Tesis sobre el tema "Alleles"

Cancer is a leading cause of death worldwide. This somatic cell genetic disease is characterized by progressive accumulation of mutations in multiple genes. An important characteristic of cancer cells is an increased rate of gains and losses of chromosomes, termed Chromosomal Instability (CIN). One of the frequently mutated genes in a variety of cancers is FBXW7 (F-Box and WD repeat domain-containing 7), encoding the substrate-recognition component of a ubiquitin ligase complex. Fbxw7 targets a number of oncoproteins such as, Cyclin E, c-Myc, Notch1 and Aurora A for ubiquitin mediated degradation. Inactivation of FBXW7 has been linked to CIN in cancer cell lines. The majority of cancer-associated mutations in FBXW7 are monoallelic, missense substitutions whose phenotypic effects are difficult to predict. Interestingly, most of the mutations in FBXW7 cluster at three mutational hotspots, Arg465, Arg479 and Arg505. Located at β propeller-tip, these residues are critical for interaction with the Fbxw7 substrates. This study investigates the functional consequences of the substitutions at these residues. We individually tested the functional status of the R465C, R479Q and R505C variants of FBXW7 in three colorectal cancer cell lines in an HCT116 background. These cell lines had both, one or none of the alleles of FBXW7 inactivated by homologous recombination. Our data shows that the cell lines producing R465C, R479Q or R505C variants of FBXW7 failed to degrade Cyclin E, one of the major targets of FBXW7. These cell lines also exhibited a CIN phenotype, observed as an increase in the frequency of abnormal anaphases. These results show that mutations R465C, R479Q and R505C occurring in FBXW7 cause loss of function of the protein and act as dominant negative mutations.

This study describes the development and validation of mutagenized cloned DNA constructs, which correspond to the polymorphic regions of the class II region of the HLA complex. The constructs were used to verify the allelic specificity of primers and probes in polymerase chain reaction (PCR)-based HLA typing assays such as Sequence Specific Primers (SSP) and Sequence Specific Oligonucleotide Probes (SSOP). The constructs consisted of the entire polymorphic region of exon 2 of class II HLA allele sequences that included primer annealing sites or probe hybridization sites. An HLA allele sequence was inserted into a plasmid, cloned, then mutagenized to match a specific HLA allele, and finally, the correct clone was verified by bidirectional sequencing of the insert. Thus, the construct created a cloned reference DNA sample for any specific allele, and can be used to validate the accuracy of various molecular methodologies.

DNA topological stress impedes normal DNA replication. If topological stress is allowed to build up in front of the replication fork, the fork rotates to overcome the stress, leading to formation of DNA pre-catenanes. The formation of DNA pre-catenanes is therefore a marker of DNA topological stress. In this study, I have examined how transcription linked DNA topological stress impacts on fork rotation and on endogenous DNA damage. Transcription, similar to replication, affects the topology of the DNA; and collision between the two machineries is likely to lead to high levels of DNA topological stress. I found that the frequency of fork rotation during DNA replication, increases with the number of genes present on a plasmid. Interestingly, I also found that this increase in pre-catenation is dependent on the cohesin complex. Cohesin and transcription are known to be linked, as transcription leads to the translocation of cohesin along budding yeast DNA away from its loading sites. Cohesin plays a major role in establishing chromosomal structure, influencing gene expression and genetic inheritance. In this work, I have analysed the relationship between cohesin and the generation of topological stress and found that topological stress associated with cohesin can lead to DNA replication stress and DNA damage.

Thesis (M.S.) -- University of Maryland, College Park, 2003.<br>Thesis research directed by: Dept. of Cell Biology and Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Thesis (MSc)--Stellenbosch University, 2003.<br>ENGLISH ABSTRACT: This study describes: 1. The isolation of total RNA and mRNA from Angora goat adrenals. 2. Synthesis and nucleotide sequence alignment of Angora goat CYPI7 cDNA. Two DNA sequences were produced, identifying two CVP 17 alleles in an Angora goat from the Swartland district. 3. The development of a CYPI7 genotype test for Angora goats. 4. Genotyping of Angora goats and Boer goats with the developed genotype test. S. Mapping of the substituted amino acids in the amino terminal of CVP 17 to a specific CYPI7 genotype. 6. Partial synthesis and alignment of Angora goat genomic nucleotide CYPI7 sequences.<br>AFRIKAANSE OPSOMMING: Hierdie studie beskryf: 1. Die isolering van totale RNA en mRNA van Angorabok byniere. 2. Sintese en nukleotied volgorde oplyning van Angorabok CYP17 eDNA. Twee DNA volgordes is geproduseer, en so is twee CYP17 allele in 'n Angorabok van die Swartland omgewing geïdentifiseer. 3. Die ontwikkeling van 'n CYP17 genotipe toets vir Angorabokke. 4. Genotipering van Angorabokke en Boerbokke met die ontwikkelde genotipe toets. 5. Korrelering van die omgeruilde aminosure in die aminoterminaal van CYPl7 met 'n spesifieke genotipe. 6. Gedeeltelike sintese en oplyning van Angorabok genomiese CYPl7 nukleotied volgordes.

Persistence of lactase into adulthood varies in frequency worldwide and is attributable to several different single nucleotide changes in an enhancer of the LCT gene. One of these is at particularly high frequencies in Europeans and several others have been found elsewhere. However, information about their worldwide distribution is patchy. 2056 DNA samples from populations of Europe, Asia and the Middle East were sequenced to examine the distribution of allelic variants of the LCT enhancer region. It was confirmed that -13910*T is also the predominant allele around the periphery of Europe, and that this allele extends as far as the South and East of the Arabian Peninsula. Other alleles appear to have spread out of Africa or Arabia and most variation was found in the Middle East. No new common alleles were found that were likely to be causal. In previous studies four lactase persistence associated nucleotide substitutions (-13910*T, -14010*C, -13915*G and -13907*G) have been studied functionally. In this thesis four additional enhancer alleles were examined using enhancer/promoter construct transfections and electrophoretic mobility shift assays. Three enhancer variants alter transcription factor binding in vitro and/or reporter-gene expression. Bioinformatic tools and specific antibodies were used to assist in identifying the transcription factors involved. The results show that different mechanisms lead to a disruption of the normal down-regulation of lactase in adult life. Known haplotypic markers were assessed on an overlapping set of samples and a greater number of flanking markers was typed in an extended region of 1.8 Mb around LCT, in order to chart the evolutionary relationships and extent of historic recombination of the chromosomes carrying the derived alleles, as well as those th at do not. All of the functional alleles tested have longer extended haplotypes than their ancestral counterparts, but so also does the derived variant at position -958, a haplotype marker for the B haplotype. The finding of an extended region of high linkage disequilibrium in all populations, and an extended B haplotype, is discussed in relation to the methods to study selection. Because phenotypic studies suggest missing functional variants, the immediate promoter and a part of intron 2 of LCT were also selected for sequencing. No serious candidates were found in intron 2. Two alleles in the immediate promoter were studied by transfections but there was little evidence for any in vitro effect of these variants.

Late Blight disease, caused by Phytophthora infestans, is the most significant threat to potato production world-wide. Identifying and deploying more durable host resistance to P. infestans is a promising way forward to sustain the production of potato. To achieve this goal, it is important to seek key pathogen components that are essential for infection and which, upon detection by the host, trigger a resistance response. One such potential key pathogen molecule is the RXLR-containing effector Avr3a. Avr3a is highly up-regulated during infection and is also required for P. infestans pathogenicity. To date, all P. infestans isolates studied contain Avr3a alleles E80M103 and/or K80I103. However, a study of Avr3a diversity in the Toluca Valley, Mexico, has identified additional alleles such as K80I103L139, K80I103H133, E80M103H133 and E80M103G124. Functional studies of these alleles were conducted as part of this thesis, which also include the Avr3a paralogs Pex147-2 and Pex147-3. By examining the amino acid changes in relation to the established protein structure, it was determined that all alterations within the Avr3a variants occur at surface exposed amino acids. The change R124G that leads to Avr3aEMG is located in the a-helix loop 3 and the changes Q133H and M139L (Avr3aKIH, Avr3aEMH and Avr3aKIL) locate to a helix 4. Whereas amino acid substitutions in PEX147-3 only affect surface exposed residues, amino acid changes that occur in PEX147-2 involves a ‘buried’ amino acid that is key to structure and stability. Indeed, with the exception of PEX147-2, all Avr3a variants and PEX147-3 are stable upon transient expression in planta and in yeast cells. In terms of host recognition, the protein products of the Avr3a alleles derived from Avr3aKI are recognised by the cognate host resistance gene product R3a whereas those derived from Avr3aEM evade recognition. Similarly, PEX147-3 is recognised by R3a but PEX147-2 is not. In addition to host recognition, virulence functions of these alleles and paralogs have been elucidated. INF1 and AVR4/CF-4 induced cell death responses, which are dependent on the host defence protein CMPG1, are suppressed by Avr3aKI, Avr3aKIH, Avr3aKIL, Avr3aEM and Avr3aEMG but not by Avr3aEMH. All Avr3a variants interact with and stabilise the host E3 ubiquitin ligase CMPG1 to various degrees in planta and this interaction was found to be weakest for Avr3aEMH. Interestingly, PEX147-3, which did not interact with or stabilise CMPG1, could only suppress INF1 cell death but not CF-4/AVR4 elicited responses. A P. infestans isolate, CS12, which was stably silenced for Avr3aEM expression and subsequently shown to be compromised in virulence on the normally susceptible host Nicotiana benthamiana, was used for in planta complementation studies. As shown previously, upon transient expression in planta prior to infection with C12, Avr3aKI and Avr3aEM successfully restore pathogenicity. Similar levels of virulence re-establishment were only observed for Avr3KI derived alleles Avr3aKIH and Avr3aKIL but not for alleles derived from Avr3aEM. This study concludes that Avr3aEM is currently the only form of the essential effector that is fully functional and evades recognition by the known resistance gene product R3a. This functionality is the likely reason that 70% of all studied isolates in the Toluca Valley are homozygous for Avr3aEM. This form of the effector is therefore a suitable target for identifying more durable resistances.

Breast cancer is the most common malignancy affecting women in the Western world. At present, several risk factors have been identified and, among them, the most significant is a positive family history for the disease due to the presence of inherited predisposing germline alterations. Despite the discovery of many susceptibility genes, among which the highly penetrant BRCA1 and BRCA2 are the most relevant, more than 70% of the genetic predisposition to breast cancer remains unexplained. During my three-years Ph.D., I used different analytical approaches to investigate DNA alterations and loci that could be relevantly implicated in the aetiology and progression of hereditary breast cancers. As first project on non informative BRCA1/2 families, I was involved in the assessment of the pathogenetic role of the BRCA1 p.Val1688del allele, which recurs in the Northeast Italy. Unclassified variants (UVs) provide a considerable challenge in the clinical management of high-risk families. Indeed, the absence of conclusive data on UVs pathogenicity, due to the lack of an evident deleterious effect on protein function, prevents their use in the identification of individuals predisposed to breast cancer development. To assess the pathogenetic role of BRCA1 p.Val1688del, we used the integrated multifactorial likelihood model described by Goldgar et al. (2004). Several independent evidences were derived from epidemiologic and linkage studies as well as histopathology, LOH and evolutionary conservation analyses, in 12 families carrying the p.Val1688del allele. All the evidences were than properly evaluated to obtain, from each, the probability of the observed data given that the variant is a deleterious mutation, against the probability of the observed data under the hypothesis that the variant is neutral. The likelihood ratios were integrated to obtain a combined odd of variant causality, resulting in a clear assessment of the BRCA1 p.Val1688del pathogenicity. Indeed, the final integrated odd of 349,000:1 in favor of disease causality largely exceeds the established cut off 1,000:1 needed to state the deleterious effect of the variant. The second project, described herein, focused on the identification of molecular alterations involved in non-BRCA1/BRCA2 breast cancers. To this purpose, I performed a comprehensive genomic profile of the breast cancer cell line HCC1500, which was established from a patient who probably harboured a predisposing germline mutation affecting genes other than BRCA1 and BRCA2. Indeed, despite the early age of disease development and a positive family history for breast and colon cancer, we did not detect any deleterious BRCA1, BRCA2 or TP53 alteration. The HCC1500 cell line was analysed by two complementary approaches: i) a novel combined strategy named GINI (Gene Identification by NMD Inhibition; Noesie and Dietz, 2001) for the identification of nonsense mutations at the transcriptome level, and ii) a copy number alteration (CNA) analysis that was achieved by array-based high-density SNP genotyping. This approach led to the identification of genes specifically mutated by either point mutations or major genomic rearrangements. In particular, we identified an intronic substitution in the PNLIPRP3 gene, which is predicted to create a PTC by altering pre-mRNA splicing. Moreover, high-resolution CNA analysis allowed the detection of small homozygously deleted regions as well as focal amplifications, involving single or few genes. A gene selection based on the specific biological function and/or type of mutation and/or previously reported involvement in cancer pathways, allowed the definition of a relatively small number of candidate breast cancer genes that will deserve further and more specific investigation. Finally, among the minor research projects that I will not discuss in my thesis, I was also directly involved in studies carried out by our research group as part of an international consortium, named CIMBA (Consortium of Investigators of Modifiers of BRCA1/2), whose aim is the identification of genetic factors implicated in the modification of BRCA1 and BRCA2 penetrance (Chenevix-Trench et al., 2007). Candidate single nucleotide polymorphisms (SNPs), most of which derived from genome-wide association studies, were genotyped in 213 BRCA1/2 mutation carriers from our cohort by the taqman allele discrimination technique, and subsequently statistically analyzed together with the data obtained from more than 30 study groups world-wide. The genotyping of SNP alleles in thousands of BRCA1/BRCA2 mutation carriers provide the necessary statistical power for the identification of significant association between common SNPs with typical low effects and specific subclasses of individuals at higher risk. These studies were carried out on more than 9400 BRCA1 and 5600 BRCA2 mutation carriers and allowed the identification of a significant association between rs3817198 in LPS1 and rs13387042 at 2q35 (already found to be associated with increased breast cancer risks in the general population) and increased breast cancer risk in BRCA2 and both BRCA1 and BRCA2 mutation carriers, respectively. The identification of genetic modifiers of breast cancer risk not only will favor a better understanding of breast cancer predisposition and pathogenesis but will also allow more precise cancer risk estimates and will represent a useful tool for the design of new therapeutic approaches.<br>Il tumore della mammella e’ la neoplasia più frequente nelle donne dell’Occidente. Ad oggi sono stati identificati alcuni fattori di rischio per lo sviluppo della neoplasia mammaria, ma il maggiormente significativo è la presenza di storia familiare, che è associata alla presenza nei membri della famiglia di alterazioni germinali predisponenti. Fino ad oggi sono stati identificati alcuni geni responsabili dell’aumento di rischio per lo sviluppo del tumore della mammella, tra cui i più rilevanti sono i geni ad alta penetranza BRCA1 e BRCA2. Tuttavia più del 70% dell’eccesso di rischio che si riscontra in casi familiari di tumore della mammella rispetto la popolazione generale non trova ad oggi un riscontro in alterazioni genetiche predisponenti. Durante il mio dottorato mi sono occupata dell’identificazione di alterazioni genetiche coinvolte nella predisposizione e progressione del tumore eredo-familiare della mammella in cui sono state escluse mutazioni chiaramente patogenetiche dei geni BRCA1/BRCA2. In un primo progetto ho partecipato alla caratterizzazione del ruolo patogenetico della variante BRCA1 p.Val1688del, identificata come variante di incerto significato clinico (Unclassified Variant, UV) e riscontrata frequentemente in pazienti provenienti dalla regione Veneto. In assenza di un chiaro effetto deleterio sulla funzionalità della proteina, le UV non possono essere utilizzate per l’identificazione e la sorveglianza degli individui ad alto rischio per lo sviluppo della neoplasia mammaria. Allo scopo di chiarire il ruolo patogenetico della delezione p.Val1688del, abbiamo utilizzato un approccio multi fattoriale, descritto da Golgar et al. (2004), attraverso il quale evidenze di diversa natura vengono integrate per derivare un rapporto di probabilità in favore della patogenicità o neutralità della variante. A questo scopo, i membri di 12 famiglie portatrici della variante sono stati analizzati per ottenere dati quali la co-segregazione della variante con il fenotipo tumorale, la co-presenza di mutazioni patogenetiche nel gene BRCA1, l’istopatologia del tumore, la perdita di eterozigosi (LOH) e la conservazione filogenetica del residuo aminoacidico alterato. Assunta l’indipendenza delle evidenze raccolte, i rapporti di probabilità associati a ciascuna evidenza sperimentale e clinica sono stati combinati, ottenendo un valore di 349000:1 in favore del ruolo patogenetico della variante, che supera di molto il cut off di 1000:1 stabilito per poter classificare la variante come alterazione predisponente. Il secondo progetto di cui mi sono occupata riguarda la definizione del profilo genetico della linea cellulare di carcinoma mammario HCC1500, derivata da una paziente con probabile tumore eredo-familiare della mammella. Nonostante l’età precoce di insorgenza e la storia familiare di tumore della mammella e del colon, il coinvolgimento dei geni BRCA1, BRCA2 e TP53 è stato escluso mediante screening mutazionale. La linea HCC1500 è stata quindi analizzata mediante due approcci complementari: i) l’identificazione di mutazioni nonsenso mediante l’utilizzo di un’approccio di analisi recentemente descritto chiamato GINI (Gene Identification by NMD Inhibition; Noesie and Dietz, 2001), e ii) l’identificazione di alterazioni numeriche mediante l’utilizzo di array per la genotipizzazione di un elevato numero di SNP, una recente tecnologia che permette un’elevata risoluzione di analisi. Mediante questo approccio è stato possibile identificare singoli geni mutati con specifiche alterazioni puntiformi o a causa di più estesi riarrangiamenti genomici. In particolare, nel gene PNLIPRP3 abbiamo identificato una sostituzione intronica che, modificando lo splicing del pre-mRNA, potrebbe dare origine ad un trascritto con mutazione troncante. Inoltre, l’elevata risoluzione dell’analisi per lo studio delle alterazioni cromosomiche numeriche, ci ha permesso di identificare singoli o pochi geni coinvolti in delezioni in omozigosi, amplificazioni di piccole regioni e riarrangiamenti intragenici derivanti da eventi di amplificazione o delezione. Sulla base di criteri quali la funzione biologica e mutazioni ritenute rilevanti per la selettività con cui alterano specifiche regioni codificanti, abbiamo selezionato alcuni geni che, prioritariamente ad altri, andrebbero confermati e analizzati in maggior dettaglio con ulteriori studi.. Un terzo progetto che ho seguito direttamente, ma che comunque non tratterò nella mia tesi, riguarda l’identificazione di fattori genetici coinvolti nella modificazione del rischio per i soggetti portatori di mutazione BRCA1 e BRCA2. Tale progetto si inserisce nell'ambito di un consorzio internazionale, denominato CIMBA (Consortium of Investigators of Modifiers of BRCA1/2; (Chenevix-Trench et al., 2007). SNP identificati prevalentemente attraverso studi di associazione genome-wide sono stati genotipizzati in 213 carriers di mutazione BRCA1/2 della nostra casistica mediante discriminazione allelica con sonde TaqMan. L’analisi statistica è stata effettuata sui dati genotipici provenienti da più di 30 gruppi di lavoro, permettendo la raccolta di dati derivanti da migliaia di individui. In questo modo è possibile ottenere la potenza statistica necessaria per l’identificazione dell’associazione tra polimorfismi con debole effetto e un aumentato rischio di sviluppo del tumore della mammella in specifiche sottoclassi di soggetti già portatori di mutazione nei geni ad alta penetranza BRCA1/2. Effettuati su più di 9400 e 5600 carriers di mutazione rispettivamente di BRCA1 e BRCA2, tali studi hanno permesso di identificare un’associazione significativa tra gli SNP rs3817198 (LPS1) e rs13387042 (2q35) ed un aumentato rischio in soggetti portatori di mutazioni BRCA2, e, nel secondo caso BRCA1 o BRCA2. L’identificazione e studio di fattori genetici modificatori permetterà di acquisire una più approfondita conoscenza della predisposizione e patogenesi del tumore della ereditario mammella, ma anche di stimare con migliore precisione il rischio di sviluppo della neoplasia così come di fornire utili informazioni per l'identificazione di nuovi approcci terapeutici.

One of the proteins that lie at the heart of the DNA Damage Response (DDR) is Topoisomerase II-binding protein I (TopBP1). TopBP1 was initially identified and has been extensively studied in the yeast model organisms. However, the lack of readily available tools, including genetically defined mutant cell lines, has rendered the characterisation of TopBP1 in higher eukaryotes more challenging. Sequence information obtained from the characterisation of the gallus gallus TopBP1 mRNA revealed a different splicing pattern at the 5'end to the one reported in the Genome Browser. Our assembled TopBP1 mRNA sequence containing a novel open reading frame (ORF) enabled the creation of a conditional knockout cell line of TopBP1 in DT40, which has been impossible with the use of the annotated cDNA sequence. Thus the avian TopBP1 ORF identified herein contained the necessary function(s) to sustain viability of DT40 cells in the absence of the endogenous protein. Additionally, the establishment of an isogenic set of stable cell lines from the chicken B cell line DT40 by targeted deletion of the TopBP1 alleles revealed a gene dosage-dependent reduction of the TopBP1 protein levels and functions. This work establishes a novel gene-dosage system that can be used for the knock in of point mutations within the endogenous TopBP1 locus. Using this system, a novel characterisation of knock-in point mutants of the ATR Activation Domain (AAD) of TopBP1 was carried out, providing in vivo evidence of its DDR function(s). Finally, a stably integrated overexpression system (SIOS) capable of producing increased amounts of a protein of interest has been established in DT40 cells. SIOS represents an easy to use versatile system for various experimental purposes in the field of DT40. The work presented in this thesis represents a novel characterisation of the avian TopBP1 mRNA and the TopBP1 protein and its functions. This is crucial to gain insight into the mechanistics of the DDR network and the genetic instability characterising cancer development.

Gene regulation id controlled by transcription factor proteins that bind to specific DNA sequences, known as transcription factor binding sites (TFBSs). Combinations of transcription factors working, co-operatively in cis-regulatory modules (CRMs), play a role in regulating gene expression. Current computational methods for TFBS prediction cannot distinguish between functional and non-functional sites, and predict very large numbers of false positives. The thesis focuses on the development of a novel computational model, based on artificial neural networks (ANNs), for the identification of functional TFBSs, and the CRMs within which they operate in the human genome. Datasets of 12,239 experimentally verified true positive (TP) TFBSs and 130,199 false positive (FP) TFBSs were extracted using a combination of position weight matrices from the JASPAR database and experimentally verified sites from the Encyclopedia of DNA elements (ENCODE). A number of machine learning alsgorithms were tested using a range of genetic information including gene expression, necleosome positioning, DNA methylation states and DNA entropy. The best model, that gave a mean area under the curve under a receiver operator characteristic curve of 0.800, was based on a feedforward ANN using backpropagation. This model was then used to predict functional TFBSs in a number of gene sets from the human genome. The predictions, combined with experimentally proven TFBSs from ENCODE, were used to investigate combinatorial patterns of TFBSs operating in CRMs. CRM patterns have been analysed in disease-associated genes located in linkage disequilibrium blocks containing SNPs obtained from Genome Wide Association Studies (GWAS). The potential for the model to make functional TFBS predictions to aid in the annotation of orphan genes of unknown function is discussed. In addition this thesis presents computational work on a number of smaller published studies.

A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation by the protein kinase ATM. H2AX is unevenly distributed throughout chromatin and is rapidly phosphorylated to form γH2AX up to 2 megabases either side of DSBs. Studies in yeast systems have shown that while γH2A can spread in cis surrounding the break site, it can also spread in trans onto unbroken chromosomes located in close spatial proximity. Although the majority of data in the current literature presents the well characterised in cis spread of γH2AX, there are strong indications that it can also occur in trans in mammalian systems; analogous to the findings shown in yeast. This thesis lays out the steps taken to develop a novel system to address the spatial distribution of γH2AX around a nascent DSB. Since the first published live imaging experiments of the dynamics of chromatin by in vivo single particle tracking there has been extensive investigation into the regulation and biological function of movement of damaged DNA. In yeast, a relative consensus exists that DSB induction increases the movement of a DSB. In contrast to yeast however, data published of DSB movement in higher eukaryotes has been controversial, caused by conflicting results. Here, I developed a cell-based system, and utilised timelapse live cell imaging to show that a chromosomal locus containing a single endonuclease-induced DSB shows confined movement in comparison to an undamaged locus. Furthermore, this confined movement of a damaged locus is compounded by treatment with an ATM kinase inhibitor but not a DNA-PKcs kinase inhibitor, suggesting that the kinase activity of ATM and not the kinase activity of DNA-PKcs plays a significant role in the dynamics of DSBs.

Hundreds of thousands of putative small ORFs (smORFs) sequences are present in eukaryotic genomes, potentially coding for peptides less than 100 amino acids. smORFs have been deemed non-coding on the basis of their high numbers and their small size that makes it extremely challenging to assess their functionality both bioinformatically and biochemically. The recently developed Ribo-Seq technique, which is the deep sequencing of ribosome footprints, has generated significant controversy by showing extensive translation of smORFs outside of annotated protein coding regions, including putative non-coding RNAs.. Our lab adapted the Ribo-Seq technique by combining it with the polysome fractionation in order to assess smORF translation in Drosophila S2 cells. This thesis provides a high-throughput assessment of smORF translation in Drosophila melanogaster by firstly implementing complementary techniques such as transfection-tagging and Mass spectrometry methods in order to provide an independent corroboration of the S2 cell data (Chapter 3). Secondly, the in order to expand the catalogue of smORFs that are translated, I significantly improve upon the yield and sequencing efficiency of the Poly-Ribo-Seq protocol while adapting it to Drosophila embryos and then implementing it across embryogenesis divided in to Early, Mid and Late stages (Chapter 4). Currently, there is still a lot of debate in the field with regards to Ribo-Seq data analysis, and various computational metrics have been developed aimed at discerning 'real' translation events to background noise. Chapter 5 explores some of the metrics developed and establishes a translation cut-off suitable for designating small ORFs as translated. Altogether, the improvements introduced to the protocol and my data analysis shows the translation of 500 annotated smORFs, 500 smORFs in long non-coding RNAs and 5,000 uORFs, of which only one-third of each type of smORF has previous evidence of translation. These findings strengthen the establishment of smORFs as a distinct class of genes that significantly expand the protein coding complement of the genome.

USA soybean germplasm has a narrow genetic base that could be augmented by alleles from the wild species Glycine soja which positively influence agronomic traits. The objective of this study was to identify such alleles for yield and yield component QTL (quantitative trait loci). Two populations of 150 BC2F4 lines were generated from a mating between recurrent parent Glycine max 7499 and donor parent Glycine soja PI 245331 with one line in each population tracing back to the same BC2 plant. Population A was used for the QTL identification analysis and population B was used for the QTL verification test. The population A lines were genotyped at 120 SSR marker loci and one phenotype marker, covering a total map length of 1506 cM in 20 linkage groups with an average interval size of 12.5 cM. There were nine putative QTL significantly (Pandlt;0.0001, LODandgt;3.0) associated with yield and yield component traits across 3 environments. One QTL for seed yield was identified using the combined data; the G. soja allele at satt511 on LG-A1 was associated with increased seed yield (LOD=4.3) with an additive yield effect of 190 235 kg ha-1 depending on the QTL analysis method. The phenotypic variance accounted for by the QTL at satt511 was 12%. This QTL also provided a significant yield increase across environments in the validation population; lines that were homozygous for the G. soja allele at satt511 demonstrated a 6.3% (P=0.037) yield increase over lines that were homozygous for the G. max allele. One seed filling period QTL was identified at satt335 (LOD=4.0) on LG-F with an additive effect of +1 day. This QTL also provided a +1 day additive effect (LOD=3.3) on maturity. These results demonstrate the potential of using exotic germplasm to improve soybean yield.

Collagen is the major protein of bone, and an intronic polymorphism of the COLIA1 gene is associated with low bone mineral density (BMD) and with osteoporotic vertebral fracture. This polymorphism has not been extensively studied in hip fracture. We screened a cohort of 661 females with osteoporotic hip fracture in comparison with 483 controls, and carried out a similar analysis in 169 males. Genotyping was carried out for the COLIA1 <I>sp1</I> and VDR <I>Bsm1</I> polymorphisms. The COLIA 1 <I>Sp1</I> polymorphism was found to be an independent risk factor for hip fracture among females, with 'ss' homozygotes being at increased risk (OR=2.772; 95% CI: 1.11-693; p=0.031). No such difference was seen in relation to VDR genotype. Hip geometry is a risk factor for hip fracture. We investigated the relationship between COLIA1 <I>Sp1 </I> alleles and femoral neck geometry by carrying out measurements in pelvic radiographs from 153 patients with hip fracture, and in DXA scan printouts from 183 normal subjects. While no difference was seen between hip axis length and femoral neck width between COLIA1 genotypes, a wider femoral neck angle (approx. 2°) was seen in individuals expressing the 's' allele (p=0.001; GLM ANOVA). Such an increase in angle may have unfavourable biomechanical consequences in the event of a fall. We also investigated the effect of etidronate therapy on BMD in relation to COLIA1 genotype in a cohort of 108 post-menopausal women using a randomised-controlled trial. Individuals expressing the 's' allele had a heterogeneous BMD response at the hip as compared with 'SS' homozygotes. In general 'Ss' heterozygotes responded poorly to etidronate at the hip, but this differential response was not seen at the lumbar spine. The COLIA1 <I>Sp1</I> polymorphism thus has complex effects on the structure and function of bones as studied at the femoral neck.

Blastocystis sp. é um protozoário comumente encontrado em amostras fecais de humanos e animais, envolto por aspectos patogênicos e zoonóticos ainda controversos. Estudos recentes têm demonstrado a distribuição dos subtipos (STs) de Blastocystis sp., porém são escassos os relatos sobre a sua caracterização molecular na América Latina, sobretudo no Brasil. O objetivo do presente estudo foi investigar os STs presentes em isolados fecais de indivíduos acompanhados no Hospital das Clínicas de São Paulo da Faculdade de Medicina da Universidade de São Paulo (HC/FMUSP). Para tanto, amostras fecais positivas para Blastocystis sp. diagnosticadas na Seção de Parasitologia do Laboratório Central (HC/FMUSP) foram utilizadas para o isolamento de DNA. A reação em cadeia da polimerase (PCR) foi realizada utilizando iniciadores específicos para a subunidade 18S do DNA ribossomal de Blastocystis sp. A reação de sequenciamento dos produtos de PCR foi realizada, as sequências de DNA obtidas foram alinhadas e comparadas com outras sequências da base de dados GenBank e MLST. Foram identificados os STs (ST1, ST2, ST3 e ST6), sendo o ST3 o mais prevalente entre os isolados humanos seguido pelo ST1. Os alelos de número 34 e 36 foram os mais frequentes. Em conclusão, estes resultados contribuem para a caracterização molecular e a distribuição dos STs de Blastocystis sp. em amostras de fezes humanas no Brasil.<br>Blastocystis sp. is an organism described as enteroparasite protozoan, commonly found in stool samples from humans. Several subtypes have been described in humans, but pathogenic potential and aspects epidemiological are still controversial. The aim of the present study was to investigate Blastocystis subtypes (STs) from patients of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC/FMUSP), Brazil. Blastocystis sp. positive stool samples diagnosed in Section of Parasitology of Central Laboratory (HC/FMUSP) were used for DNA isolation. Polymerase chain reaction (PCR) was performed using specific primers targeting the small subunit of rRNA gene. Direct DNA sequencing of PCR products was performed, and the DNA sequences were aligned and compared to other sequences present in GenBank and MLST database. Four STs were identified (ST1, ST2, ST3 and ST6), where the ST3 was the most prevalent ST among human isolates followed by ST1. Allele nos. 34 and 36 were the most frequent. Another important finding is the presence of ST6, rarely detected in human isolates. In conclusion, our results contribute to the molecular characterization and distribution of Blastocystis sp. STs in human stool specimens in Brazil.

The rate at which an organism ages, as well as the onset of senescence are determined by many factors. Different species, as well as different strains of the same species, have characteristic lifespans. This study is an investigation of three strains of Drosophila melanogaster, to test their influences on ageing and senescence. Temperature-sensitive putative allele mutants [formula omitted], [formula omitted], and [formula omitted], and wild-type Oregon-R (control) Drosophila melanogaster were examined for patterns of age dependent behaviour loss over the course of their adult lifespans. Longevity, geotaxis, phototaxis, and motor activity were examined at both the permissive temperature, 22°C, and the restrictive temperature, 29°C. [formula omitted] displayed a longevity and behaviour loss pattern similar to the wild-type strain at 22°C. At 29°C, longevity was significantly reduced compared to wild-type, and the pattern of age-dependent behaviour loss was compressed into a shorter time frame. This pattern of behaviour loss was consistent with that expected from a mutation which increases the rate of ageing (Leffelaar and Grigliatti, 1984). The lifespan of [formula omitted] at 22°C was reduced, when compared with Oregon-R, but the behaviour loss pattern was similar. At 29° C the flies died rapidly, with almost total, immediate, behaviour loss. Survival curves at 22°C, 25°C, and 29°C adjusted for the rate of living were coincident. Flies of the type [formula omitted], isolated in a separate screen as flight-reduced, demonstrated differences from [formula omitted] and [formula omitted] in longevity curve shape as well as behaviour. Lifespan was reduced at both 22°C and 29°C, and although behaviour differed slightly in young flies, the behaviour loss pattern was similar to that of [formula omitted]. The order of severity of effects of the restrictive temperature from the least affected allele to the most affected was [formula omitted], [formula omitted], and [formula omitted]. At 22°C, hybrid flies of type [formula omitted] / [formula omitted] and [formula omitted] / [formula omitted] exhibited lifespans comparable to Oregon-R, the latter being longer lived than either parental strain. The hybrid strain [formula omitted] / [formula omitted] was demonstrated to have longevity intermediate between parent types, and reduced with respect to Oregon-R. Geotactic behaviour was reduced in all three hybrids at 22°C, but phototaxis and motor activity were similar to that of wild-type flies. At 29°C all hybrid strains could be said to demonstrate intermediate lifespan, between females of the generating parent strains. Complementation did not occur, and thus all mutants under study were confirmed to be alleles. Behaviour was reduced in [formula omitted] / [formula omitted] and [formula omitted] / [formula omitted] at 29°C, but not in [formula omitted] / [formula omitted]. Flies of type Deficiency/mutant were found to have greatly reduced lifespans at both 22°C and 29°C. The order of severity of effects was Df/[formula omitted], Df/[formula omitted], and Df/[formula omitted], with the last being the most severely affected, with deformities noted at both temperatures. This result confirms the cytological location of these mutants relative to their genetically determined position. The focus of action of the mutant gene [formula omitted] was mapped two ways by means of gynandromorph analyses. The drop-dead behaviour was mapped against a background map constructed for this study. This behaviour appears to map to the ventral thoracic region, and most likely involves presumptive mesodermal tissue. The paralysis behaviour noted in these flies at 29°C was then mapped separately for each leg. Three foci were found. All three appear to map to presumptive nervous tissue. Involvement of nerve and muscle tissues would not be surprising, considering the behaviours noted earlier. The discussion involves speculation as to the precise function of all three [formula omitted] genes.<br>Science, Faculty of<br>Zoology, Department of<br>Graduate

Thesis (M.S.)--University of California, San Diego, 2008.<br>Title from first page of PDF file (viewed July 8, 2008). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 26).

An estimated 50% of all people carry the stomach bacterium Helicobacter pylori (H. pylori). This organism is responsible for gastric problems like gastritis and gastric ulcers, and is one of the major causes of gastric cancer worldwide. Large numbers of people carry this organism asymptomatically and many questions remain about why serious symptoms develop in a subset of infected humans. These extremely recombinant bacteria may take different evolutionary trajectories in different people, and some genomic changes may be associated with gastric cancer. To test this, and learn more about the genetics of cancer-associated H. pylori, different approaches were used. First, evolution of H. pylori populations was investigated looking at both core and accessory genomes and revealed traces of the long and complex history of the Americas in the bacterial genomes, as well as a similar evolution in core and accessory genome. This was the first time accessory genome of H. pylori was studied that way. Secondly, evolution occurring in the bacterial genome during colonisation of a single host was studied in mice model. This analysis revealed small changes during the passage from a human host to a mice host, and during the long-term colonisation of mice stomach. Then a Genome Wide Association Study (GWAS) approach was applied to a large isolate collection sampled across Europe comprising strains isolated from cancer patients and strains from asymptomatic or gastritis-suffering patients. This approach identified 11 polymorphisms in 9 genes (3 cagPAI genes, babA, hpaA, 1 outer membrane protein coding gene HP1055 and 3 other core genes (HP0747, HP0709 and HP0468)) associated with cancer and a preliminary risk score was built to identify high risk strains. Finally, variations observed among clinical isolates of H. pylori from European patients with different pathologies in terms of motility and ability to trigger cytokine production in two types of cells were quantified. Motility variations were not associated with the disease type, but a link was observed for cytokine production. This was compared to genomic variations, confirming the role of known genomic factors such as cagPAI genes and sheding light to possible functions of a number of new genes.

Repair of damage to the DNA is essential for the maintenance of genomic stability, both during embryonic development and normal growth. The cell has therefore evolved a complex array of interconnected pathways to ensure the appropriate response to DNA damage is initiated, such as cell cycle checkpoint arrest, activation of DNA repair pathways or induction of apoptotic processes. These co-ordinated signal transduction pathways have been termed the DNA damage response (DDR). A previous study showed that ATR-dependent damage responses were frequently defective in cell lines from patients with Microcephalic Primordial Dwarfism (MPD) disorders. In this thesis I have further characterised ATR–dependent damage response signalling in several cell lines from patients with various MPD disorders. I have shown that novel mutations in PCNT, which encodes a structural centrosomal protein, result in an MPD disorder and have characterised the associated ATRdependent DNA damage responses. I also contributed to the identification of mutations in ORC1, encoding a component of the DNA replication Origin Recognition Complex, in further MPD patients and examined origin licensing and Sphase progression in the patient derived cell lines. As a novel finding, I observed defects in the ATR-dependent G2/M checkpoint response in these cells. Additionally, I have characterised novel mutations in ATRIP, a gene encoding the obligate partner of ATR, in Seckel Syndrome patients, denoting a novel genetic defect in this condition. Finally, I have explored the role of PLK1 and AurA kinase in ATRdependent G2/M checkpoint control and provided compelling evidence of misregulation of this pathway in various MPD-patient derived cell lines. Collectively these data provide important functional insights into the genetic defects that cause MPD disorders and further explore the link between defective ATR-dependent damage response signalling and microcephaly.

The INO80 complex is a large ATPase chromatin remodeller which contains 15 accessory subunits in S.cerevisiae. Its subunits include the highly conserved ATPases Ruvb1 and Ruvb2, the actin-related proteins Arp5, Arp8, Act1 and Arp4, Actin, and a number of IES (I̱noE̱ighty S̱pecific) subunits Ies1, Ies2, Ies3, Ies4, Ies5 and Ies6, in addition to subunits Nhp10 and Taf14. All 15 of the accessory subunits are assembled around a catalytic core component known as Ino80. The INO80 complex has roles in transcription, DNA repair, replication, and chromosome segregation. These roles are in addition to its traditional nucleosome remodelling activities and the dispacement of H2A.Z from chromatin. Recent studies in S. cerevisiae have identified the subunit Ies6 as a critical component of the INO80 complex. Deletion of IES6, which encodes the small accessory subunit, clearly mimics the deletion f the gene encoding the catalytic subunit, INO80. Surprisingly, only one domain within Ies6 has been formally identified based on sequence analysis. This domain belongs to the L1_C class of domains. Such domains are commonly associated with DNA binding activity and transcription factors. This stud has further characterised the Ies6 subunit both genetically and biochemically. Genetically, it has demonstrated that single point mutations at regions of proposed subunit-subunit interaction between the Arp5 or Rvb2 subunits, or within the YL1_C are not sufficient to disrupt Ies6 function. However, expression of a double point mutation, ies6(K114E/Y125A), in combination with rad50 deletion, caused a sensitivity to replication inhibition, but not chromosome segregation inhibition, indicating a potential separation of function in this utant due to the loss due of only one of the biological functions of Ies6. Biochemically, we have confirmed that DBA binding capacity of Ies6 resides within the YL_C domain. In addition, although it has been demonstrated that the removal of H2A.Z acetylation exacerbates the increase in cellular ploidy observed in ies6 null cells, we found that overall levels of H2A.Z acetylation were not influenced by the loss of Ies6. This indicates that the role of H2A.Z acetylation in chromosome segregation may only affect ploidy status upon the loss of Ies6. In addition, work on the R2TP complex (which contains the INO80 APases Ruvb1/Ruvb2, and subunits Tah1 and Phi1) has revealed the recruitment mechanism for the molecular chaperone, Hsp90, and the telomere length regulation protein, Tel2. Together, the R2TP complex, Hsp90 and Tel2 promote the stabilisation and maturation of multi-protein complexes. These include Phosphatidylinositol 3-kinase-related kinases (PIKKs, a family of kinases involved i Serine and Threonine phosphorylation), subunits of the INO80 complex and subunits of the SWR1 chromatin remodelling complex (a partner comlex to INO80 that incorporates H2A.Z into chromatin).

Over the last decade, major advances in the development and application of microarray-based comparative genomic hybridisation (aCGH) technology have significantly contributed to our understanding of Genomic Disorders. My aims here were to provide insight into the genotype to phenotype relationships of three Genomic Disorders; CUL4B-deleted X-Linked Mental Retardation (XLMR), Wolf-Hirschhorn Syndrome (WHS) and 16p11.2 Copy Number Variant Disorder. CUL4B encodes a structural component of the Cullin-RING-ligase 4-containing class of E3 ubiquitin ligases. CUL4B-deleted XLMR represents a syndromal form of mental retardation whereby patients exhibit other clinical features aside from the MR, such as seizures, growth retardation and disrupted sexual development. I used CUL4B-deleted patient-derived cell lines to investigate the impacts of CUL4B loss on mitochondrial function. I have shown that loss of CUL4B is associated with a distinct set of mitochondrial phenotypes, identifying CUL4B-deleted XLMR as a disorder associated with mitochondrial dysfunction. Furthermore, I have uncovered a reciprocal relationship between CUL4B and Cereblon, providing evidence of a potential role for the CUL4-CRBN E3 ligase complex in maintaining mitochondrial function. Deletion or duplication of the 16p11.2 region is associated with macro-/microcephaly respectively. Here, I have evaluated the cellular consequences of 16p11.2 CNV, specifically with regards KCTD13 expression, DNA replication and checkpoint activation. WHS is typically caused by a small hemizygous telomeric deletion of the 4p16.1 region. Haploinsufficiency of 4p16.1 is associated with microcephaly, growth retardation and complex developmental abnormalities. I investigated the impacts of LETM1 copy number change in WHS patient-derived cells. Here, I have shown that copy number change of LETM1 specifically segregates with mitochondrial dysfunction, likely underlying the seizure phenotype exhibited by the large subgroup of WHS patients whose deletions incorporate LETM1 as well as the rarer instances of the reciprocal duplication. In this thesis I use patient-derived cell lines from three Genomic Disorders as a fundamental tool providing new pathomechanistic insight into the clinical presentation of these conditions.

Mutations are the ultimate source of new genetic information and they can be neutral, harmful or beneficial. The ultimate fate of all mutations is either to be lost or to eventually become fixed in a population. In this thesis I investigate genome wide traces of natural selection in eukaryotes. I focus on the most common type of mutations, point mutations, in protein coding genes. I investigated whether there is adaptive evolution in 11 plant species comparisons by applying an extension of the McDonald Kreitman (MK) test and found little evidence of adaptive evolution. However, most of the investigated plant species have low effective population sizes (Ne) and the rate of adaptive evolution is thought to be correlated to Ne. I therefore extended my study using additional data from mammals, drosophilids and yeast to investigate the relationship between the rate of adaptive evolution and Ne. I found a highly significant correlation between the rate of adaptive evolution relative to the rate of neutral evolution (!a) and Ne. It has been proposed that evidence of adaptive evolution can be an artifact of fluctuating selection. I simulated a model of fluctuating selection, in which the average strength of selection acting upon mutations is zero. Under this model adaptive evolution is inferred using MK-type tests. However, the mutations which become fixed are on average positively selected. The signal of adaptive evolution is therefore genuine. Ne can not only vary between species but also across genomes. However, how much variation there is, and whether this affects the efficiency of natural selection, is unknown. I analysed 10 species and show that variation in Ne is widespread. However, this variation is limited, amounting to a few fold variation in Ne between most genomic regions. This is never-the-less sufficient to cause variation in the efficiency of selection.

Hox genes encode a family of evolutionarily conserved transcription factors involved in the activation of diverse cell differentiation programs along the antero-posterior axis of animals. Hox gene expression is controlled by a complex set of regulatory mechanisms which are still not fully understood. Despite this, misregulation of Hox gene expression can lead to severe developmental abnormalities and various forms of disease. This work addresses the way in which small non-coding RNAs (microRNAs, miRNAs) regulate Hox gene expression and function during development. To do this we use the Drosophila Hox gene Ultrabithorax (Ubx) as a paradigm for Hox gene function. Using a suite of genetic methods we first uncover a novel regulatory interaction between Drosophila Ubx and the miR-310C family of miRNAs during the development of the haltere, a small dorsal appendage involved in flight control. We also show that this miRNA cluster is required to fine tune Ubx expression. Furthermore, our data provides insight into the role played by Ubx during appendage development. Secondly, using a next generation RNA sequencing approach, we identify the full repertoire of miRNAs present in two serially homologous appendages of Drosophila – the wing and haltere. Our results show that these morphologically distinct appendages have divergent miRNA profiles, including miRNAs which display appendage-specific expression patterns. In addition, combining these profiles with available transcriptomic data enabled us to study how miRNAs are integrated into the Ubx gene regulatory networks that govern haltere development. This analysis suggests that haltere miRNAs reinforce the regulatory programmes installed by Ubx during haltere development. Our work therefore contributes to the understanding of the regulatory function of miRNAs during development and sheds light on the ways in which Hox gene expression can contribute to the formation of complex morphological structures.

Maintenance of the genome is essential for life to prosper. Regular insults to the genome are sustained by all cellular life and can foster genetic instability if left unrepaired. The most lethal genetic damage is a double strand break (DSB), the cleavage of the phosphate backbone on both strands of the DNA double helix. Two main pathways exist which provide mechanisms for coping with DSBs; precise repair utilising the identical sister chromatid as a template to recreate the broken segment (homologous recombination; HR), and direct fusion of the broken ends in the absence of an intact template (nonhomologous end joining; NHEJ). NHEJ was first characterised in eukaryotes, and an analogous system has been found to exist in bacteria during the past decade. The bacterial NHEJ pathway is composed of four key proteins; the DNA end binding Ku homodimer, a DNA Ligase, a DNA polymerase and a phosphoesterase (PE). The first results chapter of this thesis details the identification of an orthologous set of proteins in the archaeon Methanocella paludicola, and their subsequent isolation and characterisation. The second results chapter expands on the individual activities of the proteins by combining them, and asserting the ability of archaeal NHEJ to join discontinuous ends in vitro. The role of the PE has been unclear in the bacterial system, but in vitro assays described here suggest that the enzyme plays a role in processing NHEJ intermediates formed by the NHEJ polymerase. The PE is found to optimise repair intermediates for ligation, and to reverse potentially genotoxic DNA strand displacements. The final results chapter investigates the structural aspects of the archaeal NHEJ enzymes. Together these studies establish a functional NHEJ system in an archaeon for the first time, and expand our knowledge of the bacterial system by proposing a standard model of archaeo--‐prokaryotic NHEJ.

The purpose of protein homeostasis (proteostasis) is to maintain proteome integrity, thereby promoting viability at both the cellular and organism levels. Exposure to a range of acute stresses often produces misfolded proteins, which present a challenge to maintaining proteostatic balance. The accumulation of misfolded proteins can lead to the formation of potentially toxic protein aggregates, which are characteristic of a number of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Therefore, a number of protein quality control pathways exist to promote protein folding by molecular chaperones or target terminally misfolded proteins for degradation via the ubiquitin proteasome system or autophagy. Within the cytosol the mechanisms responsible for targeting substrates for proteasomal degradation remain to be fully elucidated. In this thesis, we established and employed thermosensitive model substrates to screen for factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations in Saccharomyces cerevisiae. Using a genome- wide flow cytometry based screen we identified the prefoldin chaperone subunit Gim3 as well as the E3 ubiquitin ligase Ubr1. An absence of Gim3 leads to the accumulation of model substrates in cytosolic inclusions and their delayed degradation. We propose that Gim3 promotes degradation by maintaining substrate solubility. In the course of screening for factors involved in degradative protein quality control, we identified secondary mutations in the general stress response gene WHI2 among a number of E3 ligase deletion strains. We demonstrate that an absence of WHI2 is responsible for the observed impairment in the proteolytic degradation of Guk1-7. We propose a link between mutations in WHI2 to a deficiency in the Msn2/4 transcriptional response, thereby altering the cell’s capacity to degrade misfolded cytosolic proteins. Collectively, the data in this thesis generated with the Guk1-7 model substrate underscores how changes in the elaborate protein quality control network can perturb proteostasis. Given that proteostasis is altered in a number of diseases ranging from cancer to ageing, identifying the factors that mediate protein quality control and understanding the interplay between members of the proteostatic network are important not only for understanding the basic biological processes but also for potential therapeutic applications.<br>Science, Faculty of<br>Graduate

Brassica napus L. (rapeseed) is an economically important oilseed crop worldwide, having uses in both food and non-food sectors. Its industrial applications are linked to the natural occurrence of erucic acid (EA, C22:1), together with other fatty acids in its seeds. EA is a valuable fatty acid that could be derived into products such as erucamide, brassylic acid and pelargonic acid having a wide range of industrial applications such as plasticizers, slip additives, lubricants, pharmaceuticals, biodiesel and many more. EA biosynthesis is controlled by Bna.FAE1s (FATTY ACID ELONGASES 1) in B. napus. In addition, low levels of polyunsaturated fatty acids (PUFAs) are desirable in the oil for the industry as it increases the thermal stability of the oil. PUFAs biosynthesis is controlled by Bna.FADs (FATTY ACID DESATURASES) in B. napus. The present study was aimed to underpin the new loci affecting the EA biosynthesis by using the associative transcriptomics approach and to study the influence of Bna.FAD2 family on the erucic acid (or very long chain fatty acids, VLCFAs) and polyunsaturated fatty acids levels. Although new loci influencing the erucic acid levels were not found from the present study but a unique specification – high erucic acid rapeseed in the low polyunsaturates (HELP) background was developed by introducing partially functional Bna.FAD2 family from EMS mutagenized mutants to the high erucic acid rapeseed background. HELP lines showed the influence of partially functional Bna.FAD2 alleles in the fatty acid compositions, ~8% increase in the erucic acid (60%) and VLCFAs (66%) levels as compared to the parental high erucic parents having functional Bna.FAD2 family. Polyunsaturates content of less than 7% was found in these HELP lines. HELP oil is anticipated to be a valuable industrial oil that could contribute significantly to reduce the processing costs and serve as a renewable environment-friendly industrial resource with no toxicity.

To date, germline mutations in known high-penetrance genes, mainly BRCAI and BRCA2, and in moderate- and low-penetrance genes are responsible for approximately 30- 35% of breast cancer familial clustering, leaving the majority of them unexplained. In addition, the variability of the risk conferred by BRCAI and BRCA2 mutations suggests the presence of genetic modifiers of this risk. Therefore, the identification and characterization of as many as possible of genetic factors is crucial for risk prediction in members of breast cancer families. In this context, the aim of this thesis was firstly to investigate the role of the two Fanconi Anemia (FA) genes PALB2 and SLX4 as breast cancer predisposing loci. In the PALB2 screening, I observed a frequency of deleterious mutation of 2.1 % in familial cases recruited in cancer centers in Milan. Interestingly, I also identified the recurrent mutation c. l 027C > T, detected with 10-fold increased frequency in cases from Bergamo with respect to those ascertained in Milan, suggesting a founder effect. On the contrary. the SLX 4 analysis failed to identify any clearly deleterious mutation, excluding a major role of this gene in breast cancer susceptibility in the Italian population. In addition, I genotyped the candidate low-risk rs895819 polymorphism, located in the gene coding for miR-27a, to evaluate its role in reducing breast cancer risk, previously reported in the German population. No such an association was observed in our sample set. Finally, I investigated the role of the CASPS rs3834129 ins/del polymorphism as a genetic modifier in Italian BRCA1 and BRCA2 mutation carries and I observed an association of this SNP with increased breast cancer risk only in individuals carrying BRC1 mutations. In conclusion, our investigation contributed to assess the role of candidate predisposing loci and genetic modifiers of breast cancer risk, providing further knowledge on the susceptibility to this disease.

Maintenance of genome integrity and stability is fundamental for any form of life. This is complicated as DNA is highly reactive and always under attack from a wide range of endogenous and exogenous sources which can lead to different damages in the DNA. To preserve the integrity of DNA replication, cells hav evolved a variety of DNA repair pathways. DNA damage tolerance mechanisms serve as the last line of defence to rescue the stalled replications forks. TLS, error-prone type of DNA damage tolerance, acts to bypass DNA lesions and allows continuation of DNA replication. Surprisingly majority of archaeal species lack canonical TLS polymerases. This poses a question as to how archaea restart stalled replication in the absence of TLS or lesion repair pathways. This thesis establishes that archaeal replicative primase (PriS/L), a member of the archaeo-eukaryotic primase (AEP) superfamily, possessing both primase and polymerase activities, is able to bypass the most common oxidative damages and highly distorting lesions caused by UV radiation. It has been postulated that archaeal replicative polymerases (Pol B and Pol D family Pols) can bind tightly to the deaminated bases uracil and cause replication fork stalling four bases prior to dU. A specific mechanism for resuming replication of uracil containing DNA by PriS/L is suggested in this thesis. In this thesis, we also reported how the enzymatic activities of archaeal PriS/L are regulated. Here, it is demonstrated that in contrast to archaeal replicative polymerases, single-strand binding proteins (RPA) significantly limit the polymerase activity of PriS/L. The remaining results chapter interrogates the possible interactions between PriS/L and RPA. Finally, the attempts to reconstitute an archaeal CMG complex in vitro, with the aim of shedding light on the role of archaeal replicative primase in replication-specific TLS are described.