Bipolar affective disorder is a severe mood disorder, which is characterised by episodes of mania and depression. The aetiology of bipolar disorder remains elusive, with little known about the underlying biological, anatomical, or biochemical effects. However, family, twin and adoption studies provide evidence for a strong genetic component to the disorder. Due to the high heritability, familial clustering, and common population prevalence of the illness, molecular genetic studies can be implemented to identify bipolar disorder susceptibility genes. This thesis investigated the candidate gene serotonin 2A receptor (HTR2A), which lay within a region on chromosome 13q14 previously identified by bipolar disorder genome-wide linkage scans. Significant association was found with bipolar disorder and a SNP within intron 2 of HTR2A in an Australian case-control cohort. Haplotype association analysis identified a 5-SNP protective haplotype within HTR2A. Conducting a new genome-wide linkage scan on 35 Australian bipolar disorder pedigrees found significant evidence for linkage on chromosome 15q25-26. Subsequent fine-mapping of the region verified the linkage peak with a significant maximum multipoint LOD score of 4.58. Haplotype analysis, based on pedigree-specific, identical-by-descent allele sharing, supported the location of a bipolar susceptibility gene within a 6.2Mb confidence interval. The candidate gene sialyltransferase 8B (ST8SIA2), which had previously shown association with SNPs within the gene’s promoter region and schizophrenia in two independent Asian cohorts, lies within the chromosome 15q25-26 locus. Failing to replicate the association found with these specific SNPs, and without finding association with two additional SNPs in an upstream conserved putative regulatory region, a fine-mapping association study was conducted across the entire 6.2Mb interval. The strongest association signals were observed at SNPs 16kb upstream from and within the fourth intron of ST8SIA2. A specific bipolar disorder risk haplotype was identified for ST8SIA2, and this was also observed to be over-represented in a cohort of Australian schizophrenia cases. This finding suggests that the ST8SIA2 gene, for which strong developmental regulation was observed, may be a shared susceptibility gene for both bipolar disorder and schizophrenia. In summary, this thesis has provided evidence identifying both HTR2A and ST8SIA2 as bipolar disorder susceptibility genes.

The global prevalence of type 2 diabetes (T2DM) and obesity is increasing, with obesity the most important predisposing factor contributing to the development of T2DM. Epidemiological and genetic evidence supports a major genetic component in both multifactorial and heterogeneous disorders. The identification of disease susceptibility genes in humans could greatly assist in the elucidation of underlying pathophysiological mechanisms and allow the development of more effective preventative and therapeutic strategies for these conditions. Three candidate genes, uncoupling proteins 2 and 3 (UCP2; UCP3) and calpain 10 (CAPN10), are proposed and the rationale for their selection discussed. Gene variants were identified in UCP2 and UCP3. These variants were tested for association with T2DM, obesity and intermediate quantitative traits in a South Indian population and family collection, and also a cohort of British obese case/control subjects. No variant was associated with T2DM. However, investigations revealed positive associations with a UCP2 3'UTR 45bp Ins/Del and a novel UCP3 promoter variant (-55C/T) with variation in body mass (BMI) and fat distribution (WHR) respectively. The results support the view that uncoupling proteins may influence weight gain and hence progression to obesity/T2DM. A significant correlation with plasma leptin levels and the UCP2 Ins/Del variant might indicate one potential mechanism whereby weight could be modulated by uncoupling proteins. A linkage study in affected sibling pairs of North European descent, was negative for the putative T2DM susceptibility gene region, NIDDMI. In contrast, haplotypes of four sequence variants of a T2DM susceptibility gene (CAPN10) identified in this region positively associated with T2DM in a South Indian population. In conclusion, these investigations provide evidence that the three genes studied may contribute to susceptibility for development of T2DM or obesity. However, the findings are in agreement with the most likely genetic model for non-Mendelian complex diseases, that many genes are involved in determining susceptibility to disease with no single gene capable of determining the overall disease phenotype.

Doctor of Philosophy / Department of Plant Pathology / Frank White / The genus Xanthomonas consists of bacterial species causing economically important plant diseases in major crops. In a wide variety of Xanthamonas species, the transcription activator-like (TAL) effectors (proteins) are synthesized and secreted into host cells, whereby they enter the plant nucleus. TAL effectors bind specific host gene promoters, inducing the expression of the targeted genes, which in some cases leads to either resistance or an enhanced state of disease susceptibility. The TAL effectors in individual Xanthomanas species and their targets in host plants have been characterized in relatively few cases. The premier example is the induction of any one member of a clade of sugar transporter genes in rice by TAL effectors of the bacterial blight pathogen X. oryzae pv. oryzae, where induction of the susceptibility (S) genes was shown to be required for the disease process. TAL effector genes are present in a wide variety of Xanthomonas species other than X. oryzae pv. oryzae. My dissertation focuses on the characterization of the TAL effectors in the citrus bacterial canker (CBC) and soybean bacterial pustule pathosystems. In CBC, CsLOB1 was identified as the S gene targeted by multiple major TAL effectors from CBC causal strains. Furthermore, another two members in family of citrus LBD family, although not identified as targets in the field, can serve as S genes in CBC. Initial analysis of bacterial pustule disease of soybean indicates that the TAL effector TAL2 of X. axonopodis pv. glycines is a virulence effector and associated with the expression of two candidate S genes, which encode a member of the ZF-HD transcription factors and a member of aluminum activated malate transporter family. These studies will enhance our understanding of plant-bacterial interactions and evolution of disease susceptibility, and also inform development of durable disease resistant crop varieties.

O diabetes mellitus do tipo 1 (DM1) é uma doença autoimune que se desenvolve a partir da ação combinada de múltiplos fatores genéticos e ambientais, sendo caracterizada pela perda seletiva das células produtoras de insulina nas ilhotas pancreáticas em indivíduos geneticamente susceptíveis. O HLA de classe II, localizado no cromossomo humano 6p21.3, representa uma das regiões genômicas mais importantes associadas ao DM1, embora evidências apontem para a participação de diversos outros loci na susceptibilidade à doença. Essas regiões cromossômicas poderiam apresentar genes funcionalmente ativos com perfis transcricionais semelhantes ao camundongo Mus musculus, muito utilizado como modelo animal para o estudo de doenças humanas. Para testar esta hipótese, foi realizada análise dos perfis transcricionais de linfócitos periféricos provenientes de pacientes com DM1 e camundongos NOD (Non-obese diabetic) diabéticos, focando os genes situados em regiões de susceptibilidade. Foram utilizados dados de microarrays do genoma funcional completo da plataforma Agilent (Whole genome one-color Agilent 4x44k) de dezenove pacientes e oito controles, e de camundongos NOD pré-diabéticos e diabéticos. A linhagem NOD foi utilizada no estudo, pois representa um modelo experimental para o estudo do diabetes autoimune e desenvolve a doença espontaneamente. Para a análise dos dados de microarrays foi utilizado o software GeneSpring GX e os programas Cluster e TreeView. A modulação transcricional dos genes foi estabelecida comparando-se os pacientes com os controles, e os animais diabéticos com os pré-diabéticos. Os loci de susceptibilidade ao DM1 humano foram definidos utilizando-se uma tabela curada disponível no banco T1DBase (http://t1dbase.org) e seus correspondentes murinos, segundo o banco de dados Homology Maps (http://www.ncbi.nlm.nih.gov/projects/homology/maps/). Foram considerados para o estudo apenas os pares de homólogos com expressão em linfócitos humanos e murinos conforme os dados disponíveis no banco BioGPS (http://biogps.org). Avaliamos se os genes murinos estavam situados em regiões de susceptibilidade (Idd) ao DM1 do camundongo, utilizando-se o T1DBase. Todos os genes humanos selecionados com homologia com camundongo foram mapeados quanto à localização cromossômica, buscando-se por regiões de sintenia entre as duas espécies por meio da ferramenta Synteny disponível no banco de dados Ensembl Genome Browser (http://www. ensembl.org/). Os pares de homólogos situados em regiões sintênicas foram então verificados quanto à similaridade de sequência de DNA e identidade protéica entre humano e camundongo, utilizando-se dados do HomoloGene (http://www.ncbi.nlm.nih. gov/homologene/). Foram analisados 463 genes humanos, dos quais 73 apresentaram correspondência em camundongo e expressão em linfócitos T. Dos 73 genes identificados, 31 deles apresentaram mesma modulação de fold change entre as duas espécies, com 12 mapeados em regiões de susceptibilidade murinas (Idd). Dos doze genes em regiões Idd, 4 se apresentaram induzidos: APOM (Apom), COL11A2 (Col11a2), HLA-DOB (H2-Ob) e PRR3 (Prr3); e 8 reprimidos: CYP21A2 (Cyp21a1), STK19 (Stk19), PHTF1 (Phtf1), RSBN1 (Rsbn1), CDSN (Cdsn), TRIM39 (Trim39), VARS2 (Vars2) e IL21 (Il21). Foram identificados 59 genes em regiões de sintenia humano-camundongo, com 58 apresentando similaridade na sequência de DNA acima de 70% entre as duas espécies, e identidade de sequência de aminoácidos das respectivas proteínas variando de 61,4 a 99,7%. Esses resultados demonstram que a maioria dos genes estudados apresenta conservação funcional, como indicado pelo alto grau de identidade das proteínas. Além disso, evidenciou-se um compartilhamento de perfis de expressão de genes em regiões de susceptibilidade ao DM1 humano e murino, contribuindo para um melhor conhecimento das semelhanças entre o modelo animal (linhagem NOD) e o diabetes autoimune humano. / Type 1 diabetes (T1D) is a common autoimmune disease that arises from multiple genetic and environmental risk factors. It is characterized by selective loss of insulin-producing -cells in the pancreatic islets in genetically susceptible individuals. The HLA class II locus on human chromosome 6p21.3 represents one of the most important genomic regions associated with T1D but there are evidences for the participation of several others along the chromosomes, which also contribute to disease susceptibility. These chromosomal regions may harbor functional genes with transcriptional profiles similar to those presented by the mouse Mus musculus, an animal model highly used in researches of human diseases. To test this hypothesis, it was performed a transcriptome profiling analysis of peripheral lymphocytes from T1D patients and diabetic NOD (Non-obese diabetic) mice focusing those genes positioned in chromosomal susceptibility regions. To perform this analysis, whole genome one-color Agilent 4x44k microarrays were used from 19 patients and 8 controls and from pre-diabetic and diabetic NOD mice. NOD strain was used since It is a well established mouse model for T1D. GeneSpring GX software and Cluster and TreeView programs were applied for microarray data analysis. The transcriptional modulation was established by comparisons of diabetic patients versus controls and diabetic versus pre-diabetic animals. The human T1D susceptibility loci were defined using a curated human dataset available in T1DBase (http://t1dbase.org) and mouse counterparts according to Homology Maps database (http://www.ncbi.nlm.nih.gov/projects/homology/maps/). For the present study we considered only homolog gene pairs with expression in human and mouse lymphocytes taking into account data available in BioGPS database (http://biogps.org). Following these procedures, mouse genes were checked for chromosome location in order to subserve the establishment of syntenic regions. The human-mouse syntenic regions were defined using Synteny tool from Ensembl Genome Browser (http://www.ensembl.org/). The homolog gene pairs located in human-mouse syntenic regions were checked for DNA sequence similarity and protein identity according to HomoloGene data (http://www.ncbi.nlm.nih.gov/homologene/). The 463 human genes were analyzed with 73 presenting murine counterparts and expression in lymphocytes. From these, 31 genes presented same fold change modulation in both species, with 12 of them mapped in murine diabetes susceptibility regions (Idd). The 12 genes in Idd regions showed different transcriptional modulations, 4 featured up-regulation: APOM (Apom), COL11A2 (Col11a2), HLA-DOB (H2- Ob) e PRR3 (Prr3); and 8 down-regulation: CYP21A2 (Cyp21a1), STK19 (Stk19), PHTF1 (Phtf1), RSBN1 (Rsbn1), CDSN (Cdsn), TRIM39 (Trim39), VARS2 (Vars2) e IL21 (Il21). 51 genes were identified in human-mouse syntenic regions with 58 presenting DNA sequence similarity above 70% and protein identity ranging from 61,4 to 99,7%. These results show that most genes studied present functional conservation, as indicated by the high degree of identity of the proteins. Additionally, it was observed shared expression profiles between human and murine T1D susceptibility regions. These results contribute to a better understanding of similarities between the animal model (NOD mouse strain) and human autoimmune diabetes.

O diabetes mellitus do tipo 1 (DM1) é uma doença autoimune que se desenvolve a partir da ação combinada de múltiplos fatores genéticos e ambientais, sendo caracterizada pela perda seletiva das células produtoras de insulina nas ilhotas pancreáticas em indivíduos geneticamente susceptíveis. O HLA de classe II, localizado no cromossomo humano 6p21.3, representa uma das regiões genômicas mais importantes associadas ao DM1, embora evidências apontem para a participação de diversos outros loci na susceptibilidade à doença. Essas regiões cromossômicas poderiam apresentar genes funcionalmente ativos com perfis transcricionais semelhantes ao camundongo Mus musculus, muito utilizado como modelo animal para o estudo de doenças humanas. Para testar esta hipótese, foi realizada análise dos perfis transcricionais de linfócitos periféricos provenientes de pacientes com DM1 e camundongos NOD (Non-obese diabetic) diabéticos, focando os genes situados em regiões de susceptibilidade. Foram utilizados dados de microarrays do genoma funcional completo da plataforma Agilent (Whole genome one-color Agilent 4x44k) de dezenove pacientes e oito controles, e de camundongos NOD pré-diabéticos e diabéticos. A linhagem NOD foi utilizada no estudo, pois representa um modelo experimental para o estudo do diabetes autoimune e desenvolve a doença espontaneamente. Para a análise dos dados de microarrays foi utilizado o software GeneSpring GX e os programas Cluster e TreeView. A modulação transcricional dos genes foi estabelecida comparando-se os pacientes com os controles, e os animais diabéticos com os pré-diabéticos. Os loci de susceptibilidade ao DM1 humano foram definidos utilizando-se uma tabela curada disponível no banco T1DBase (http://t1dbase.org) e seus correspondentes murinos, segundo o banco de dados Homology Maps (http://www.ncbi.nlm.nih.gov/projects/homology/maps/). Foram considerados para o estudo apenas os pares de homólogos com expressão em linfócitos humanos e murinos conforme os dados disponíveis no banco BioGPS (http://biogps.org). Avaliamos se os genes murinos estavam situados em regiões de susceptibilidade (Idd) ao DM1 do camundongo, utilizando-se o T1DBase. Todos os genes humanos selecionados com homologia com camundongo foram mapeados quanto à localização cromossômica, buscando-se por regiões de sintenia entre as duas espécies por meio da ferramenta Synteny disponível no banco de dados Ensembl Genome Browser (http://www. ensembl.org/). Os pares de homólogos situados em regiões sintênicas foram então verificados quanto à similaridade de sequência de DNA e identidade protéica entre humano e camundongo, utilizando-se dados do HomoloGene (http://www.ncbi.nlm.nih. gov/homologene/). Foram analisados 463 genes humanos, dos quais 73 apresentaram correspondência em camundongo e expressão em linfócitos T. Dos 73 genes identificados, 31 deles apresentaram mesma modulação de fold change entre as duas espécies, com 12 mapeados em regiões de susceptibilidade murinas (Idd). Dos doze genes em regiões Idd, 4 se apresentaram induzidos: APOM (Apom), COL11A2 (Col11a2), HLA-DOB (H2-Ob) e PRR3 (Prr3); e 8 reprimidos: CYP21A2 (Cyp21a1), STK19 (Stk19), PHTF1 (Phtf1), RSBN1 (Rsbn1), CDSN (Cdsn), TRIM39 (Trim39), VARS2 (Vars2) e IL21 (Il21). Foram identificados 59 genes em regiões de sintenia humano-camundongo, com 58 apresentando similaridade na sequência de DNA acima de 70% entre as duas espécies, e identidade de sequência de aminoácidos das respectivas proteínas variando de 61,4 a 99,7%. Esses resultados demonstram que a maioria dos genes estudados apresenta conservação funcional, como indicado pelo alto grau de identidade das proteínas. Além disso, evidenciou-se um compartilhamento de perfis de expressão de genes em regiões de susceptibilidade ao DM1 humano e murino, contribuindo para um melhor conhecimento das semelhanças entre o modelo animal (linhagem NOD) e o diabetes autoimune humano. / Type 1 diabetes (T1D) is a common autoimmune disease that arises from multiple genetic and environmental risk factors. It is characterized by selective loss of insulin-producing -cells in the pancreatic islets in genetically susceptible individuals. The HLA class II locus on human chromosome 6p21.3 represents one of the most important genomic regions associated with T1D but there are evidences for the participation of several others along the chromosomes, which also contribute to disease susceptibility. These chromosomal regions may harbor functional genes with transcriptional profiles similar to those presented by the mouse Mus musculus, an animal model highly used in researches of human diseases. To test this hypothesis, it was performed a transcriptome profiling analysis of peripheral lymphocytes from T1D patients and diabetic NOD (Non-obese diabetic) mice focusing those genes positioned in chromosomal susceptibility regions. To perform this analysis, whole genome one-color Agilent 4x44k microarrays were used from 19 patients and 8 controls and from pre-diabetic and diabetic NOD mice. NOD strain was used since It is a well established mouse model for T1D. GeneSpring GX software and Cluster and TreeView programs were applied for microarray data analysis. The transcriptional modulation was established by comparisons of diabetic patients versus controls and diabetic versus pre-diabetic animals. The human T1D susceptibility loci were defined using a curated human dataset available in T1DBase (http://t1dbase.org) and mouse counterparts according to Homology Maps database (http://www.ncbi.nlm.nih.gov/projects/homology/maps/). For the present study we considered only homolog gene pairs with expression in human and mouse lymphocytes taking into account data available in BioGPS database (http://biogps.org). Following these procedures, mouse genes were checked for chromosome location in order to subserve the establishment of syntenic regions. The human-mouse syntenic regions were defined using Synteny tool from Ensembl Genome Browser (http://www.ensembl.org/). The homolog gene pairs located in human-mouse syntenic regions were checked for DNA sequence similarity and protein identity according to HomoloGene data (http://www.ncbi.nlm.nih.gov/homologene/). The 463 human genes were analyzed with 73 presenting murine counterparts and expression in lymphocytes. From these, 31 genes presented same fold change modulation in both species, with 12 of them mapped in murine diabetes susceptibility regions (Idd). The 12 genes in Idd regions showed different transcriptional modulations, 4 featured up-regulation: APOM (Apom), COL11A2 (Col11a2), HLA-DOB (H2- Ob) e PRR3 (Prr3); and 8 down-regulation: CYP21A2 (Cyp21a1), STK19 (Stk19), PHTF1 (Phtf1), RSBN1 (Rsbn1), CDSN (Cdsn), TRIM39 (Trim39), VARS2 (Vars2) e IL21 (Il21). 51 genes were identified in human-mouse syntenic regions with 58 presenting DNA sequence similarity above 70% and protein identity ranging from 61,4 to 99,7%. These results show that most genes studied present functional conservation, as indicated by the high degree of identity of the proteins. Additionally, it was observed shared expression profiles between human and murine T1D susceptibility regions. These results contribute to a better understanding of similarities between the animal model (NOD mouse strain) and human autoimmune diabetes.

<p>Systemic lupus erythematosus (SLE) is a complex autoimmune disease with unknown etiology. The aim of this thesis was to identify susceptibility regions through genetic mapping, using model-based linkage analysis on nuclear and extended SLE multicase families.</p><p>In the first paper we performed a genome scan on 19 genetically homogenous Icelandic and Swedish families. One region at 2q37 was identified with a significant linkage with contribution from both populations (Z=4.24). Five other regions 2q11, 4p13, 9p22, 9p13 and 9q13 showed suggestive linkage (Z>2.0).</p><p>In the second paper, 87 families from 10 different countries were analysed only for chromosome 1. One region at 1q31 showed significant linkage (Z=3.79) with contribution from families from all populations, including Mexicans and Europeans. Four other regions 1p36, 1p21, 1q23, and 1q25, showed levels of suggestive linkage. Linkage for most regions was highly dependent on what population was used, which indicated strong genetic heterogeneity in the genetic susceptibility for SLE.</p><p>In the two last papers, we used the positional candidate gene strategy, in order to investigate candidate genes in two regions linked to SLE. For the Bcl-2 gene (at 18q21) we could not detect any association with SLE using three different markers. However, when we investigated the tightly linked low-affinity family of FcγR genes (at 1q23), we could find association for two risk alleles in the FcγRIIA and FcγRIIIA genes. The risk alleles were transmitted to SLE patients on one specific haplotype and therefore are not independent risk alleles.</p><p>The results show that model-based linkage analysis is a strong approach in the search for susceptibility genes behind complex diseases like SLE.</p>

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with unknown etiology. The aim of this thesis was to identify susceptibility regions through genetic mapping, using model-based linkage analysis on nuclear and extended SLE multicase families. In the first paper we performed a genome scan on 19 genetically homogenous Icelandic and Swedish families. One region at 2q37 was identified with a significant linkage with contribution from both populations (Z=4.24). Five other regions 2q11, 4p13, 9p22, 9p13 and 9q13 showed suggestive linkage (Z&gt;2.0). In the second paper, 87 families from 10 different countries were analysed only for chromosome 1. One region at 1q31 showed significant linkage (Z=3.79) with contribution from families from all populations, including Mexicans and Europeans. Four other regions 1p36, 1p21, 1q23, and 1q25, showed levels of suggestive linkage. Linkage for most regions was highly dependent on what population was used, which indicated strong genetic heterogeneity in the genetic susceptibility for SLE. In the two last papers, we used the positional candidate gene strategy, in order to investigate candidate genes in two regions linked to SLE. For the Bcl-2 gene (at 18q21) we could not detect any association with SLE using three different markers. However, when we investigated the tightly linked low-affinity family of FcγR genes (at 1q23), we could find association for two risk alleles in the FcγRIIA and FcγRIIIA genes. The risk alleles were transmitted to SLE patients on one specific haplotype and therefore are not independent risk alleles. The results show that model-based linkage analysis is a strong approach in the search for susceptibility genes behind complex diseases like SLE.

Pain is a major public health issue throughout the world. Increased understanding of the different forms of pain and identification of susceptibility genes could contribute to improved treatments. The main aims of this thesis were to identify the underlying genetic causes of pain by studying two large families affected with migraine and pain insensitivity, respectively. Migraine is one of the most common neurovascular disorders, affecting over 12% of the western population. The genetic contribution to migraine is about 50% according to family and twin studies. To identify novel susceptibility loci for migraine, we performed a genome-wide screen in a large family with migraine from northern Sweden. Linkage analysis revealed significant evidence of linkage (LOD=5.41) on chromosome 6p12.2-p21.1. A predisposing haplotype spanning 10 Mb was inherited with migraine in all affected members of the pedigree. Further fine-mapping of multiple SNP markers restricted the disease critical region to 8.5 Mb. Nine candidate genes were sequenced, revealing no disease-associated polymorphisms in SLC29A1, CLIC5, PLA2G7, IL17, SLC25A27 and TNFRSF21, but rare novel polymorphisms segregating with the disease haplotype in EFHC1, RHAG and MEP1A. EFHC1 has recently been shown to be involved in epilepsy, which is interesting considering the link between migraine and epilepsy. However, association analysis of EFHC1 revealed no difference between patients and controls, suggesting that this gene is not a risk factor for migraine. The combination of the two polymorphisms in RHAG and MEP1A could, however, not be found in any control individuals, indicating that they might be involved in genetic predisposition to migraine in this family. Disorders with reduced pain sensitivity are very rare, since pain perception is essential for survival. A number of disorders have still been identified with pain insensitivity and peripheral nerve degeneration as major clinical signs, including the hereditary sensory and autonomic neuropathies (HSAN). In order to identify novel susceptibility genes for HSAN V, we performed a genome-wide screen in a large consanguineous pedigree from a small village in northern Sweden. A homozygous region identical-by-descent was identified on chromosome 1p11.2-p13.2 in the three most severely affected patients. Subsequent analysis of candidate genes revealed a missense mutation in a conserved region of the nerve growth factor beta (NGFB) gene, causing a drastic amino acid change (R211W) in the NGF protein. NGF is important for the development and maintenance of the sympathetic and sensory nervous system and is therefore likely to be involved in disease. Functional analysis revealed that mutant NGF failed to induce neurite outgrowth and cell differentiation in PC12 cells. Furthermore, almost no mutant NGF was secreted by COS-7 cells, indicating that the processing and/or secretion of the protein might be disrupted. In conclusion, these findings present a novel migraine locus on chromosome 6 and identification of two rare polymorphisms that might be risk factors for migraine. Furthermore, a mutation in NGFB was found to cause complete loss of deep pain perception, which represents a very interesting model system to study pain mechanisms.

My research was focused on understanding the importance of mammalian RAD51 paralogs in genome maintenance and suppression of tumorigenesis. The investigation carried out during this study has been addressed toward gaining more insights into the involvement of RAD51 paralogs in DNA damage signalling, repair of various types of lesions including double stranded breaks (DSBs), daughter strand gaps (DSGs), interstrand crosslinks (ICLs), and in the protection of stalled replication forks. My study highlights the molecular functions of RAD51 paralogs in Fanconi anemia (FA) pathway of ICL repair, in the ATM and ATR mediated DNA damage responses, in homologous recombination (HR), and in the recovery from replication associated lesions. My research also focused on the development of a novel photoinducible ICL agent for targeted cancer therapy. The thesis has been divided into following sections as follows: Chapter I: General introduction that describes about DNA damage responses and the known functions of RAD51 paralogs across species in DNA repair and checkpoint The genome of every living organism is susceptible to various types of DNA damage and mammalian cells are evolved with various DNA damage surveillance mechanisms in response to DNA damages. In response to DNA damage, activated checkpoints arrest the cell cycle progression transiently and allow the repair of damaged DNA. Upon completion of DNA repair, checkpoints are deactivated to resume the normal cell cycle progression. Defective DNA damage responses may lead to chromosome instability and tumorigenesis. Indeed, genome instability is associated with several genetic disorders, premature ageing and various types of cancer in humans. The major cause of chromosome instability is the formation of DSBs and DSGs. Both DSBs and DSGs are the most dangerous type of DNA lesions that arise endogenously as well as through exogenous sources such as radiations and chemicals. Spontaneous DNA damage is due to generation of reactive oxygen species (ROS) through normal cellular metabolism. Replication across ROS induced modified bases and single strand breaks (SSBs) leads to DSGs and DSBs, respectively. Such DNA lesions need to be accurately repaired to maintain the integrity of the genome. To understand the various cellular responses that are triggered after different types of DNA damage and the possible roles of RAD51 paralogs in these processes, chapter I of the thesis has been distributed in to multiple sections as follows: Briefly, the initial portion of the chapter provides a glimpse of various types of DNA damage responses and repair pathways to deal with the lesions arising from both endogenous as well as exogenous sources. Owing to the vast range of cellular responses and pathways, the following section provides the detailed description and mechanisms of various pathways involved in taking care of wide range of DNA lesions from SSBs to DSBs. Subsequent section of chapter I provides a comprehensive description of maintenance of genome stability at the replication fork and telomeres. Germline mutations in the genes that regulate genome integrity cause various genetic disorders and cancer. Mutations in ATM, ATR, MRE11, NBS1, BLM and FANC (1-16), BRCA1 and BRCA2 that are known to regulate DNA damage signaling, DNA repair and genome integrity lead to chromosome instability disorders such as ataxia-telangiectasia, ATR-Seckel syndrome, AT-like disorder, Nijmegen breakage syndrome, Bloom syndrome, FA, and breast and ovarian cancers respectively. Interestingly, RAD51 paralog mutations are reported in patients with FA-like disorder and various types of cancers including breast and ovarian cancers. Mono-allelic germline mutations in all RAD51 paralogs are reported to cause cancer in addition to the reported cases of FA-like disorder with bi-allelic germline mutations in RAD51C and XRCC2. In accordance, the last section of the chapter has been dedicated to describe the genetics of breast and ovarian cancers and the known functions of tumor suppressors such as BRCA1, BRCA2 and RAD51 paralogs in the protection of genome. Despite the identification of five RAD51 paralogs nearly two decades ago, the molecular mechanism(s) by which RAD51 paralogs regulate HR and genome maintenance remain obscure. To gain insights into the molecular mechanisms of RAD51 paralogs in DNA damage responses and their link with genetic diseases and cancer, the following objectives were laid for my PhD thesis: 1) To understand the functional role of RAD51 paralog RAD51C in FA pathway of ICL repair and DNA damage signalling. 2) To dissect the ATM/ATR mediated targeting of RAD51 paralog XRCC3 in the repair of DSBs and intra S-phase checkpoint. 3) To uncover the replication restart pathway after transient replication pause and the involvement of distinct complexes of RAD51 paralogs in the protection of replication forks. 4) To design photoinducible ICL agent that can be activated by visible light for targeted cancer therapy. Chapter II: Distinct roles of FANCO/RAD51C protein in DNA damage signaling and repair: Implications for Fanconi anemia and breast cancer susceptibility RAD51C, a RAD51 paralog has been implicated in HR. However, the underlying mechanism by which RAD51C regulates HR mediated DNA repair is elusive. In 2010, a study identified biallelic mutation in RAD51C leading to FA-like disorder, whereas a second study reported monoallelic mutations in RAD51C associated with increased risk of breast and ovarian cancers. However, the role of RAD51C in the FA pathway of DNA cross-link repair and as a tumor suppressor remained obscure. To understand the role of RAD51C in FA pathway of ICL repair and DNA damage response, we employed genetic, biochemical and cell biological approaches to dissect out the functions of RAD51C in genome maintenance. In our study, we observed that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G2/M accumulation, which are hallmarks of the FA phenotype. We found that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrated that RAD51C plays a vital role in the HR-mediated repair of DSBs associated with replication. Finally, we showed that RAD51C participates in ICL and DSB induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C displayed that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor. Chapter III: ATM-and ATR-mediated phosphorylation of XRCC3 regulates DNA double-strand break-induced checkpoint activation and repair The RAD51 paralogs XRCC3 and RAD51C have been implicated in HR and DNA damage responses, but the molecular mechanism of their participation in these pathways remained obscured. In our study, we showed that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway. We found that RAD51C in CX3 complex but not in BCDX2 complex is essential for XRCC3 phosphorylation, and this modification follows end resection and is specific to S and G2 phases. XRCC3 phosphorylation was found to be required for chromatin loading and stabilization of RAD51 and HR-mediated repair of DSBs. Notably, in response to DSBs, XRCC3 participates in the intra-S-phase checkpoint following its phosphorylation and in the G2/M checkpoint independently of its phosphorylation. Strikingly, we found that XRCC3 distinctly regulates recovery of stalled and collapsed replication forks such that phosphorylation was required for the HR-mediated recovery of collapsed replication forks but is dispensable for the recovery of stalled replication forks. Together, our findings suggest that XRCC3 is a new player in the ATM/ATR-induced DNA damage responses to control checkpoint and HR-mediated repair. Chapter IV: RAD51 paralogs protect stalled forks and mediate replication restart in an FA-BRCA independent manner Mammalian RAD51 paralogs RAD51 B, C, D, XRCC2 and XRCC3 are critical for genome maintenance. To understand the crucial roles of RAD51 paralogs during spontaneously arising DNA damage, we have studied the RAD51 paralogs assembly during replication and examined the replication fork stability and its restart. We found that RAD51 paralogs are enriched onto the S-phase chromatin spontaneously. Interestingly, the number of 53BP1 nuclear bodies in G1-phase and micro-nucleation which serve as markers for under replicated lesions increases after genetic ablation of RAD51C, XRCC2 and XRCC3. Furthermore, we showed that RAD51 paralogs are specifically enriched at two major fragile sites FRA3B and FRA16D after replication fork stalling. We found that all five RAD51 paralogs bind to nascent DNA strands after replication fork stalling and protect the fork. Nascent replication tracts created before fork stalling with hydroxyurea degrade in the absence of RAD51 paralogs but remain stable in wild-type cells. This function was dependent on ATP binding at the walker A motif of RAD51 paralogs. Our results also suggested that RAD51 paralogs assemble into BCDX2 complex to prevent generation of DSBs at stalled replication forks, thereby safeguarding the pre-assembled replisome from the action of nucleases. Strikingly, we showed that RAD51C and XRCC3 in complex with FANCM promote the restart of stalled replication forks in an ATP hydrolysis dependent manner. Moreover, RAD51C R258H mutation that was identified in FA-like disorder abrogates the interaction of RAD51C with FANCM and XRCC3, and prevents fork restart. Thus, assembly of RAD51 paralogs in different complexes prevents nucleolytic degradation of stalled replication forks and promotes restart to maintain genomic integrity. Chapter V: Trans-dichlorooxovandium(IV) complex as a potent photoinducible DNA interstrand crosslinker for targeted cancer therapy Although DNA ICL agents such as MMC, cisplatin and psoralen are known to serve as anticancer drugs, these agents affect normal cells as well. Moreover, tumor resistance to these agents has been reported. We have designed and synthesized a novel photoinducible DNA crosslinking agent (ICL-2) which is a derivative of oxovanadiumterpyridine complex with two chlorides in trans position. We found that ICL-2 can be activated by UV-A and visible light to enable DNA ICLs. ICL-2 efficiently activated FA pathway of ICL repair. Strikingly, photoinduction of ICL-2 induces prolonged activation of cell cycle checkpoint and high degree of cell death in FA pathway defective cells. Moreover, we showed that ICL-2 specifically targets cells that express pathological RAD51C mutants. Our findings suggest that ICL-2 can be potentially used for targeted cancer therapy in patients with gene mutations in FA and HR pathway.

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