Urofacial syndrome (UFS; MIM# 236730) is a rare autosomal recessive condition characterised by urinary bladder and bowel voiding dysfunction with a pathognomonic abnormality of facial movement with expression. UFS can be caused by biallelic putative loss-of-function mutations in HPSE2, which encodes heparanase 2. Failure to discover HPSE2 mutations in all cases of UFS suggests genetic heterogeneity. The urinary tract features of UFS overlap those seen in the spectrum of non-syndromic non-neurogenic voiding dysfunction and vesicoureteric reflux (VUR). This overlap suggests there may be some aspects of pathogenesis in common. The project aimed to define the genotypic and phenotypic spectrum associated with mutations in HPSE2 by Sanger sequencing and multiplex ligation-dependent probe amplification (MPLA) in newly referred cases of UFS and making comparison to a review of mutations and phenotypes seen in the literature. This work discovered five further families with HPSE2 associated UFS increasing known mutations whilst, reinforced that this is an under-recognised condition and emphasised the previously under-reported feature of facial weakness. The failure to discover HPSE2 mutations in all cases referred provided further evidence of genetic heterogeneity. The project also aimed to discover further genes associated with UFS. Autozygosity mapping and whole exome sequencing was carried out in cases of UFS without mutations in HPSE2. This led to the recognition that UFS is also caused by biallelic putative loss-of-function mutations in LRIG2 encoding the leucine-rich repeats and immunoglobulin-like domains 2 (LRIG2) protein in three families. Failure to identify LRIG2 mutations in all HPSE2 negative families suggests further genetic heterogeneity. To address the question of whether the pathogenesis of UFS overlaps more common conditions with a similar spectrum of urinary tract abnormalities I aimed to examine whether pathogenic variants in HPSE2 and LRIG2 were seen in these phenotypes. Unexpectedly this led to the discovering of further families affected by UFS but failed to show an association of variants in UFS genes with non-syndromic urinary tract abnormalities. However, variants of potential interest were discovered. As part of work toward understanding the pathogenesis of UFS and designing a model to test the pathogenesis of sequence variants expression studies in a Xenopus tropicalis hpse2 knock-down model of UFS were carried out. The knock-down model provided valuable insight in to the likely pathogenesis of UFS with evidence pointing towards a congenital peripheral neuropathy with failure of correct nerve path finding. Understanding the pathogenesis of UFS has the potential to direct further research in to therapeutic intervention.

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.

Using reverse genetics, we analyzed a chikungunya virus (CHIKV) isolate of the Indian Ocean lineage lacking direct repeat (DR) elements in the 3′ untranslated region, namely DR1a and DR2a. While this deletion mutant CHIKV-∆DR exhibited growth characteristics comparable to the wild-type virus in Baby Hamster Kidney cells, replication of the mutant was reduced in Aedes albopictus C6/36 and Ae. aegypti Aag2 cells. Using oral and intrathoracic infection of mosquitoes, viral infectivity, dissemination, and transmission of CHIKV-∆DR could be shown for the well-known CHIKV vectors Ae. aegypti and Ae. albopictus. Oral infection of Ae. vexans and Culex pipiens mosquitoes with mutant or wild-type CHIKV showed very limited infectivity. Dissemination, transmission, and transmission efficiencies as determined via viral RNA in the saliva were slightly higher in Ae. vexans for the wild-type virus than for CHIKV-∆DR. However, both Ae. vexans and Cx. pipiens allowed efficient viral replication after intrathoracic injection confirming that the midgut barrier is an important determinant for the compromised infectivity after oral infection. Transmission efficiencies were neither significantly different between Ae. vexans and Cx. pipiens nor between wild-type and CHIKV-∆DR. With a combined transmission efficiency of 6%, both Ae. vexans and Cx. pipiens might serve as potential vectors in temperate regions.

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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 ψζ 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.