Microsatellites; genotyping, mutation rate and effect on disease

Microsatellites are polymorphic tracts of short tandem repeats (STRs) with one to six base-pair (bp) motifs and account for around 3% of the human genome. Just like copying by hand a text where the same word occurs many times in a row, the replication of microsatellites is error prone and frequently adds or removes one or more copies of the repeat motif. As a result, microsatellites mutate several orders of magnitude faster than unique genomic sequences and for a given microsatellite, a population can have many possible length variations. The first objective of this study was to implement a method to jointly determine the number of repeats present at each microsatellite in the genome for a large number of samples. The second goal was to make the determination of repeat numbers more computationally efficient while simultaneously increasing the detection sensitivity of heavily expanded microsatellite alleles, known as repeat expansions. Last, the software was run on two large sets of whole genome sequenced individuals, one from Iceland and the other from the UK biobank. Using the genealogy information available on the Icelandic set, de novo mutation events were detected and the effects of parental sex, age and genotypes on the types and number of mutations found in their offspring were estimated.Um það bil þrjú prósent af erfðamengi mannsins eru örtungl, en þau eru fjölbreytilegar raðir af stuttum samliggjandi endurtekningum þar sem endurtekna röðin er á bilinu einn til sex basar á lengd. Líkt og við afritun á texta þar sem sama orðið er endurtekið oft í röð, þá er villuhættan meiri við afritun örtunglaraða en við aðrar raðir erfðamengisins og afleiðingin er að endurtekningu er bætt við eða hún tapast miðað við upprunalega basaröð. Vegna þessa stökkbreytast örtungl nokkrum stærðargráðum hraðar en aðrar raðir erfðamengisins og fyrir ákveðið örtungl getur hópur af fólki haft margar mismunandi lengdarútgáfur. Fyrsta markmið verkefnisins var að hanna og skrifa hugbúnað sem gæti ákvarðað fjölda endurtekninga fyrir öll örtungl í erfðamenginu hjá mörgun einstaklingum í einu. Næst, var reikniritinu hraðað en það jafnframt gert næmara fyrir stórum útþenslu örtungla samsætum, sem geta valdið mörgum mismunandi heilkennum hjá þeim sem þær bera. Að lokum var hugbúnaðurinn notaður til að meta arfgerð allra einstaklinga í tveimur stórum þýðum, frá Íslandi annars vegar og Bretlandi hins vegar. Ættfræðiupplýsingar um íslenska þýðið voru notaðar til að greina stökkbreytingar í afkvæmum sem ekki fundust í foreldrum og stökkbreytingarnar notaðar til að meta hvernig aldur kyn og arfgerð foreldra hefur áhrif á tegund og fjölda stökkbreytinga sem þeir arfleiða afkvæmi sín að

Empirical Analysis of Schemata in Genetic Programming

Schemata and buiding blocks have been used in Genetic Programming (GP) in several contexts including subroutines, theoretical analysis and for empirical analysis. Of these three the least explored is empirical analysis. This thesis presents a powerful GP empirical analysis technique for analysis of all schemata of a given form occurring in any program of a given population at scales not previously possible for the kinds of global analysis performed. There are many competing GP forms of schema and, rather than choosing one for analysis, the thesis defines the match-tree meta-form of schema as a general language expressing forms of schema for use by the analysis system. This language can express most forms of schema previously used in tree-based GP. The new method can perform wide-ranging analyses on the prohibitively large set of all schemata in the programs by introducing the concepts of maximal schema, maximal program subset, representative set of schemata, and representative program subset. These structures are used to optimize the analysis, shrinking its complexity to a manageable size without sacrificing the result. Characterization experiments analyze GP populations of up to 501 60- node programs, using 11 forms of schema including rooted-hyperschemata and non-rooted fragments. The new method has close to quadratic complexity on population size, and quartic complexity on program size. Efficacy experiments present example analyses using the new method. The experiments offer interesting insights into the dynamics of GP runs including fine-grained analysis of convergence and the visualization of schemata during a GP evolution. Future work will apply the many possible extensions of this new method to understanding how GP operates, including studies of convergence, building blocks and schema fitness. This method provides a much finer-resolution microscope into the inner workings of GP and will be used to provide accessable visualizations of the evolutionary process

The UBE2L3 ubiquitin conjugating enzyme: interplay with inflammasome signalling and bacterial ubiquitin ligases

Inflammasome-controlled immune responses such as IL-1β release and pyroptosis play key roles in antimicrobial immunity and are heavily implicated in multiple hereditary autoimmune diseases. Despite extensive knowledge of the mechanisms regulating inflammasome activation, many downstream responses remain poorly understood or uncharacterised. The cysteine protease caspase-1 is the executor of inflammasome responses, therefore identifying and characterising its substrates is vital for better understanding of inflammasome-mediated effector mechanisms. Using unbiased proteomics, the Shenoy grouped identified the ubiquitin conjugating enzyme UBE2L3 as a target of caspase-1. In this work, I have confirmed UBE2L3 as an indirect target of caspase-1 and characterised its role in inflammasomes-mediated immune responses. I show that UBE2L3 functions in the negative regulation of cellular pro-IL-1beta via the ubiquitin- proteasome system. Following inflammatory stimuli, UBE2L3 assists in the ubiquitylation and degradation of newly produced pro-IL-1b. However, in response to caspase-1 activation, UBE2L3 is itself targeted for degradation by the proteasome in a caspase-1-dependent manner, thereby liberating an additional pool of IL-1beta which may be processed and released. UBE2L3 therefore acts a molecular rheostat, conferring caspase-1 an additional level of control over this potent cytokine, ensuring that it is efficiently secreted only in appropriate circumstances. These findings on UBE2L3 have implications for IL-1-driven pathology in hereditary fever syndromes, and autoinflammatory conditions associated with UBE2L3 polymorphisms.Open Acces

Mecanismos moleculares de regulación alostérica de la enzima IMP deshidrogenasa

Tesis por compendio de publicaciones[ES] La tesis de la que trata este resumen es un compendio de publicaciones. En relación al primer artículo, varias observaciones realizadas en el laboratorio llevaron a plantear la hipótesis de que, en condiciones de crowding macromolecular, las IMPDHs humanas ensamblan in vitro formando estructuras micrométricas semejantes a los cytoophidia. En el artículo, se propuso validar esta hipótesis y caracterizar la dinámica de ensamblaje de cytoophidia en relación con la inhibición alostérica por nucleótidos de guanina. Respecto al segundo artículo del compendio, los dinucleósidos polifosfatos son moléculas ubicuas en las que dos nucleósidos están unidos por una cadena de dos a siete fosfatos. Se ha descrito que el dinucleósido polifosfato Ap4A puede unirse a las IMPDHs bacterianas, ejerciendo un efecto semejante al del ATP [37 y 38]. Un análisis detallado de la estructura de la IMPDH unida a GDP, permitió plantear la hipótesis de que los dinucleósidos polifosfatos podrían unirse de manera simultánea a los dos sitios canónicos de los dominios Bateman. Por ello, en este trabajo se propuso corroborar esta hipótesis y evaluar si los dinucleósidos polifosfatos podrían tener efecto sobre el interruptor conformacional que controla la actividad catalítica de las IMPDHs. Finalmente, la elevada conservación del dominio Bateman y sus sitios canónicos de unión de nucleótidos en las IMPDHs procariotas llevó a plantear la hipótesis de que, muy probablemente, estas enzimas también se controlan de manera alostérica por nucleótidos de purina. En el tercer artículo se propuso analizar los mecanismos de regulación alostérica de la IMPDH de organismos procariotas, así como caracterizarlos desde un punto de vista estructural y funcional

Challenges in biomedical data science: data-driven solutions to clinical questions

Data are influencing every aspect of our lives, from our work activities, to our spare time and even to our health. In this regard, medical diagnosis and treatments are often supported by quantitative measures and observations, such as laboratory tests, medical imaging or genetic analysis. In medicine, as well as in several other scientific domains, the amount of data involved in each decision-making process has become overwhelming. The complexity of the phenomena under investigation and the scale of modern data collections has long superseded human analysis and insights potential

Molecular assessment of parasite infection within socioeconomically important UK salmonid populations

Salmo trutta (Brown trout) is a native salmonid species to the United Kingdom. The economy generated via fish farming and eco-tourism has aided isolated communities in job creation and tourism income. It is particular true in highland communities in Scotland, where parasite infection has threatened the health of Scottish salmonids. The work presented in this thesis assessed parasite infection of wild brown trout populations in the northwest highlands of Scotland. Initial analysis of fish health screened subpopulations within the Gairloch region for infective parasite species. Eye fluke Diplostomum spp. was highly prevalent throughout the system with all lochs populations harboring infection. Through molecular analysis the species was confirmed as D. baeri which, when compared to other European isolates indicated highly diverse species complex infecting freshwater fish across the continent, most likely through definitive bird host. A cestode species that infected 4 trout subpopulations across the system was the medically relevant Diphyllobothrium dendriticum. Using molecular species identification techniques, it was the first finding of the parasite within UK freshwater fish populations. In response to differential parasite infection adaptive immunogenic traits were also observed within trout host subpopulations. The MHC II related gene Satr-DAB variability was higher in populations with differential parasite species infection suggesting the diversity of infection maintains MHC diversity within the population. The evasion of immune recognition to achieve high levels of infection is key to sustained parasite infection. The highly infective parasite in the Gairloch system, D. baeri, utilizes intracellular antigens Tetraspanins and Venom allergen-like antigenic proteins. These antigens were isolated using genomic techniques to highlight potential vaccine targets in aquaculture. The body of work presented here has furthered the knowledge of the highly infective D. baeri and provided molecular methodologies to identify medically relevant D. dendriticum. Genomic analysis of trout population immunogenics and parasite antigenic factors provides key knowledge to further conservation stocking methods and sustainable aquaculture practice

Identification of inhibitors of A to l editing

RNA-editing ist ein konservierter Mechanismus zur Diversifizierung der RNA von der genetisch codierten Information. Adenosin zu Inosin (A zu I) Editing bewirkt eine Informationsänderung an der RNA von A zu G und wird durch Doppelsträngige RNA spezifische Adenosin Deaminasen (ADARs)positionsabhängig katalysiert. Die biologische Bedeutung von Editing ist weitreichend und umfasst antivirale Mechanismen, alternatives Splicing, selektive Codonumwandlung, als auchModulierung der microRNA Prozessierung. Defekte in der Regulation des RNA Editings werden mit verschiedenen neuronalen Erkrankungen des Menschen in Verbindung gebracht wie Depression und Epilepsie. Auch wurde vermindertes RNA Editing als Eigenschaft von Tumorgewebe identifiziert. Bislang wird angenommen, dass die Aktivität von ADAR2 über RNA Splicing und post-translationelle Modifikationen reguliert wird. Um potentielle Modulatoren der ADAR2 Proteinaktivität zu identifizieren, wurde ein Editing System in Hefe konstruiert. Dieses System enthält ein mutiertes URA3 Gen, in dessen 5‘ Region ein UAG Stop Codon liegt, dieses jedoch durch ADAR2 in ein codierendes Tryptophan Codon editiert wird. Die Konvertierung dieses internen Stop Codons gestattet in Folge die Expression von URA3. Durch Anwesenheit des Selektionsmittels 5-FOA (5-Fluororotsäure) wird nun das Wachstum der URA3 exprimierenden Zellen jedoch verhindert. Um Editing inhibierende Proteine zu finden, wurde eine humane cDNA Bibliothek in das Systemtransformiert. Durch 5-FOA können Kolonien nur aus jenen Transformanten hervorgehen, in denen ADAR2 Editing inhibiert wurde. DiecDNA möglicher Editinginhibitoren wurde aus den gewonnenen Transformanten isoliert. Um die Relevanz potentieller ADAR2 Inhibitoren in Säugerzellen zu bestimmen, wurde für die Sekundäranalyse ein Zellkulturreportersystem in Säugerzellen etabliert und eingesetzt. Letztlich konnten drei RNA bindende Proteine, RPS-14 (kleine ribosomale Untereinheit, Protein 14), SFRS-9 (Splicing Faktor, Arginin/Serin-reich 9), und DDX-15 (DEAH-box protein 15), als wahrscheinliche Editing-Inhibitoren identifiziert werden. Mittels Kolokalisations- und Koimmunopräzipitationsexperimenten konnten RPS-14 und SFRS-9 als ADAR2-Interaktoren identifiziert werden. Weiters konnte für RPS-14, SFRS-9 und DDX-15 eine Beteiligung in der Editing-Inhibierung an endogenen ADAR2 Substraten nachgewiesen werden. Zudem konnte die Editing inhibierende Aktivität von DDX-15im Fadenwurm Caenorhabditis elegans wiedergefunden werden, Indiz für einen konservierten Mechanismus. Unsere Resulate lassen daher auf die erfolgreiche Identifikation von drei bisher unbekannten Inhibitoren des ADAR2 vermittelten RNA Editings, RPS-14, SFRS-9 und DDX-15, schliessen.RNA editing is a conserved posttranscriptional mechanism diversifying RNA and proteins from limited genetic information. A to I editing is carried out by ADARs (Bass). Editing has profound effect in various biological processes as anti-viral defense, alternation of splicing, codon changes and modulation of microRNA processing. Editing deregulation has been linked to CNS diseases as depression and epilepsy. Recently, global hypoediting has been found in tumor tissues indicating a correlation between editing and cancer. Until now, splicing and post translational modifications are proposed to modulate ADAR2 activity. To identify potential cellular modulators of ADAR 2 activity a yeast-editing assay was established. In this assay, an amber stop codon located in a stem loop in the 5’ region of the URA3 gene can be edited into a tryptophan codon by ADAR2. Conversion of the amber codon to a tryptophan codon thereby allows URA3 expression. However, addition of the drug 5-FOA selectively kills the cells expressing URA3. To identify cellular proteins that inhibit editing, a human cDNA library was transformed into the reporter strain. In the presence of 5-FOA only colonies that fail to express URA3, i.e colonies in which editing was inhibited could grow. A tissue culture reporter assay was established for secondary screening of the potential inhibitors in the mammalian system. Three RNA binding candidate proteins RPS-14 (small subunit ribosomal protein 14), SFRS-9 (splicing factor, arginine/serine rich 9), and DDX-15 (DEAH-box protein 15, pre-mRNA processing factor) were isolated from this screen as inhibitors of editing in mammalian cells. Further co-localization and co-immunoprecipitation studies are done in order to dissect the mode of interaction. RPS-14 and SFRS-9 were found to interact with ADAR2. RPS-14, SFRS-9, and DDX-15 show editing inhibition on endogenous substrates. DDX-15 entailed ADAR2 activity inhibition is conserved in Caenorhabditis elegans when compared to other RNA helicases. Our results indicate that RPS-14, SFRS-9 and DDX-15 are potential cellular inhibitors of ADAR2 mediated editing, proving the functionality of the candidates in mammalian cells

A novel role for the transcriptional modulator NusA in DNA repair/damage tolerance pathways in Escherichia coli

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2009.Includes bibliographical references.All organisms must contend with the consequences of DNA damage, induced by a variety of both endogenous and exogenous sources. Mechanisms of DNA repair and DNA damage tolerance are crucial for cellular survival after DNA damage. Translesion DNA synthesis (TLS) is one such mechanism of DNA damage tolerance which utilizes a specialized translesion DNA polymerase capable of catalyzing DNA synthesis on imperfect templates. There are two TLS polymerases present in Escherichia coli encoded by the dinB (Pol IV) and umuDC (Pol V) gene products. While TLS polymerases provide a variety of benefits to the cell, it is important that they are properly regulated as they have reduced fidelity on undamaged DNA compared to replicative DNA polymerases. Here I present evidence that the essential transcriptional modulator NusA associates with TLS polymerases in E. coli both physically, as noted for DinB, and genetically, with DinB and the umuDC gene products. Mutation of nusA renders cells sensitive to DNA damaging agents and produces phenotypes reminiscent of mutants with altered DNA processing. Moreover, I report that the nusAll mutation completely eliminates the formation of adaptive mutants, revealing that nusA+ function is required for cells to adapt and mutate in response to stress. Though the phenomenon of adaptive mutagenesis also requires dinB+, my data suggest that the role for nusA in adaptive mutagenesis extends beyond an interaction with DinB.(cont.) Furthermore, I report that NusA in addition to having a role in transcription elongation is also important for promoting survival after DNA damage. Phenotypes of nusA mutants are more exaggerated than those of TLS polymerase mutants. Genetic interactions of nusA+ with the nucleotide excision repair pathway suggest that nusA+ may play a role in a new class of NusA-dependent transcription coupled repair. Moreover, I have isolated RNA polymerase mutants with altered ability to survive after DNA damage, and this altered ability is absolutely dependent on nusA+ and uvrA+. The completion of translesion DNA synthesis requires both the insertion of a nucleotide opposite the adducted template base and extension from that position by several subsequent nucleotide additions. We present evidence that DinB is specialized to perform strikingly proficient extension after insertion opposite an N2-dG lesion. Our data indicate that cellular survival is coupled to completion of TLS and regulation of these precise steps in vivo is genetically complex and involves the toxin-antitoxin module MazEF and the iron import protein TonB.by Susan E. Cohen.Ph.D