Denna projektrapport är avsedd att vara ett hjälpmedel för SiCell, en del av SciLifeLab Uppsala som ska bli Europas första plattform för enkelcellgenomik till hösten 2013. SiCell har bett projektgruppen om undersökningar gällande ett Laboratory Information Management System, LIMS. På svenska ett datahanteringsystem för laboratorier. Ett sådant system skulle kunna effektivisera SiCells verksamhet. Undersökningarna har resulterat i en kravspecifikation som ett LIMS för SiCell ska uppfylla och en sammanställning av tillgängliga mjukvaror som bäst uppfyller dessa krav. Screensaver, MISO och Gnomex är de tre gratisprogram med öppen källkod som hamnade högst upp i listan. Inget av dem uppfyller alla krav men med modifieringar av programmerare tros detta vara möjligt. SiCell bad också om lägre prioriterade undersökningar av några av de metoder som används inom plattformen. Cellysering, Alternativa amplifieringsmetoder och transkriptomik har undersökts av projektgruppen. Detta resulterade i en sammanställning av vilka alternativ som finns och vad som är under utveckling inom respektive område.

Protein ubiquitination is essential for the proper functioning of many eukaryotic cellular processes. The cleavage of ubiquitin chains from ubiquitinated proteins is performed by deubiquitinating enzymes, of which there are 16 in the Ubp (ubiquitin specific protease) group in the budding yeast Saccharomyces cerevisiae. The goal of my thesis has been to examine the biological roles and molecular functions of these enzymes using a combination of proteomic and molecular genetic approaches. As part of a large collaborative effort, interacting protein partners of the Ubps were isolated through affinity purification of tagged proteins, followed by protein identification by mass spectrometry. Purification of tagged Ubp6 led to the identification of the 19S proteasome complex, along with a novel subunit, Sem1. As the human homologue of Sem1 was previously identified as being associated with a protein involved in the repair of DNA double-strand breaks, I examined the possible role of Sem1 in DNA damage repair. A deletion of Sem1 and other 19S subunits resulted in hypersensitivity to various DNA damaging drugs, implicating the 19S complex in the process of DNA repair. iii I also found that purified Ubp2 interacted stably with the ubiquitin ligase Rsp5 and the protein Rup1. UBP2 interacts genetically with RSP5, indicating a functional relationship, while Rup1 facilitates the physical tethering of Ubp2 to Rsp5. Using the uracil permease Fur4, a Rsp5 substrate, as a model reporter, I found that ubp2Δ cells exhibited a temporal stabilization of Fur4 at the plasma membrane following the induction of endocytosis, implicating Ubp2 in protein sorting, specifically at the multivesicular body. In order to understand the role of Ubp2, I examined the effect of Ubp2 on Rsp5 function. I found that Rsp5, similar to its mammalian homologues, is auto-ubiquitinated in vivo, and that Ubp2 is able to directly deubiquitinate Rsp5 in vitro. Moreover, the presence of a substrate or Rup1 both resulted in increased autoubiquitination, implying an auto-inhibitory mechanism of Rsp5 regulation. Taken together, the data presented in this thesis implicate deubiquitinating enzymes in interesting and varied roles in the cell, and suggest a novel mechanism for the modulation of Rsp5-dependent trafficking processes.

<p>In the post-genomics era, the sheer volume of data is overwhelming without appropriate tools for data integration and analysis. Studying genomic sequences in the context of other related genomic sequences, i.e. comparative genomics, is a powerful technique enabling the identification of functionally interesting sequence regions based on the principal that similar sequences tend to be either homologous or provide similar functionality.</p> <p>Costs associated with full genome sequencing make it infeasible to sequence every genome of interest. Consequently, simple, smaller genomes are used as model organisms for more complex organisms, for instance, Mouse/Human. An annotated model organism provides a source of annotation for transcribed sequences and other gene regions of the more complex organism based on sequence homology. For example, the gene annotations from the model organism aid interpretation of expression studies in more complex organisms.</p> <p>To assist with comparative genomics research in the Arabidopsis/Brassica (Thale-cress/Canola) model-crop pair, a web-based, graphical genome browser (BioViz) was developed to display short Brassica genomic sequences in the context of the Arabidopsis model organism genome. This involved the development of graphical representations to integrate data from multiple sources and tools, and a novel user interface to provide the user with a more interactive web-based browsing experience. While BioViz was developed for the Arabidopsis/Brassica comparative genomics context, it could be applied to comparative browsing relative to other reference genomes.</p> <p>BioViz proved to be an valuable research support tool for Brassica / Arabidopsis comparative genomics. It provided convenient access to the underlying Arabidopsis annotation, allowed the user to view specific EST sequences in the context of the Arabidopsis genome and other related EST sequences. In addition, the limits to which the project pushed the SVG specification proved influential in the SVG community. The work done for BioViz inspired the definition of an opensource project to define standards for SVG based web applications and a standard framework for SVG based widget sets.</p>

Systematic genomic studies in the budding yeast S. cerevisiae have greatly increased our capacity to conduct functional profiling of the eukaryotic genome. I describe a new method that makes use of yeast “Barcoder” strains to uniquely tag strains in a yeast overexpression collection (FLEX) and to systematically examine the effects of gene overexpression on cell fitness. This novel system is compatible with the so-called Synthetic Genetic Array (SGA) method, which automates yeast genetics and allows introduction of marked query alleles of interest into arrayed collections of yeast mutants. I identified SDL interactions for two key regulatory kinases, Dun1 and Mck1, using my system. I also used my array and approach to identify SDL interactions for Dun1 that are only manifest in the presence of DNA damage. These studies demonstrate the utility of the pooled SGA-SDL method for systematic discovery of condition-specific genetic interactions in conserved biological pathways.

Systematic genomic studies in the budding yeast S. cerevisiae have greatly increased our capacity to conduct functional profiling of the eukaryotic genome. I describe a new method that makes use of yeast “Barcoder” strains to uniquely tag strains in a yeast overexpression collection (FLEX) and to systematically examine the effects of gene overexpression on cell fitness. This novel system is compatible with the so-called Synthetic Genetic Array (SGA) method, which automates yeast genetics and allows introduction of marked query alleles of interest into arrayed collections of yeast mutants. I identified SDL interactions for two key regulatory kinases, Dun1 and Mck1, using my system. I also used my array and approach to identify SDL interactions for Dun1 that are only manifest in the presence of DNA damage. These studies demonstrate the utility of the pooled SGA-SDL method for systematic discovery of condition-specific genetic interactions in conserved biological pathways.

Recent advances in DNA sequencing technology have enabled entire genomes to be sequenced quickly and accurately, resulting in an exponential increase in the number of organisms whose genome sequences have been elucidated. While the genome sequence of a given organism represents an important starting point in understanding its physiology, the functions of the protein products of many genes are still unknown; as such, computational methods for studying protein function are becoming increasingly important. In addition, this wealth of genomic information has created an unprecedented opportunity to compare the protein content of different organisms; among other applications, this can enable us to improve taxonomic classifications, to develop more accurate diagnostic tests for identifying particular bacteria, and to better understand protein content relationships in both closely-related and distantly-related organisms.<p> This thesis describes the design, evaluation, and use of a program called Proteome Subtraction and Intersection (PSI) that uses an idea called genome subtraction for discovering protein-phenotype relationships and for characterizing differences in protein content in groups of organisms. PSI takes as input a set of proteomes, as well as a partitioning of that set into a subset of "included" proteomes and a subset of "excluded" proteomes. Using reciprocal BLAST hits, PSI finds orthologous relationships among all the proteins in the proteomes from the original set, and then finds groups of orthologous proteins containing at least one orthologue from each of the proteomes in the "included" subset, and none from any of the proteomes in the "excluded" subset.<p> PSI is first applied to finding protein-phenotype relationships. By identifying proteins that are present in all sequenced isolates of the genus <i>Lactobacillus</i>, but not in the related bacterium <i>Pediococcus pentosaceus</i>, proteins are discovered that are likely to be responsible for the difference in cell shape between the lactobacilli and <i>P. pentosaceus</i>. In addition, proteins are identified that may be responsible for resistance to the antibiotic gatifloxacin in some lactic acid bacteria.<p> This thesis also explores the use of PSI for comparing protein content in groups of organisms. Based on the idea of genome subtraction, a novel metric is proposed for comparing the difference in protein content between two organisms. This metric is then used to create a phylogenetic tree for a large set of bacteria, which to the author's knowledge represents the largest phylogenetic tree created to date using protein content. In addition, PSI is used to find the proteomic cohesiveness of isolates of several bacterial species in order to support or refute their current taxonomic classifications.<p> Overall, PSI is a versatile tool with many interesting applications, and should become more and more valuable as additional genomic information becomes available.

Expansion of common bean (<i>Phaseolus vulgaris</i> L.) crops in the northern Great Planes has been hampered due to the lack of cultivars demonstrating sufficient vitality under low temperature conditions. <i>Phaseolus angustissimus</i> L., a wild bean species, has been previously shown to possess the ability to survive low temperatures in field trials. Freezing tolerance experiments under controlled conditions resulted in P. angustissimus demonstrating a greater capacity for freezing tolerance than <i>P. vulgaris</i>, as all P. vulgaris plants studied were dead at -2.5oC while most P. angustissimus plants treated to the same conditions survived. Exposure to chilling temperatures over five days resulted in stunted growth in both species, but the cultivated bean suffered more compared to the wild bean, as noted by a marked loss in tissue water content over the first three days of chilling. Interspecific macroarray hybridizations of a cDNA library from cold acclimated Medicago sativa L. using cDNAs derived from non-chilled and three-day chilled <i>P. vulgaris</i> and <i>P. angustissimus</i> plants showed that <i>P. vulgaris</i> showed more changes in gene expression after three days of chilling. Also, <i>P. vulgaris</i> showed a general trend towards down-regulation of the transcripts sampled on the third day of chilling compared to <i>P. angustissimus</i>. RT-PCR experiments were conducted using cDNAs from plant tissues exposed to various durations of chilling to confirm the results from the macroarray experiment. These time-course RT-PCR experiments revealed expression patterns across various chilling durations in genes identified from the macroarray. Data from these experiments suggest that <i>P. vulgaris</i> and <i>P. angustissimus </i> seedlings respond differently to low temperature exposure, and that some of the changes in <i>P. angustissimus</i> transcripts monitored here may be useful for researchers in better understanding how Phaseolus species can respond better to chilling temperatures.

Protein ubiquitination is essential for the proper functioning of many eukaryotic cellular processes. The cleavage of ubiquitin chains from ubiquitinated proteins is performed by deubiquitinating enzymes, of which there are 16 in the Ubp (ubiquitin specific protease) group in the budding yeast Saccharomyces cerevisiae. The goal of my thesis has been to examine the biological roles and molecular functions of these enzymes using a combination of proteomic and molecular genetic approaches. As part of a large collaborative effort, interacting protein partners of the Ubps were isolated through affinity purification of tagged proteins, followed by protein identification by mass spectrometry. Purification of tagged Ubp6 led to the identification of the 19S proteasome complex, along with a novel subunit, Sem1. As the human homologue of Sem1 was previously identified as being associated with a protein involved in the repair of DNA double-strand breaks, I examined the possible role of Sem1 in DNA damage repair. A deletion of Sem1 and other 19S subunits resulted in hypersensitivity to various DNA damaging drugs, implicating the 19S complex in the process of DNA repair. iii I also found that purified Ubp2 interacted stably with the ubiquitin ligase Rsp5 and the protein Rup1. UBP2 interacts genetically with RSP5, indicating a functional relationship, while Rup1 facilitates the physical tethering of Ubp2 to Rsp5. Using the uracil permease Fur4, a Rsp5 substrate, as a model reporter, I found that ubp2Δ cells exhibited a temporal stabilization of Fur4 at the plasma membrane following the induction of endocytosis, implicating Ubp2 in protein sorting, specifically at the multivesicular body. In order to understand the role of Ubp2, I examined the effect of Ubp2 on Rsp5 function. I found that Rsp5, similar to its mammalian homologues, is auto-ubiquitinated in vivo, and that Ubp2 is able to directly deubiquitinate Rsp5 in vitro. Moreover, the presence of a substrate or Rup1 both resulted in increased autoubiquitination, implying an auto-inhibitory mechanism of Rsp5 regulation. Taken together, the data presented in this thesis implicate deubiquitinating enzymes in interesting and varied roles in the cell, and suggest a novel mechanism for the modulation of Rsp5-dependent trafficking processes.

Genomic microarray analysis is rapidly replacing conventional chromosome analysis by molecular karyotyping due to the significant increase in the power to detect causative CNVs. Here, we extensively validated the HumanHap550 and Human610-Quadv1_B Illumina platforms for potential diagnostic application by using patients with undiagnosed intellectual disability (ID). The first and foremost goal of our application study was to use these arrays for reliable genome wide detection of rare CNVs in patients of three different cohorts: 1) patients with unexplained intellectual disability 2) patients with unknown diffuse congenital hyperinsulinism (CHI) and 3) a family with a distinctive diagnosis of Holt-Oram syndrome (HOS). We showed that SNP-based arrays allow the detection of intragenic deletions and duplications. The identification of a disease-CNV affecting only a single gene allowed us to consider that particular gene as a candidate for intellectual disability. This was the case for three unrelated patients with moderate intellectual disability, global developmental delay, and severe speech and language disorders in which a de novo deletion encompassing solely the FOXP1 gene was detected. To prove further the causality of the FOXP1 deletion following-up investigations were based on a screening of the entire coding region of FOXP1 for nucleotide changes in a panel of 883 probands with intellectual disability. Eight non-synonymous coding changes, three synonymous and nine non-coding variants were identified. In addition to the de novo cases of ID, also patients suffering from an autosomal recessive form of ID were found in our cohort. We detected three partial heterozygous deletions of the COH1 gene at locus 8q22 which is mutated in Cohen syndrome. After sequencing the entire coding region and the exon/intron boundaries of COH1 we identified a stop mutation, a frameshift and two missense mutations in the remaining allele, respectively. Therefore, three compound heterozygous mutations were identified in the COH1 gene, thus providing a distinctive Cohen Syndrome diagnose to three unrelated patients of our ID cohort. We studied the genetic basis of a rare human autosomal disorder such as diffuse Congenital Hyperinsulinsm (CHI) in a cohort of 40 patients with inconspicuous mutation screening of ABCC8 and KCNJ11 genes. Chromosomal abnormalities detected by SNP oligonucleotide arrays accounted for 20% of the studied cases. The most interesting rearrangement was a 970kb deletion at the chromosomal band 1p31.1 which was found to encompass the PTGER3 and ZRANB2 genes and the last exon of the NEGR1 gene. We hypothesized that the haploinsufficiency of PTGER3 gene induces a 50% reduction of the stimulation by PGE2, thus diminishing the inhibition of glucose-stimulated insulin secretion (GSIS) and resulting in elevated insulin secretion. The screening for point mutations in the candidate gene PTGER3 did not reveal any pathogenic variant neither in the second allele of the patient in which a de novo deletion was detected nor in a cohort of 39 unrelated patients with unexplained CHI. Instead we identified a novel polymorphic variant which was also detected in 18 individuals of our control cohort. CNV analysis in a family with both atypical Holt-Oram syndrome and additional mammary glands was performed allowing the detection of a contiguous heterozygous duplication at the chromosomal band 12q24.21. The maximal duplication size could be estimated as aproximately 345,6kb including the whole coding region of the TBX5 and TBX3 genes. Gene dosage assessment at specific genetic loci demonstrated the cosegregation of the duplication and the Holt-Oram syndrome/supernumerary mammary glands phenotype in this pedigree, this being a strong indicator of its pathogenecity. Up to date, this is the first report of a heterozygous duplication encompassing both TBX5 and TBX3 genes, and consequently the first report of a combined phenotype of Holt-Oram syndrome and supernumerary mammary glands.

Genetic and genomic data often contain unobservable or missing information. Applications of probabilistic models such as mixture models and hidden Markov models (HMMs) have been widely used since the 1960s to make inference on unobserved information using some observed information demonstrating the versatility and importance of these models. Biological applications of mixture models include gene expression data, meta-analysis, disease mapping, epidemiology and pharmacology and applications of HMMs include gene finding, linkage analysis, phylogenetic analysis and identifying regions of identity-by-descent. An important statistical and informatics challenge posed by modern genetics is to understand the functional consequences of genetic variation and its relation to phenotypic variation. In the analysis of whole-exome sequencing data, predicting the impact of missense mutations on protein function is an important factor in identifying and determining the clinical importance of disease susceptibility mutations in the absence of independent data determining impact on disease. In addition to the interpretation, identifying co-inherited regions of related individuals with Mendelian disorders can further narrow the search for disease susceptibility mutations. In this thesis, we develop two probabilistic models in application of genetic and genomic data with missing information: 1) a mixture model to estimate a posterior probability of functionality of missense mutations and 2) a HMM to identify co-inherited regions in the exomes of related individuals. The first application combines functional predictions from available computational or {\it in silico} methods which often have a high degree of disagreement leading to conflicting results for the user to assess the pathogenic impact of missense mutations on protein function. The second application considers extensions of a first-order HMM to include conditional emission probabilities varying as a function of minor allele frequency and a second-order dependence structure between observed variant calls. We apply these models to whole-exome sequencing data and show how these models can be used to identify disease susceptibility mutations. As disease-gene identification projects increasingly use next-generation sequencing, the probabilistic models developed in this thesis help identify and associate relevant disease-causing mutations with human disorders. The purpose of this thesis is to demonstrate that probabilistic models can contribute to more accurate and dependable inference based on genetic and genomic data with missing information.