phenol-Chloroform-Extraktion 3.2.15 Pulsfeldgelelektrophorese 3.2.16 Quantitative Real-Time-PCR (qRT-PCR) 3.2.17 RNA-Isolation aus eukaryotischen Zellen und Geweben 3.2.18 Southern Blot 3.3 Mikrobiologische Methoden 3.3.1 Kultivierung von Bakterien 3.3.2 Hitzschock-Transformation von E.coli 3.3.3 Kryokonservierung 3.4 Proteinbiochemische Methoden 3.4.1 CBA (cytokine bead assay) 3.4.2 Proteinbestimmung 3.4.3 Immunpräzipitation mit Caspase-1 p10 (M-20) Antikörper von Santa Cruz 3.4.4 Immunpräzipitation mit AntiFlag M2 Affinity Gel von Sigma 3.4.5 Diskontinuierliche SDS-Polyacrylamid-Gelelektrophorese (SDS-PAGE) 3.4.6 Western Blot 3.4.7 Immunchemische Detektion von Proteinen 3.5 Zellbiologische Methoden 3.5.1 Bestimmung der Zellzahl 3.5.2 Isolierung und Generierung von BMDCs 3.5.3 Kryokonservierung von Zellen 3.5.4 Kultivierung von Zelllinien 3.6 Tierexperimentelle Methoden 3.6.1 Genotypisierung 3.6.2 Haltung 3.6.3 Hautbiopsie 3.6.4 Retroorbitale Blutentnahme und Herzpunktion 3.6.5 Injektion von LPS 3.6.6 Organentnahme 4 Ergebnisse 4.1 Generierung eines Casp1C284A-BAC-Transgens 4.1.1 Identifikation und Verifizierung des BAC-Klons 4.1.2 Klonierung des Casp1C284A-BAC-Transgens 4.1.2.1 Entfernung der wt-loxP-Stelle vom Vektorrückrat 4.1.2.2 Insertion eines Flag-Tags 4.1.2.3 Einführung der Punktmutation C284A 4.1.3 „Shaving“ des generierten Casp1C284A-BAC-Konstrukts 4.1.4 Aufreinigung des Casp1C284A-BAC-Konstrukts und Pronukleusinjektion 4.1.5 Genotypisierung der transgenen Casp1C284A Gründertiere 4.2 Charakterisierung der transgenen Casp1C284A-Tiere (tg) 4.2.1 In vitro-Stimulation von BMDCs mit LPS 4.2.2 In vivo-Stimulation der transgenen Casp1C284A-Tiere 4.3 Generierung eines konditionalen Casp1C284A-Konstrukts 4.3.1 Klonierung des Targetingvektors pSerc_Casp1C284A_Flag und des Kontroll-Targetingvektors pSerc_Casp1_Flag 4.3.2 Screening von ES-Zellen 4.3.3 Genotypisierung der Chimäre und Verpaarung mit PGK-Cre Tieren 4.3.4 Expressionsanalyse der R26_Casp1C284A del-Tiere 4.3.5 In vivo-Stimulation der R26_Casp1C284A del-Tiere 5 Diskussion 5.1 Entwurf eines Mausmodells für ICE-Fieber 5.2 Konstruktion des Casp1C284A-BAC-Transgens 5.3 Identifizierung von Casp1C284A-transgenen Tieren 5.4 Funktionelle Analyse des Casp1C284A-BAC-Transgens 5.5 Konstruktion des konditionalen Mausmodells R26_Casp1C284A 5.6 Aktivierung des ki-Mausmodells R26_Casp1C284A durch Cre-mediierte Rekombination in vivo 5.7 Funktionelle Analyse des ki-Mausmodells R26_Casp1C284A 5.8 Relevanz eines Mausmodells für den Patienten 6 Zusammenfassung 7 Literaturverzeichnis 8 Anhang A. Abbildungsverzeichnis B. Tabellenverzeichnis<br>Problem: In order to recapitulate the effects of the CASP1 variants found in the patients a mouse model should be generated. The analysis of material from the patients unfortunately is very restricted and therefore the generation of a mouse model represents the best alternative to see if the in vitro hypothesis of the IFG group really applies to an in vivo situation. Results: To generate a transgenic mouse model the artificial variant Casp1C284A was inserted into a BAC to enable a natural expression and regulation of Casp1C284A. This mutation results in a disruption of the active centre and to a complete loss of the enzymatical activity of caspase-1. After two pronuclei injections we received 180 pubs of TG mice. Only three of them harboured transgenic sequences and only one animal in the F1 generation harboured the complete Casp1C284A sequence. Expression analyses of the offspring of this mouse revealed no basal transcriptional expression of the transgene. Hence, protein expression could not be detected in unstimulated cells. However, stimulation with LPS upregulated transcription and low-level translation of Casp1C284A in BMDCs and an elevated secretion of the proinflammatory cytokines TNF-α and IL-6 was detected as well. Likewise, after in vivo stimulation of transgenic mice with LPS i.p. the drop of body temperature was significantly enhanced in comparison to the control mice. And also the level of the proinflammatory cytokines was increased. Furthermore, a conditional R26_Casp1C284A construct allowing a temporal or a celltype specific expression of the caspase-1 with the central mutation was generated. Positive screened ES cell clones were injected into blastocysts and thereafter chimera could be identified. An embryonic lethality due to the integration of the enzymatically inactive caspase-1 could be excluded by the crossing with ubiquitious expressing PGK-Cre mice. All the corresponding mice were alive and a basal transcriptional as well translational expression was demonstrated. Concordantly with the results of the transgenic mice the conditional R26_Casp1C284A mice showed an enhanced drop of the body temperature in comparison to control mice after stimulation with sublethal dose of LPS in vivo. Likewise, a trend to an elevated secretion of TNF-α and IL-6 was observed. Conclusion: With the generated transgenic Casp1C284A as well as the conditional R26_Casp1C284A animal models we showed that an inactive variant of the procaspase-1 could result in a proinflammatory cytokine response and a development of an increased inflammation of a whole organism. Thus, these data support the previous postulated model of the IFG group for proinflammatory effects induced by variants of procaspase-1 with reduced enzymatic activity. Hence, the mouse models established in this work are suited for further analysis of the pathomechanism in the patients with ICE fever. For instance the cellular mechanism could be examined if the inflammation is provoked by an increased interaction of the mutated procaspase-1 with the kinase RIP2 and therefore the NFκB activation is increased, respectively. Also a further medicinal inhibition of the RIP2 signaling or other therapeutic testings in this mouse models are conceivable.:Inhaltsverzeichnis Abkürzungsverzeichnis 1 Einleitung 1.1 Das angeborene und adaptive Immunsystem 1.2 Sensoren des angeborenen Immunsystems 1.2.1 Membran-gebundene Rezeptoren 1.2.2 Intrazelluläre PRRS 1.2.3 Inflammasome als Multiproteinkomplexe 1.2.4 Das NLRP3-Inflammasom 1.2.5 Metabolische Krankheiten, die mit Inflammasom-Aktivität assoziiert werden 1.2.6 Inflammasom-assoziierte autoinflammatorische Erkrankungen 1.2.6.1 Das Cryoporin-assoziierte periodische Syndrom (CAPS) 1.2.6.2 Familian Mediterranean Fever (FMF) 1.2.6.3 Pyogenic arthritis, pyoderma gangrenosum and acne syndrome (PAPA) 1.3 Caspase-1 1.3.1 Caspasen im Allgemeinen 1.3.2 Der Caspase-1 Gen-Lokus und das Caspase-1 Gen 1.3.3 Das Caspase-1 Protein 1.3.4 Funktionen von Caspase-1 1.3.4.1 Prozessierung der Zytokine pro-IL-1ß und pro-IL-18 1.3.4.2 Induktion von Pyroptose 1.3.4.3 Aktivierung der Caspase-1 durch ER-Stress 1.3.4.4 Aktivierung des Transkriptionsfaktors NFκB 1.3.4.5 weitere Funktionen 1.3.5 Caspase-1 Genvarianten und Entzündung 1.4 Mausmodelle 1.4.1 NLRP3 1.4.2 Pyrin 1.4.3 Caspase-1 2 Zielsetzung 3 Material und Methoden 3.1 Material 3.1.1 Antibiotika 3.1.2 Antikörper 3.1.3 Bakterienstämme 3.1.4 BAC-Klon 3.1.5 Plasmide 3.1.6 Zelllinie 3.1.7 Chemikalien und Substanzen 3.1.8 Enzyme 3.1.9 Größenstandards 3.1.10 Oligonukleotide 3.1.11 Kommerzielle Kits 3.1.12 Puffer und Lösungen 3.1.13 Mauslinien 3.1.14 Medien 3.1.15 Geräte 3.1.16 Software 3.2 Molekularbiologische Methoden 3.2.1 Agarosegelelektrophorese 3.2.2 Amplifikation von Nukleinsäuren mittels Polymerasekettenreaktion 3.2.3 Aufreinigung von DNA-Fragmenten aus Agarosegelen 3.2.4 Aufreinigung von BAC-DNA mittels Elektroelution 3.2.5 Aufreinigung von Subklon-Plasmid-DNA für die Elektroporation von ES-Zellen 3.2.6 Bestimmung der Konzentration und der Reinheit von DNA und RNA 3.2.7 c-DNA-Synthese 3.2.8 Dephosphorylierung von 5´-Phosphatresten 3.2.9 DNA-Isolierung 3.2.9.1 DNA-Isolierung aus Bakterienzellen 3.2.9.2 DNA-Isolierung aus eukaryotischen Zellen 3.2.10 DNA-Sequenzierung 3.2.11 Enzymatische Restriktionsspaltung von DNA-Fragmenten 3.2.12 Homologe Rekombination von DNA-Sequenzen 3.2.13 Ligation von DNA-Fragmenten mit linearisierten Vektoren 3.2.14 Phenol-Chloroform-Extraktion 3.2.15 Pulsfeldgelelektrophorese 3.2.16 Quantitative Real-Time-PCR (qRT-PCR) 3.2.17 RNA-Isolation aus eukaryotischen Zellen und Geweben 3.2.18 Southern Blot 3.3 Mikrobiologische Methoden 3.3.1 Kultivierung von Bakterien 3.3.2 Hitzschock-Transformation von E.coli 3.3.3 Kryokonservierung 3.4 Proteinbiochemische Methoden 3.4.1 CBA (cytokine bead assay) 3.4.2 Proteinbestimmung 3.4.3 Immunpräzipitation mit Caspase-1 p10 (M-20) Antikörper von Santa Cruz 3.4.4 Immunpräzipitation mit AntiFlag M2 Affinity Gel von Sigma 3.4.5 Diskontinuierliche SDS-Polyacrylamid-Gelelektrophorese (SDS-PAGE) 3.4.6 Western Blot 3.4.7 Immunchemische Detektion von Proteinen 3.5 Zellbiologische Methoden 3.5.1 Bestimmung der Zellzahl 3.5.2 Isolierung und Generierung von BMDCs 3.5.3 Kryokonservierung von Zellen 3.5.4 Kultivierung von Zelllinien 3.6 Tierexperimentelle Methoden 3.6.1 Genotypisierung 3.6.2 Haltung 3.6.3 Hautbiopsie 3.6.4 Retroorbitale Blutentnahme und Herzpunktion 3.6.5 Injektion von LPS 3.6.6 Organentnahme 4 Ergebnisse 4.1 Generierung eines Casp1C284A-BAC-Transgens 4.1.1 Identifikation und Verifizierung des BAC-Klons 4.1.2 Klonierung des Casp1C284A-BAC-Transgens 4.1.2.1 Entfernung der wt-loxP-Stelle vom Vektorrückrat 4.1.2.2 Insertion eines Flag-Tags 4.1.2.3 Einführung der Punktmutation C284A 4.1.3 „Shaving“ des generierten Casp1C284A-BAC-Konstrukts 4.1.4 Aufreinigung des Casp1C284A-BAC-Konstrukts und Pronukleusinjektion 4.1.5 Genotypisierung der transgenen Casp1C284A Gründertiere 4.2 Charakterisierung der transgenen Casp1C284A-Tiere (tg) 4.2.1 In vitro-Stimulation von BMDCs mit LPS 4.2.2 In vivo-Stimulation der transgenen Casp1C284A-Tiere 4.3 Generierung eines konditionalen Casp1C284A-Konstrukts 4.3.1 Klonierung des Targetingvektors pSerc_Casp1C284A_Flag und des Kontroll-Targetingvektors pSerc_Casp1_Flag 4.3.2 Screening von ES-Zellen 4.3.3 Genotypisierung der Chimäre und Verpaarung mit PGK-Cre Tieren 4.3.4 Expressionsanalyse der R26_Casp1C284A del-Tiere 4.3.5 In vivo-Stimulation der R26_Casp1C284A del-Tiere 5 Diskussion 5.1 Entwurf eines Mausmodells für ICE-Fieber 5.2 Konstruktion des Casp1C284A-BAC-Transgens 5.3 Identifizierung von Casp1C284A-transgenen Tieren 5.4 Funktionelle Analyse des Casp1C284A-BAC-Transgens 5.5 Konstruktion des konditionalen Mausmodells R26_Casp1C284A 5.6 Aktivierung des ki-Mausmodells R26_Casp1C284A durch Cre-mediierte Rekombination in vivo 5.7 Funktionelle Analyse des ki-Mausmodells R26_Casp1C284A 5.8 Relevanz eines Mausmodells für den Patienten 6 Zusammenfassung 7 Literaturverzeichnis 8 Anhang A. Abbildungsverzeichnis B. Tabellenverzeichnis

碩士<br>國立臺灣大學<br>微生物學研究所<br>98<br>It has been widely accepted that invertebrates contain only innate immunity but no adaptive immunity. The innate immunity includes phagocytosis, encapsulation, melanization and secretion of antimicrobial peptides (AMPs). There are five AMPs called Attacin, Cecropin, Defencin, Diptericin and Gambicin in the mosquito Aedes aegypti and these AMPs are regulated by Toll or Imd pathway. In mosquitoes, vitellogenesis and metamorphosis are important life cycle that can be researches decreasing vectore-borne diseases. Previous cDNA microarray analysis revealed that CAS1, ELMO and F-Box/LRR are simultaneously up-regulated with Inhibitor of Apoptosos 2 (IAP2), an important regulator of IMD pathway, upon bacteria challenge in Anopheles gambiae. CAS1 was associated with acetylation in hunan and fungus－Cryptococcus neoformans. It is also called acetyltransferase. ELMO was shown to be involved in D. melanogaster development and cytoskeleton stability. It was demonstrated to affect phagocytosis in mammals. Previous research showed that F-box may play important role in protein-protein interaction and it is an important factor in ubiquitylation. Therefore, we ought to explore the functions of CAS1, ELMO and F-box in the mosquito A. aegypti. First, we made use of RNA interference (RNAi) technique to silence the mRNA expression of ELMO and F-box in A. aegypti, followed by the challenge of Staphylococcus aureus or Escherichia coli. The survival assay was performed to analyze the mosquito resistance to bacteria. Our results revealed that Aedes aegypti showed resistance to S. aureus in the absence of ELMO and F-Box. Therefore, we speculated that ELMO and F-box may serve as negative regulators in Toll pathway. Next, the expression of Cecropin A, a downstream target of Toll pathway, was examined. The results showed that silencing of ELMO resulted in the over-expression of Cecropin A upon S. aureus challenge, suggesting that ELMO negatively regulateof the expression of Cecropin A. Then we made use of FITC-labeled bacteria to observe the effect of phagocytosis in A. aegypti. The result showed that silencing of ELMO can increase the phagocytic ability to S. aureus in the mosquito. It’s suggested that ELMO played a negative role in phagocytosis to Gram positive bacteria. In addition, we made use of RNAi technique to silence the mRNA expression of CAS1 and F-box in A. aegypti, followed by feeding blood meal. The result showed that silencing of CAS1 decreased vitellogenin expression and egg production. Finally we use RNAi technique to silence the ELMO and F-box in larvalstage of Aedes aegypti and calculate the ratio of mosquito emergence. The result revealed that silencing of F-box resulted in the decrease of metamorphosis rate in larval stage and metamorphosis genes (broad,E75b, JHA15) expression in adult mosquitoes.