Tesis sobre el tema "DSBF"

This study set out to investigate the impact of inhibiting bacterial enzymes on pathogen growth and virulence as an alternative antimicrobial strategy to antibiotics. Results support the hypothesis that DsbA enzyme inhibition is a robust anti-virulence strategy that can 'disarm but not kill bacteria' using uropathogenic Escherichia coli as the model pathogen. These findings have enhanced the pharmacological importance of DsbA as an anti-virulence drug target and contribute to ongoing research that aims to develop DsbA inhibitors against different bacterial pathogens with diverse DsbA enzymes.

DSB Mobile is a small Peruvian software developing company based in Lima. DSB Mobile specializes in the development of both mobile and web applications and has worked with major companies such as Samsung, Claro & Entel. The company is composed of the General Manager, Zico Herrera, a sales manager, operations manager and both fulltime and contract based software developers that are hired based on the current demand for service. DSB Mobile has established a strong reputation and brand in Peru and is now looking to expand outside of Peru where they can introduce their software products into international markets. In their aspiration to internationalize, DSB Mobile is seeking to find out not only the most profitable markets for their company but also markets that would best align with DSB Mobiles mission. The solution to their question of expansion was to determine the best markets using a variety of both quantitative and qualitative factors. In using an IT Competitiveness report that was done by the British Software Alliance this was used as a reference point to determine the best ranked countries for IT competitiveness and the best countries to conduct business in base off of important IT indicators. Combined with software data statistics in terms of charges per project and cost of IT Consultants, this further narrowed down the scope to the most attractive, profitable and mutually beneficial market for DSB Mobile. The implementation plan proposed involved two market lines namely, the North American market line and the European Market line. The proposed solution takes into consideration different scenarios, one of them being the moderate work balance of 1 project per month, giving a total cost of $391,065 per year with a profit of $180,736. The Gantt chart outlined is intended to guide the company with the step by step implementation of this international expansion and prepare them to execute this plan in the most efficient and effective manner
DSB Mobile es una pequeña empresa peruana de desarrollo de software con sede en Lima. DSB Mobile se especializa en el desarrollo de aplicaciones móviles y web y ha trabajado con grandes empresas como Samsung, Claro y Entel. La compañía está compuesta por el Gerente General, Zico Herrera, un gerente de ventas, un gerente de operaciones y desarrolladores de software a tiempo completo y algunos que se contratan en base a la demanda actual de servicio. DSB Mobile ha establecido una fuerte reputación y marca en Perú y ahora está buscando expandirse fuera de Perú donde puedan introducir sus productos de software en los mercados internacionales. En su aspiración de internacionalización, DSB Mobile está tratando de descubrir no sólo los mercados más rentables para su empresa, sino también los mercados que mejor se alinean con la misión DSB Mobile. La solución a su problema de expansión fue determinar los mejores mercados utilizando una variedad de factores tanto cuantitativos como cualitativos. Al utilizar un informe de competitividad de TI que fue realizado por la British Software Alliance, se utilizó como punto de referencia para determinar los países mejor clasificados para la competitividad de TI y los mejores países para llevar a cabo negocios en base de importantes indicadores asociados a estos. Combinado con estadísticas de datos de software en términos de gastos por proyecto y coste de consultores en TI, esto permitió reducir aún más el alcance para obtener un mercado más atractivo, rentable y mutuamente beneficioso. El plan de implementación propuesto involucró dos líneas de mercado, a saber, la línea del mercado norteamericano y la línea del mercado europeo. La solución propuesta posee diferentes escenarios; por ejemplo, el escenario con trabajo moderado consta de 1 proyecto por mes y tiene un costo total de $391,065 por año obteniendo así una rentabilidad de $180,736. El gráfico de Gantt esbozado pretende guiar a la compañía con la implementación paso a paso de esta expansión internacional y prepararlos para ejecutar este plan de la manera más eficiente y efectiva
Tesis

Klebsiella pneumoniae is a clinically important bacterial pathogen causing urinary tract infections and pneumonia which can progress to more severe disease. With the increase in antimicrobial resistance globally, K. pneumoniae infections have become difficult to treat, with many antibiotics now ineffective. Therefore, it is imperative that we investigate new and effective treatments for these drug resistant infections. This thesis describes the identification and characterization of multiple diverse disulphide bond enzymes in K. pneumoniae that have potential as novel therapeutic targets.

DNA中の二本鎖切断(DSB)に対するアスコルビン酸(AA)およびDMSOの保護効果を、蛍光顕微鏡による巨大DNA(T4 DNA; 166kbp)の単分子観察によって評価した。凍結／解凍の状態に対して3つの異なる形態の放射源、可視光、γ線、および超音波の環境下にさらした。1‐プロパノールと2‐プロパノールの間で異なる効果が表れた。ゲノムDNA分子の高次構造の変化は、1−プロパノールを用いると、長軸長が濃度60％で最小を示し、次にアルコール含有量の増加と共に増加する傾向があることを見出した。一方、2−プロパノールを用いると、長軸長はアルコール含有量の増加と共にほぼ単調な減少を示した。
The protective effect of ascorbic acid (AA) and DMSO against double-strand breaks (DSBs) in DNA was evaluated by single-molecule observation of giant DNA (T4 DNA; 166kbp) through fluorescence microscopy. Samples were exposed to three different forms of radiation: visible light, γ-ray, and ultrasound or freeze/thawing. The change of the higher-order structure of genomic DNA molecules in the presence of alcohols by use of single DNA observation with fluorescence microscopy, by focusing our attention to unveil the different effect between 1-propanol and 2-propanol.
博士(工学)
Doctor of Philosophy in Engineering
同志社大学
Doshisha University

La quasi-totalite des proteines d'interet economique est constituee de proteines extracellulaires dont l'un des elements essentiel de stabilite et de repliement est la presence de ponts disulfure. Les strategies utilisees pour la production recombinante de ces proteines impliquent leur expression dans le periplasme de bacteries hotes. Dans le periplasme d'e. Coli, les enzymes du systeme dsb catalysent la formation et l'isomerisation des ponts disulfure. Dans des conditions d'expression normale, elles permettent le repliement correct des proteines naturelles. En revanche, la surexpression de proteines heterologues dans le periplasme conduit souvent a l'agregation ou a la degradation des proteines, en raison de l'accumulation de formes a ponts disulfure non formes ou non natifs. L'amelioration par ingenierie des proprietes de ces catalyseurs s'avere donc essentielle pour la production en grande quantite de proteines correctement repliees. Dsba, le principal agent de controle du potentiel oxydoreducteur periplasmique, constitue une cible ideale pour ce type d'etude. Afin de mieux comprendre ses proprietes catalytiques, l'etude de ses caracteristiques structurales et dynamiques a fait l'objet de ce travail. Dans la premiere partie, l'etude structurale comparative des formes oxydee et reduite a permis d'identifier les differences d'environnement electronique entre ces deux etats redox. Dans la seconde partie, l'etude d'un complexe covalent entre dsba et un peptide substrat a fourni une description detaillee des interactions entre les deux partenaires. Enfin, la mobilite au sein des trois formes (oxydee, reduite, complexee) a ete etudiee par la technique d'echange proton-deuterium. Au contraire de la structure, qui apparait tres similaire dans les differentes formes, la flexibilite de dsba varie largement entre les trois formes. Les informations obtenues au cours de cette etude fournit les elements de base pour la conception de nouveaux catalyseurs derives de dsba.

Tiivistelmä. Tässä työssä esitellään National Instrumentsin USRP ohjelmistoradio NI2900-laitemalli ja sen käyttö MATLAB-Simulink ympäristössä. Työssä esitellään ohjelmistoradion toimintamalli ja toteutetaan modulaattori ja demodulaattori amplitudimodulaatiolle sekä kaksisivukaistamodulaatiolle käyttäen USRP:n Simulink:lle suunniteltuja lohkoja. Tuloksena saadaan moduloituja ja -demoduloituja signaaleja sekä niillä toteutettavia lohkokaavioita, joiden malleja voidaan käyttää toteuttaessa muita modulaatiomenetelmiä samassa ympäristössä. Lopuksi pohditaan USRP toiminnan toteutuksen käyttökokemusta Simulink-ympäristössä.Implementation of digital AM- and DSB modulations in Simulink-environment using USRP software defined radio. Abstract. In this research paper the USRP NI-2900 software defined radio developed by National Instruments is represented and implemented in MATLAB-Simulink environment. The basic functionalities of software defined radio are represented and digital modulators and demodulators for amplitude and double sideband modulations are implemented using USRPs blocks in Simulink. As a result, modulated and demodulated signals are generated with the designed blocks charts, and the device models can be used for further implementations for another modulation techniques. Finally, the implementation of USRP in Simulink-environment is considered in respect of accessibility.

In eukaryotic cells, ionizing radiation (IR) induces damages to proteins, lipids and DNA, directly or indirectly, as a result of free radical formation. Among the numerous types of DNA lesions, the double-strand breaks (DSBs) are particularly important, since an inefficient or inaccurate repair may lead to cell death or genomic instability. The presence of DSBs leads to a complex DNA damage response, consisting in a cascade of cellular events, which involve sensing the damage, signal transduction to the effectors of DNA repair, cell cycle arrest and apoptosis induction. In mammals, a very early step in the cellular response to DSBs is the phosphorylation of the histone H2AX (γ-H2AX) at the sites of DNA damage by members of the phosphatidylinositol-3-OH kinase (ATM, DNA-PK and ATR). This event plays a critical role in the recruitment of signaling–repair proteins (i.e 53BP1, Mre11, Rad50, Nbs1) to the sites of damage to form the ionizing radiation-induced foci (IRIF), which contain hundreds to thousands of proteins. DNA damage and repair can be quantified in individual cells by monitoring the kinetics of formation and disappearance of IRIF that accumulate at sites of DSBs; in particular, the rate of loss of γ-H2AX foci correlates with the progression of DSB repair. Cell signaling events in response to ionising radiation depend on environmental conditions occurring during DNA repair, besides genetic and physiological features of the biological systems. For this reason we have studied and compared the human cell response to IR in different gravity conditions, normal gravity as on Earth and reduced gravity as in space environment where exposure to cosmic radiation during space missions is associated to the reduction of gravitational force, which is approximately 10-4-10-6g. Indeed, space environment is characterized by the presence of ionizing radiation in the form of charged atomic particles travelling at close to the speed of light, which represents the most significant factor limiting humans’ ability to participate in long-duration space missions, and also by a condition of weightlessness called microgravity. As reported in literature, microgravity effects on astronauts include: immune cell function suppression, skeletal muscle atrophy, cardiovascular problems and loss of calcium and minerals from bone. In-flight cell cultures and ground models of microgravity showed inhibition of lymphocyte proliferation, suppression or alteration in cytokine secretion, modifications of cytoskeleton, and also increase of chromosome aberrations and apoptosis. The question whether radiation effects are influenced by microgravity is still open and it is an important point in the risk estimation of space missions. In our experiments, microgravity condition was obtained in laboratory using of the bioreactor “Rotating Wall Vessel” (Synthecon, Inc., Houston, Texas), which simulates the weightlessness, an aspect of spaceflights. This condition is called “modeled microgravity” (MMG). In the first part of this project, DSB rejoining was investigated in human PBL irradiated with γ-rays and incubated in 1g or MMG during repair time. Formation and disappearance of γ-H2AX foci were monitored at various times after irradiation by in situ immunofluorescence; in the same samples the apoptotic index and the DNA fragmentation were determined, the last one was measured by pulsed-field gel electrophoresis (PFGE) and the fraction of DNA released in the gel (FR) was considered as a measure of DSBs. Results obtained provided evidences that MMG incubation during repair time affected cell survival, apoptosis and delayed DSB rejoining, increasing the genotoxic effects of ionising radiation. On the base of these evidences, we investigated if ionizing radiation and modeled microgravity could have an effect on cells focusing on the expression profile of microRNAs: negative regulators of gene expression. MicroRNAs (miRNAs) are a recently discovered class of small (~22nt) endogenously expressed translational-repressor RNAs that play a key role in many cellular pathways. In animal cells, these molecules bind to complementary sequences in the 3’-untraslated region (3’UTR) of the target messenger RNAs (mRNAs) of protein-coding genes, to direct their translational repression. For this reason, miRNAs have been implicated in numerous biological processes including developmental timing, cell fate decisions, cell death and proliferation, stem cell function, tumorigenesis and disease. Aim: The aims of this project were: i) to analyze the efficiency of DNA repair occurring in modeled microgravity culture conditions focusing on DSBs repair kinetics; ii) to investigate if ionizing radiation and modeled microgravity could have a synergistic action on human cells by comparing radio-responsive miRNA in ground gravity and MMG. Activity carried out: The presence of nuclear γ-H2AX foci and the apoptotic index were monitored by in situ immunofluorescence and western blot in not- and γ-irradiated Peripheral Blood Lymphocytes (PBL), at 0.5, 2, 6 and 24h from irradiation, in 1g and MMG. In the same samples was studied the DSBs repair analyzing the fraction of DNA released (FR) after Pulsed-field gel electrophoresis (PFGE), this value was calculated either by measuring optical density of DNA migrating in the gel and by quantity of DNA (ng) retained in the plug/wells. MiRNA expression profile of human PBL, irradiated with γ-rays (0.2-2Gy) and incubated in MMG and in parallel 1g condition, was examined through “Human miRNA microarray Kit V2” (Agilent) and quantitative real-time PCR (RT-qPCR). In addition, we used “Whole Human Genome Oligo Microarray” (Agilent) to determine the gene expression profile, in the same PBL analysed for miRNA profiling, with the aim to identify the most likely miRNA targets by integration of miRNA and mRNA expression data in an anti-correlation analysis. Finally, to identify biological pathways most involved in radiation cell response we performed a Gene Ontology (GO) analysis, on significant anti-correlated target genes identifying biological pathways significantly enriched (P<0.05). Results and conclusions: Results obtained in the study of nuclear γ-H2AX foci in irradiated PBL showed that the mean number of foci/nucleus during a short-repair time was comparable in 1g and MMG. At later times the decrease of foci number was significantly different; indeed, PBL incubated in 1g at 24h after irradiation showed 2 foci/nucleus, instead those incubated in MMG showed a mean of 6.4 foci/nucleus at the same time-points. To verify whether the disappearance of γ-H2AX foci correlated with the rejoining of double strand breaks, we subjected irradiated PBL, incubated in 1g or MMG, to PFGE assay. We found that in cells incubated in MMG, FR was higher than in 1g (77% vs. 33% at 2 h and 50% vs. 17% at 6 h, respectively). Summarizing, MMG probably affects the chromatin structure modulation that occurs after DSB formation, decreasing the efficiency of DNA repair. Thus, DSB rejoining that is almost completed in few hours in normal culture conditions, could take more time in MMG. In second part of the project we focused on miRNA expression profile analysis of γ- irradiated PBL incubated in 1g and MMG. Our results showed that radiation affected miRNA expression profile according to the dose and the time after irradiation, in both gravity conditions. Exposure to γ-rays in 1g altered miRNA expression profile at early and late time points (4h and 24h), with more responsive miRNAs at 24h after high irradiation dose (2Gy). In particular, the 20 radio-responsive miRNAs common to 0.2Gy and 2Gy of treatment showed a time dependent expression pattern, with a general down-regulation at 4h and up-regulation at 24h after irradiation. Human PBL incubated in MMG after γ-irradiation showed miRNA expression profile alteration higher at 24h than at 4h, in both irradiation doses. Interestingly, in non-irradiated PBL, 24h of MMG incubation altered the expression profile of 42 miRNA species respect to 1g. At the end, comparing the miRNA expression profiles of γ-irradiated PBL incubated 24h in the two different gravity conditions, we individuated miRNAs specifically expressed during repair time in MMG; these miRNA species were probably altered by the combined action of IR and MMG in a way that is dose dependent. In order to figure out the mechanism by which miRNAs can modulate a certain biological function in response to IR, gene expression profiles were analysed on the same PBL samples used to assess miRNA expression levels, in 1g and MMG. The anti-correlation analysis was performed between differentially expressed mRNAs and deregulated miRNAs to investigate the putative miRNA target genes. Finally, Gene Ontology analysis was conducted on significant anti-correlated target genes with the aim of identifying the biological categories which they belong to. From our results it arose that few genes were activated in irradiated PBL incubated 24h either in 1g or MMG and most of them were gravity-specific. In 2Gy PBL incubated in 1g a great number of deregulated genes belonged to the DNA Damage Response (DDR) categories: apoptosis, response to wounding and response to DNA damage. These categories were not found in 2Gy PBL incubated in MMG, where instead were altered biological processes involved in regulation of development, cell differentiation/activation, immune system, cytokine production and hemopoiesis; they were all characterized by a general gene down-regulation. In addition, we focused on significantly anti-correlated genes of DDR pathway, activated in the two gravity conditions, to highlight the differences between 1g and MMG. We hypothesize that the smaller number of radio-responsive miRNAs in MMG can operate an unscheduled regulation of the expression level of transcripts usually not targeted. The scenario proposed in this work is that modeled microgravity incubation, following ionizing radiation exposure (simulated space environment), could affect the appropriate radiation cell response of human lymphocytes reducing the efficiency of DNA repair. To better investigate miRNA biological functions in the simulated space environment, it has been necessary to focus our studies on miRNA target validation and functional analysis. For this reason, the PhD program was carried out for seven months in the laboratory of Prof. Riccardo Dalla-Favera at “Institute for Cancer Genetics”, Columbia University (New York, USA), to acquire some expertise in molecular biology techniques and their applications to the study of microRNAs.
Le radiazioni ionizzanti (IR), colpendo le cellule degli organismi eucarioti è in grado di provocare danni a proteine, lipidi e molecole di DNA, in modo diretto o indiretto come risultato della formazione di radicali liberi. Tra i numerosi tipi di danno al DNA, le rotture a doppio filamento o double-strand breaks (DSBs) rappresentano il tipo di lesione più grave, dal momento che una riparazione inefficiente o non accurata può portare a morte cellulare o instabilità genomica. La presenza di DSBs induce una complessa risposta al danno al DNA che vede coinvolti una serie di eventi cellulari quali: la rilevazione del danno, la trasduzione del segnale agli effettori della riparazione, l’arresto del ciclo cellulare e l’induzione di apoptosi. Nei mammiferi, una delle risposte cellulari più precoci dopo l’induzione di una doppia rottura è la fosforilazione dell’istone H2AX (γ-H2AX) in corrispondenza del sito di danno, che avviene per opera delle fosfatidilinositolol-3-OH-chinasi (ATM, DNA-PK and ATR). Questo evento sembra essere importante nel reclutamento di fattori di segnalazione del danno e di proteine coinvolte nella riparazione delle DSBs nei siti danneggiati (i.e 53BP1, Mre11, Rad50, Nbs1), dando origine a ionizing radiation-induced foci (IRIF), che possono essere costituiti da migliaia di queste molecole proteiche. Monitorando la cinetica di formazione e scomparsa degli IRIF, che si accumulano nei siti danneggiati, è possibile analizzare il danno al DNA e la sua riparazione; in particolare, è stato osservato che la diminuzione dei foci di γ-H2AX correla con la progressione della riparazione delle DSBs. Gli eventi di segnalazione attivati in risposta alle radiazioni ionizzanti dipendono, oltre che dalle caratteristiche genetiche e fisiologiche del sistema biologico osservato, anche dalle condizioni ambientali presenti durante la riparazione del DNA. Per questa ragione abbiamo analizzato e confrontato la risposta cellulare umana alle IR in condizioni diverse di gravità, normale come sulla Terra (1g) e ridotta come nell’ambiente spaziale; in quest’ultimo l’esposizione ai raggi cosmici a cui l’uomo è soggetto durante le missioni spaziali e associata alla riduzione della forza di gravità. L’ambiente spaziale è caratterizzato dalla presenza di radiazioni ionizzanti, nella forma di particelle atomiche cariche che rappresentano il più importante fattore limitante la lunga permanenza dell’uomo nello spazio, ma anche dalla condizione di assenza di peso, che prende il nome di microgravità (10-4–10-6g). In letteratura sono stati riportati alcuni effetti della microgravità osservati in astronauti di ritorno dai voli spaziali, questi riguardano: la soppressione del sistema immunitario, l’atrofia muscolare, problemi cardiovascolari e la demineralizzazione e decalcificazione ossea. Cellule mantenute in coltura durante le missione spaziali e modelli a terra della microgravità mostrano inibizione della proliferazione dei linfociti, soppressione o alterazione della secrezione di citochine, modificazioni del citoscheletro e anche incremento delle aberrazioni cromosomiche e apoptosi. Pertanto, capire se gli effetti della radiazione ionizzante possano essere influenzati dalla microgravità rimane un punto di rilevante importanza nella valutazione dei rischi durante le missioni spaziali. In questo lavoro, la microgravità è stata simulata in laboratorio usando il bioreattore “Rotating Wall Vessel” (Synthecon) messo a punto nei laboratori della NASA a Houston; questo strumento permette di riprodurre un aspetto dei voli spaziali che è l’assenza di peso, condizione che prende il nome di “modeled microgravity” (MMG). Nella prima parte di questo progetto è stata studiata la riparazione delle DSBs in linfociti umani irradiati con raggi gamma e mantenuti durante il tempo di riparazione in 1g o MMG. La formazione e la scomparsa dei foci dell’istone γ-H2AX è stata monitorata a diversi tempi dall’irradiazione mediante immunofluorescenza; nei medesimi campioni è stato anche analizzato l’indice apoptotico e la frammentazione del DNA, quest’ultimo con la tecnica della pulsed-field gel electrophoresis (PFGE) in cui la frazione di DNA rilasciata nel gel (FR) è considerata una misura delle DSBs. I risultati ottenuti confermano che l’incubazione in MMG durante il tempo di riparazione influenza la sopravvivenza cellulare, l’apoptosi e ritarda la riparazione delle DSBs, incrementando l’effetto genotossico delle radiazioni ionizzanti. Sulla base delle osservazioni fatte, si è passati a studiare se la IR e la MMG possono avere un’azione sinergica sulle cellule analizzando i profili di espressione dei microRNAs: regolatori negativi dell’espressione genica. I microRNAs (miRNAs) sono una classe di corti RNA (~22nt) endogeni, che svolgono un ruolo chiave in molti processi cellulari poiché reprimono l’espressione dei mRNA target. Nelle cellule animali, queste molecole vanno a reprimere la traduzione dei geni codificanti proteine legandosi a sequenze complementari nelle regioni non tradotte al 3’ terminale (3’UTR) dei mRNA. Per questo motivo i miRNAs sono coinvolti in numerosi processi biologici come: lo sviluppo, la proliferazione cellulare, l’apoptosi, la funzionalità delle cellule staminali e la tumorigenesi. Scopo: Questo progetto si proponeva di: i) analizzare l’efficienza di riparazione del DNA in condizione di microgravità simulata (MMG), puntando l’attenzione alla cinetica di riparazione delle DSBs; ii) capire se la radiazione ionizzante e la microgravità simulata possono avere un’azione sinergica in cellule umane, confrontando i miRNA radio-responsivi nelle due condizioni di gravità (1g e MMG) Attività svolta: La presenza di foci nucleari dell’istone γ-H2AX e l’indice apoptotico sono stati monitorati in linfociti umani irradiati con raggi γ e non, incubati in 1g e MMG. Negli stessi campioni è stata studiata la riparazione delle DSBs analizzando la frazione di DNA rilasciata (FR) dopo Pulsed-field gel electrophoresis (PFGE). In seguito, usando l’approccio dei microarray con “Human miRNA microarray Kit V2” (Agilent) e della real-time qPCR, sono stati analizzati i profili di espressione dei miRNAs in linfociti umani irradiati con raggi γ e incubati in 1g e MMG. Impiegando poi i microarrays “Whole Human Genome Oligo Microarray” (Agilent) per gli stessi campioni di cellule, è stato possibile determinare i profili di espressione genica; allo scopo di identificare i probabili mRNA target dei miRNA radio-responsivi i dati di espressione dei miRNA e dei mRNA sono stati integrati in un’analisi di anticorrelazione. Infine, per studiare i processi biologici maggiormente coinvolti nella risposta cellulare alle radiazioni ionizzanti è stata eseguita una Gene Ontology analysis (GO) applicata ai miRNA-mRNA target significativamente anti-correlati. Risultati e conclusioni: I risultati ottenuti dallo studio dei foci dell’istone γ-H2AX in PBL irradiati mostrano che il numero medio di foci/nucleo a tempi brevi di riparazione nelle due condizioni di gravità è comparabile. Al contrario, per tempi lunghi, la diminuzione del numero di foci è significativamente differente; infatti, a 24h dall’irradiazione i PBL incubati in 1g presentano 2 foci/nucleo, mentre quelli in MMG 6.4 foci/nucleo. Per verificare che la scomparsa dei foci di γ-H2AX fosse correlata con la riparazione delle DSBs è stata utilizzata la tecnica della PFGE. La cinetica di riparazione delle DSBs è stata analizzata in PBL irradiati e incubati in 1g e MMG; nelle cellule incubate in MMG il contenuto di DNA frammentato era maggiore rispetto alla 1g (FR 77% vs. 33% a 2 h e FR 50% vs. 17% a 6 h, rispettivamente). Probabilmente la MMG influisce sulle modificazioni strutturali della cromatina che avvengono in risposta alla DSBs, diminuendo l’efficienza di riparazione; pertanto, la riparazione delle DSBs che in 1g avviene in poche ore, richiede più tempo in MMG. Nella seconda parte del progetto sono stati analizzati i profili di espressione di miRNA in PBL irradiati con raggi γ e incubati in 1g e MMG. Dai risultati ottenuti è emerso che la radiazione altera i profili di espressione dei miRNA in modo dose e tempo dipendente, in entrambe le condizioni di gravità. L’esposizione ai raggi gamma in 1g altera i profili di espressione dei miRNA, sia a tempi brevi che lunghi, con maggior numero di miRNA radio-responsivi a 24h dopo esposizione alla dose maggiore (2Gy). Dal confronto dei profili di espressione di miRNA in PBL irradiati e mantenuti 24h nelle due condizioni di gravità vengono individuati miRNAs espressi in modo specifico durante l’incubazione in MMG; questi miRNAs vengono probabilmente alterati dall’azione combinata della IR con la MMG con effetto dose-dipendente. Anche le cellule non irradiate ma mantenute 24h in MMG presentano 42 miRNA deregolati rispetto alla 1g. Per far luce sul meccanismo col quale i miRNAs possono modulare alcuni processi biologici in risposta alle radiazioni ionizzanti, sono stati analizzati i profili di espressione di mRNAs negli stessi campioni per i quali sono stati ricavati i profili dei miRNAs. L’analisi di anti-correlazione tra i miRNA e i mRNA differenzialmente espressi e l’analisi computazionale con PITA hanno permesso di predire geni target dei miRNA. Infine, è stata eseguita la Gene Ontology analisi su geni target significativamente anti-correlati, allo scopo di identificare le categorie biologiche di appartenenza. Dai nostri risultati è emerso che alcuni geni sono attivati in PBL irradiati e incubati 24h sia in 1g che MMG, molti di loro sono gravità-specifici. In cellule irradiate con 2Gy e incubate in 1g un grande numero di mRNAs alterati appartiene alle categorie della risposta al danno al DNA (DDR): apoptosi, risposta allo stress, risposta al danno al DNA. Queste categorie non sono risultanti dall’analisi dei PBL irradiati e mantenuti in MMG, dove invece sono alterati processi coinvolti nel differenziamento e attivazione cellulare, sistema immunitario, produzione di citochine ed emopoiesi; tutte caratterizzate da una sostanziale down-regolazione genica. Questo studio fornisce prove che la MMG associata alla radiazione ionizzante porta ad una non appropriata risposta al danno al DNA in linfociti umani, dovuta probabilmente alla perdita di miRNAs radio-responsivi coinvolti nella DDR. Per meglio studiare le funzioni biologiche dei miRNAs in condizione di microgravità simulata è stato necessario puntare l’attenzione sulla validazione dei messageri target predetti e sull’analisi funzionale. Per questa ragione il programma finale di dottorato è stato svolto presso il laboratorio del Prof. Riccardo Dalla-Favera all’“Institute for Cancer Genetics” (Columbia University, New York, USA), per un periodo di sette mesi, allo scopo di acquisire competenze di biologia molecolare che vengono applicate allo studio dei microRNAs.

The study of measurement invariance in polytomous items that targets individual score levels is known as differential step functioning (DSF; Penfield, 2007, 2008). DSF methods provide specific information describing the manifestation of the invariance effect within particular score levels and therefore serve a diagnostic role in identifying the individual score levels involved in the item's invariance effect. The analysis of DSF requires the creation of a set of dichotomizations of the item response variable. There are two primary approaches for creating the set of dichotomizations to conduct a DSF analysis. The first approach, known as the adjacent categories approach, is consistent with the dichotomization scheme underlying the generalized partial credit model (GPCM; Muraki, 1992) and considers each pair of adjacent score levels while treating the other score levels as missing. The second approach, known as the cumulative approach, is consistent with the dichotomization scheme underlying the graded response model (GRM; Samejima, 1997) and includes data from every score level in each dichotomization. To date, there is limited research on how the cumulative and adjacent categories approaches compare within the context of DSF, particularly as applied to a real data set. The understanding of how the interpretation and practical outcomes may vary given these two approaches is also limited. The current study addressed these two issues. This study evaluated the results of a DSF analysis using both the adjacent categories and cumulative dichotomization schemes in order to determine if the two approaches yield similar results and interpretations of DSF. These approaches were applied to data from a polytomously scored alternate assessment administered to children with significant cognitive disabilities. The results of the DSF analyses revealed that the two approaches generally led to consistent results, particularly in the case where DSF effects were negligible. For steps where significant DSF was present, the two approaches generally guide analysts to the same location of the item. However, several aspects of the results rose questions about the use of the adjacent categories dichotomization scheme. First, there seemed to be a lack of independence of the adjacent categories method since large DSF effects at one step are often paired with large DSF effects in the opposite direction found in the previous step. Additionally, when a substantial DSF effect existed, it was more likely to be significant using the cumulative approach over the adjacent categories approach. This is likely due to the smaller standard errors that lead to greater stability of the cumulative approach. In sum, the results indicate that the cumulative approach is preferable over the adjacent categories approach when conducting a DSF analysis.

Homologous recombination (HR) during meiosis is initiated by programmed DNA double strand breaks (DSBs). Some of these DSBs are repaired to give crossovers (COs), which connect maternal and paternal homologous chromosomes and thus ensure proper segregation during meiosis I. In contrast, HR in mitotic cells forms mostly noncrossovers (NCOs); this prevents deleterious genome rearrangement and loss of heterozygosity. Therefore, meiotic HR is regulated to enrich for COs, but much remains to be understood regarding the basis of this regulation. Meiotic cells express unique HR proteins, and these global factors might facilitate the distinct regulation of meiotic HR. In addition, meiosis-specific proteins localize to the chromosome axis, and these proteins interact with the meiotic Spo11 complex during DSB formation. Thus, the substrate for meiotic HR forms in a unique local chromatin context, and is then acted upon by unique global recombination factors. In order to better understand the balance between local and global influences, we studied the meiotic repair of DSBs formed by the VMA1-derived endonuclease (VDE). Repair of these breaks, which form independent of the meiotic chromosome axis, should still be influenced by global cell wide meiotic recombination proteins, but may or may not be influenced by the localized meiotic chromosome axis. We studied repair of two VDE DSBs: one located in a region that is "hot" for Spo11 DSBs and is enriched for axis proteins; and one in a Spo11 DSB "cold" region that is not enriched for axis proteins. VDE DSBs are repaired at both loci to produce NCOs in excess over COs, but more COs are formed at the hot-spot locus. The hot-spot also accumulated more joint molecules (JMs), which are the intermediates of CO formation. In addition, CO formation shows different resolvase dependence at the two loci. Hot-spot COs are Mlh3-dependent but largely independent of the "mitotic" Mus81-Mms4 structure-selective nuclease; whereas cold-spot COs are Mlh3-independent but show dependence on Mus81- Mms4. Finally, in spo11-Y135F cells lacking genome wide meiotic DSBs, VDEinitiated COs are reduced at both loci, which now display an identical NCO-CO ratio. This effect is partially attributable to the lack of pairing in these cells, as ectopic repair of VDE DSBs in SPO11 cells also have a similar NCO-CO ratio. Thus, COs in meiosis require a specific global recombination environment. COs are also influenced by factors that act locally, such that VDE initiated COs at a hot-spot and a cold-spot are reminiscent of meiosis and mitosis respectively.

L'ataxie spinocérébelleuse avec neuropathie axonale (SCAN1) est un rare syndrome neurodégénératif récessif associé à une atrophie cérébelleuse et à une neuropathie périphérique. Il est causé par une mutation homozygote dans le gène tyrosyl-ADN phosphodiestérase-1 (TDP1) (A1478G). Cette mutation entraîne une substitution de l'histidine 493 par une arginine (H493R) dans le site catalytique de TDP1, ce qui conduit à une diminution de l’activité de TDP1. TDP1 hydrolyse la liaison entre une extrémité 3' de l'ADN et une tyrosine au sein d'un complexe de clivage de la topoisomérase I (TOP1cc) piégé sur la chromatine. TDP1 n'excise pas seulement les TOP1cc piégés, mais répare également d'autres lésions bloquant l'extrémité 3', notamment les 3'-phosphoglycolates qui résultent d’une oxydation. Toutefois, le mécanisme par lequel la mutation TDP1 H493R entraine le phénotype SCAN1, qui est associé avec la mort des neurones post-mitotiques, est peu connu. Les cassures double-brin (DSBs) de l'ADN sont peu fréquentes mais parmi les lésions génomiques les plus sévères. Un défaut de réparation peut induire la mort cellulaire, et elles ont été impliquées dans la pathogénèse de nombreuses maladies humaines, incluant des syndromes neurodégénératifs. Mon objectif de doctorat a été de déterminer si le phénotype SCAN1 pouvait être lié à une accumulation de DSBs dans des cellules non réplicatives portant la mutation TDP1 H493R. Les seuls modèles disponibles pour étudier l'impact de cette mutation étaient des lignées de cellules lymphoblastoïdes provenant de patients SCAN1 comparées à celles d'individus sains. Nous avons donc généré des modèles de cellules d'ostéosarcome U2OS homozygotes pour TDP1 H493R ou TDP1 KO en utilisant la technique CRISPR-Cas9. Nous avons également généré des cellules primaires de poumon WI38 hTERT TDP1 KO. Nous avons observé que les cellules TDP1 H493R et TDP1 KO accumulent des DSBs endogènes, principalement dans la phase G1 du cycle cellulaire par rapport à la phase S. Une augmentation de DSBs a également été observée après déplétion de TDP1 par siRNA dans les cellules WI38 hTERT quiescentes, suggérant la nature réplication-indépendante de ces DSBs. Le traitement des cellules TDP1 H493R et TDP1 KO par la camptothécine, qui induit spécifiquement des TOP1ccs, suggère en outre que l'accumulation de DSBs pourrait être liée au défaut d'élimination des TOP1ccs. Ensuite, nous nous sommes demandé si l'accumulation de DSBs dans ces cellules pouvait être liée à une augmentation de la production de ces DSBs et/ou à un défaut dans leur réparation. Notamment, les structures R-loops qui se forment lors de la transcription peuvent induire des DSBs dans les cellules non réplicatives. Nous avons montré que la déficience en TDP1 modulait les R-loops sur certains gènes, ce qui soulève la possibilité de leur implication dans la formation de DSBs. L'analyse de la réparation des DSBs après traitement par la camptothécine a révélé que les cellules TDP1 H493R et TDP1 KO étaient toutes deux défectueuses dans la réparation des DSBs en G1 mais pas en S, la mutation TDP1 H493R ayant l'effet le plus prononcé. Ces résultats suggèrent que les DSBs pourraient s'accumuler spécifiquement dans les cellules déficientes en TDP1 qui ne répliquent pas en raison d'une réparation défectueuse de ces cassures. L’ensemble des notre résultats apporte de nouvelles informations sur l'étiologie du syndrome neurodégénératif SCAN1. Ce travail a été soutenu par une bourse de doctorat dans le cadre du programme universitaire franco-italien VINCI 2016
Spinocerebellar ataxia with axonal neuropathy (SCAN1) is a rare recessive neurodegenerative syndrome associated with cerebellar atrophy and peripheral neuropathy. It is caused by a homozygous missense mutation in the tyrosyl-DNA phosphodiesterase-1 (TDP1) gene (A1478G). This results in a substitution of histidine for arginine-493 (H493R) in the TDP1 catalytic site, leading to a reduced TDP1 activity. TDP1 hydrolyses the bond between a DNA 3’-end and a tyrosyl moiety within a trapped topoisomerase I cleavage complex (TOP1cc). TDP1 not only excises trapped TOP1ccs but also processes other 3’-end-blocking lesions, including 3’-phosphoglycolates that result from oxidation. Even so, how TDP1 H493R mutation promotes the SCAN1 phenotype, which is associated with the death of post-mitotic neurons, is unclear. DNA double-strand breaks (DSBs) are infrequent but among the most harmful genomic lesions. Their defective repair can induce cell death, and they have been implicated in the pathogenesis of several human diseases, including neurodegenerative syndromes. Hence, my Ph.D. objective was to investigate whether the SCAN1 phenotype could be related to an accumulation of DSBs in non-replicating cells harboring the H493R mutation of TDP1. The only available models to study the impact of TDP1 H493R mutation were lymphoblastoid cell lines derived from SCAN1 patients compared to those of healthy individuals. Hence, we have generated models of osteosarcoma U2OS cells homozygous for TDP1 H493R or TDP1 KO employing the CRISPR-Cas9 technique. We have also generated primary lung WI38 hTERT fibroblasts TDP1 KO. We found that both TDP1 H493R and TDP1 KO cells accumulate endogenous DSBs, primarily in the G1 phase of the cell cycle compared to S phase. A similar increase of DSBs was observed in quiescent WI38 hTERT cells following depletion of TDP1 with siRNA, suggesting the replication-independent nature of DSBs. Treatment of TDP1 H493R and TDP1 KO cells with camptothecin to induced trapped TOP1ccs, further suggests that accumulation of DSBs could be related to the defective removal of TOP1ccs. Next, we asked whether DSB accumulation in those cells could be related to an increase in DSB production and/or a defect in their repair. Notably, R-loop structures that form co-transcriptionally can induce DSBs in non-replicating cells. We found that TDP1 deficiency modulated R-loop levels at some gene loci, raising the possibility of their implication in DSB formation. Analysis of DSB repair following camptothecin treatment revealed that both TDP1 H493R and TDP1 KO cells were defective in the repair of DSBs in G1 but not in S, with TDP1 H493R having the most pronounced effect. These results suggest that DSBs would accumulate specifically in TDP1-deficient cells that do not undergo replication, due to a defective repair of those breaks. Together, our results provide insights on the etiology of the SCAN1 neurodegenerative syndrome. This work was supported by a PhD fellowship under the French-Italian University VINCI Program 2016

Dipeptidyl peptidase 9 (DPP9) is an emerging cancer associated protease, which has unclear roles in hepatocellular carcinoma (HCC). DPP9 has important roles in DNA Double-Strand Break Repair (DSBR), inflammasome and immunoregulation, metabolism, and disease pathogenesis. Here we used hepatocyte-specific gene knock-out and enzymatic deficiency strategies to characterise the roles of DPP9 in the context of DNA Double-Strand Breaks (DSBs) and HCC. DPP9 gki (DPP9 enzyme-negative gene-knock-in; DPP9S729A/S729A) Mouse Embryonic Fibroblasts (MEFs) had been derived from DPP9S729A/S729A mice. Compared to DPP9 wild-type (wt) MEFs, DPP9 enzymatically deficient MEFs were hypersensitive to DSBs. DPP9 localisation was not changed following DSBs regardless of its enzyme activity. The chromatin-associated DPP9 did not quantitatively change following DSBs. DPP9 hepatocyte-specific depleted male C57Bl/6J mice and littermate control mice were treated with Diethylnitrosamine (DEN) and Thioacetamide (TAA), and were provided with an atherogenic High Fat Diet (HFD) until the age of 28 weeks. The hepatocyte-specific DPP9 depletion was not lethal. In this model, mice with hepatocyte-specific DPP9 depletion had reduced mass of the liver and subcutaneous adipose tissue, and lower fasting plasma glucose. These mice developed similar levels of HCC, inflammation and steatosis as the control group by histology assessment. These mice with hepatocyte-specific DPP9 depletion showed increased levels of active caspase-1 and of the autophagy marker beclin1. These mice did not have significantly different levels of the DSB marker γH2AX. Following the success of a near-infrared fluorescent protein (iRFP720) based ovarian cancer monitoring system in our collaborative laboratory, the iRFP720 fluorescent cellular tag integration was also successfully performed on a mouse liver cancer cell line (Hepa 1-6). Such fluorescent liver cancer cell line could be used in the future for the development of an orthoptic HCC model. In conclusion, DPP9 in the hepatocytes does not drive cancer formation. Increased caspase-1 activation and autophagy regulation was observed in mice with hepatocyte-specific DPP9 depletion. DPP9 might have a role in glucose metabolism regulation, which needs further investigation. DPP9 could be therapeutic target in the liver via intrahepatic regulation of inflammasome activation, autophagy and metabolism.

Meiosis is a cell division in which one diploid parent cell produces four haploid daughter cells. Accurate alignment and segregation of homologous chromosomes during metaphase is critical for a successful meiotic division and viable gametes. Three concomitant events are required for a successful meiotic division: chromosome pairing, synapsis and recombination. Recombination is initiated by programmed induction of DNA double-strand breaks (DSBs). Interchromosomal repair of meiotic DSBs can form a crossover leading to genetic diversity by modifying linkage groups. Crossovers also tether the homologous chromosomes and help resist the tension of the first meiotic spindle. Controlled recombination is required for a successful meiotic division and segregation, however, recombination has to be tightly regulated. This work investigates the roles of Te11,Rad6 and Srs2 during meiotic homologous DSB repair. Tel1 is protein kinase required for initiating a signalling cascade in response to many forms of DNA damage. Tel1 has also been proven to function during meiosis and has been shown in some conditions to initiate a signalling cascade after the initiation of meiotic DSBs. In this work Tel1 is shown to influence the early stages of DSB repair during meiosis, however this is not in response to the formation of Spo11-DSB. Recombination ensures genetic variation and correct homologue alignment during meiosis I therefore is extremely important and tightly controlled. Srs2 is known to be a negative regulator of recombination and is important for normal sporeulation and viability in yeast. Analysis of an experimental site specific DSB (made by VDE) and at natural Spo11-DSBs indicates that in the absence of Srs2 the rate of repair can be increased at Spo11-DSBs and decreased at the VDE-DSB. One potential role for Srs2 during meiosis is to dismantle recombination intermediates formed between the sister chromatids. Rad6 is required for wild type amounts of Spo11-DSB formation. This work investigated the VDE-DSB repair in the absence of Rad6, and discovered that Rad6 has a role in the initiation of repair. Rad6 ubiquitinates histone H2B, and further analyses suggest that this modification is required for repair at the VDEDSB. Each of the genes studied is required for wild type repair of VDE-DSBs and Spo11-DSBS, even though they come from widely different functional groups. This illustrates the diversity of cellular pathways controlling the initiation and regulation of meiotic recombination.

Orientadores: Beatriz Gomes Guimarães, Jose Antonio Brum
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin
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Resumo: As oxidoredutases de pontes dissulfeto da família DsbA são responsáveis pela catálise da formação de pontes dissulfeto em proteínas secretadas para o periplasma, participando do processo de enovelamento de fatores de virulência de diversos organismos. É a proteína com maior potencial de oxidação atualmente caracterizada e tal propriedade é associada às interações eletrostáticas envolvendo resíduos de seu sítio ativo, que apresenta um arranjo Cys-Pro-His-Cys altamente conservado. A bactéria fitopatogênica Xylella fastidiosa possui dois genes adjacentes que codificam duas oxidoredutases pertencentes à família das DsbAs (XfDbsA e XfDbsA2). Embora a XfDbsA conserve o arranjo CPHC, a XfDbsA2 possui a substituição do resíduo histidina, descrito como essencial à atividade da enzima, por alanina (CPAC). Visando a caracterização estrutural e funcional destas proteínas, a estrutura cristalográfica da XfDsbA foi determinada a 1,9 Å de resolução e um modelo por homologia da XfDsbA2 foi construído. Além disso os potenciais de oxidação das enzimas foram determinados por medidas de fluorescência. A estrutura da XfDsbA revelou a presença de um peptídeo ligado próximo a região do sítio ativo em um dos monômeros mostrando, pela primeira vez em uma estrutura a alta resolução, o provável modo de interação da DsbA com um substrato. Os ensaios funcionais revelaram que as DsbAs de X. fastidiosa apresentam potenciais redox similares e ligeiramente superiores ao da homóloga de Escherichia coli. Embora trabalhos sobre a importância do arranjo CPHC têm associado o alto potencial redox das DsbAs à presença do resíduo histidina no sítio ativo, os resultados obtidos para a XfDsbA2 mostraram que a substituição do resíduo de histidina por alanina não afeta seu potencial redox. A análise das interações envolvendo resíduos do sítio ativo mostrou diferenças importantes entre XfDsbA, XfDsbA2 e suas homólogas de E. coli e Vibrio cholerae. Ensaios funcionais com mutantes foram realizados em busca da identificação dos resíduos que possam compensar a ausência da histidina em XfDsbA2. Os resultados obtidos fornecem novas informações sobre o mecanismo molecular dessa família de enzimas
Abstract: Disulfide oxidoreductase DsbA catalyzes disulfide-bond formation in proteins secreted to the periplasm and has been related to the folding process of virulence factors in many organisms. It is the most oxidizing of the thioredoxin-like proteins and DsbA redox power is understood in terms of the electrostatic interactions involving the active site motif CPHC. The plant pathogen Xylella fastidiosa has two chromosomal genes encoding two oxidoreductases belonging to the DsbA family and, in one of them, the canonical motif CPHC is replaced by CPAC. Aiming at the structural and functional characterization of X. fastidiosa DsbAs, the crystal structure of XfDsbA was solved at 1.9 Å resolution and the XfDsbA2 homology model was calculated. We also determined the redox potential of both enzymes by means of fluorescence experiments. The crystal structure of the XfDsbA revealed an electron density corresponding to an 8-mer peptide interacting with the hydrophobic groove on the surface of the monomer C next to the active site. This modeled peptide shows at first time in a high-resolution crystal structure the probable mode of interaction between DsbA and a substrate. Furthermore, the results presented in this work surprisingly show that, despite the absence of the active site histidine in XfDsbA2, both proteins have similar redox potentials. In addition, the structure of XfDsbA revealed critical differences in the interactions involving the active site residues. Biochemical assays with XfDsbA mutants were performed in order to investigate the residues which may be responsible for compensate for the lack of the conserved histidine in XfDsbA2. The results presented contribute to the understanding of DsbA molecular mechanism
Doutorado
Física da Matéria Condensada
Doutor em Ciências

L’ADN cellulaire qu’il soit nucléaire ou mitochondrial est constamment soumis à l’action de stress d’origine exogène ou même endogène à la base d’altérations plus ou moins profondes de sa structure. Ces modifications chimiques sont très variées et peuvent aller de l’oxydation d’une base aux cassures double-brins de la molécule d’ADN. Ces dernières sont considérées comme les dommages les plus agressifs pour la cellule car peuvent conduire à la perte d’information et donc à la mort cellulaire. Parmi les systèmes de surveillance de la stabilité du génome figure la Poly(ADP-ribosyl)ation (PARylation). Cette modification post-traductionnelle est assurée essentiellement par les protéines PARP et PARG et est caractérisée par l’incorporation des polymères d’ADP ribose (pADPr) sur des protéines cibles. La PARylation constitue un élément clé dans plusieurs voies de maintien de l’intégrité génomique (BER, NHEJ, HR). La PARylation est aussi décrite au niveau de la mitochondrie mais son rôle dans la gestion des DSBs de l’ADNmt n’est pas connu. Le travail, objet de cette thèse, consiste à étudier le rôle de la PARylation dans le cas des DSB au niveau général chez la drosophile et ensuite de comprendre les mécanismes de gestion des DSB mitochondriales et évaluer l’implication de la PARylation dans ce processus. Nos résultats montrent que : (1) le comportement de la PARylation ne varie pas au cours du processus de cassures et de réparation de l’ADN nucléaire, alors que l’expression des ARNm de PARP-I et PARP-II augmente durant la phase de réparation ; (2) les cassures de l’ADN mitochondrial, induites par la bléomycine, entraînent une augmentation du nombre de copies de l’ADNmt. Cette augmentation transitoire de la quantité de l’ADNmt est observée durant la phase des dommages et retourne à la valeur initiale durant la phase de la réparation. Ce comportement semble être régulé par PARP. L’ensemble de ces résultats suggère que la réparation des DSBs est indépendante de la PARylation au niveau nucléaire mais que la présence de PARP est importante. De plus, PARP semble avoir un rôle dans la régulation de la réplication de l’ADNmt en réponse à un stress génotoxique
Both nuclear and mitochondrial DNA alterationsarise following exposure to environmental and endogenous stresses. These genomic alterations are various, ranging from base oxidation to DNA strand breaks, single- and double-strand breaks. These damages are highly detrimental to the cell because they can lead to loss of genetic information and thus to cell death. However, cells have developed various mechanisms to counteract this biological issue and to lead up to a complex DNA damage response (DDR). The Poly (ADP- ribosyl) ation (PARylation) is among these DDR systems. This post-translational modification is mainly carried out by PARP and PARG proteins and is characterized by the incorporation of polymers of ADP-ribose on target proteins. The majority of the PARylationfunctions are related to cellular stress response, particulary in response to genomic damages where it is implicated in many DNA integrity pathways such as Base Excision Repair, Non Homologous End Joining and Homologous Recombination. In contrast to the nucleus, PARylation is also described in the mitochondria but its role in mtDNA integrityis still a heavily debate issue, particularly in case of mtDNA DSBs.To understand it, we used Drosophila model wherePARP-B isoform (human PARP-1 ortholog) is the only enzymatically active form in Drosophila PARP family. The aim of this thesis is to study the role of PARylation in response to DSBs induction in nucleus and mitochondrial DNAand then to understand the mechanisms involved in mtDNA integrity and to evaluate the role of PARylation in this process. Our results show that PARylation level remains stable during DSBs induction and also during repair process,contrary to what is shown in Human cells.However, PARP-I and PARP-II mRNA expression increase during repair period. In mitochondria compartment,our data show an increase of mtDNA copy number in presence of mtDNA DSBs. This increased level returns to normal during repair period and seems to be dependent on PARP. All these results suggest that DSBs repair is PARylation independent at the nuclear level but that the presence of PARP is important. In addition, PARP appears to have a role in the regulation of mtDNA replication in response to genotoxic stress

Our group previously demonstrated that when a DNA double-strand break (DSB) occurs, RNA polymerase II (RNAPII) is recruited to the exposed DNA ends and allows the by-directional synthesis of transcripts derived from exposed DNA ends that we called damage-induced long non-coding RNAs (dilncRNAs). Recently we reported that a DSB also recruits the full transcriptional machinery, in particular the preinitiation complex (PIC), just as commonly recruited at canonical RNA polymerase II-driven transcriptional promoters. This, blurs the distinction between a DSB and a transcriptional promoter. dilncRNAs were defined as non-coding as most of our genome is not coding for polypeptides and thus, DSB will most often transcribe non coding regions of DNA. However, what would be the consequences of a DSB occurring upstream of a gene unit, lacking a promoter but carrying all other features of a coding sequence, such as an open reading frame (ORF) and a poly(A) signal? Would the ensuing dilncRNA actually be a coding transcript? In other words: can a DSB trigger the synthesis of a protein? Therefore, the aim of this project is to test if a DSB appropriately positioned at the transcriptional start site (TSS) of an otherwise silent gene may assemble a functional promoter, triggering RNA synthesis which leads to protein expression. I have carried out such studies in artificial cell systems in which I have engineered promoter-less reporter genes. In addition, I have tested whether a DSB is sufficient to re-express a candidate tumour suppressor gene, HIC-1 (hyper-methylated in cancer 1), that is commonly silenced by promoter methylation. In more in detail, I tested my working hypothesis in three different systems. As a proof of concept, I first used two reporter gene-based systems: a stable clonal cell line of HeLa cells bearing an integrated lentiviral construct encoding for an enhanced green fluorescent protein (EGFP) ORF lacking its transcriptional promoter and enhancer region, and an immortalized mouse embryonic fibroblast (MEF) cell line carrying a promoter-less enhanced yellow fluorescent protein (EYFP) integrated by homologous recombination in the locus Rosa26 of the mouse genome. To induce sites-specific DSBs, I employed the CRISPR/Cas9 technology. Single guide RNAs targeting the GFP or EYFP transcriptional start site (TSS) were cloned in a lentiviral vector and cells were transduced with such a vector expressing them together with Cas9 to test the impact of DSB induction on the expression of the reporter gene. The second system takes advantage of MDA MB 231, a human breast cancer cell line, in which HIC1 endogenous gene is actively silenced by promoter DNA methylation. The goal, here, is to infect these cells with a CRISPR/Cas9 lentiviral vector to induce a DSB at the TSS of HIC1 gene and monitor the synthesis of a mature messenger RNA. These experiments suggested that a DSB, appropriately positioned at a gene TSS, triggers the transcription of RNAs that are polyadenylated, exported in the cytoplasm and translated into a functional protein. This study could be relevant to identify a novel mechanism of transcriptional regulation based on DSB at promoters. This approach could also be used to activate gene expression in genetic disease settings, or to activate the expression of tumor suppressor genes that are silenced in tumors.

Orientadores: Anete Pereira de Souza, Ricardo Aparício
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Xylella fastidiosa é uma bactéria responsável por inúmeras doenças de plantas em culturas economicamente importantes ao redor do mundo, incluindo a clorose variegada dos citros. Após a infecção de seu hospedeiro, as células de X. fastidiosa é apta a formarem uma estrutura de biofilme que bloqueia os vasos xilemáticos, levando a uma condição de estresse hídrico na planta hospedeira e desencadeando o desenvolvimento da doença. Tendo como estímulo a relevância econômica da citricultura para o Brasil e, visando reduzir os prejuízos provocados pelos problemas fitossanitários que acometem esta cultura, foi realizado um consórcio de pesquisa com o intuito de se conhecer completamente o genoma da linhagem 9a5c de X. fastidiosa. Inúmeras proteínas associadas com patogenicidade, adaptação e sobrevivência bacteriana foram identificadas, incluindo XfDsbC (proteína disulfeto isomerase), Xf5'-Nt (5'-nucelotidase), XfTolB (proteína de translocação B) e XfPal (lipoproteína associada ao peptidoglicano) que foram caracterizadas neste estudo. Empregando ferramentas de caracterização de proteínas, aspectos funcionais e estruturais destas quatro proteínas alvos foram avaliados. Dentre os resultados destaca-se a imunodetecção de XfDsbC, Xf5'-Nt, XfTolB e XfPal durante as diferentes fases de formação e desenvolvimento do biofilme de X. fastidiosa, que é tido como o principal mecanismo de patogenicidade deste fitopatógeno, confirmando a predição inicial de tais proteínas como associadas à patogenicidade bacteriana. Adicionalmente, resultados funcionais e estruturais revelaram detalhes finos do papel biológico desempenhado por cada uma das proteínas estudadas. Juntos, os resultados apresentados neste trabalho contribuem para o melhor entendimento de patogenicidade bacteriana, especialmente com respeito ao fitopatógeno X. fastidiosa
Abstract: Xylella fastidiosa is a plant pathogen bacterium responsible for numerous economically important crops diseases around the world, including the citrus variegated chlorosis. Following the host infection, the X. fastidiosa cells are able to form a biofilm structure which block the xylem vessels, leading to a hydric stress condition in the host plant and triggers the disease development. Given the economic relevance of citriculture for Brazil and in order to reduce the damage caused by phytosanitary problems that affect the citrus production, a research consortium was established with the aim to elucidate the complete genome sequence of the X. fastidiosa 9a5c strain. Numerous proteins associated with bacterial pathogenicity, adaptation and survival have been identified, including XfDsbC (protein disulfide isomerase), Xf5'-Nt (5'-nucleotidase), XfTolB (protein translocation B) and XfPal (peptidoglycan-associated lipoprotein) which were characterized in this study. Using tools for protein characterization, structural and functional aspects of these four protein targets were evaluated. Among the results, we highlight the immunodetection of XfDsbC, Xf5'-Nt, XfTolB and XfPal during the different stages of X. fastidiosa biofilm formation and development which is considered the primary mechanism of pathogenicity of this pathogen. These findings, confirming the initial prediction that relates such proteins as associated with bacterial pathogenicity. Additionally, structural and functional results revealed accurate details of the biological role played by each protein studied. Taken together, the findings presented in this study contribute to a better understanding of bacterial pathogenesis, especially with regard to the plant pathogen X. fastidiosa
Doutorado
Genetica de Microorganismos
Doutor em Genetica e Biologia Molecular

L’instabilità genomica è una delle caratteristiche principali delle cellule tumorali e può essere causata da difetti nella riparazione del DNA, dal mancato arresto del ciclo cellulare e dalla perdita della protezione telomerica all’estremità dei cromosomi, che porta alla degradazione e alla fusione delle estremità. Tra i vari tipi di danno al DNA, le rotture della doppia elica del DNA (Double-Strand Break o DSB) rappresentano una delle lesioni più pericolose, poiché possono causare mutazioni o riarrangiamenti cromosomici. In presenza di DSBs, le cellule eucariotiche attivano un checkpoint, dipendente dalle protein chinasi Tel1/ATM e Mec1/ATR, che arresta il ciclo cellulare finché il danno non è stato riparato. Mec1/ATR è attivata dal DNA a singolo filamento (ssDNA) ricoperto da RPA che si forma dopo il processamento nucleolitico (resection) delle estremità del DSB. Una simile risposta è attivata anche quando le estremità naturali dei cromosomi eucariotici perdono la loro protezione, generando delle estremità simili ad un DSB che vengono riconosciute dal checkpoint e dai meccanismi di riparazione. Questa protezione è fornita da complessi nucleoproteici specializzati, chiamati telomeri. Il DNA telomerico è costituito da sequenze ripetute ricche in G che terminano con una coda a singolo filamento sporgente in 3’ (detta coda G), la quale è importante per l’estensione dei telomeri ad opera della telomerasi. Diverse proteine, tra cui il complesso CST, sono necessarie al mantenimento della struttura e della lunghezza dei telomeri sia in lievito che nei mammiferi. Recenti dati sperimentali indicano che i fattori che processano l’RNA hanno un ruolo fondamentale nella stabilità del genoma e nel metabolismo telomerico, anche se il meccanismo è ancora poco compreso. In questa tesi abbiamo dimostrato che in Saccharomyces cerevisiae le proteine che degradano l’RNA Xrn1, Rrp6 e Trf4 promuovono l’attivazione di Mec1/ATR facilitando la formazione di DNA a singolo filamento ricoperto da RPA ai DSB. Inoltre, Xrn1 e Rrp6 sono necessarie per attivare il checkpoint anche ai telomeri deprotetti a causa del malfunzionamento di Cdc13, una delle subunità del complesso CST coinvolto nella protezione dei telomeri. Xrn1 facilita la formazione di DNA a singolo filamento ai DSBs promuovendo il caricamento del complesso MRX, mentre come svolga questa funzione ai telomeri deprotetti rimane ancora da chiarire. Al contrario, la generazione di ssDNA ai DSBs non è influenzata dalla mancanza di Rrp6 o Trf4, ma la loro assenza ostacola il reclutamento di RPA e quindi di Mec1 al sito di danno. L’inattivazione di Rrp6 e Trf4 non influenza né l’associazione di Rad51/Rad52 ai DSB né la riparazione della rottura attraverso la ricombinazione omologa (Homologous Recombination o HR), suggerendo che la piena attivazione di Mec1 richieda più DNA a singolo filamento ricoperto da RPA di quanto ne sia richiesto per la riparazione attraverso la ricombinazione omologa. Infine, Xrn1, regolando negativamente il trascritto di RIF1, è coinvolto nel mantenimento della lunghezza dei telomeri promuovendo l’associazione di Cdc13 indipendentemente dalla formazione di DNA a singolo filamento. In conclusione, i nostri risultati forniscono un nuovo collegamento tra il processamento dell’RNA e il mantenimento della stabilità del genoma.
Genome instability is one of the most pervasive characteristics of cancer cells. It can be due to DNA repair defects, failure to arrest the cell cycle and loss of telomere-end protection that lead to end-to-end fusion and degradation. Among the many types of DNA damage, the DNA Double Strand Break (DSB) is one of the most severe, because it can cause mutations and chromosomal rearrangements. Eukaryotic cells respond to DSBs by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR, in order to arrest the cell cycle until DSBs are repaired. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA) that arises upon nucleolytic degradation (resection) of the DSB. A similar checkpoint response is triggered when the natural ends of eukaryotic chromosomes lose their protection, resembling and being recognized as DSBs. This protection is provided by specialized nucleoprotein complexes called telomeres. Telomeric DNA consists of repetitive G-rich sequences that terminate with a 3’-ended single-stranded overhang (G-tail), which is important for telomere extension by telomerase. Several proteins, including the CST complex, are necessary to maintain telomere structure and length in both yeast and mammals. Emerging evidences indicate that RNA processing proteins play critical, yet poorly understood, roles in genomic stability and telomere metabolism. We provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 facilitate activation of Mec1/ATR by promoting the generation of RPA-coated ssDNA at intrachromosomal DSBs. Xrn1 and Rrp6 are also required to activate a Mec1/ATR-dependent checkpoint at uncapped telomeres due to loss of the CST component Cdc13. Xrn1 promotes checkpoint activation by facilitating the generation of ssDNA at both DSBs and uncapped telomeres. Xrn1 exerts this function at DSBs by promoting the loading of the MRX complex, whereas how it does at uncapped telomeres remains to be determined. By contrast, DSB resection is not affected by the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Finally, we demonstrate that Xrn1 maintains telomere length by promoting the association of Cdc13 to telomeres independently of ssDNA generation and exerts this function by downregulating the RIF1 transcript. Our results provide novel links between RNA processing and genome stability.

L’instabilità genomica è una delle principali caratteristiche delle cellule tumorali e può essere generata da danni al DNA o da stress replicativi. Le rotture della doppia elica di DNA, Double Strand Breaks-DSBs, sono tra i danni più pericolosi che le cellule devono affrontare. In risposta ai DSBs, le cellule attivano un meccanismo molto conservato noto come checkpoint da danno al DNA, il cui effetto primario è quello di bloccare il ciclo cellulare fino a quando la rottura non è stata riparata. L’attivazione del checkpoint è dovuta alle chinasi apicali Tel1 e Mec1 che fosforilano e attivano le chinasi effettrici Rad53 e Chk1. I DSBs possono essere riparati mediante la ricombinazione omologa che inizia con la degradazione nucleolitica-resection- dell’estremità della rottura catalizzata dal complesso MRX e da Sae2. In seguito, le nucleasi Exo1 e Dna2, insieme all’elicasi Sgs1, catalizzano la formazione di lunghi tratti di DNA a singolo filamento. La resection è controllata negativamente dal complesso Ku, che inibisce Exo1, e dalla proteina di checkpoint Rad9, il cui meccanismo di regolazione non è noto. In lievito, l’assenza di Sae2 genera un difetto di resection che è responsabile dell’attivazione persistente del checkpoint dipendente da Tel1 e da Rad53. Per via di questo difetto, mutanti sae2 sono sensibili ad agenti genotossici che inducono DSBs. Tuttavia, la causa del difetto di resection e come l’attivazione incontrollata del checkpoint contribuiscano al fenotipo di sensibilità non è ancora noto. Per questo abbiamo cercato altri meccanismi che regolano l’inizio della resection, identificando mutazioni extrageniche in grado di sopprimere le sensibilità di cellule sae2. Abbiamo quindi isolato tre alleli SGS1-G1298R, rad53-Y88H e tel1-N2021D, in grado di sopprimere non solo le sensibilità ma anche il difetto di resection di mutanti sae2. La soppressione mediata da Sgs1-G1298R dipende da Dna2 e non da Exo1. Inoltre, l’azione di Sgs1-G1298R non solo sopprime il difetto di resection di cellule sae2 ma aumenta anche l’efficienza del processo rispetto ad un ceppo selvatico, a causa della resistenza all’inibizione mediata da Rad9. Infatti, Rad9 regola negativamente il reclutamento di Sgs1 alle estremità della lesione. Quando l’azione inibitoria di Rad9 viene meno, la richiesta del complesso MRX e di Sae2 nell’inizio della resection è ridotta. Rad53-Y88H e Tel1-N2021 sono varianti con perdita di funzione in grado di sopprimere le sensibilità di cellule sae2, in maniera dipendente da Sgs1-Dna2. Inoltre, anche l’assenza dell’attività chinasica di Rad53 e Tel1 permette di ottenere lo stesso fenotipo di soppressione che, tuttavia, non è dovuto al ruolo delle stesse nel blocco del ciclo cellulare. Infatti, queste mutazioni diminuiscono la quantità di Rad9 legato al DSB. Ciò facilita l’azione dell’elicasi Sgs1 e della nucleasi Dna2, sopprimendo così il difetto di resection di cellule sae2. Tali dati portano ad ipotizzare che l’attivazione persistente del checkpoint Tel1 e Rad53 dipendente causi un aumento del reclutamento dell’inibitore Rad9 nell’intorno della lesione che, a sua volta, è responsabile del difetto di resection e delle sensibilità di cellule sae2. Gli stress replicativi inducono il blocco della forca di replicazione e il processo di resection può essere un valido meccanismo per risolverlo. A questo proposito, abbiamo dimostrato che l’assenza dell’inibizione mediata da Rad9 compromette la risposta agli stress replicativi di cellule difettive nell’attività chinasica di Mec1, attraverso la degradazione delle forche bloccate in maniera dipendente da Sgs1 e Dna2. Tale funzione protettiva di Rad9 è indipendente dalla sua funzione nel checkpoint ma dipende principalmente dall’interazione di Rad9 con la proteina Dpb11. Per questo, abbiamo ipotizzato che Rad9 sia in grado di regolare la resection non solo al DSB ma anche alle forche di replicazione bloccate.
Genome instability is an hallmark of cancer cells and can be due to DNA damage or replication stress. DNA double strand breaks (DSBs) are the most dangerous type of damage that cells have to manage. In response to DSBs, cells activate an highly conserved mechanism known as DNA damage checkpoint (DDC), whose primary effect is to halt the cell cycle until the damage is repaired. DDC is activated by the apical kinases Tel1/ATM and Mec1/ATR, which phosphorylate and activate the effector kinases Rad53/CHK2 and Chk1/CHK1. The Homologous Recombination (HR)-mediated repair of a DSB starts with the nucleolytic degradation (resection) of the 5’ ends to create long ssDNA tails. In Saccharomyces cerevisiae, resection starts with an endonucleolytic cleavage catalyzed by the MRX complex together with Sae2. More extensive resection relies on two parallel pathways that involve the nucleases Exo1 and Dna2, together with the helicase Sgs1. Resection must be tightly controlled to avoid excessive ssDNA creation. The Ku complex and the checkpoint protein Rad9 negatively regulate resection. While Ku inhibits Exo1, Rad9 restrains nucleolytic degradation by an unknown mechanism. The absence of Sae2 impairs DSB resection and causes prolonged MRX binding at DSB that leads to persistent Tel1 and Rad53-dependent DNA damage checkpoint. SAE2 deleted strains are sensitive to DSBs inducing agents, like camptothecin (CPT). This sensitivity has been associated to the resection defect of sae2∆ cells, but what causes this resection defect and if the enhanced checkpoint signaling contributes to the DNA damage sensitivity of sae2∆ cells is unknown. For these reasons, we tried to identify other possible mechanisms regulating MRX/Sae2 requirement in DSB resection by searching extragenic mutations that suppressed the sensitivity to DNA damaging agents of sae2Δ cells. We identified three mutant alleles (SGS1-G1298R, rad53-Y88H and tel1-N2021D) that suppress both the DNA damage hypersensitivity and the resection defect of sae2∆ cells. We show that Sgs1-G1298R-mediated suppression depends on Dna2 but not on Exo1. Furthermore, not only Sgs1-G1298R suppresses the resection defect of sae2∆ cells but also increases resection efficiency even in a wild type context by escaping Rad9-mediated inhibition. In fact, Rad9 negatively regulates the binding/persistence of Sgs1 at the DSB ends. When inhibition by Rad9 is abolished by the Sgs1-G1298R mutant variant, the requirement for MRX/Sae2 in DSBs resection is reduced. Rad53-Y88H and Tel1-N2021 are loss of function mutant variants that suppress sae2∆ cells sensitivity in a Sgs1-Dna2 dependent manner. Furthermore, abolishing Rad53 and Tel1 kinase activity results in a similar suppression phenotype which does not involve the escape from the checkpoint mediated cell cycle arrest. Rather, defective Rad53 or Tel1 signaling bypasses Sae2 function in DSBs resection by decreasing the amount of Rad9 bound at DSBs. This increases the Sgs1-Dna2 activity that, in turn, can compensate for the lack of Sae2. We propose that persistent Tel1 and Rad53 checkpoint signaling in sae2∆ cells causes DNA damage hypersensitivity and defective DSB resection by increasing the amount of Rad9 that, in turn, inhibits Sgs1-Dna2. Replication stress can induce fork stalling and controlled resection can be a relevant mechanism to allow repair/restart of stalled replication forks. We show that loss of the inhibition that Rad9 exerts on resection exacerbates the sensitivity to replication stress of Mec1 defective yeast cells by exposing stalled replication forks to Dna2-dependent degradation. This Rad9 protective function is independent of checkpoint activation and relies mainly on Rad9-Dpb11 interaction. We propose that Rad9 not only regulates the action of Sgs1-Dna2 at DSBs but also at stalled replication forks, supporting cell viability when the S-phase checkpoint is not fully functional.

Increased mutagenesis is a hallmark of cancers. On the other hand, this can trigger the generation of polymorphisms and lead to evolution. Lately, it has become clear that one of the major sources of increased mutation rates in the genome is chromosomal break formation and repair. A variety of factors can contribute to the generation of breaks in the genome. A paradoxical source of breaks is the sequence composition of the genomic DNA itself. Eukaryotic and prokaryotic genomes contain sequence motifs capable of adopting secondary structures often found to be potent inducers of double strand breaks culminating into rearrangements. These regions are therefore termed fragile sequence motifs. Here, we demonstrate that in addition to being responsible for triggering chromosomal rearrangements, inverted repeats and GAA/TTC repeats are also potent sources of mutagenesis. Repeat-induced mutagenesis extends up to 8 kb on either side of the break point. Remarkably, error-prone repair of the break by Polζ reconstitutes the repeats making them a long term source of mutagenesis. Despite its negative connotations for genome stability, the mechanisms underlying the unstable nature of double strand break repair pathways are not known. Previous studies have demonstrated that break induced replication (BIR), a mechanism employed to repair broken chromosomes with only one repairable end, is highly mutagenic, undergoes frequent template switching and often yields half-crossovers. In the work presented here, we show that the instabilities inherent to BIR can be attributed to its unusual mode of synthesis. We determined that BIR proceeds via a migrating bubble with long stretches of single-stranded DNA and culminates with conservative inheritance of the newly synthesized DNA. We propose that the mechanisms described here might be important for generation of repair-associated mutagenesis in higher organisms. Secondary structure forming repeats like inverted repeats have been found to be enriched in cancer cells. These motifs often constitute chromosomal rearrangement hot-spots and demonstrate the phenomenon of kataegis. This study provides a mechanistic insight into how such breakage-prone motifs contribute to hypermutability of cancer genomes.

An efficient DNA damage response is critical for maintaining the integrity of the mammalian genome, and ensuring the accurate transfer of genetic information between generations. Of particular biological relevance are DNA double strand breaks (DSB), which if repaired incorrectly may contribute to carcinogenesis. Review of contemporary literature has led to the identification of protein interactions and transcriptional events, tightly associated with the mammalian DSB response. Characteristics of selected events have been manipulated, with the notion of developing a reporter system that offers a sensitive and rapid method of detecting DSB in living mammalian cell models. Work presented here provides a quantitative evaluation of DSB generation in various mammalian cell lines, following chemical and irradiation treatment, and highlights the limitations of currently used markers. A series of recombinant proteins comprising peptide interacting domains, which exhibit altered spatio-temporal dynamics in relation with each other following DSB induction, are proposed as potential reporters of damage in mammalian cells. Novel gene constructs have been engineered that encode these peptide interacting domains, sandwiched between fluorescence-resonance-energy transfer (FRET) capable proteins. DSB specific events are predicted to induce peptide interactions that may be tracked in real time, by monitoring alterations in the fluorescent properties of such a recombinant protein. In an alternative approach, the transcriptional up-regulation of RAD52 mRNA following DSB induction was extended to whole cells. Optimisation of a fluorescent molecular beacon probe complementary to mammalian RAD52 mRNA is described, and data obtained in mammalian cells following DSB induction supports the notion that RAD52 is actively transcribed as part of the DSB response.

Thesis (M.S.)--University of Missouri-Columbia, 2006.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 30, 2007) Includes bibliographical references.

O ácido cis-11-metil-2-dodecenóico (DSF), substância utilizada como autoindutor nos sistemas de Quorum Sensing da bactéria Xanthomonas axonopodis pv. citri, foi utilizado como um modelo de estudos para a análise de moléculas secretadas por populações celulares utilizando técnicas eletroanalíticas. Cultivos celulares provenientes de três linhagens diferentes da bactéria Xanthomonas axonopodis pv. citri foram utilizados para os estudos. Uma linhagem selvagem (WT), que produzia DSF em quantidades naturalmente observadas, e duas linhagens geneticamente modificadas. Em uma linhagem (ΔC) a modificação genética resultava em elevada produção de DSF, para a segunda (ΔF) o resultado era a anulação da produção do autoindutor. As principais técnicas analíticas utilizadas foram: voltametria cíclica, eletroforese capilar e cromatografia líquida (com detecção UV-Vis e acoplada a espectrometria de massas), sendo a última para efeitos de comparação. Procedimentos de extração líquido-líquido com solventes orgânicos foram realizados como etapas de pré-tratamento de amostras, visando diminuir a quantidade de interferentes e realizar uma pré-concentração do analito. Três metodologias principais foram adotadas. Uma primeira voltada para a diferenciação dos cultivos celulares, analisando-se ou os sobrenadantes provenientes dos cultivos ou os extratos resultantes dos pré-tratamentos desses sobrenadantes, tendo como principal foco a identificação de um sinal diferencial entre os cultivos que correspondesse às quantidades de DSF, ou seja, inexistente para a linhagem ΔF, de alta intensidade para linhagem ΔC e de intensidade intermediária para a linhagem WT. A segunda metodologia foi baseada no uso de uma molécula modelo para realizar experimentos e obter dados como parâmetros físico-químicos, o ácido láurico (ácido dodecanóico) foi escolhido por sua semelhança estrutural e facilidade de obtenção, curvas de calibração e experimentos de separação de moléculas com estrutura semelhante foram realizados com a técnica de eletroforese capilar. Uma terceira metodologia foi desenvolvida utilizando o padrão do ácido cis-11-metil-2-dodecenóico (DSF), curvas de calibração foram obtidas com o padrão isolado apresentando ótimas linearidades, tanto com a técnica de eletroforese capilar quanto com cromatografia líquida com detecção UV-Vis, experimentos de adição de padrão às amostras também foram executados. Os experimentos realizados com as amostras não apresentaram os resultados esperados, os problemas foram associados principalmente à quantidade de interferentes na amostra e a baixa concentração do analito nos cultivos, sendo que os métodos de extração realizados não foram suficientes para solucioná-los. O insucesso da técnica de voltametria cíclica foi associado ao envenenamento do eletrodo, devido à alta quantidade de compostos orgânicos presentes na amostra, e dúvidas relacionadas à eletroatividade do DSF. As dificuldades encontradas com a técnica de eletroforese capilar foram associadas à alta força iônica da amostra, presença de interferentes e baixa concentração do analito nos cultivos. Mesmo os experimentos realizados com cromatografia líquida acoplada a detecção com espectrometria de massas não atenderam às expectativas, dificultando a elucidação dos problemas encontrados até então e impossibilitando o desenvolvimento de uma metodologia que atendesse aos objetivos propostos.
Cis-11-methyl-2-dodecenoic acid, also known as DSF (autoinducer of the Quorum Sensing system used by the bacteria Xanthomonas axonopodis pv. citri) was explored as a study model for the cell secreted molecules analysis by electroanalytical techniques. Cell cultures from three different cell lines of the bacteria Xanthomonas axonopodis pv. citri were used for this study. A wild type (WT), which produced DSF in naturally observed quantities and two genetically modified ones. One of the lines (ΔC) had a genetic modification that resulted in an elevated production of DSF, and the other (ΔF) had a modification that stopped DSF production. Main analytical techniques used were: cyclic voltammetry, capillary electrophoresis and liquid chromatography (with UV-Vis detection and coupled with mass spectrometry), this last one was used with comparison purposes. Liquid-liquid extractions with organic solvents were realized as sample pre-treatment steps aiming the reduction of interfering species and analyte pre-concentration. Three main methodologies were adopted. One based in the differentiation of cell cultures analyzing or the cell culture supernatants or the resulting extracts from the pre-treatment steps. The main focus was the identification of a differential signal between the cultures that corresponded to the DSF quantities, in other words, null for the ΔF line, high intensity for the ΔC line and a an intermediary intensity for the WT line. The second methodology was based in experiments with a model molecule to obtain some data such as physical-chemistry parameters. Lauric acid (dodecanoic acid) was chosen for its structural similarity and easily acquisition. Calibration curves and similar structure molecules separation experiments were realized using capillary electrophoresis. Third and last methodology was developed using standard cis-11-methyl-2-dodecenoic acid (DSF). Calibration curves were obtained with the isolated standard showing great linearities using both capillary electrophoresis and liquid chromatography with UV-Vis detection. Experiments of standard addiction to the samples were also realized. Experiments using the samples did not show the expected results. Problems found were mainly associated with high amount of interfering species and low analyte concentration at the cultures, extraction methods used were not enough to solve this. The failures with the cyclic voltammetric techniques were associated to electrode poisoning, due to the high amount of organic compounds present at the sample, and DSF lack of electroactivity. Difficulties found with capillary electrophoresis were associated with the samples high ionic strength, presence of interfering species and low analyte concentration. Even liquid chromatography coupled with mass spectrometry experiments didn\'t attended the expectations. Making it difficult to elucidate the problems and not allowing the development of a methodology that could achieve the proposed objectives.

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Este trabalho trata da realização de um circuito integrado analógico para a conversão de sinais com modulação em amplitude de banda dupla (Double Sideband ou DSB) para modulação de banda simples (Single Sideband ou SSB). Implementado por circuitos de tempo discreto a capacitores chaveados, utiliza-se de um filtro com resposta infinita ao impulso (Infinite Input Response ou IIR) para compor um transformador de Hilbert como alternativa a implementações digitais, que se aproveitam da grande capacidade de processamento paralelo dos circuitos digitais para a obtenção do transformador de Hilbert por meio de filtros com resposta finita ao impulso (Finite Impulse Response ou FIR) de ordem elevada. Fabricado em tecnologia CMOS de 180 nm com capacitores do tipo metal-metal (MiM), a adoção de filtros estruturalmente passa-tudo reduz significativamente a sensibilidade do conversor ao descasamento de capacitores. Para alimentação de 1,8 V e sinais diferenciais de até 1 V, resultados experimentais mostram que o conversor atinge taxa de rejeição de imagem (Image Rejection Ratio ou IRR) maior que 39,5 dB para modulação Lower Sideband (LSB) e 38,0 dB para modulação Upper Sideband (USB) para sinais de entrada na faixa de 25% a 75% da frequência da portadora, valores estes superiores a propostas analógicas anteriores e comparáveis a propostas digitais do estado da arte em circuitos integrados. Com área de silício de 1,09 mm2, o conversor consome apenas 17,7 mW para frequência de amostragem de 1 MHz enquanto sua IRR apresentou desvio padrão de apenas 0,5 dB dentre 20 amostras avaliadas.
The realization of an analog integrated circuit for conversion of Double-Sideband (DSB) amplitude-modulated signals into Single-Sideband (SSB) is presented. Implemented by discrete-time switched-capacitor circuits, it adopts an Infinite Impulse Response (IIR) filter to realize a Hilbert transformer as alternative to digital implementations which take advantage of high processing capacity from parallel digital circuits to obtain the Hilbert transformer by means of high-order Finite Impulse Response (FIR) filters. Fabricated in a 180 nm CMOS technology with metal-metal (MiM) capacitors, the use of structurally all-pass filters greatly reduces the converter’s sensitivity to capacitor mismatch. For 1.8 V power supply and 1 V differential input/output signals, experimental results show the converter achieves Image Rejection Ratio (IRR) greater than 39.5 dB for Lower-Sideband (LSB) modulation and 38.0 dB for Upper-Sideband (USB) modulation for input signals ranging from 25% to 75% of the carrier frequency. These figures are higher than previous analog circuit proposals and comparable to digital implementations of state-of-the-art integrated circuits. Its silicon area is 1.09 mm2 and the converter consumes only 17.7 mW for 1 MHz sampling frequency while its IRR presents standard deviation of only 0.5 dB among 20 chip samples.

Many Gram-negative bacterial pathogens such as Shigella and adhesive invasive Escherichia coli (AIEC) cause infections characteristic of hyperinflammation. These infections require antimicrobial therapy. However, due to the widespread emergence of multiple drug-resistant strains, alternative strategies must be sought to combating infectious diseases. It has been shown that natural compounds such as propolin D are able to control Shigella growth inside host cells. Geraniol is another natural product which has a chemical structure similar to the side chain of propolin D, which possesses properties potentially useful for antimicrobial therapy. qPCR analysis revealed that propolin D caused extensive bacterial envelopE stress, as indicated by a changed expression of key bacterial genes involved in stress responses. Propolin D also enhanced the autophagy activity of the host cells; the intracellular growth of S. sonnei was significantly reduced in wild type HEK293 cells but not changed in ATG5 knockout cells. Propolin D was unable to enhance septin cage as intracellular S. sonnei formed actin tails in the presence of propolin D; septin cage would restrict formation of actin tails. Geraniol has been shown to target the major virulence regulator, DsbA, which is vital for Shigella’s survival in the reducing host cell cytosol. Geraniol and geranylxvacetate inhibited DsbA function in vitro; wild type DsbA efficiently reduced fluorescently labelled Di-E-GSSG whereas a mutant protein, DsbA33G, was less potent in this in vitro assay. By supplementing acidic and nutrient-poor medium with geraniol the growth of S. sonnei and AIEC strains was severely inhibited. Geraniol was effective in protecting of Galleria. mellonella larva from S. sonnei and AIEC infection. The Galleria mellonella larvae were highly tolerant to geraniol – indicating the great potential of geraniol for future in vivo and clinical studies. In light of previous reports that geraniol synergistically works with antibiotics and induces IL-10 from macrophages, it was concluded that geraniol holds great potential in treating Shigella and AIEC infections.

DNA double strand breaks (DSBs), which may occur during DNA replication or due to the action of genotoxic agents, are extremely dangerous DNA lesions as they can cause chromosomal rearrangements and cell death. Therefore, accurate DSB repair is vital for genome stability and cell survival. Two main mechanisms serve to repair DNA DSBs: non-homologous end joining, which re-ligates DNA ends together, and homologous recombination (HR), which restores broken DNA using homologous sequence as a template for repair. One-ended DSBs are a subject for the specialised HR-dependent repair pathway known as break-induced replication (BIR). At low frequency, DNA breaks can also be healed by telomerase, which normally extends telomeres at natural chromosome ends, but may also add de novo telomeres to DSBs due to their similarity to chromosome ends. De novo telomere addition is a deleterious event, which is effectively inhibited by the nuclear Pif1 (nPif1) helicase phosphorylated at the TLSSAES motif in response to DNA damage. In this study, it is reported that the same regulatory motif of nPif1 is also required for DSB repair via BIR. The requirement of the nPif1 TLSSAES sequence in BIR is dependent on the functional DNA damage response (DDR). Thus, nPif1 phosphorylation by the DDR machinery might mediate the role of nPif1 in BIR. In contrast, the nPif1 regulatory motif is not essential for BIR at telomeres in cells lacking telomerase. These observations indicate that the mechanism of nPif1 function in DSB repair via BIR and in BIR at telomeres might be different. In this work, a protocol for nPif1 pull-down was optimized to reveal the mechanism of the phosphorylation-dependent nPif1 functions in cells undergoing DNA repair, i. e. the mechanism of nPif1-mediated inhibition of de novo telomere addition and promoting DSB repair via BIR. In future, this protocol can be used to dissect the role of nPif1 in DNA repair through the identification of its potential interacting partners. The Srs2 helicase negatively regulates HR via dismantling Rad51 filaments. According to preliminary data from the laboratory of Sveta Makovets, Srs2 also promotes de novo telomere addition at DSBs in a Rad51-dependent manner. The work presented here establishes that Srs2 is dispensable for telomerase-mediated addition of TG1-3 repeats to DSBs. Instead, Srs2 is required for the reconstitution of the complementary DNA strand after telomerase action, thus ensuring the completion of de novo telomere addition. Overall, this study demonstrates that recombination-dependent DSB repair and de novo telomere addition share common regulatory components, i. e. the nPif1 helicase phosphorylated in response to DNA damage and the Srs2 helicase. Phosphorylated nPif1 promotes DSB repair via BIR in addition to its known role in inhibition of telomerase at DSBs, whereas Srs2 uses its well established ability to remove Rad51 from ssDNA to promote the restoration of dsDNA and thus to complete de novo telomere addition.

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Este trabalho trata da realização de um circuito integrado analógico para a conversão de sinais com modulação em amplitude de banda dupla (Double Sideband ou DSB) para modulação de banda simples (Single Sideband ou SSB). Implementado por circuitos de tempo discreto a capacitores chaveados, utiliza-se de um filtro com resposta infinita ao impulso (Infinite Input Response ou IIR) para compor um transformador de Hilbert como alternativa a implementações digitais, que se aproveitam da grande capacidade de processamento paralelo dos circuitos digitais para a obtenção do transformador de Hilbert por meio de filtros com resposta finita ao impulso (Finite Impulse Response ou FIR) de ordem elevada. Fabricado em tecnologia CMOS de 180 nm com capacitores do tipo metal-metal (MiM), a adoção de filtros estruturalmente passa-tudo reduz significativamente a sensibilidade do conversor ao descasamento de capacitores. Para alimentação de 1,8 V e sinais diferenciais de até 1 V, resultados experimentais mostram que o conversor atinge taxa de rejeição de imagem (Image Rejection Ratio ou IRR) maior que 39,5 dB para modulação Lower Sideband (LSB) e 38,0 dB para modulação Upper Sideband (USB) para sinais de entrada na faixa de 25% a 75% da frequência da portadora, valores estes superiores a propostas analógicas anteriores e comparáveis a propostas digitais do estado da arte em circuitos integrados. Com área de silício de 1,09 mm2, o conversor consome apenas 17,7 mW para frequência de amostragem de 1 MHz enquanto sua IRR apresentou desvio padrão de apenas 0,5 dB dentre 20 amostras avaliadas.
The realization of an analog integrated circuit for conversion of Double-Sideband (DSB) amplitude-modulated signals into Single-Sideband (SSB) is presented. Implemented by discrete-time switched-capacitor circuits, it adopts an Infinite Impulse Response (IIR) filter to realize a Hilbert transformer as alternative to digital implementations which take advantage of high processing capacity from parallel digital circuits to obtain the Hilbert transformer by means of high-order Finite Impulse Response (FIR) filters. Fabricated in a 180 nm CMOS technology with metal-metal (MiM) capacitors, the use of structurally all-pass filters greatly reduces the converter’s sensitivity to capacitor mismatch. For 1.8 V power supply and 1 V differential input/output signals, experimental results show the converter achieves Image Rejection Ratio (IRR) greater than 39.5 dB for Lower-Sideband (LSB) modulation and 38.0 dB for Upper-Sideband (USB) modulation for input signals ranging from 25% to 75% of the carrier frequency. These figures are higher than previous analog circuit proposals and comparable to digital implementations of state-of-the-art integrated circuits. Its silicon area is 1.09 mm2 and the converter consumes only 17.7 mW for 1 MHz sampling frequency while its IRR presents standard deviation of only 0.5 dB among 20 chip samples.

Neisseria meningitidis est le principal agent responsable de méningites bactériennes. Les interactions hôte-pathogène dépendent du repliement correct de nombreuses protéines de surface, qui nécessite souvent la formation de ponts disulfures. Chez les bactéries à Gram-négatif, la synthèse de ces ponts est catalysée par l'oxydoréductase de thiol-disulfure DsbA. N. Meningitidis possède trois gènes qui codent pour trois DsbA actives : DsbA1, DsbA2 et DsbA3. DsbA1 et DsbA2 sont des lipoprotéines impliquées dans la virulence alors que DsbA3 est une enzyme soluble périplasmique non reliée à la virulence. Les travaux de cette thèse se rapportent aux caractérisations biochimiques de ces trois enzymes et structurales de DsbA1 et DsbA3. DsbA1 et DsbA3 adoptent le repliement classique de DsbA d'Escherichia coli. La caractéristique la plus étonnante partagée par ces trois enzymes est leur exceptionnel pouvoir oxydant. Avec un potentiel redox de -80 mV, les DsbA de Neisseria sont les enzymes de la famille des thiorédoxines les plus oxydantes connues à ce jour. En accord avec cela, les études de stabilité thermales indiquent que leur forme réduite est extrêmement stable. Pour chacune de ces enzymes, les études montrent que le résidu Thréonine, retrouvé dans la région du site actif, joue un rôle clé dans la détermination de cet extraordinaire pouvoir oxydant. L'ensemble de ces résultats montrent comment des résidus situés en dehors du motif actif CXXC peuvent influencer le potentiel redox de membres de la famille des thiorédoxines. Ils montrent également que le phénotype associé à DsbA3 chez N. Meningitidis ne peut être expliqué par une différence d'activité redox ou de structure
Neisseria meningitidis is an invasive bacterial pathogen causing life-threatening infection. Host-pathogen interactions depend on the correct folding of many surface-exposed proteins, which often requires disulfide bond formation. In Gram-negative bacteria, the synthesis of disulfide bonds is catalyzed by the thiol-disulfide oxidoreductase DsbA. N. Meningitidis possesses three genes encoding three active DsbA (DsbA1, DsbA2 and DsbA3). DsbA1 and DsbA2 are lipoproteins involved in the virulence while DsbA3 is a soluble periplasmic protein non related to the virulence. This work reports the biochemical characterisation of the three neisserial enzymes and the crystal structures of DsbA1 and DsbA3. DsbA1 and DsbA3 adopt the classical Escherichia coli DsbA fold. The most striking feature shared by all three proteins is their exceptional oxidizing power. With a redox potential of -80 mV, they are the most oxidizing thioredoxin-like enzymes known to date. For each of these enzymes, the threonine residue found within the active site region plays a key role in dictating this extraordinary oxidizing power. Consistent with these findings, thermal studies indicate that their reduced form is also extremely stable. This result highlights how residues located outside the CXXC motif may influence the redox potential of members of the thioredoxin family. In addition, this functional and structural study shows that the phenotype associated with DsbA3 in N. Meningitidis cannot be explained by a difference of redox activity

Nous montrons que la structure RMN, en solution, du domaine N-terminal de DsbD de Neisseria meningitidis (nDsbD) présente, à l’état réduit, un repliement de type immunoglobuline avec un site actif adoptant une conformation fermée. Toutefois, l’analyse des mouvements internes du squelette peptique de nDsbD montre que la région dite « couvercle » de la protéine et qui protège les résidus actifs dans les formes réduite et oxydée, est dotée de mouvements internes. Cela démontre les capacités intrinsèques d’ajustement structural de nDsbD. Nous montrons aussi que les structures RMN, en solution, du domaine N-terminal de PilB de N. meningitidis (NterPilB) sous ses deux formes, réduite et oxydée, présentent un repliement de type thiorédoxine. Ces deux formes, très proches d’un point de vue structural, apparaissent comme étant globalement rigides. Par conséquent, la boucle FLHE, caractéristique de NterPilB et bordant le site actif de la protéine, ne dévoile pas de nouveaux indices structuraux et/ou dynamiques traduisant son implication dans la spécificité de substrat. Finalement, nous montrons, grâce à l’étude structurale et dynamique, en solution, d’un complexe entre nDsbD et NterPilB de N. meningitidis, que nDsbD fait preuve d’une grande adaptabilité à l’état complexé. La région « couvercle » s’ouvre pour venir se positionner au dessus de l’hélice a qui contient les cystéines actives de NterPilB. Par contre, la boucle FLHE de NterPilB ne semble pas intervenir dans la stabilisation du complexe. Nous proposons que des phénomènes dynamiques puissent faciliter d’une part, l’adaptabilité relative des deux partenaires dans le complexe, et d’autre part, la dissociation finale de ces derniers
We show, on one hand, that the NMR solution structure of DsbD N-terminal domain from Neisseria meningitidis (nDsbD) displays, in its reduced state, an immunoglobulin fold with a closed conformation of its active site. Nonetheless, our backbone dynamics study shows that the cap-loop region of the protein, which covers active residues in both oxidized and reduced forms, displays internal motions. This illustrates the inner structural adjustment capacities of nDsbD. On the other hand, we show that NMR solution structures of the oxidized and reduced forms of N. meningitidis NterPilB display a thioredoxin-like fold. These two structures appear to be very similar and globally rigid. Consequently, the NterPilB characteristic FLHE loop, which covers one edge of the active site, does not reveal new structural and/or dynamics properties for its involvement in the substrate specificity. Finally, we point out, from the structural and dynamics study of a complex between nDsbD and NterPilB from N. meningitidis, that nDsbD exhibits a powerful adaptability in its complex state. Its cap-loop region opens and comes over the a helix containing the NterPilB active cysteines. In contrast, the NterPilB FLHE loop does not seem to play a role in the complex stabilization. We propose that internal dynamics should facilitate, on one hand, the relative adaptability between the two partners of the complex and, on the other hand, their subsequent dissociation

The study of natural variation is a principle component of biology. One process that affects levels of natural variation is meiotic recombination—the process by which homologous chromosomes break and interchange genetic information with one another during the formation of gametes. Surprisingly, this factor that shapes levels of natural variation across the genome itself presents with a great deal of variation. That variation manifests itself at many levels: within genomes, between individual organisms, across populations, and among species. The factors and mechanisms responsible for the non-random patterning of recombination events across the genome remain particularly elusive in most cases. Herein, I utilize a combination of bioinformatic and molecular genetic approaches to better explain recombination patterning. I explore several factors that are now known to contribute to the distribution of recombination events across genomes. In particular, I demonstrate that transcriptional activity during meiosis is associated with, and partially predictive of crossing over events in Drosophila melanogaster. Additionally, I present a model which is capable of accounting for approximately 40% of the variation in crossover rates in Drosophila based on the localization of several previously identified DNA motifs. Lastly, I present preliminary data describing how recombination patterns are altered under naturally stressful conditions, a key insight that is necessary for uniting our findings at one level of variation with the many others. These findings support a multifactorial model for crossover distribution that includes both genetic and epigenetic factors and will further progress the field in developing a comprehensive understanding of recombination localization.

L'instabilità del genoma è una delle caratteristiche delle cellule tumorali e può essere causata da difetti nella riparazione del DNA. In particolare, le rotture del doppio filamento del DNA (DSBs) sono lesioni altamente citotossiche che possono formarsi accidentalmente durante la replicazione del DNA o in seguito all'esposizione ad agenti genotossici, e devono essere correttamente riparate al fine di garantire la stabilità genomica. Per far fronte a queste lesioni del DNA, le cellule eucariotiche attivano la risposta al danno del DNA (DDR) e utilizzano due meccanismi principali per la riparazione dei DSBs: l’unione terminale non omologa (NHEJ) e la ricombinazione omologa (HR). La risposta cellulare ai DSBs ha inizio con il reclutamento dei complessi Ku e MRX/MRN alle due estremità rotte di un DSB. Inoltre, il complesso MRX recluta al DSB anche Tel1/ATM, una chinasi coinvolta nel checkpoint da danno al DNA. Tel1, a sua volta, consente di promuovere e stabilizzare l'associazione del complesso MRX sia ai DSBs che ai telomeri in un ciclo a feedback positivo. Ku, MRX/MRN e Tel1/ATM sono anche necessari per mantenere la lunghezza dei telomeri, strutture nucleoproteiche specializzate situate alle estremità dei cromosomi eucariotici. Il DNA telomerico deve inoltre essere distinto dalle estremità dei DSBs intracromosomici attraverso diversi complessi proteici, i quali vengono reclutati ai telomeri al fine di prevenire l'attivazione della DDR. Nel lievito S. cerevisiae, Rif2 e Rap1 costituiscono due delle principali proteine che compongono tali complessi. Sia Rif2 che Rap1 contrastano l'attivazione di Tel1, la degradazione nucleolitica e l’unione terminale non omologa ai telomeri. Rif2 sembra esercitare tutte queste funzioni inibendo l'associazione del complesso MRX al DNA telomerico; tuttavia, restava ancora da determinare come Rap1 controllasse negativamente l'attività di MRX alle estremità del DNA. Nella prima parte del mio dottorato di ricerca, ho contribuito a dimostrare che Rif2 contrasta l'associazione del complesso MRX sia ai DSBs che ai telomeri in modo dipendente da Rap1. Rap1, a sua volta, può inibire le funzioni di MRX in modo sia dipendente sia indipendente da Rif2, e le funzioni di Rap1 alle estremità del DNA sono influenzate dalle modalità con cui questa proteina lega il DNA. In merito al NHEJ, una questione importante è rappresentata dal mantenimento delle estremità di un DSB in stretta prossimità fra loro, necessario per consentire una corretta rilegatura. Questa funzione è chiamata end-tethering e sebbene alcuni dati in E.coli abbiano suggerito un coinvolgimento del complesso Ku in questo meccanismo di controllo, restava ancora da chiarire quale fosse il suo esatto ruolo nell’end-tethering. Nella seconda parte del mio dottorato, ho quindi studiato questa problematica tramite la generazione di una variante mutante della proteina Ku70 in grado di aumentare la persistenza del complesso Ku ai DSBs. La caratterizzazione dell'allele ku70-C85Y ha consentito di dimostrare che il complesso Ku promuove l’end-tethering del DNA e la mutazione C85Y migliora tale funzione aumentando la ritenzione di Ku in stretta prossimità alle estremità di un DSB. Inoltre, la funzione svolta da Ku nel DSB end-tethering è regolata da Tel1/ATM, che antagonizza tale funzione del complesso Ku limitandone la persistenza alle estremità dei DSBs. Poiché la presenza del complesso Ku alle estremità dei DSBs impedisce l'accesso delle nucleasi di resezione, la regolazione dell'associazione di Ku alle estremità rotte del DNA mediata da Tel1 fornisce un importante livello di controllo nella scelta tra il meccanismo di NHEJ e di HR, suggerendo una nuova funzione di Tel1 nella risposta al danno al DNA. Tutti questi risultati hanno contribuito a chiarire i meccanismi molecolari che modulano la riparazione del DNA in risposta ai DSBs, con un focus specifico sulle funzioni e sulla regolazione dei complessi MRX e Ku.
Genome instability is one of the hallmarks of cancer cells and it can be caused by DNA repair defects. Among several types of DNA damage, DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can form accidentally during DNA replication or upon exposure to genotoxic agents. DSBs must be repaired to avoid loss of genetic information and to ensure genomic stability. Eukaryotic cells repair DSBs by activating the DNA damage response (DDR) and by using two main mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). The cellular response to DSBs is initiated by the recruitment of Ku (Ku70-Ku80) and MRX/N (Mre11-Rad50-Xrs2/Nbs1) complexes at the two DSB broken ends. MRX in turn recruits Tel1/ATM, a kinase involved in the DNA damage checkpoint, a surveillance mechanism that couples DSB repair and cell-cycle progression. Tel1 allows to promote and stabilize MRX association at both DSBs and telomeres in a positive feedback loop. Ku, MRX/MRN, and Tel1/ATM are also required to maintain the length of telomeres, specialized nucleoprotein complexes at the ends of eukaryotic chromosomes. Furthermore, telomeric DNA must be distinguished from intrachromosomal DSBs ends through different protein complexes, which are recruited to telomeres in order to prevent DDR activation. In S. cerevisiae, Rif2 and Rap1 are two of the main proteins that compose these complexes. Both Rif2 and Rap1 counteract Tel1 activation, nucleolytic degradation, and NHEJ at telomeres. Rif2 appears to exert all these functions by inhibiting MRX association with telomeric DNA, however how Rap1 negatively controls MRX activity at DNA ends remained to be determined. In the first part of my PhD, I contributed to show that Rif2 counteracts MRX association at both DSBs and telomeres in a Rap1-dependent manner. Rap1 in turn can inhibit MRX functions in a Rif2-dependent and -independent manner, and Rap1 functions at DNA ends are influenced by its DNA binding mode. An important issue in NHEJ is the maintenance of the DSB ends in close proximity to allow their correct re-ligation. This function is called end-tethering and some data in E.coli suggested an involvement of the Ku complex in this control mechanism. However, a Ku role in end-tethering remained to be determined. In the second part of my PhD, I investigated this issue by generating a Ku70 mutant variant that increases Ku persistence at DSBs. The characterization of the ku70-C85Y allele has allowed to show that the Ku complex promotes DSB end-tethering and the C85Y mutation enhances this bridging function by increasing Ku retention very close to the DSB ends. The function of Ku in DSB end-tethering is also regulated by Tel1/ATM, which antagonizes this Ku function by limiting Ku persistence at the DSB ends. As the presence of Ku at the DSB ends prevents the access of resection nucleases, the Tel1-mediated regulation of Ku association with the DSB ends provides an important layer of control in the choice between NHEJ and HR mechanism, suggesting a new function of Tel1 in the DNA damage response. All these findings contributed to elucidate the molecular mechanisms that modulate DNA repair and maintain genome stability in response to DSBs, with a specific focus on the functions and regulation of MRX and Ku complexes.

Le rotture del doppio filamento del DNA (DSB) sono tra le lesioni del DNA le più gravi. Se non adeguatamente riparati, i DSB potrebbero portare alla perdita di informazioni genetiche e all'instabilità del genoma, che è uno dei tratti distintivi delle cellule tumorali. Le cellule eucariotiche riparano i DSB mediante il non-homologous end joining (NHEJ), che ricongiunge direttamente le estremità rotte del DNA e la ricombinazione omologa (HR), che utilizza la sequenza di DNA omologa per riparare il DSB. L'HR richiede una degradazione nucleolitica delle estremità, in un processo chiamato resection. In Saccharomyces cerevisiae, il complesso MRX (Mre11, Rad50 e Xrs2), aiutato da Sae2, avvia il processamento delle estremità del DSB eseguendo un taglio sulle estremità 5'. Questo taglio, catalizzato dalla subunità Mre11, consente l'accesso alle nucleasi Exo1 e Dna2. Nel NHEJ, le due estremità devono essere collegate per consentire la loro corretta riparazione. Questa funzione, chiamata end tethering, dipende dalla subunità Rad50, che lega e idrolizza l'ATP. Una transizione da uno stato legato all'ATP a uno stato di taglio post-idrolisi regola le attività di associazione e processamento del DNA di MRX. Il complesso MRX è essenziale anche nell'attivazione del checkpoint perché recluta la chinasi del checkpoint Tel1 al DSB. In questa tesi, abbiamo studiato le funzioni e la regolazione del complesso MRX nella riparazione dei DSB. Abbiamo trovato degli alleli mre11 che sopprimono l'ipersensibilità delle cellule sae2Δ agli agenti genotossici. Le mutazioni nell'N-terminale di Mre11 sopprimono il difetto di resection delle cellule sae2Δ riducendo l'associazione di MRX e Tel1 al DSB. La ridotta persistenza di Tel1 potenzia l'attività di resection di Dna2 diminuendo l'associazione di Rad9 al DSB. Al contrario, le mutazioni di mre11 localizzate nel C-terminale non necessitano di Sae2 nel tethering ma non nella resection, possibilmente destabilizzando la conformazione aperta di Mre11 - Rad50. Questi risultati mostrano l'esistenza di domini Mre11 strutturalmente distinti che supportano la resistenza agli agenti genotossici mediando diversi processi. L'attivazione di Tel1 in vitro da parte di MRX richiede il legame dell'ATP a Rad50. In questa tesi, descriviamo due alleli, mre11-S499P e rad50-A78T, che influenzano l'attivazione di Tel1 senza compromettere le funzioni MRX nella riparazione DSB. Queste due varianti riducono l'interazione Tel1-MRX portando a una bassa associazione Tel1 ai DSB che ne riduce l'attivazione. Le simulazioni di dinamica molecolare mostrano che il sub-complesso MR wild-type legato all'ATP rimane in una conformazione 'chiusa', mentre la presenza di ADP porta alla destabilizzazione del dimero Rad50 e dell'associazione Mre11-Rad50, entrambi gli eventi sono richiesti per la transizione conformazionale MR ad uno stato aperto. Al contrario, MRA78T provoca un'apertura del complesso anche se legato all'ATP, indicando che il difetto di attivazione di Tel1 causato da MRA78T risulta dalla destabilizzazione dello stato conformazionale legato all'ATP. La mancanza di Sae2 aumenta la persistenza di MRX ai DSB e all'attivazione dei checkpoint. In questa tesi, dimostriamo anche che la proteina telomerica Rif2, che stimola l'idrolisi dell'ATP da parte di Rad50, inibisce l'attività dell'endonucleasi Mre11 ed è responsabile dell’aumento di MRX ai DSB nelle cellule sae2Δ. Abbiamo identificato un residuo di Rad50 che è importante per l'interazione Rad50-Rif2 e l'inibizione mediata da Rif2 della nucleasi Mre11. Questo residuo altera l'interazione Mre11-Rad50. Proponiamo che Sae2 stimoli l'attività endonucleasica di MRX stabilizzando lo stato di taglio, mentre Rif2 lo inibisce antagonizzando il legame di Sae2 e stabilizzando una conformazione di MR che non è adatta al taglio.
DNA double strand breaks (DSBs) are among the most severe DNA lesions. If not properly repaired, DSBs could lead to loss of genetic information and genome instability, which is one of the hallmarks of cancer cells. Eukaryotic cells repair DSBs by non-homologous end joining (NHEJ), which directly re-ligates the DNA broken ends, and homologous recombination (HR), which uses the intact homologous DNA sequence as a template to repair the DSB. HR requires a nucleolytic degradation of the broken DNA ends, in a process called resection. In Saccharomyces cerevisiae, the MRX (Mre11, Rad50 and Xrs2) complex, aided by Sae2, initiates resection of the DSB ends by performing an endonucleolytic cleavage on the 5’-ended strands. This cleavage, catalyzed by the Mre11 subunit, allows the access of Exo1 and Dna2 nucleases that elongate the ssDNA ends. In NHEJ, the two broken ends need to be physically connected to allow their correct religation. This function, called end tethering, depends on the Rad50 subunit, which binds and hydrolyses ATP. A transitions between an ATP-bound state to a post-hydrolysis cutting state regulates MRX DNA binding and processing activities. The MRX complex is also essential in DNA damage checkpoint activation because it recruits the checkpoint kinase Tel1 at the break site. In this thesis, we studied functions and regulation of the MRX complex in DSB repair. We found mre11 alleles that suppress the hypersensitivity of sae2Δ cells to genotoxic agents. The mutations in the Mre11 N-terminus suppress the resection defect of sae2Δ cells by lowering MRX and Tel1 association to DSBs. The diminished Tel1 persistence potentiates Dna2 resection activity by decreasing Rad9 association to DSBs. By contrast, the mre11 mutations localized at the C-terminus bypass Sae2 function in end-tethering but not in DSB resection, possibly by destabilizing the Mre11–Rad50 open conformation. These findings unmask the existence of structurally distinct Mre11 domains that support resistance to genotoxic agents by mediating different processes. In vitro Tel1 activation by MRX requires ATP binding to Rad50, suggesting a role for the MR subcomplex in Tel1 activation. In this thesis, we describe two separation-of-functions alleles, mre11-S499P and rad50-A78T, which we show to specifically affect Tel1 activation without impairing MRX functions in DSB repair. Both Mre11-S499P and Rad50-A78T reduce Tel1–MRX interaction leading to low Tel1 association at DSBs that reduces Tel1 activation. Molecular dynamics simulations show that the wild type MR subcomplex bound to ATP lingers in a tightly ‘closed’ conformation, while ADP presence leads to the destabilization of Rad50 dimer and of Mre11–Rad50 association, both events being required for MR conformational transition to an open state. By contrast, MRA78T undertakes complex opening even if Rad50 is bound to ATP, indicating that defective Tel1 activation caused by MRA78T results from destabilization of the ATP- bound conformational state. The lack of Sae2 increases MRX persistence at DSBs and checkpoint activation. In this thesis, we also show that the telomeric protein Rif2, which stimulates ATP hydrolysis by Rad50, inhibits the Mre11 endonuclease activity and is responsible for the increased MRX retention at DSBs in sae2Δ cells. We identified a Rad50 residue that is important for Rad50-Rif2 interaction and Rif2-mediated inhibition of Mre11 nuclease. This residue is located nearby a Rad50 surface that binds Sae2 and is important to stabilize the Mre11-Rad50 interaction in the cutting state. We propose that Sae2 stimulates MRX endonuclease activity by stabilizing the cutting state, whereas Rif2 inhibits it by antagonizing Sae2 binding to Rad50 and stabilizing a MR conformation that is not competent for DNA cleavage. The results described in this PhD thesis contribute to the understanding of the molecular mechanisms supporting functions and regulation of the MRX complex at DSBs.

This dissertation presents fragment screening studies against two human proteins Siah1 and SENP1, which function in post-translational modification pathways. Siah1 is an E3 ubiquitin ligase that functions as a scaffold to transfer ubiquitin bound to an E2 ubiquitin-conjugating enzyme to a substrate as part of the ubiquitination pathway. SENP1 is a cysteine protease that catalyses two essential reactions in the SUMO pathway. It processes pre-SUMO proteins to their mature form and removes SUMO from the target proteins. Siah1 interactions with other proteins involve large surface areas, while SENP1 has a small active site, making it hard to identify ligands for these proteins. The fragment-based approach has emerged as a complementary method to high-throughput screening of finding novel small molecules. The main aim of the study was to examine whether fragment screening would identify any ligands against these targets. Chapter 1 introduces post-translational modifications and presents fragment-based approach used in drug discovery. Chapter 2 describes the experimental methods used. The results from fragment screening against Siah1 using SPR and DSF are reported in chapter 3. The chapter also presents the structure of Siah1 refined to 1.95 Å that displays new parts of the structure, previously missing due to the absence of reliable electron density. Chapter 4 contains results from the fragment screens against SENP1 using DSF and NMR. The crystal structure of SENP1 was determined with a number of improvements made over earlier structures. Besides performing fragment screening, the binding between Siah1 interacting proteins reported in the literature and Siah1 was investigated. A number of Siah1 binding partners were successfully expressed and purified as described in chapter 5. One of those, SIP showed a clear interaction with Siah1, as observed by the shift on a size exclusion column of the complex relative to the individual protein species. Siah1 was reported to collaborate with PEG3 in the regulation of ß-catenin degradation. A SCAN domain, located at the N-terminus of PEG3, was tested for binding using gel filtration chromatography and NMR, but no interaction was observed. PEG3 was used in the crystallographic studies and a structure of its SCAN domain was solved using molecular replacement and refined to 1.95 Å. The structure of PEG3-SCAN domain revealed a stable homodimer with an extensive dimerization interface. The structure of a zinc-dependent cytosolic carboxypeptidase from Burkholderiacenocepacia was determined and is reported in chapter 6. This work was a side project assessing a new refinement strategy, which involved the use of the automated protocols embedded in the PDB_REDO server. The structure revealed that carboxypeptidase is a tetramer and provides details of its active site, whose spatial conformation of residues supports the notion that the protein might function as a deglutamylase.