Diese Arbeit untersucht den Einsatz von keramischen Materialien in der Trinkwasseraufbereitung mittels Filtration und fokussiert dabei die Entfernung von Viren. Herkömmliche, auf Kieselgur basierende Tiefenfilter (Filterkerzen) mit Porengrößen im unteren Mikrometerbereich, werden hinsichtlich ihres Rückhaltevermögens gegenüber Kolloiden (Viren sowie Polystyrolpartikel) untersucht, um deren Einsatzfähigkeit in der Entfernung von Mikroorganismen im Allgemeinen abschätzen zu können. Ferner wird gezeigt, wie durch ein einfaches Verfahren solche Filter modifiziert werden können, um auch kleinste Viren mit ca. 30 nm Durchmessern aus dem Rohwasser zu entfernen. Die Zugabe von MgO während der Granulierungsstufe im Herstellungsprozess der Filterkerzen bewirkt eine erhebliche Verbesserung des Virenrückhalts bis zu über 99.9999%. Die experimentellen Ergebnisse wurden dabei mit theoretischen Modellen verglichen, um Aussagen über die Mechanismen der Virenentfernung treffen zu können.:Contents Chapter I Introduction 1 Chapter II Removal or inactivation of microorganisms, in particular viruses, for drinking water purposes with focus on small-scale, decentralised systems: A literature review 7 II. I Physical and chemical treatments 8 II. II Filtration processes 10 II. III Conclusions 15 Chapter III Mechanisms of adsorption in depth filtration 17 III.I Surface charge and the electrical double layer 18 III.II van der Waals interactions 22 III.III DLVO theory 23 III.IV Non-DLVO forces 25 III.V Extended DLVO Theory 27 Chapter IV Virus adsorption studies 29 IV.I A literature review 30 IV.I.I Virus concentration by adsorption-elution 33 IV.I.II Improved virus adsorption in filtration 35 IV.II The electrostatic enhanced adsorption approach 37 Chapter V Viruses 39 V.I Literature review 40 V.I.I Structure and morphology 40 V.I.II The viral life cycle 41 V.I.III Human pathogenic viruses in the aquatic environment 42 V.II Experimental 46 V.II.I The choice of viruses for adsorption studies 46 V.II.II Propagation and enumeration of the bacteriophages 48 V.II.III Characterisation of bacteriophages 51 V.III Results and discussion 54 V.III.I Production of high-titre and high-purity phage stocks 54 V.III.II Characteristics of bacteriophages 59 V.III.III Detection of a viral contaminant - the ‘Siphophage’ 64 Chapter VI The diatomaceous earth-based depth filter 69 VI.I Literature review 70 VI.I.I Diatomaceous earth 70 VI.I.II Retention of microorganisms in the DE-based depth filter 71 VI.II Experimental 73 VI.II.I Manufacturing the depth filter 73 VI.II.II Physical characterisation 74 VI.II.III Performing filter retention tests 75 VI.II.IV Latex retention test 76 VI.II.V Studying adsorption kinetics in a batch experiment 79 VI.II.VI Applying (X-)DLVO theory 80 VI.III Results and discussion 83 VI.III.I Characterisation of the depth filter 83 VI.III.II Latex removal in the depth filter 86 VI.III.III Filter performance on virus removal 94 VI.III.IV Batch-sorption experiments 99 VI.IV Summary and conclusions 102 Chapter VII The magnesium oxide modified depth filter 103 VII.I Experimental 104 VII.I.I Choice of the adsorbent material 104 VII.I.II Manufacturing the MgO-modified filter and characterisation methods 105 VII.II Results and discussion 106 VII.II.I The adsorbent: Magnesium oxide powder 106 VII.II.II Physical characterisation of modified depth filters 108 VII.II.III Virus removal in depth filters containing MgO 113 VII.II.IV Ageing behaviour of MgO modified filters 118 VII.II.V Discussion on the removal mechanisms 130 VII.III Summary and conclusions 134 Chapter VIII Summary, conclusions and outlook 135 VIII.I Summary and conclusions 136 VIII.II Outlook 137 Abbreviations, symbols and physical constants 139 Reference list 142

In recent decades, most countries of the world have experienced a shortage of water and increase its rate of consumption. Today, every country in the world are interested in this problem by trying to find alternatives to address this shortage. One solution is reuse greywater (GW) for irrigation after treatment. GW is all water generated from Household except toilet water. The risks associated with the reuse of these waters are the presence of pathogens that can infect humans, animals and plants. In this thesis focused on studying treatment by slow sand filtration and the survival of representatives of pathogens, such as E. Coli, P. aeruginosa , E. Faecalis and Bacteriophage MS2 which could be found in the greywater. The study factors was a physico-chemicals factors such as; temperature (6±2,23±2,42±2°c), salinity (1.75 and 3.5% Nacl), oxygen (aerobic and anaerobic condition), nutrient ( rich media , 50%: 50% salt and poor media ), light with photocatalysis ( UV and Visible lights) and slow sand filter (Egyptian desert sand and swimming pool sand). A combination of high temperature, sunlight and photocatlysis are mainly responsible for the rapid decline of bacteria and MS2 coliphage. Slow sand filter have clearly less influence on the survival of bacteria in the greywater, but it effective to decline turbidity and COD for short times.

No description available.

<p>ASF/SF2 is an essential splicing factor, required for constitutive splicing, and functioning as a regulator of alternative splicing. ASF/SF2 is modular in structure and contains two amino-terminal RNA binding domains (RBD1 and RBD2), and a carboxy-terminal RS domain. The results from my studies show that the different activities of ASF/SF2 as a regulator of alternative 5’ and 3’ splice site selection can be attributed to distinct domains of ASF/SF2.</p><p>I show that ASF/SF2-RBD2 is both necessary and sufficient to reproduce the splicing repressor function of ASF/SF2. A SWQDLKD motif was shown to be essential for the splicing repressor activity of ASF/SF2. In conclusion, this study demonstrated that ASF/SF2 encodes for distinct domains responsible for its function as a splicing enhancer (the RS domain) or a splicing repressor (the RBD2) protein. Using a model transcript containing two competing 3’ splice sites it was further demonstrated that the activity of ASF/SF2 as a regulator of alternative 3’ splice site selection was directional: i.e. resulting in RS or RBD1 mediated activation of upstream 3’ splice site selection while simultaneously causing an RBD2 mediated repression of downstream 3’ splice site usage.</p><p>In alternative 5’ splice site selection, the RBD2 alone was sufficient to reproduce the activity of the full-length protein as an inducer of proximal 5’ splice site usage, while RBD1 had the opposite effect and induced distal 5’ splice site selection. The conserved SWQDLKD motif and the RNP-1 type RNA recognition motif in ASF/SF2-RBD2 were both essential for this induction. The activity of the ASF/SF2-RBD2 domain as a regulator of alternative 5’ splice site was shown to correlate with the RNA binding capacity of the domain.</p><p>Collectively, my results suggest that the RBD2 domain in ASF/SF2 plays the most decisive role in the alternative 5’ and 3’ splice site regulatory activities of ASF/SF2.</p>

ASF/SF2 is an essential splicing factor, required for constitutive splicing, and functioning as a regulator of alternative splicing. ASF/SF2 is modular in structure and contains two amino-terminal RNA binding domains (RBD1 and RBD2), and a carboxy-terminal RS domain. The results from my studies show that the different activities of ASF/SF2 as a regulator of alternative 5’ and 3’ splice site selection can be attributed to distinct domains of ASF/SF2. I show that ASF/SF2-RBD2 is both necessary and sufficient to reproduce the splicing repressor function of ASF/SF2. A SWQDLKD motif was shown to be essential for the splicing repressor activity of ASF/SF2. In conclusion, this study demonstrated that ASF/SF2 encodes for distinct domains responsible for its function as a splicing enhancer (the RS domain) or a splicing repressor (the RBD2) protein. Using a model transcript containing two competing 3’ splice sites it was further demonstrated that the activity of ASF/SF2 as a regulator of alternative 3’ splice site selection was directional: i.e. resulting in RS or RBD1 mediated activation of upstream 3’ splice site selection while simultaneously causing an RBD2 mediated repression of downstream 3’ splice site usage. In alternative 5’ splice site selection, the RBD2 alone was sufficient to reproduce the activity of the full-length protein as an inducer of proximal 5’ splice site usage, while RBD1 had the opposite effect and induced distal 5’ splice site selection. The conserved SWQDLKD motif and the RNP-1 type RNA recognition motif in ASF/SF2-RBD2 were both essential for this induction. The activity of the ASF/SF2-RBD2 domain as a regulator of alternative 5’ splice site was shown to correlate with the RNA binding capacity of the domain. Collectively, my results suggest that the RBD2 domain in ASF/SF2 plays the most decisive role in the alternative 5’ and 3’ splice site regulatory activities of ASF/SF2.

Die Genexpression ist ein stochastischer Prozess, dessen strenge Regulation einen ungestörten Zellzyklusverlauf ermöglicht. Jeglicher Stress löst eine Neuprogrammierung der Expression und somit einen Stillstand des Zellzyklus aus. Um ein besseres Verständnis des eukaryotischen Zellzyklus zu erlangen, wurde in dieser Arbeit die Fluoreszenzmikroskopie einzelner Zellen (S.cerevisiae) mit stochastischer Modellierung der Hauptregulatorgene des G1/S-Übergangs (SIC1, CLN2, CLB5) kombiniert. Mithilfe des MS2-CP-Systems wurden mRNA-Level von SIC1 in lebenden Zellen bestimmt und verschiedene Transportwege von SIC1-mRNA visualisiert. RNA-FISH in Kombination mit genetischen und morphologischen Markierungen ermöglichte es, die absolute Quantifizierung von SIC1-, CLN2- und CLB5-mRNA in allen Zyklusphasen vorzunehmen. Die Auswirkung von Osmostress, in Hinblick auf eine transkriptionale Verzerrung, wurde untersucht. Basierend auf den experimentellen-Daten wurde ein stochastisches Model entwickelt, dass die Expression von SIC1, CLN2 und CLB5 mRNA und Proteinlevel in Abhängigkeit von Osmostress über den gesamten Zellzyklus hinweg abbildet. Die Modellierung ermöglichte eine in silico Synchronisation und somit die Extraktion kinetischer Parameter. Die Expression der beobachteten Gene wurde im Verlauf des Zellzyklus nicht ein- und ausgeschaltet, stattdessen kam es zu Phasen hoher oder niedriger Expression. Niedriger SIC1 Expression gewährleistete niedriger Sic1 Protein Verzerrung und robustes G1/S Timing. CLN2 und CLB5 zeigten ein maximales Expressionslevel in G1 und auch eine erhöhte Expression in der späten Mitose. Osmostress induzierte einen langanhaltenden Effekt auf die Transkription und die Dauer der Zellzyklusphasen. Der hier vorgestellte Ansatz ermöglichte quantitative Einblicke in die Genexpression und zeitliche Koordination des Zellzyklus von S.cerevisiae. Einige der hier beobachteten Regulationsmechanismen könnten allgemeine Gültigkeit im eukaryotischen Zellzyklus besitzen. / Gene expression is a stochastic process and its appropriate regulation is critical for cell cycle progression. Cellular stress response requires expression reprogramming and cell cycle arrest. Time-resolved quantitative methods on single cells are needed to understand eukaryotic cell cycle in context of noisy gene expression and external perturbations. We applied single-cell fluorescence microscopy and stochastic modeling to SIC1, CLN2 and CLB5, the main G1/S regulators in S. cerevisiae. Using MS2-CP system we estimated SIC1 mRNA levels and visualized different types of transport for SIC1 mRNA particles in living cells. With RNA-FISH combined to genetic and morphological markers we monitored absolute numbers of mRNA and transcriptional noise over cell cycle phases with and without osmostress. Stochastic modeling enabled in silico synchronization, the extraction of kinetic parameters as well as expanded the static mRNA data into time courses for mRNAs, proteins and their noise. Based on our experimental data we developed a stochastic model of G1/S timing centered on SIC1 and a second one for the entire cell cycle involving SIC1, CLN2 and CLB5 and the response to osmostress. All three genes exhibited basal expression throughout cell cycle enlightening that transcription is not divided in on and off but rather in high and low phases. A low SIC1 transcript level ensured a low protein noise and a robust timing of the G1/S transition. CLN2 and CLB5 showed main expression peaks in G1 as well as an expression upshift in late mitosis. Osmostress induced different periods of transcriptional inhibition for CLN2 and CLB5 and long-term impact on cell cycle phase duration. Our approach disclosed detailed quantitative insights into gene expression and cell cycle timing, not available from bulk experiments. Importantly some regulation mechanisms specific to SIC1, CLN2 and CLB5 might be generalized to other genes as well as to other organisms.

Telomeres are nucleoprotein structures that cap the physical ends of eukaryotic chromosomes. They consist of repetitive DNA sequences 5’-TTAGGG-3’ assembled with proteins which form the shelterin complex. This complex protects the ends of chromosomes by inhibiting DNA repair pathways at telomeres and avoid their recognition as double-strand breaks. Telomeres have been identified as a transcriptionally silent zone until 2007 when a noncoding RNA called TERRA (TElomeric Repeat containing RNA) transcribed from telomeres was discovered. This RNA gave rise to many questions: How is TERRA regulated? How is TERRA expressed? Does TERRA interact with proteins, DNA or RNA? After several studies, we know that TERRA is frequently expressed in cancer cells and it interacts with a large proteome. Nevertheless, its specific function remains unknown. In this thesis, we studied this RNA in human cancer cells using live-cell microscopy which allowed us to get information on TERRA’s dynamics, localization and its interactome. Moreover, we used single-molecule imaging on TERRA 15q labeled by the MS2-GFP system, which allowed the visualization of TERRA transcripts. This study resulted in the discovery of two types of TERRA population from telomere 15q: one of the population is characterized by the formation of clusters and a second population is constituted of unique molecules more dynamic in the nucleus. Finally, in order to better understand TERRA’s functions, we developed a new approach which consists on immunoprecipitating TERRA using the MS2 stem-loops as a tag to identify TERRA-interacting proteins such as the telomeric factor TRF2 or RNA-binding proteins like hnRNP -A1 or FUS. / Les télomères forment les extrémités des chromosomes chez les eucaryotes. Ces séquences répétées en tandem 5’-TTAGGG-3’ font partie d’un complexe nucléoprotéique appelé shelterin. En effet, cet assemblage de protéines télomériques permet la protection des extrémités des chromosomes, permettant à celles-ci de ne pas être reconnues comme des cassures dans l’ADN et d’activer les voies de réparation de l’ADN. Les télomères ont longtemps été reconnus comme étant des zones de transcription inactives, ce jusqu’en 2007 lorsqu’une équipe de recherche découvrit un ARN non codant appelé TERRA (Telomeric Repeat containing RNA). Ce dernier a suscité de nombreuses questions : quel est le rôle de cet ARN? Comment est-il exprimé et régulé? Interagit-il avec d’autres facteurs cellulaires? Les différentes recherches menées sur cet ARN ont permis de conclure que celui-ci était fréquemment induit dans les cellules de cancer, que ses partenaires d’interactions sont nombreux, mais que ses fonctions restent encore mal définies. Par ailleurs, ces différentes études ont toujours été ou presque réalisées sur des cellules fixées, sur une population totale d’ARN télomérique TERRA. Afin d’apporter de nouvelles réponses et de mieux caractériser cet ARN, nous avons étudié ce transcrit dans des cellules de cancer humain en utilisant la technique de microscopie en temps réel, qui permet de récolter des données sur la dynamique, la localisation de cet ARN et ses éventuels partenaires. De plus, nous nous sommes intéressés à des molécules uniques de TERRA issues du télomère 15q en exploitant la technique de marquage avec des tiges-boucles MS2 (MS2-GFP). Cette étude de microscopie a permis de découvrir deux types de population de l’ARN TERRA 15q : une population caractérisée par des assemblages d’ARN dit clusters (agrégats d’ARN) et une population plus singulière qui semble avoir une diffusion plus importante dans le noyau de la cellule. Par ailleurs, l’expression de l’ARN TERRA semble être différente d’un type cellulaire à un autre et nous avons donc cherché à connaître le niveau d’expression de cet ARN au sein de la lignée étudiée au cours de ce projet de recherche. Enfin, afin de découvrir de nouveaux rôles pour cet ARN, nous avons développé une approche de co-immunoprécipitation de l’ARN TERRA pour identifier des interactions avec des protéines du complexe shelterin comme TRF2, ou des protéines liant l’ARN comme hnRNP-A1 ou encore FUS.

As high-throughput biology experiments generate increasing amounts of data, the field is naturally turning to data-driven methods for the analysis and extraction of novel insights. These insights into biological systems are crucial for understanding disease progression, drug targets, treatment development, and diagnostics methods, ultimately leading to improving human health and well-being, as well as, deeper insight into cellular biology. Biological data sources such as the genome, transcriptome, proteome, metabolome, and metagenome provide critical information about biological system structure, function, and dynamics. The focus of this licentiate thesis is on proteomics, the study of proteins, which is a natural starting point for understanding biological functions as proteins are crucial functional components of cells. Proteins play a crucial role in enzymatic reactions, structural support, transport, storage, cell signaling, and immune system function. In addition, proteomics has vast data repositories and technical and methodological improvements are continually being made to yield even more data. However, generating proteomic data involves multiple steps, which are prone to errors, making sophisticated models essential to handle technical and biological artifacts and account for uncertainty in the data. In this licentiate thesis, the use of machine learning and probabilistic methods to extract information from mass-spectrometry-based proteomic data is investigated. The thesis starts with an introduction to proteomics, including a basic biological background, followed by a description of how massspectrometry-based proteomics experiments are performed, and challenges in proteomic data analysis. The statistics of proteomic data analysis are also explored, and state-of-the-art software and tools related to each step of the proteomics data analysis pipeline are presented. The thesis concludes with a discussion of future work and the presentation of two original research works. The first research work focuses on adapting Triqler, a probabilistic graphical model for protein quantification developed for data-dependent acquisition (DDA) data, to data-independent acquisition (DIA) data. Challenges in this study included verifying that DIA data conformed with the model used in Triqler, addressing benchmarking issues, and modifying the missing value model used by Triqler to adapt for DIA data. The study showed that DIA data conformed with the properties required by Triqler, implemented a protein inference harmonization strategy, and modified the missing value model to adapt for DIA data. The study concluded by showing that Triqler outperformed current protein quantification techniques. The second research work focused on developing a novel deep-learning based MS2-intensity predictor by incorporating the self-attention mechanism called transformer into Prosit, an established Recurrent Neural Networks (RNN) based deep learning framework for MS2 spectrum intensity prediction. RNNs are a type of neural network that can efficiently process sequential data by capturing information from previous steps, in a sequential manner. The transformer self-attention mechanism allows a model to focus on different parts of its input sequence during processing independently, enabling it to capture dependencies and relationships between elements more effectively. The transformers therefore remedy some of the drawbacks of RNNs, as such, we hypothesized that the implementation of MS2-intensity predictor using transformers rather than RNN would improve its performance. Hence, Prosit-transformer was developed, and the study showed that the model training time and the similarity between the predicted MS2 spectrum and the observed spectrum improved. These original research works address various challenges in computational proteomics and contribute to the development of data-driven life science. / Allteftersom high-throughput experiment genererar allt större mängder data vänder sig området naturligt till data-drivna metoder för analys och extrahering av nya insikter. Dessa insikter om biologiska system är avgörande för att förstå sjukdomsprogression, läkemedelspåverkan, behandlingsutveckling, och diagnostiska metoder, vilket i slutändan leder till en förbättring av människors hälsa och välbefinnande, såväl som en djupare förståelse av cell biologi. Biologiska datakällor som genomet, transkriptomet, proteomet, metabolomet och metagenomet ger kritisk information om biologiska systems struktur, funktion och dynamik. I licentiatuppsats fokusområde ligger på proteomik, studiet av proteiner, vilket är en naturlig startpunkt för att förstå biologiska funktioner eftersom proteiner är avgörande funktionella komponenter i celler. Dessa proteiner spelar en avgörande roll i enzymatiska reaktioner, strukturellt stöd, transport, lagring, cellsignalering och immunsystemfunktion. Dessutom har proteomik har stora dataarkiv och tekniska samt metodologiska förbättringar görs kontinuerligt för att ge ännu mer data. Men för att generera proteomisk data krävs flera steg, som är felbenägna, vilket gör att sofistikerade modeller är väsentliga för att hantera tekniska och biologiska artefakter och för att ta hänsyn till osäkerhet i data. I denna licentiatuppsats undersöks användningen av maskininlärning och probabilistiska metoder för att extrahera information från masspektrometribaserade proteomikdata. Avhandlingen börjar med en introduktion till proteomik, inklusive en grundläggande biologisk bakgrund, följt av en beskrivning av hur masspektrometri-baserade proteomikexperiment utförs och utmaningar i proteomisk dataanalys. Statistiska metoder för proteomisk dataanalys utforskas också, och state-of-the-art mjukvara och verktyg som är relaterade till varje steg i proteomikdataanalyspipelinen presenteras. Avhandlingen avslutas med en diskussion om framtida arbete och presentationen av två original forskningsarbeten. Det första forskningsarbetet fokuserar på att anpassa Triqler, en probabilistisk grafisk modell för proteinkvantifiering som utvecklats för datadependent acquisition (DDA) data, till data-independent acquisition (DIA) data. Utmaningarna i denna studie inkluderade att verifiera att DIA-datas egenskaper överensstämde med modellen som användes i Triqler, att hantera benchmarking-frågor och att modifiera missing-value modellen som användes av Triqler till DIA-data. Studien visade att DIA-data överensstämde med de egenskaper som krävdes av Triqler, implementerade en proteininferensharmoniseringsstrategi och modifierade missing-value modellen till DIA-data. Studien avslutades med att visa att Triqler överträffade nuvarande state-of-the-art proteinkvantifieringsmetoder. Det andra forskningsarbetet fokuserade på utvecklingen av en djupinlärningsbaserad MS2-intensitetsprediktor genom att inkorporera self-attention mekanismen som kallas för transformer till Prosit, en etablerad Recurrent Neural Network (RNN) baserad djupinlärningsramverk för MS2 spektrum intensitetsprediktion. RNN är en typ av neurala nätverk som effektivt kan bearbeta sekventiell data genom att bevara och använda dolda tillstånd som fångar information från tidigare steg på ett sekventiellt sätt. Självuppmärksamhetsmekanismen i transformer tillåter modellen att fokusera på olika delar av sekventiellt data samtidigt under bearbetningen oberoende av varandra, vilket gör det möjligt att fånga relationer mellan elementen mer effektivt. Genom detta lyckas Transformer åtgärda vissa nackdelar med RNN, och därför hypotiserade vi att en implementation av en ny MS2-intensitetprediktor med transformers istället för RNN skulle förbättra prestandan. Därmed konstruerades Prosit-transformer, och studien visade att både modellträningstiden och likheten mellan predicerat MS2-spektrum och observerat spektrum förbättrades. Dessa originalforskningsarbeten hanterar olika utmaningar inom beräkningsproteomik och bidrar till utvecklingen av datadriven livsvetenskap. / <p>QC 2023-05-22</p>

Background: Humanmilk oligosaccharides (HMOs) support and concurrently shape the neonatal immune system through various mechanisms. Thereby, they may contribute to lower incidence of infections in infants. However, there is limited evidence on the role of individual HMOs in the risk of otitis media (OM), as well as lower and upper respiratory tract infections (LRTI and URTI, respectively) in children up to 2 years. Objective: To investigate whether individual HMO concentrations measured at 6 weeks of lactation were associated with risk of OM, LRTI or URTI up to 2 years in breastfed infants. Associations with OM, LRTI and URTI were determined for the most prominent human milk oligosaccharides including 13 neutral, partly isomeric structures (trioses up to hexaoses), two acidic trioses, and lactose. Design: HMO measurements and physician reported data on infections were available from human milk samples collected at 6 weeks postpartum (n = 667). Associations of HMOs with infections were assessed in crude and adjusted models using modified Poisson regression. Results: Absolute concentrations (median [min, max], in g/L) of 2′-fucosyllactose (2′-FL) tended (p = 0.04) to be lower, while lacto-N-tetraose (LNT) was higher in the milk for infants with OM in the 1st year of life (p = 0.0046). In the milk of secretor mothers, LNT was significantly higher in the milk for infants with OM (RR [95% CI]: 0.98 [0.15, 2.60]) compared to infants without OM (RR [95% CI]: 0.76 [0.14, 2.90]) at 1 year (p = 0.0019). No statistically significant milk group differences and associations were observed for OM, LRTI, and URTI (p > 0.0031). Conclusion: Our findings suggest that neither prominent neutral individual HMOs (ranging from 2′-FL to LNDFHs) nor acidic human milk sialyllactoses or lactose are significantly associated with a reduced or increased risk of infections in infants up to 2 years of age. Further research is needed to determine whether specific HMOs could potentially reduce the incidence or alleviate the course of distinct infections in early life.

Please find the referenced videos attached / The physical ends of eukaryotic chromosomes consist of repetitive DNA sequences, which are associated with specialized proteins forming a nucleoprotein structure essential for the integrity of the linear chromosomes, and are known as telomeres. Telomerase is an enzyme responsible for the maintenance of the telomeric repeats at the end of the chromosomes. Telomerase is a ribonucleoprotein, which contains a catalytic subunit that possesses reverse transcriptase activity, and a RNA subunit that acts as a template, since it possess the telomeric repeat sequences necessary to amplify telomere ends. Telomeres are transcribed in most eukaryotes into a non-coding RNA know as TERRA (Telomeric repeats-containing RNA). It has been proposed that TERRA may act as a regulator of telomere homeostasis, and as an inhibitor of telomerase, however, its specific function is still unknown. In Saccharomyces cerevisiae, TERRA is rapidly degraded by the 5’-3’ Rat1 exonuclease, which has hampered its study by classic biochemical experiments in yeast. In this thesis, we report the use of cytological approaches to study TERRA in budding yeast. Two different approaches were used for this purpose: the fluorescent in-situ hybridization (FISH) and the labeling of TERRA by the MS2-GFP system, which allow the visualization of TERRA transcripts form a single telomere in living cells. With these two approaches, we observed that TERRA is expressed from a single telomere and accumulates as a single perinuclear foci, in a small percentage of cells population. We also demonstrate that TERRA expression occurs due to telomere shortening. We demonstrate that TERRA interacts in vivo with the telomerase RNA (TLC1) in yeast. Telomere elongation depends on the action of several telomerase molecules that are visible as clusters, which associate with telomeres in late S phase in yeast, and mammalian cells. In adidition, we show that TERRA stimulates the nucleation of telomerase clusters. By performing time course experiments of TERRA and TLC1 RNA in live cells, we observed that TERRA acts as a scaffold for generating telomerase clusters, which are then recruited in late S phase to the telomere from which TERRA molecules originated. The recruitment of TERRA to its telomere of origin is dependent on factors that control telomerase recruitment at telomeres like: Mre11, Tel1 and the yKu complex. We propose that a short telomere expresses TERRA to assemble and organize telomerase molecules, which later on allows their recruitment at the short telomere, where elongation is needed. Finally we showed an up-regulation of TERRA, and telomerase RNA TLC1, accompanied by a predominant cytoplasmic localization as cell growth progresses from exponential growth to diauxic shift, and stationary phase. In these conditions, TERRA foci co-localize with TLC1 RNA foci, suggesting that the function of TERRA as a scaffold molecule to generate telomerase cluster is necessary for this yeast cell growth phases. / Les télomères à l’extrémité des chromosomes constituent une structure d’ADN et de protéines essentielle à l’intégrité de ces chromosomes. La télomérase est l’enzyme responsable du maintien des répétitions télomériques à l’extrémité des chromosomes. Cette enzyme est constituée d’une sous-unité catalytique, qui possède une activité de transcriptase réverse, et d’une sous-unité d’ARN, qui fourni la matrice nécessaire à la synthèse des répétitions télomériques. Les ARN contenant des répétions télomériques (ou Telomeric repeats-containing RNA; TERRA) constitue une nouvelle classe d’ARN non-codants transcrits à partir des télomères et conservée chez la plupart des eucaryotes. TERRA a été proposé d’agir comme un régulateur de l‘homéostasie des télomères et comme inhibiteur de la télomérase, mais sa fonction spécifique reste inconnue. De plus, chez la levure Saccharomyces cerevisiae, TERRA est rapidement dégradé par l’exonucléase 5’-3’ Rat1, ce qui complique l’étude de cet ARN par les méthodes biochimiques classiques. Dans cette thèse, nous rapportons l‘utilisation d’une approche cytologique pour étudier TERRA dans les cellules de levures. Deux approches sont utilisées : l’hybridation in situ en fluorescence (FISH) et l’étiquetage de TERRA à l’aide du système MS2-GFP, qui nous permet de visualiser l’expression de TERRA transcrit d’un seul télomère dans des cellules vivantes. Avec ces deux approches, nous observons que TERRA exprimé à partir d’un seul télomère s’accumule dans un faible nombre de cellules, sous la forme d’un focus périnucléaire. De plus, nous montrons que TERRA est exprimé lorsque son télomère raccourcit. Par immunoprécipitation, nous montrons que TERRA interagit in vivo avec l’ARN de la télomérase de levure, TLC1. L’élongation des télomères dépend de l‘action de multiples molécules de télomérase, qui sont visibles sous la forme de clusters de télomérases, qui s‘associent en phase S avec les télomères chez la levure et les cellules de mammifère. Nous démontrons que TERRA stimule la nucléation de ces clusters de télomérase. Par imagerie en temps réel de TERRA et de l’ARN TLC1, nous observons que TERRA agit comme molécule d’échafaudage pour générer des clusters de télomérases, qui sont par la suite recrutés, en phase S, au télomère duquel TERRA a été exprimé. Le recrutement d’un focus de TERRA à son télomère d’origine dépend des facteurs contrôlant le recrutement de la télomérase aux télomères : Mre11, Tel1 et le complexe yKu. Nous proposons qu’un télomère court exprime TERRA pour assembler et organiser les molécules de télomérase, afin que celles-ci soit puissent être recrutées au télomère court pour permettre son élongation. Enfin, nous observons une surexpression de l’ARN de la télomérase TLC1 et de TERRA, ainsi qu’une accumulation cytoplasmique de ceux-ci sous la forme de foci, lorsque la cellule passe de la phase de croissance exponentiel à la phase diauxique, puis à la phase stationnaire. Dans ces conditions, les foci d’ARN TLC1 colocalisent avec les foci de TERRA, suggérant que la fonction de TERRA comme molécule d’échafaudage pour générer des foci de télomérase est aussi nécessaire durant ces phases du cycle de croissance des levures.