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Biochemical Characterization of Proteins that Interact with RNAYe, Xuan January 2020 (has links)
No description available.
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Investigation of Innate Immune Responses in Eptesicus Bat Cells via Comprehensive Analysis / 網羅的な分析によるEptesicus属コウモリ培養細胞における自然免疫反応の解明Lin, Hsien-Hen 23 March 2022 (has links)
京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24052号 / 生博第478号 / 新制||生||63(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 朝長 啓造, 教授 野田 岳志, 教授 今吉 格 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM
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Phage display to identify functional resistance mutations to RigosertibFilipovic, Nedim 01 January 2017 (has links)
In vitro protein selection has had major impacts in the field of protein engineering. Traditional screens assay individual proteins for specific function. Selection, however, analyzes a pool of mutants and yields the best variants. Phage display, a successful selection technique, also provides a reliable link between variant phenotype and genotype. It can also be coupled with high throughput sequencing to map protein mutations; potentially highlighting vital mutations in variants. We propose to apply this technique to cancer therapy. RAF, a serine/threonine kinase, is critical for cell regulation in mammals. RAF can be activated by oncogenic RAS, found in over 30% of cancers, to drive cancer proliferation. Rigosertib, a benzyl styryl sulfone in phase III clinical trials for myelodysplastic syndrome (MDS), is an inhibitor of the RAS binding domain (RBD) in RAF. Phage display can be used to select RAF mutants for RAS binding affinity, in the presence of Rigosertib. High-throughput sequencing of these variants can provide a means of anticipating, and mapping resistance to this anti-cancer drug.
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A Comparative Analysis Of The Moose Rumen Microbiota And The Pursuit Of Improving Fibrolytic Systems.Pellegrini, Suzanne Ishaq 01 January 2015 (has links)
The goal of the work presented herein was to further our understanding of the rumen microbiota and microbiome of wild moose, and to use that understanding to improve other processes. The moose has adapted to eating a diet of woody browse, which is very high in fiber, but low in digestibility due to the complexity of the plant polysaccharides, and the presence of tannins, lignin, and other plant-secondary compounds. Therefore, it was hypothesized that the moose would host novel microorganisms that would be capable of a wide variety of enzymatic functions, such as improved fiber breakdown, metabolism of digestibility-reducing or toxic plant compounds, or production of functional metabolites, such as volatile fatty acids, biogenic amines, etc.
The first aim, naturally, was to identify the microorganisms present in the rumen of moose, in this case, the bacteria, archaea, and protozoa. This was done using a variety of high-throughput techniques focusing on the SSU rRNA gene (see CHAPTERS 2-5). The second aim was to culture bacteria from the rumen of the moose in order to study their biochemical capabilities (see CHAPTERS 6-7). The final aim was to apply those cultured bacterial isolates to improve other systems. Specifically, bacteria from the rumen of the moose was introduced to young lambs in order to colonize the digestive tract, speed the pace of rumen development, and improve dietary efficiency (see CHAPTER 8).
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Structure et activité des Archaea planctoniques dans les écosystèmes aquatiques / Structure and activity of planktonic Archaea in aquatic ecosystemsHugoni, Mylène 31 October 2013 (has links)
Les Archaea planctoniques contribuent de façon significative aux grands cycles biogéochimiques dans les écosystèmes aquatiques, néanmoins la structure des communautés actives ainsi que leurs variations saisonnières sont encore largement méconnues. En outre, la découverte de l’implication des Archaea dans le cycle de l’azote (Ammonia Oxidizing Archaea ou AOA), plus particulièrement dans le processus de nitrification a considérablement modifié la perception d’un processus autotrophe réalisé uniquement par des bactéries (Ammonia Oxidizing Bacteria ou AOB). Dans les écosystèmes marins, la large distribution des AOA suggère que ces microorganismes joueraient un rôle prépondérant dans le cycle de l’azote néanmoins, ces observations ne sont pas généralisables à l’ensemble des écosystèmes aquatiques en raison de leur grande diversité et/ou d'un manque d'informations et d’études sur certains d'entre eux. Ainsi, les objectifs de ce projet étaient i) d’étudier la structure spatiale et temporelle des communautés d’Archaea actives dans des écosystèmes aquatiques contrastés en termes d’apports anthropiques et/ou de gradients de salinité (lac, estuaire, milieu côtier) ; ii) de déterminer la contribution relative des Archaea au processus d’oxydation de l’ammonium, en comparaison avec celle des bactéries ; et iii) de mieux comprendre les paramètres environnementaux qui pourraient déterminer l’établissement des communautés d’AOA ou d’AOB. / Aquatic Archaea are important players among microbial plankton and significantly contribute to biogeochemical cycles, especially nitrogen, but details regarding their community structure and seasonal activity and dynamics remain largely unexplored. In marine ecosystems, the widespread distribution of Ammonia Oxidizing Archaea (AOA) suggests that they probably play a major role in nutrients cycling. However, we cannot generalize these observations to all aquatic ecosystems because of their high diversity and/or a lack of information and studies on these organisms for some of these ecosystems. More precisely, lacustrine and coastal ecosystems were less studied while they are potentially subjected to strong anthropogenic impacts. Moreover, notable differences in terms of diversity and activity between marine and freshwater communities can be expected, considering the specific environmental parameters of each ecosystem. The objectives of this thesis were: i) to study the archaeal community structure across a temporal scale and assess the diversity of archaeal communities and AOA in diverse aquatic ecosystems along anthropogenic and/or salinity gradient (lacustrine, estuarine and coastal ecosystems); ii) to determine their relative contribution in ammonia oxidation, compared to Ammonia Oxidizing Bacteria (AOB) by looking at their spatial and temporal distribution and activity, and iii) to explore more precisely the environmental parameters that could drive AOA and/or AOB establishment.
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Phenotypic evolution and adaptive strategies in marine phytoplankton (Coccolithophores)Šupraha, Luka January 2016 (has links)
Coccolithophores are biogeochemically important marine algae that interact with the carbon cycle through photosynthesis (CO2 sink), calcification (CO2 source) and burial of carbon into oceanic sediments. The group is considered susceptible to the ongoing climate perturbations, in particular to ocean acidification, temperature increase and nutrient limitation. The aim of this thesis was to investigate the adaptation of coccolithophores to environmental change, with the focus on temperature stress and nutrient limitation. The research was conducted in frame of three approaches: experiments testing the physiological response of coccolithophore species Helicosphaera carteri and Coccolithus pelagicus to phosphorus limitation, field studies on coccolithophore life-cycles with a method comparison and an investigation of the phenotypic evolution of the coccolithophore genus Helicosphaera over the past 15 Ma. Experimental results show that the physiology and morphology of large coccolithophores are sensitive to phosphorus limitation, and that the adaptation to low-nutrient conditions can lead to a decrease in calcification rates. Field studies have contributed to our understanding of coccolithophore life cycles, revealing complex ecological patterns within the Mediterranean community which are seemingly regulated by seasonal, temperature-driven environment changes. In addition, the high-throughput sequencing (HTS) molecular method was shown to provide overall good representation of coccolithophore community composition. Finally, the study on Helicosphaera evolution showed that adaptation to decreasing CO2 in higher latitudes involved cell and coccolith size decrease, whereas the adaptation in tropical ecosystems also included a physiological decrease in calcification rates in response to nutrient limitation. This thesis advanced our understanding of coccolithophore adaptive strategies and will improve our predictions on the fate of the group under ongoing climate change.
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High-throughput Detection Of Potentially Active L1 Elements In Human GenomesJanuary 2014 (has links)
The active human retrotransposon L1 is the most prevalent human retroelement, constituting 17% of the mass of the human genome and contributing significantly to mutagenesis. L1 mutagenizes human genomes in a number of ways including insertional mutagenesis of itself and other retrotransposons, creating of DNA double strand breaks, and induction of non-allelic homologous recombination. Through these processes, the activity of L1 is responsible for approximately 0.5% of all new genetic diseases. All L1-derived mutagenesis stems from the activity of a small number of intact full-length L1 loci that remain capable of mobilization. A smaller subset of these active L1s are called hot L1s and are responsible for the vast majority of all L1 activity. Hot L1s are polymorphic in the population and represent evolutionarily recent L1 insertion events. Here, we show that potentially active full length L1 elements are more prevalent in individual genomes than previously believed. We find that the typical individual likely harbors approximately 60 active and 50 hot L1s. However, we also find that there is significant variation between individuals in numbers of potentially active L1s. As a result, the mutagenic burden associated with L1 likely varies between individuals. / acase@tulane.edu
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Understanding the Noise : Spliceosomal snRNA ProfilingConze, Lei Liu January 2012 (has links)
The concept of the gene has been constantly challenged by new discoveries in the life sciences. Recent challenging observations include the high frequency of alternative splicing events and the common transcription of non-protein-coding-RNAs (ncRNAs) from the genome. The latter has long been considered noise in biological systems. Multiple lines of evidence from genomic studies indicate that alternative splicing and ncRNA play important roles in expanding proteome diversity in eukaryotes. Here, the aim is to find the link between alternative splicing and ncRNAs by studying the expression profile of the spliceosomal snRNAs (U snRNA). Spliceosomal snRNAs are essential for pre-mRNA splicing in eukaryotes. They participate in splice site selection, recruitment of protein factors and catalyzing the splicing reaction. Because of this, both the abundance and diversity of U snRNAs were expected to be large. In our study we deeply analyzed the U snRNA population in primates using a combination of bioinformatical, biochemical and high throughput sequencing approaches. This transcriptome profiling has revealed that human, chimpanzee and rhesus have similar U snRNA populations, i.e. the vast majority of U snRNAs originate from few well-defined gene loci and the heterogeneity observed in U snRNA populations was largely due to the presence of SNPs at these loci. It seems that the gene loci that could potentially encode a significantly heterogeneous population of U snRNAs are mostly silent. Only few minority transcripts were detected in our study, and among them three U1-like snRNAs might play a role in the regulation of alternative splicing by recognizing non-canonical splicing sites. Mutations of U snRNA have been shown to impact the splicing process. Therefore, our study provides a reference to study the biological significance of SNPs in U snRNA genes and their association with diseases.
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Interpreting the human transcriptomeWerne Solnestam, Beata January 2015 (has links)
The human body is made of billions of cells and nearly all have the same genome. However, there is a high diversity of cells, resulted from what part of the genome the cells use, i.e. which RNA molecules are expressed. Rapid advances within the field of sequencing allow us to determine the RNA molecules expressed in a specific cell at a certain time. The use of the new technologies has expanded our view of the human transcriptome and increased our understanding of when, where, and how each RNA molecule is expressed. The work presented in this thesis focuses on analysis of the human transcriptome. In Paper I, we describe an automated approach for sample preparation. This protocol was compared with the standard manual protocol, and we demonstrated that the automated version outperformed the manual process in terms of sample throughput while maintaining high reproducibility. Paper II addresses the impact of nuclear transcripts on gene expression. We compared total RNA from whole cells and from cytoplasm, showing that transcripts with long, structured 3’- and 5’-untranslated regions and transcripts with long protein coding sequences tended to be retained in the nucleus. This resulted in increased complexity of the total RNA fraction and fewer reads per unique transcript. Papers III and IV describe dynamics of the human muscle transcriptome. For Paper III, we systematically investigated the transcriptome and found remarkably high tissue homogeneity, however a large number of genes and isoforms were differentially expressed between genders. Paper IV describes transcriptome differences in response to repeated training. No transcriptome-based memory was observed, however a large number of isoforms and genes were affected by training. Paper V describes a transcript profiling protocol based on the method Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification. We designed the method for a few selected transcripts whose expression patterns are important for detecting breast cancer cells, and optimized the method for single cell analysis. We successfully detected cells in human blood samples and applied the method to single cells, confirming the heterogeneity of a cell population. / Människokroppen är uppbyggd av miljarder celler och nästan alla innehåller samma arvsmassa. Trots detta finns det många olika celler med olika funktioner vilket är en följd av vilken del av arvsmassan som cellerna använder, dvs vilka RNA-molekyler som finns i varje cell. Den snabba utvecklingen av sekvenseringstekniker har gjort det möjligt att studera när, var och hur varje RNA-molekyl är uttryckt och att få en djupare förståelse för hur människans celler fungerar. Arbetet som presenteras i denna avhandling fokuserar på analys av RNA-molekyler i människans celler. I artikel I beskriver vi en automatiserad metod för att förbereda cellprov för RNA-sekvensering. Det automatiserade protokollet jämfördes med det manuella protokollet, och vi visade att det automatiserade protokollet överträffade det manuella när det gällde provkapacitet samtidigt som en höga reproducerbarheten behölls. I artikel II undersökte vi effekterna som RNA-molekyler från en del av cellen (cellkärnan) har på den totala mängden uttryckta RNA-molekyler. Vi jämförde RNA från hela cellen och från en del av cellen (cytoplasman) och visade att RNA-molekyler med långa och strukturerade 3'- och 5'-otranslaterade regioner och RNA-molekyler med långa proteinkodande sekvenser tenderade att hållas kvar i cellkärnan till en högre grad. Detta resulterade i en ökad komplexitet av RNA-molekylerna i hela cellen, medan vi i cytoplasma-fraktionen lättare kunde hitta de korta och svagt uttryckta RNA-molekyler. I Artikel III och IV studerar vi RNA-molekyler i människans skelettmuskler. I artikel III visar vi att andelen RNA-molekyler uttryckta i skelettmuskler är väldigt lika mellan muskler och mellan olika personer, men att ett stort antal RNA-molekyler var uttryckta i olika nivåer hos kvinnor och män. Artikel IV beskriver RNA-nivåer som svar på upprepade perioder av uthållighetsträning. Artikel V beskriver en metod för att studera ett fåtal utvalda RNA-molekyler. Vi valde RNA-molekyler vars uttryck är viktigt vid analys av bröstcancerceller, och optimerade metoden för analys av enskilda celler. Vi analyserade cancerceller från blodprov och använde metoden för att titta på RNA-nivåer i enskilda celler från en grupp av celler och visade på skillnader i RNA-nivåer inom gruppen. / <p>QC 20150115</p>
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Étude génomique de l'interférence entre la réplication et la transcription comme source du stress réplicatif. / Genome-wide study of the interference between DNA replication and transcription as a source of replication stressPadioleau, Ismaël 24 November 2017 (has links)
L’activation d’oncogènes entraine une prolifération aberrante des cellules, un stress réplicatif et des cassures de l’ADN. Un lien a été établi entre l’instabilité génomique résultant des cassures et l’inhibition de checkpoints entrainant l’accumulation de mutations et finalement le cancer (Halazonetis et al. 2008). Cependant, les mécanismes liant ces différents évènements n’ont pas encore été caractérisés. Notre hypothèse est que la prolifération incontrôlée des cellules augmente les incidents dus aux conflits entre les polymérases responsables de la réplication et celles responsables de la transcription. Lors de la rencontre des deux polymérases, l’accumulation de surenroulements positifs de l’ADN induit un blocage des fourches de réplication. Ceci crée des zones de fragilité, notamment dues à l’exposition d’ADN simple brin, et pourrait être à l’origine des cassures observées chez les cellules tumorales. Pour valider cette hypothèse, les biologistes de l'équipe ont étudié plusieurs lignées de cellules HeLa dans lesquelles les conflits réplication-transcription sont augmentés et j'ai réalisé l'analyse bioinformatique des approches génomiques suivantes :-DRIP-seq pour la détection des R-loops, une structure double brin hybride ADN/ARN qui se forme lors de la transcription, exposant ainsi un brin d’ADN simple brin.- ChIP-seq de γ-H2AX, une marque d’histone indiquant les cassures de l’ADN.-ChIP-seq de phospho-RPA (S33), un substrat de la kinase ATR au niveau des fourches bloquées. Pour chaque expérience, nous avons utilisé une lignée contrôle et deux lignées dans lesquelles TOP1 et ASF/SF2 sont appauvries avec un shRNA inductible (shTOP1 et shASF). La Topoisomérase I (TOP1) est une enzyme qui relaxe les surenroulements de l’ADN. Le complexe ASF/SF2 est un facteur d’épissage responsable entre autres de l’assemblage des mRNP (ribonucleoprotein particles) au moment de la transcription, qui limitent la formation des R-loops. L’analyse bioinformatique de ces données, ainsi que d'autres données de la littérature, m'a permis d'identifier des régions à risque du génome, localisées en aval de gènes fortement transcrits et répliqués précocement en phase S par des fourches progressant en sens opposé à la transcription. J’ai également observé que les gènes impliqués dans le cancer sont surreprésentés dans ces régions à risque. / Oncogenes activation promotes aberrant cell proliferation, increasing replication stress and DNA damage. It has been proposed that genomic instability leads to checkpoints inhibition and promotes cancer development (Halazonetis et al. 2008). However, the link between aberrant proliferation, replication stress and DNA breaks is still unclear. We hypothesized that aberrant proliferation leads to more incident due to DNA and RNA polymerases encounter and stalling. When the two polymerases encounter, the accumulation of positive-supercoiled DNA between two polymerases induces fork stalling, resulting in the formation of fragile structures such as single-stranded DNA (ssDNA). These ssDNAs formed at stalled forks could be a source for DNA breaks, promoting the development of cancer cells. To validate this hypothesis, biologists from our team have worked on HeLa cell lines with increased replication-transcription conflicts. I perform the bioinformatics analysis of the following genomic data:- DRIP-seq: R-Loops positioning on genome using immunoprecipitation on DNA/RNA hybrids.-γ-H2AX ChIP-Seq: Gamma-H2AX is an histone mark found at DNA breaks.-pRPA ChIP-Seq : Positioning of stalled forks using the substrate of ATR kinase, phospho-RPA (S33) as a marker.Each data was produced on control cells and two cell lines where TOP1 and ASF/SF2 were depleted by as inducible shRNA (shTOP1 and shASF). Topoisomerase 1 is a topological enzyme that unwinds DNA when supercoiling accumulates. ASF/SF2 is part of the splicing complexes that processes mRNP (messenger ribonucleoprotein particles) to prevent the accumulation of R-loops during transcription. Using these data and others from literature, I determined that regions having higher risk to induce replication stress are located downstream of highly transcribed and early replicated genes, and preferentially with head-on collision between DNA and RNA polymerases. I also revealed that cancer-related genes are enriched in these regions of the genome.
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