The 23 pairs of chromosomes comprising the human genome are intricately folded within the nucleus of each cell in a manner that promotes efficient gene regulation and cell function. Consequently, active gene rich regions are compartmentally segregated from inactive gene poor regions of the genome. To better understand the mechanisms driving compartmentalization we investigated what would occur if this system was disrupted. By digesting the genome to varying sizes and analyzing the fragmented 3D structure over time, our work revealed essential laws governing nuclear compartmentalization. At a finer resolution within compartments, chromatin forms loop structures capable of regulating gene expression. Genome wide association studies have identified numerous single nucleotide polymorphisms (SNPs) associated with the neuropsychiatric disease schizophrenia. When these SNPs are not located within a gene it is difficult to gain insight into disease pathology; however, in some cases chromatin loops may link these noncoding schizophrenia risk variants to their pathological gene targets. By generating 3D genome maps, we identified and analyzed loops of glial cells, neural progenitor cells, and neurons thereby expanding the set of genes conferring schizophrenia risk. The binding of T-cell receptors (TCRs) to foreign peptides on the surface of diseased cells triggers an immune response against the foreign invader. Utilizing available structural information of the TCR antigen interface, we developed computational methods for successful prediction of TCR-antigen binding. As this binding is a prerequisite for immune response, such improvements in binding prediction could lead to important advancements in the fields of autoimmunity and TCR design for cancer therapeutics.

Promoter-enhancer interactions are essential for gene regulating, Capture Hi-C is a chromosome conformation capture method to map promoter-enhancer interactions at high resolution. We have Capture Hi-C data forGM12878 cells, immortalized primary B lymphocytes, in three replicates. Although Capture Hi-C maps enhancer elements together with the promoters they regulate, the overlap between enhancer datasets produced by other methods such as ChIP-seq and Capture Hi-C is lower than expected. In order to understand the reasons for lower overlap, we investigated the enhancer potential of replicated and non-replicated Capture Hi-C interactors, as well as enhancer overlapping and non-overlapping Capture Hi-C interactors. We performed a systematic comparison between our interactor and experimental regulatory and transcriptomic datasets to determine the extent of enhancer mapping. The results show replicated interactors have higher enhancer potential than non-replicated ones. However, there is evidence that interactors not overlapping with experimental validated regulatory datasets can also potentially be true enhancers.

No description available.

本研究探討高通量染色體捕捉技術 (high-throughput chromosome conformation capture, Hi-C) 實驗所產生的關聯矩陣資料之正規化方法。已知該類實驗主要用來測量染色體之間的空間距離，正規化的目的是移除資料中的系統性偏差，本文主要針對基因特徵所造成之偏差。有別於Hu等人 (2012) 所提出的「局部基因特徵正規化法」(local genome feature normalization, LGF法)，我們所提出的「二次函數正規化法」(quadratic function normalization, QF法) 建立在更為一般化的二次對數模型與負二項分配假設上。本研究透過模擬實驗以及人類淋巴細胞資料 (GSE18199) 來評估QF法的表現，並且與其他方法比較。在模擬實驗中，我們發現當模型正確時，QF法能有效消除偏差。在實例中，當基因特徵偏差被消除後，則染色體之間的相對距離在重複實驗資料之間有更為一致的結果。另一方面，我們發現實驗所採用的限制酶影響關聯矩陣的結果，而且運用這些正規化方法並不能有效消除限制酶造成的偏差。 / Recently, the high-throughput chromosome conformation capture (Hi-C) experiment is developed to explore the three-dimensional structure of genomics. To assess the chromosomal interaction, a contact matrix is produced from a Hi-C experiment. Very often, systematic technical biases appear in the contact matrix and lead to inadequate conclusions. Consequently, data normalization to remove these biases is essential and necessary prior advanced inference. In this research, we propose the so-called quadratic function normalization method, which is a modification of the local genome feature normalization (Hu et al., 2012) by considering a more general model. Simulation studies are conducted to evaluate the proposed method. When the model assumption holds, the proposed method has adequate performance. Further, a Hi-C data set of a human lymphoblastoid cell GSE18199 is employed for a comparison of our method and two existing methods. It’s observed that normalization improves the reproducibility between experimental replicates. However, the effect of normalization is lean in eliminating the bias of restriction enzymes.

<p>ASSA AB develops different types of products that can be used in or around doors. ASSA offers a complete door solution to the customers. Many of these products are linked together and work for a complete formation of access control. ASSA has recently developed a new communications bus for some of their products. One advantage of the new communication bus is that each product should be able to offer different kinds of added values to the customers.</p><p>This master thesis main focus is to find a model to describe the predicted life length of ASSA:s motor lock called 811C50, which includes a study that determine which environmental parameters that can affects the motor locks life length. This master thesis is also a pre-study for further work of an existing motor lock.</p><p>The chosen test model, <em>full parameter test, leads to a life length equation. This equation shall later on be implemented in the motor locks so the motor locks itself can predict its own life length. To make the equation completed different parameters, that can affect the motor locks life length, are needed. This can be implemented through different technological tests and to succeed with the test, right test equipment is needed. No establish test equipment was available, which required that new test equipment, which could test all motor locks, were build. On the bases from interviews with engineers at ASSA it was established that the following parameters probably can affect the motor locks life length: temperature, air humidity, dust, corrosion, side load and vibration. Not until certain factors and coefficients have been determined, for the equation, can the equation be used. </em></p> / <p>ASSA AB är ett företag som utvecklar olika typer av produkter som kan användas i eller runt en dörr. ASSA erbjuder en komplett dörrlösning till kunderna. Många av de här produkterna är sammankopplade och arbetar tillsammans för att komplett passersystem. ASSA har nyligen utvecklat en ny kommunikationsbuss för några av deras produkter. En fördel med den nya kommunikationsbussen är att varje produkt kan ge ett ökat mervärde för slutkunden.</p><p>Denna avhandling har som huvudsyfte att hitta en modell som kan beskriva livslängden för ASSA:s motorlås, 811C50. Till detta görs en studie för att undersöka vilka miljö parametrar som kan påverka motorlåsets livslängd. Denna avhandling kan även ses som en förstudie där målet är att vidareutveckla det befintliga motorlåset.</p><p>Den valda modellen, full variabel test, leder till en livslängdsekvation. Denna ekvation kommer sedan att bli implementerad i motorlåset så att låset själv kan prediktera sin egen livslängd. För att göra ekvationen komplett behövs olika parameterr som kan påverka motorlåsets livslängd identifieras. Detta kan ske genom olika tekniska test och för att lyckas med testen krävs rätt testutrustning. Ingen befintlig testutrustning fanns tillgänglig vilket resulterade i en ny testutrustning som kunde testa alla motorlås utvecklades. Utifrån intervjuer med ingenjörer på ASSA kunde det fastställas att följande parameterr troligtvis kan påverka motorlåsets livslängd: temperatur, luftfuktighet, damm, korrosion, listtryck och vibrationer. Inte förens koefficienter, till ekvationen, har blivit fastställda är ekvationen färdig att användas.</p>

ASSA AB develops different types of products that can be used in or around doors. ASSA offers a complete door solution to the customers. Many of these products are linked together and work for a complete formation of access control. ASSA has recently developed a new communications bus for some of their products. One advantage of the new communication bus is that each product should be able to offer different kinds of added values to the customers. This master thesis main focus is to find a model to describe the predicted life length of ASSA:s motor lock called 811C50, which includes a study that determine which environmental parameters that can affects the motor locks life length. This master thesis is also a pre-study for further work of an existing motor lock. The chosen test model, full parameter test, leads to a life length equation. This equation shall later on be implemented in the motor locks so the motor locks itself can predict its own life length. To make the equation completed different parameters, that can affect the motor locks life length, are needed. This can be implemented through different technological tests and to succeed with the test, right test equipment is needed. No establish test equipment was available, which required that new test equipment, which could test all motor locks, were build. On the bases from interviews with engineers at ASSA it was established that the following parameters probably can affect the motor locks life length: temperature, air humidity, dust, corrosion, side load and vibration. Not until certain factors and coefficients have been determined, for the equation, can the equation be used. / ASSA AB är ett företag som utvecklar olika typer av produkter som kan användas i eller runt en dörr. ASSA erbjuder en komplett dörrlösning till kunderna. Många av de här produkterna är sammankopplade och arbetar tillsammans för att komplett passersystem. ASSA har nyligen utvecklat en ny kommunikationsbuss för några av deras produkter. En fördel med den nya kommunikationsbussen är att varje produkt kan ge ett ökat mervärde för slutkunden. Denna avhandling har som huvudsyfte att hitta en modell som kan beskriva livslängden för ASSA:s motorlås, 811C50. Till detta görs en studie för att undersöka vilka miljö parametrar som kan påverka motorlåsets livslängd. Denna avhandling kan även ses som en förstudie där målet är att vidareutveckla det befintliga motorlåset. Den valda modellen, full variabel test, leder till en livslängdsekvation. Denna ekvation kommer sedan att bli implementerad i motorlåset så att låset själv kan prediktera sin egen livslängd. För att göra ekvationen komplett behövs olika parameterr som kan påverka motorlåsets livslängd identifieras. Detta kan ske genom olika tekniska test och för att lyckas med testen krävs rätt testutrustning. Ingen befintlig testutrustning fanns tillgänglig vilket resulterade i en ny testutrustning som kunde testa alla motorlås utvecklades. Utifrån intervjuer med ingenjörer på ASSA kunde det fastställas att följande parameterr troligtvis kan påverka motorlåsets livslängd: temperatur, luftfuktighet, damm, korrosion, listtryck och vibrationer. Inte förens koefficienter, till ekvationen, har blivit fastställda är ekvationen färdig att användas.

Des variations importantes du surenroulement de l’ADN peuvent être générées durant la phase d’élongation de la transcription selon le modèle du « twin supercoiled domain ». Selon ce modèle, le déplacement du complexe de transcription génère du surenroulement positif à l’avant, et du surenroulement négatif à l’arrière de l’ARN polymérase. Le rôle essentiel de la topoisomérase I chez Escherichia coli est de prévenir l’accumulation de ce surenroulement négatif générée durant la transcription. En absence de topoisomérase I, l’accumulation de ce surenroulement négatif favorise la formation de R-loops qui ont pour conséquence d’inhiber la croissance bactérienne. Les R-loops sont des hybrides ARN-ADN qui se forment entre l’ARN nouvellement synthétisé et le simple brin d’ADN complémentaire. Dans les cellules déficientes en topoisomérase I, des mutations compensatoires s’accumulent dans les gènes qui codent pour la gyrase, réduisant le niveau de surenroulement négatif du chromosome et favorisant la croissance. Une des ces mutations est une gyrase thermosensible qui s’exprime à 37 °C. La RNase HI, une enzyme qui dégrade la partie ARN d’un R-loop, peut aussi restaurer la croissance en absence de topoisomérase I lorsqu’elle est produite en très grande quantité par rapport à sa concentration physiologique. En présence de topoisomérase I, des R-loops peuvent aussi se former lorsque la RNase HI est inactive. Dans ces souches mutantes, les R-loops induisent la réponse SOS et la réplication constitutive de l’ADN (cSDR). Dans notre étude, nous montrons comment les R-loops formés en absence de topoisomérase I ou RNase HI peuvent affecter négativement la croissance des cellules. Lorsque la topoisomérase I est inactivée, l’accumulation d’hypersurenroulement négatif conduit à la formation de nombreux R-loops, ce qui déclenche la dégradation de l’ARN synthétisé. Issus de la dégradation de l’ARNm de pleine longueur, des ARNm incomplets et traductibles s’accumulent et causent l’inhibition de la synthèse protéique et de la croissance. Le processus par lequel l’ARN est dégradé n’est pas encore complètement élucidé, mais nos résultats soutiennent fortement que la RNase HI présente en concentration physiologique est responsable de ce phénotype. Chose importante, la RNase E qui est l’endoribonuclease majeure de la cellule n’est pas impliquée dans ce processus, et la dégradation de l’ARN survient avant son action. Nous montrons aussi qu’une corrélation parfaite existe entre la concentration de RNase HI, l’accumulation d’hypersurenroulement négatif et l’inhibition de la croissance bactérienne. Lorsque la RNase HI est en excès, l’accumulation de surenroulement négatif est inhibée et la croissance n’est pas affectée. L’inverse se produit Lorsque la RNase HI est en concentration physiologique. En limitant l’accumulation d’hypersurenroulement négatif, la surproduction de la RNase HI prévient alors la dégradation de l’ARN et permet la croissance. Quand la RNase HI est inactivée en présence de topoisomérase I, les R-loops réduisent le niveau d’expression de nombreux gènes, incluant des gènes de résistance aux stress comme rpoH et grpE. Cette inhibition de l’expression génique n’est pas accompagnée de la dégradation de l’ARN contrairement à ce qui se produit en absence de topoisomérase I. Dans le mutant déficient en RNase HI, la diminution de l’expression génique réduit la concentration cellulaire de différentes protéines, ce qui altère négativement le taux de croissance et affecte dramatiquement la survie des cellules exposées aux stress de hautes températures et oxydatifs. Une inactivation de RecA, le facteur essentiel qui déclenche la réponse SOS et le cSDR, ne restaure pas l’expression génique. Ceci démontre que la réponse SOS et le cSDR ne sont pas impliqués dans l’inhibition de l’expression génique en absence de RNase HI. La croissance bactérienne qui est inhibée en absence de topoisomérase I, reprend lorsque l’excès de surenroulement négatif est éliminé. En absence de RNase HI et de topoisomérase I, le surenroulement négatif est très relaxé. Il semble que la réponse cellulaire suite à la formation de R-loops, soit la relaxation du surenroulement négatif. Selon le même principe, des mutations compensatoires dans la gyrase apparaissent en absence de topoisomérase I et réduisent l’accumulation de surenroulement négatif. Ceci supporte fortement l’idée que le surenroulement négatif joue un rôle primordial dans la formation de R-loop. La régulation du surenroulement négatif de l’ADN est donc une tâche essentielle pour la cellule. Elle favorise notamment l’expression génique optimale durant la croissance et l’exposition aux stress, en limitant la formation de R-loops. La topoisomérase I et la RNase HI jouent un rôle important et complémentaire dans ce processus. / Important fluctuations of DNA supercoiling occur during transcription in the frame of the “twin supercoiled domain” model. In this model, transcription elongation generates negative and positive supercoiling respectively, upstream and downstream of the moving RNA polymerase. The major role of bacterial topoisomerase I is to prevent the accumulation of transcription-induced negative supercoiling. In its absence, the accumulation of negative supercoiling triggers R-loop formation which inhibits bacterial growth. R-loops are DNA/RNA hybrids formed during transcription when the nascent RNA hybridizes with the template strand thus, leaving the non-template strand single stranded. In cells lacking DNA topoisomerase I, a constant and selective pressure for the acquisition of compensatory mutations in gyrase genes reduces the negative supercoiling level of the chromosome and allows growth. One of these mutations is a thermosensitive gyrase expressed at 37 °C. The overexpression of RNase HI, an enzyme that degrades the RNA moiety of an R-loop, is also able to correct growth inhibition in absence of topoisomerase I. In the presence of topoisomerase I, R-loops can also form when RNase HI is lacking. In these mutants, R-loop formation induces SOS and constitutive stable DNA replication (cSDR). In our study, we show how R-loops formed in cells lacking topoisomerase I or RNase HI can affect bacterial growth. When topoisomerase I is inactivated, the accumulation of hypernegative supercoiling inhibits growth by causing extensive R-loop formation which, in turn, can lead to RNA degradation. As a result of RNA degradation, the accumulation of truncated and functional mRNA instead of full length ones, is responsible for protein synthesis inhibition that alters bacterial growth. The mechanism by which RNA is degraded is not completely clear but our results strongly suggest that RNase HI is involved in this process. More importantly, the major endoribonuclease, RNase E, is not involved in RNA degradation because RNA is degraded before its action. We show also that there is a perfect correlation between RNase HI concentration, the accumulation of hypernegative supercoiling and bacterial growth inhibition. When RNase HI is in excess, no accumulation of hypernegative supercoiling and growth inhibition are observed. The opposite is true when RNase HI is at its wild type level. By preventing the accumulation of hypernegative supercoiling, the overproduction of RNase HI inhibits extensive R-loop formation and RNA degradation, thus, allowing growth. In absence of RNase HI (rnhA) and in presence of topoisomerase I, R-loops are also responsible for an inhibition in gene expression, including stress genes such as rpoH and grpE. The inhibition of gene expression is not related to RNA degradation as seen in absence of topoisomerase I but it is rather related to a reduction in gene expression. In absence of RNase HI, the diminution of genes expression is responsible for a reduction in the cellular level of proteins, which negatively affects bacterial growth and bacterial survival to heat shock and oxydative stress. Additional mutations in RecA, the protein that activates SOS and cSDR after R-loop formation in rnhA, do not correct this phenotype in rnhA. Thus, SOS and cSDR are not directly involved in the inhibition of gene expression in the absence of RNase HI. In absence of topoisomerase I, growth inhibition resumes when hypernegative supercoiling is reduced. When compared to wild type strains, DNA is very relaxed in absence of RNase HI and topoisomerase I. It seems that R-loop formation induces the relaxation of negatively supercoiled DNA. All this strongly supports the idea that negative supercoiling plays an important role in R-loop formation. Finally, our work shows how essential negative supercoiling regulation is for cell physiology. By preventing R-loop formation, regulation of negative supercoiling allows optimal gene expression, which is crucial for cellular growth and for stress survival. Both topoisomerase I and RNase HI play an important and complementary role in this process.

No description available.

No description available.

Malgré les vastes études démontrant le rôle de la conformation génomique dans le contrôle transcriptionnel, de nombreuses questions restent en suspens, et en particulier, comment ces structures chromatiniennes sont formées et maintenues. Pour mieux comprendre les liens entre l’état de la chromatine au niveau des éléments régulateurs, la topologie de la chromatine et la régulation de la transcription, nous utilisons la technique CHi-C basée sur la technologie de capture de la conformation chromosomique (3C). En utilisant deux stratégies de capture ciblant deux différentes structure chromatiniennes (les boucles chromatiniennes et les domaines topologiques), nous avons pu décrypter la structure chromatinienne associée à la différenciation des thymocytes et mettre en évidence des mécanismes de contrôle transcriptionnel de certains gènes. Les expériences futures de l’équipe vont consister à examiner les facteurs (hors transcription) qui peuvent influencer l'architecture de la chromatine, comme la liaison différentielle des CTCF, et comment ces facteurs peuvent être coordonnés par le contrôle de transcription. / Chromosome folding takes place at different hierarchical levels, with various topologies correlated with control of gene expression. Despite the large number of recent studies describing chromatin topologies and their correlations with gene activity, many questions remain, in particular how these topologies are formed and maintained. To understand better the link between epigenetic marks, chromatin topology and transcriptional control, we use CHi-C technique based on the chromosome conformation capture (3C) method. By using two capture strategies targeting two different chromatin structures (chromatin loops and topological domains), we have been able to decipher the chromatin structure associated with thymocyte differentiation and to highlight mechanisms for the transcriptional control of certain genes. Future experiments of the lab will examine mechanisms other than transcription which may influence chromatin architecture, such as differential binding of CTCF, and how these may interplay with transcriptional control and chromatin architecture.