The past few decades have witnessed a revolution in recombinant DNA and nucleic acid sequencing technologies. Recently however, technologies capable of massively high-throughout, genome-wide data collection, combined with computational and statistical tools for data mining, integration and modeling have enabled the construction of predictive networks that capture cellular regulatory states, paving the way for ‘Systems biology’. Consequently, protein interactions can be captured in the context of a cellular interaction network and emergent ‘system’ properties arrived at, that may not have been possible by conventional biology. The ability to generate data from multiple, non-redundant experimental sources is one of the important facets to systems biology. Towards this end, we have established a novel platform called ‘spotted cell microarrays’ for conducting image-based genetic screens. We have subsequently used spotted cell microarrays for studying multidimensional phenotypes in yeast under different regulatory states. In particular, we studied the response to mating pheromone using a cell microarray comprised of the yeast non-essential deletion library and analyzed morphology changes to identify novel genes that were involved in mating. An important aspect of the mating response pathway is large-scale spatiotemporal changes to the proteome, an aspect of proteomics, still largely obscure. In our next study, we used an imaging screen and a computational approach to predict and validate the complement of proteins that polarize and change localization towards the mating projection tip. By adopting such hybrid approaches, we have been able to, not only study proteins involved in specific pathways, but also their behavior in a systemic context, leading to a broader comprehension of cell function. Lastly, we have performed a novel metabolic starvation-based screen using the GFP-tagged collection to study proteome dynamics in response to nutrient limitation and are currently in the process of rationalizing our observations through follow-up experiments. We believe this study to have implications in evolutionarily conserved cellular mechanisms such as protein turnover, quiescence and aging. Our technique has therefore been applied towards addressing several interesting aspects of yeast cellular physiology and behavior and is now being extended to mammalian cells. / text

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This thesis contains information from the study of Norway spruce clones, which possess certain inheritable traits (1) of value for timber used in building constructions (i.e. higher wood basic density, elasticity, and resistance to crashing) and (2) greater wood yield. The following growth and quality traits of 90 clones in Baisogala seed orchard of Radviliškio forest enterprise were assessed: stem height and diameter, crown diameter, branching type and class of budburst. Significance of differences among the clones was estimated for all the these traits. Correlation coefficients among qualitative and quantitative traits were calculated. Owing to relatively lower radial increment and thicker annual ring, clones of brushy and flat branching types produced wood of a higher basic density and, thus, of a greater quality. A more slowly growth of clones with the above mentioned branching types was observed on their progeny as well. Therefore, if there is no need for stems of a relatively larger diameter, it would be more rational to produce Norway spruce stands of flat and brushy branching types.

Persons with severe hemophilia A exhibit significant variations in clinical phenotype; approximately 10% are milder bleeders. This study explored factors that might contribute to this inter-patient heterogeneity. Using a multi-center prospective study following boys with severe hemophilia A on a tailored primary prophylaxis regimen, age at first joint bleed, body mass index, protocol adherence and family activity level were modeled on the age at escalation from once-weekly to twice-weekly and alternate day infusions. Escalation, based on bleeding frequency, served as a surrogate marker of bleeding severity. We also assessed the roles of clinical and hemostatic parameters in predicting age at first joint bleed using a cross-sectional study. Results suggest that a delayed first joint bleed indicates later escalation (milder bleeding pattern) and that a longer lag time is associated with an earlier onset of joint bleeding. Larger studies are needed to definitively identify predictors of clinical heterogeneity in these patients.

The face of animal breeding has changed significantly over the past few decades. Traditionally, the genetic improvement of both plant and animal species focussed on the selective breeding of individuals with superior phenotypes, with no precise knowledge of the genes controlling the traits under selection. Over the past few decades, however, advances in molecular genetics have led to the identification of genetic markers associated with genes controlling economically important traits, which has enabled breeders to enhance the genetic improvement of breeding stock through linkage disequilibrium marker-assisted selection. Since the integration of marker-assisted selection into breeding programmes has not been widely documented, it is important that breeders are able to evaluate the advantages and disadvantages of marker-assisted selection, in comparison to phenotypic selection, prior to the implementation of either selection strategy. Therefore, this investigation aimed to develop deterministic simulation models that could accurately demonstrate and compare the effects of phenotypic selection and marker-assisted selection, under the assumption of both additive gene action and complete dominance at the loci of interest. Six computer models were developed using Microsoft Excel, namely 'Random Mating,' 'Phenotypic Selection,' 'Marker-Assisted Selection,' 'Selection with Dominance,' 'Direct Selection' and 'Indirect selection.' The 'Random Mating' model was firstly used to determine the effects of linkage disequilibrium between two loci in a randomly mating population. The 'Phenotypic Selection' and 'Marker-Assisted Selection' models focused primarily on examining and comparing the response to these two selection strategies over five generations and their consequent effect on genetic variation in a population when the QTL of interest exhibited additive gene action. In contrast, the 'Selection with Dominance' model investigated the efficiency of phenotypic selection and marker-assisted selection under the assumption of complete dominance at the QTL under selection. Finally, the 'Direct Selection and 'Indirect Selection' models were developed in order to mimic the effects of marker assisted selection on two cattle populations utilizing both a direct and indirect marker respectively. The simulated results showed that, under the assumption of additive gene action, marker-assisted selection was more effective than phenotypic selection in increasing the population mean, when linkage disequilibrium was present between the marker locus and the QTL under selection and the QTL captured more than 80% of the trait variance. The response to both selection strategies was shown to decrease over five generations due to the decrease in genetic variation associated with selection. When the QTL under selection was assumed to display complete dominance, however, marker-assisted selection was markedly more effective than phenotypic selection, even when a minimal amount of linkage disequilibrium was present in the population and the QTL captured only 60% of the trait variance. The results obtained in this investigation were successful in simulating the theoretical expectations of markerassisted selection. The computer models developed in this investigation have potential applications in both the research and agricultural sectors. For example, the successful application of a model developed in this investigation to a practical situation that simulated markerassisted selection, was demonstrated using data from two Holstein cattle populations. Furthermore, the computer models that have been developed may be used in education for the enhancement of students understanding of abstract genetics concepts such as linkage disequilibrium and marker-assisted selection. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

Persons with severe hemophilia A exhibit significant variations in clinical phenotype; approximately 10% are milder bleeders. This study explored factors that might contribute to this inter-patient heterogeneity. Using a multi-center prospective study following boys with severe hemophilia A on a tailored primary prophylaxis regimen, age at first joint bleed, body mass index, protocol adherence and family activity level were modeled on the age at escalation from once-weekly to twice-weekly and alternate day infusions. Escalation, based on bleeding frequency, served as a surrogate marker of bleeding severity. We also assessed the roles of clinical and hemostatic parameters in predicting age at first joint bleed using a cross-sectional study. Results suggest that a delayed first joint bleed indicates later escalation (milder bleeding pattern) and that a longer lag time is associated with an earlier onset of joint bleeding. Larger studies are needed to definitively identify predictors of clinical heterogeneity in these patients.

Background Colorectal cancer (CRC) is the second most common malignancy in developed countries. The mortality is high, with nearly half of patients dying from the disease. The primary treatment of CRC is surgery, and decisions about additional treatment with chemotherapy are based mainly on tumor stage. Novel prognostic markers that identify patients at high risk of recurrence and cancer-related death are needed. The development of CRC has been described in terms of two different pathways; the microsatellite instability (MSI) and chromosomal instability (microsatellite stable, MSS) pathway. More recently, the CpG island methylator phenotype (CIMP), characterized by frequent DNA hypermethylation, has been described as an alternative pathway of tumorigenesis. The event of DNA methylation is dependent on one-carbon metabolism, in which folate and vitamin B12 have essential functions. The purpose of this thesis was to study CIMP in CRC. The specific aims were to investigate the potential role of components of one-carbon metabolism as risk factors for this subgroup of tumors, and the prognostic importance of CIMP status, taking into consideration important confounding factors, such as MSI and tumor-infiltrating T cells. Methods CRC cases and referents included in the Northern Sweden Health and Disease Study (NSHDS, 226 cases and 437 referents) and CRC cases in the Colorectal Cancer in Umeå Study (CRUMS, n=490) were studied. Prediagnostic plasma concentrations of folate and vitamin B12 were analyzed in NSHDS. In both study groups, CIMP status was determined in archival tumor tissue by real-time quantitative PCR using an eight-gene panel (CDKN2A, MLH1, CACNA1G, NEUROG1, RUNX3, SOCS1, IGF2 and CRABP1). MSI screening status and the density of tumor-infiltrating T cells were determined by immunohistochemistry.  Results An inverse association was found between plasma concentrations of vitamin B12 and rectal, but not colon, cancer risk. We also found a reduced risk of CIMP-high and CIMP-low CRC in study subjects with the lowest levels of plasma folate. We found that patients with CIMP-low tumors in both NSHDS and CRUMS had a poorer prognosis compared with CIMP-negative, regardless of MSI screening status. We also found that MSS CIMP-high patients had a poorer prognosis compared with MSS CIMP-negative. The density of tumor-infiltrating T cells and CIMP status were both found to be independent predictors of CRC patient prognosis. A particularly poor prognosis was found in patients with CIMP-low tumors poorly infiltrated by T cells. In addition, the density of T cells appeared to be more important than MSI screening status for predicting CRC patient prognosis. Conclusion Rather than being one disease, CRC is a heterogeneous set of diseases with respect to clinico-pathological and molecular characteristics. We found that the association between risk and plasma concentration of vitamin B12 and folate depends on tumor site and CIMP status, respectively. Patient prognosis was found to be different depending on CIMP and MSI screening status, and the density of tumor-infiltrating T cells.

Leptin, a 16 (kilo Dalton) kDa hormone secreted predominantly by white adipocytes, regulates reproduction, energy intake and expenditure, and is involved in immune system function. Previous studies have identified associations between polymorphism E2FB in the leptin gene (lep) of cattle and milk quality and quantity, feed intake, and fat deposition in dairy and beef cattle though further studies have shown inconclusive results. Furthermore, indigenous South African cattle have not been involved in lep investigations or the applicability of the marker in South African beef grading systems. An investigation was conducted into the association of an SNP of a cytosine (C) to thymine (T) SNP (single nucleotide polymorphism) mutation in exon 2 of the bovine lep (leptin) gene with weight gain, body condition, carcass fat content and quality in a population of indigenous Nguni cattle (n = 70) as well as a population of exotic British Hereford cattle (n = 54). The Hereford population had higher T-allele frequencies and a lower P-value (P = 0.172) for the E2FB genotypes than the Nguni population (P = 0.958). The resulting E2FB lep genotypes CC, CT and TT did not show an association with the pre- and post-slaughter traits initial live weight (ILW), body condition score (BCS), slaughter live weight (SLW), carcass fat content (FAT), carcass conformation (CFN) or warm carcass mass (WCM) for either population though t-tests revealed an association with the CT genotype with increased ILW than TT and a significantly higher WG in the TT genotypes than the CT (P<0.05). Subsequently, differences in pre- and post-slaughter traits in both populations were largely attributable to breed differences. The Hereford population exhibited significantly higher WG, CFN, SLW, WCM and CCM (P<0.05) than the Nguni population. The Nguni displayed significantly higher ILW and BCS values when graded in terms of the commercial South African AAA feedlot system. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Atrial natriuretic factor (ANF) is a polypeptide hormone produced in heart atria, stored in atrial secretory granules and released into the circulation in response to various stimuli. Proper sorting of ANF at the level of the trans-Golgi network (TGN) is required for the storage of ANF in these specific granules, and this sorting of hormones has been found to be associated with G-proteins. Specifically, the Go protein alpha-subunit (Gαo) was established to participate in the stretch-secretion coupling of ANF, but may also be involved in the transporting of ANF from the TGN into atrial granules for storage and maturation. Based on knowledge of Gαo involvement in hormone production in other endocrine tissues, protein-protein interactions of Gαo and proANF and their immunochemical co-localization in granules, the direct involvement of these two proteins in atrial granule biogenesis is probable. In this study, mice were created using the Cre/lox recombination system with a conditional Gαo knockout in cardiocytes to study and characterize ANF production, secretion and granule formation. Deletion of this gene was successful following standard breeding protocols. Characterization and validation of cellular and molecular content of the knockout mice through mRNA levels, protein expression, peptide content, electron microscopy, and electrocardiography determined that a significant phenotypic difference was observed in the abundance of atrial granules. However, Gαo knockout mice did not significantly alter the production and secretion of ANF and only partially prevented granule biogenesis, likely due to incomplete Gαo knockout. These studies demonstrate an involvement of Gαo in specific atrial granule formation.

The aim of the thesis – to investigate the role of the inflammatory bowel disease associated genetic variants in a subset of Crohn’s disease and ulcerative colitis patients from Lithuania and Latvia and to test the relation of genetic markers to disease phenotype. The following objectives were accomplished: the associations of the inflammatory bowel disease associated single nucleotide polymorphisms in the subset of Crohn’s disease and ulcerative colitis patients were determined; the associations of the single nucleotide polymorphisms with the phenotype of the inflammatory bowel disease were evaluated; the interactions of single nucleotide polymorphisms (SNP-SNP) and their association with inflammatory bowel disease were determined; the significance of the combinations of disease associated single nucleotide polymorphisms for diagnosis of inflammatory bowel disease was evaluated. TaqMan and SNPlex genotyping methods were used in this work. The statistical data analysis consisted of: statistical study power and data quality evaluations; single marker case-control association analysis using χ2 or Fisher's exact tests, Breslow-Day test, Cochran-Mantel-Haenzsel test and post-hoc Bonferroni correction; genotype–phenotype association analysis using χ2 test and post-hoc Bonferroni correction; SNP-SNP epistasis for case-control sample using logistic regression test and post-hoc Bonferroni correction; In sicilo prediction of gene interactive network; genetic risk profile construction... [to full text] / Disertacijos tikslas – ištirti su uždegiminėmis žarnyno ligomis siejamų ge¬netinių žymenų reikšmę Krono liga bei opiniu kolitu sergantiems Lietuvos ir Latvijos ligoniams bei nustatyti žymenų ryšį su ligos fenotipu. Tikslui pasiekti, įgyvendinti šie uždaviniai: ištirta su uždegiminėmis žarnyno ligomis siejamų vieno nukleotido polimorfizmų sąsaja su Krono liga bei opiniu kolitu; nustatyta vieno nukleotido polimorfizmų sąsaja su uždegiminių žarny¬no ligų fenotipu; ištirtos vieno nukleotido polimorfizmų tarpusavio epistazinės sąvei¬kos (VNP-VNP) bei jų ryšys su uždegiminėmis žarnyno ligomis; nustatytas su liga siejamų vieno nukleotido polimorfizmų derinių reikšmingumas uždegiminių žarnyno ligų diagnostikai. Darbe taikyti SNPlex ir TaqMan genotipavimo metodai. Statistinę duomenų analizę sudarė: studijos galios bei duomenų kokybės įvertinimas; atvejo-kontrolės genų sąsajos tyrimas, taikant χ2 arba Fišerio kriterijus, Breslow-Day testą, Cochran-Mantel-Haenzsel testą bei post-hoc Bonferroni kriterijų; genų sąsajų su Krono ligos ir opinio kolito fenotipu tyrimai, naudojant χ2 bei post-hoc Bonferroni kriterijų; VNP-VNP epistazinės sąveikos įvertinimas, taikant logistinės regresijos testą bei post-hoc Bonferroni kriterijų; genų tarpusavio sąveikos tinklo in silico tyrimas; genetinės rizikos profilio sudarymas, naudojant logistinę regresinę analizę.