No description available.

Cryptosporidium is a parasite that causes gastrointestinal issues such as diarrhoea and stomach pain. The main transmission routes are through contaminated water or food, between humans and from animal to humans. Cryptosporidium infects through oocysts which contain four sporozoites that releases when entering a host and can continue to breed inside the body. Cryptosporidium can cause massive outbreaks if established in a water source used for drinking water. To prevent and detect an outbreak it´s important to trace the transmission back to the source. The GP60-gene is used to identify and subtype Cryptosporidium and is a very useful tool during contact tracing. The purpose of this study was to identify species and subtype of Cryptosporidium using nanopore sequencing. In this study the GP60-gene was amplified using a Nested PCR protocol and then sequenced using nanopore sequencing. The sequences acquired where then used to make a search in BLAST to identify the species. The GP60 subtyping method uses the hypervariable region on the GP60-gene. A series of tandem repeats are used to identify the subtype. In this study seven positive Cryptosporidium faeces samples were amplified and sequenced. Nanopore sequencing was possible for five of the seven samples with C. parvum identified in four of these samples. Targeting the GP60-gene to determine species and subtype works well for the most common human pathogen species of Cryptosporidium. Further optimization is required before the method can be implemented för diagnostic use.

S'ha estudiat la variabilitat genètica de les poblacions del nord d'Àfrica a partir de l'anàlisi de diverses regions genòmiques per tal d'entendre les poblacions analitzades d'una banda, i comprendre la dinàmica del genoma per l'altra. Els resultats obtinguts ens han permès verificar diferents hipòtesis sobre la història de les poblacions d'aquesta regió com són l'efecte paral·lel i independent de l'onada de difusió del neolític des de l'Orient Mitjà al llarg d'ambdues ribes de la Mediterrànea; i l'efecte de l'arabització. S'ha pogut estimar també la contribució genètica masculina nord africana a la península ibèrica i detectat certa contribució genètica del pobles sub-saharians a les poblacions nordafricanes. Per altra banda, el tipatge de marcadors genètics que evolucionen a velocitats diferents al cromosoma Y ha permès mostrar que el background genètic predomina sobre el background poblacional en l'estructura de la variació genètica dels microsatèl·lits en la regió no recombinant del cromosoma Y humà. / The genetic variability of the North African populations has been studied through the analysis of different genomic regions in order to understand both the analysed populations and the dynamics of the genome. The obtained results allow us to verify different hypotheses about the population history of this region including the parallel and independent effect of the Neolithic wave of advance from the Middle East and along both Mediterranean coasts; and the effect of Arabization phenomena. We also tried to estimate the North African male genetic contribution to the Iberian peninsula and detected Sub-Saharian genetic influences to the North African peoples. Moreover, the typing of genetic markers with different evolutionary rates on the Y chromosome allowed us to demonstrate that variation in microsatellites is deeply structured by genetic background on the non-recombining region of the human Y chromosome.

Megasatellites are polymorphic tandem repetitive sequences with repeat-units longer than or equal to 1000 base pairs. The novel algorithm Megasatfinder predicts megasatellites in the human genome. A structured method of analysing the algorithm is developed and conducted. The analysis method consists of six test scenarios. Scripts are created, which execute the algorithm using various parameter settings. Three nucleotide sequences are applied; a real sequence extracted from the human genome and two random sequences, generated using different base probabilities. Usability and accuracy are investigated, providing the user with confidence in the algorithm and its output. The results indicate that Megasatfinder is an excellent tool for the detection of megasatellites and that the generated results are highly reliable. The results of the complete analysis suggest alterations in the default parameter settings, presented as user guidelines, and state that artificially generated sequences are not applicable as models for real DNA in computational simulations.

<p>Megasatellites are polymorphic tandem repetitive sequences with repeat-units longer than or equal to 1000 base pairs. The novel algorithm Megasatfinder predicts megasatellites in the human genome. A structured method of analysing the algorithm is developed and conducted. The analysis method consists of six test scenarios. Scripts are created, which execute the algorithm using various parameter settings. Three nucleotide sequences are applied; a real sequence extracted from the human genome and two random sequences, generated using different base probabilities. Usability and accuracy are investigated, providing the user with confidence in the algorithm and its output. The results indicate that Megasatfinder is an excellent tool for the detection of megasatellites and that the generated results are highly reliable. The results of the complete analysis suggest alterations in the default parameter settings, presented as user guidelines, and state that artificially generated sequences are not applicable as models for real DNA in computational simulations.</p>

This thesis provides theoretical resources for the design of a new bioinformatics tool for transposon annotation with focus on their additional structural elements. There is a biological description of transposons, the mobile elements in DNA, their classification and structure. It further deals with the overview and classification of available transposon identification and annotation bioinformatics tools, description of function and implementation of a select few. Next we state the scheme of a new bioinformatics tool for LTR retrotransposon identification and annotation with a focus on extra ORFs and tandem repeats. The functionality of this new tool was tested on the A. thaliana genome. We identified 95 groups of conserved extra ORFs and 10 groups of conserved tandem repeats.

Les structures protéiques peuvent être divisées en répétitives et apériodiques, les structures apériodiques correspondant pour la plupart à des protéines globulaires. Les protéines répétitives (PRs) contiennent des unités de répétitions adjacentes, appelées séquences répétées en tandem (TRs). Les PRs sont abondantes et ont une importance fonctionnelle fondamentale. De plus de nombreuses études ont démontré l'implication des TRs dans les pathologies humaines. Ainsi, la découverte des PRs et la compréhension de leur relation séquence-structure-fonction, offrent des perspectives de recherche prometteuses.Le développement d’initiatives en génomique structurale, combiné à une meilleure adaptation des techniques de cristallographie et de RMN à l’étude des protéines non globulaires, a permis d’élucider la structure d’un nombre croissant de PRs, d’où la nécessité de mettre en place un système de classification. Les structures répétitives ont été réparties en cinq classes, principalement fondées sur la longueur des TRs: Classe I - agrégats cristallins; Classe II - structures fibreuses; Classe III - structures allongées, dont la stabilité dépend des interactions qui s’établissent entre les motifs répétés. Classe IV - structures répétitives fermées ; Classe V - structures en collier de perles. Les efforts de ces dernières années ont abouti au développement d’outils bioinformatiques utiles à la détection et l'analyse d'éléments répétitifs présents au sein des structures protéiques (3D TRs). En fonction des caractéristiques des répétitions, certaines méthodes fonctionnent mieux que d'autres, mais, jusqu’à présent, aucune ne permettait de couvrir toute la gamme des répétitions. Ce constat nous a incités à développer une nouvelle méthode, appelée détecteur de protéines en tandem (TAPO). TAPO exploite les périodicités des coordonnées atomiques ainsi que d'autres types de représentation structurale, comprenant les chaînes générées par un alphabet conformationnel, les cartes de contact entre résidus, et les arrangements en vecteurs d'éléments de structure secondaire. Actuellement, sept scores, issus des caractéristiques analysées par TAPO, sont combinés à l’aide d’une Machine à Vecteur Support pour produire un score final permettant de différencier les protéines renfermant ou non des 3D TRs. En atteignant 94% de sensibilité et 97% de spécificité pour la référence actuelle, TAPO présente des performances améliorées par rapport aux autres méthodes de pointe. Le développement de TAPO offre de nouvelles opportunités pour l’analyse à grande échelle des protéines renfermant des 3D TRs. Ainsi, notre analyse de la base de données PDB, à l’aide de TAPO, a montré que 19% des protéines contiennent des 3D TRs. L'analyse à grande échelle des structures 3D TRs dans PDB nous a également permis de découvrir plusieurs nouveaux types de structures répétitives, absents de la classification existante et dont certains sont décrits ici.Nous avons entrepris une analyse complète des 3D TRs constitutifs du Rossmann Fold (RF). Notre intérêt pour les RFs a été suscité par le fait que de nombreuses protéines RFs représentent un cas ambigüe vis à vis des structures répétitives et non répétitives. A priori, les unités hélice α - feuillet β des RFs devraient avoir une forte tendance à s’empiler et donc, à former des structures répétitives. Afin de déterminer la fréquence à laquelle les RFs forment de longues unités de répétition empilées, nous avons sélectionné, à l’aide de TAPO, des structures contenant des RFs et les avons classées. Notre analyse montre que les RFs typiques ne peuvent pas être clairement définis comme des structures répétitives mais plutôt comme des unités de structures globulaires, comptant au plus trois répétitions α-β. Des éléments de discussion seront proposés pour tenter d’expliquer cette observation surprenante. / In general, protein structures can be divided into: repetitive and aperiodic structures. Most of the aperiodic structures are globular proteins. The repetitive proteins contain arrays of repeats that are adjacent to each other, called Tandem Repeats (TRs). Proteins containing TRs are abundant and have fundamental functional importance. Numerous studies demonstrated the involvement of such TR-containing proteins in human diseases. Furthermore, genetic instability of these regions can lead to emerging infection threats. Additionally, TR-containing structures have generated significant interest with respect to protein design as they can make excellent scaffolds for specific recognition of target molecules. Therefore, the discovery of these domains, understanding of their sequence–structure–function relationship promises to be a fertile direction for research.The growth of structural genomics initiatives, in combination with improvements in crystallographic and NMR techniques aimed at non-globular proteins, has resulted in an increase in structurally elucidated TR proteins. This has necessitated the development of classification schemes. Structural repeats were broadly divided into five classes mainly based on repeat length; Class I – crystalline aggregates; Class II – fibrous structures such as collagen; Class III – elongated structures where the repetitive units require each other for structural stability such as solenoid proteins; Class IV – closed repetitive structures, such as TIM-barrels and Class V – bead on a string structures such as tandems of Ig-fold domains. Despite this progress, the majority of bioinformatics approaches have focused on non-repetitive globular proteins.In recent years, efforts have been made to develop bioinformatics tools for the detection and analysis of repetitive elements in protein structures (3D TRs). Depending on the size and character of the repeats, some methods perform better than others, but currently no best approach exists to cover the whole range of repeats. This served as a motivation for the development of our method called the TAndem PrOtein detector (TAPO). TAPO exploits, periodicities of atomic coordinates and other types of structural representation, including strings generated by conformational alphabets, residue contact maps, and arrangements of vectors of secondary structure elements. Currently, seven feature based scores produced by TAPO are combined using a Support Vector Machine, producing a score to enable the differentiation between proteins with and without 3D TRs. TAPO shows an improved performance over other cutting edge methods, achieving 94% sensitivity and 97% specificity on the current benchmark. The development of TAPO provided new opportunities for large scale analysis of proteins with 3D TRs. In accordance with our analysis of PDB using TAPO, 19% of proteins contain 3D TRs. The large scale analysis of the 3D TR structures in PDB also allows us to discover several new types of TR structures that were absent in the existing classification. Some of them are described in the thesis manuscript. This suggests that TAPO can be used to regularly update the collection and classification of existing repetitive structures. In particular, a comprehensive analysis of 3D TRs related to Rossmann Fold (RF) was undertaken. Our special interest in RFs was based on the observation that many proteins with RFs represent borderline cases between repetitive and non-repetitive structures. In principle, α-helix-β-strand units of RFs should have a strong potential to stack one over the other, forming repetitive structures. To probe the question of how frequently RFs form long arrays of stacked repeats, we selected by using TAPO known RF-containing structures and classified them. Our analysis shows that typical RFs cannot be clearly defined as repetitive, rather they are part of globular structures with up to 3 αβ-repeats. We provide some explanations for this surprising observation.

Hematopoietic stem cell transplantation (HSCT) is used to treat patient with hematological diseases such as leukemia and genetic conditions such as sickle cell anemia. After HSCT the patients are supervised for signs of relapse of disease or rejection of transplanted cells. This is done by using chimerism analysis. At the department of clinical genetics at Akademiska sjukhuset fragment analysis of short tandem repeats is used for chimerism analysis, which is to be replaced by a Next generation sequencing (NGS) based method called Devyser chimerism, which includes an IVDR labelled kit. The aim of this project was to validate the new method for chimerism analysis. DNA samples from twelve HSCT patients and their donors were analyzed with Devyser chimerism and the results were compared to the results from the current method. The sensitivity of the new method was tested by analysis of artificial chimerism samples from blood donors. The results from the comparison showed a good correlation between methods (R2 = 0,9864) and the sensitivity of the method was confirmed to be 0,1% mixed chimerism. There was some difficulty in identifying enough informative markers for re-transplanted patients two had separate donors. This is a known problem for chimerism analysis in general and not a specific problem to the new method and will not be a hindrance for the implementation of Devyser chimerism at the clinical laboratory.

No description available.

ATRX is a chromatin remodeling protein associated with X-linked Alpha-Thalassemia Mental Retardation syndrome and cancers that use the Alternative Lengthening of Telomere pathway. In the absence of ATRX there is a DNA damage response associated with telomeres and the expression of certain genes are perturbed. Recent findings (Law et al, 2010 Cell) have shown that ATRX is preferentially enriched at GC-rich tandem repeats in the genome. The mechanism for this localisation is unknown but may be related to the potential for these GC-rich tandem repeats to adopt non-B form DNA structures; ATRX has been shown to bind such structures (G4) in vitro. This study aims to understand the specific factors of the repeats that signal ATRX targeting. To address the research questions, an experimental system was developed, in which known targets, the &psi;&zeta; VNTR and telomere repeats, were inserted into an inducible ectopic gene in the 293T-Rex cell line by site-directed recombination. ATRX was found to be enriched at the ectopic repeats compared to an endogenous negative control suggesting that it is recruited by the repeats independent of its original context. Furthermore, ATRX enrichment increased upon transcription of the ectopic gene, and this was dependent on the orientation of the repeat with the non-template strand being G-rich. Interestingly, when the repeat was transcribed, the distribution of ATRX across the repeats was asymmetrical with most ATRX binding downstream of the repeat. Moreover, there was a direct correlation between the repeat size and level of ATRX bound: the longer the repeat the higher the increase in ATRX enrichment. To determine the signal for ATRX binding, assays were performed to look for features which reflected the distribution of ATRX including H3K9me3, RNA polII, G4, R loops and DNA supercoiling. R loops look to be a strong candidate for the signaling of ATRX binding.