Parvoviruses cause dreadful enteritis in waterfowls and lead to tremendous financial losses. This study aims at developing effective way to prevent waterfowl parvoviral infection. Duck parvoviruses (DPVs) and goose parvoviruses (GPVs) were isolated from organs of infected waterfowls. The presence of virus in the specimens was identified using polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) analysis. To reveal the genetic variation of viral capsid proteins (VPs), full length VPs gene were amplified and sequenced. The sequence data indicated the sequences diverge 4.1 to 4.4% among viral strains isolated during 1990 to 1999. The variant amino acids cluster in the common regions of VP3 at residues 203-266 and 482-534, which overlaps with the regions proposed to expose on the outer surfaces of parvoviral particles. These data implying that selective pressure from host immune system might play a part. The nucleotide sequences of VPs also reveal that DPV and GPV share 77 % similarity at the DNA, and 84.6% at the protein level. The most variable regions reside in the N-terminal of VP2 before the initiation codon of VP3 with 35% (19/54) amino acids divergence. This study also reveals the presence of conserved strain-specific residues in VPs and these residues seldom vary among different isolates of the same virus, suggesting that they might be important in maintaining viral structure or host specificity which worth further investigation. To investigate the antigenicity of VPs, the GPV genomic DNA encoding common region of VPs was fused in frame with glutathione S-transferase (GST) gene for the expression of GST-GPV (248-516) fusion protein in bacterial cells. Purified fusion protein was used as immunogen for the generation of rabbit anti-GPV (248-516) antiserum. The potential diagnostic usage was confirmed by the fact that this antiserum was able to differentiate between viral infected and uninfected primary embryonic fibroblast cells by immunocytochemical analysis. In addition, VPs in purified DPV and GPV virions were analyzed by Western blotting. This antiserum detected two prominent proteins bands with the molecule weight of 80 and 70 kilodaltons, which correspond to the sizes of VP1 and VP2 reported in the literature. The fact that VP1 of DPV reacts weakly with this antiserum suggests the existence of antigenic discrepancy between DPV and GPV. For the purpose of developing subunit vaccine for the control of Derzy's disease, recombinant full length VPs were expressed using both prokaryotic, GST and histidine-tagged fusion proteins, and eukaryotic, baculovirus and mammalian vero cell, expression systems. After large- scale production and purification, same amount of 4 recombinant VPs were individually used to immunize 1-week-old geese. The antibodies induced after immunization were then evaluated by enzyme-linked immunosorbent assay (ELISA). All four recombinant proteins stimulate approximately 7 to 8 folds increases of ELISA antibodies titers, and together with preliminary data of safety tests suggest a potential usage as subunit vaccine for the control of parvoviral infection.

Understanding the factors and processes that influence intraspecific genetic variation are essential to better understand evolutionary processes. In this research, I examined patterns of gene flow and their effects on the distribution of genetic variation and spatial genetic structuring at different spatial scales. I used a combination of population genetics, spatial analysis, morphometrics and phylogeography in order to understand the patterns of genetic variation and their resulting phenotypic variations in a semi-aquatic species, the muskrat (Ondatra zibethicus).

The ATP-binding cassette transporter, sub-family A, member 1 (ABCA1) mediates the major pathway for cholesterol exit from non-hepatic cells and thereby controls the rate-limiting step in the biogenesis of high density lipoprotein (HDL) particles. In humans,ABCA1 deficiency results in Tangier disease, characterized by low levels of HDL cholesterol, cellular cholesterol accumulation and increased risk for atherosclerosis. More than 100 coding variants have been described in the ABCA1 gene. We attempted to understand how both naturally occurring and engineered mutations in ABCA1 impact its role in cholesterol transport in a variety of in vitro and in vivo systems. We attempted to correlate specific genetic variants in ABCA 1 with phenotypes in patients carrying the sevariants, and used an evolutionary approach to predict which specific variants in ABCA1would impact its function. We then turned to the study of tissue-specific genetic deletion of ABCA1 in mice to study its role in HDL biogenesis, atherosclerosis and glucose metabolism. We found that intestinal ABCA1 is an important site of HDL biogenesis and that activation of intestinal ABCA1 raises HDL levels in vivo. Hepatic ABCA1, which is a major site of HDL biogenesis, was shown to significantly contribute to susceptibility to atherosclerosis. Finally, we show that ABCA1 plays an unsuspected role in B-cell function and insulin secretion. These studies have contributed to our understanding of the impact of genetic variation in ABCA1 on diverse biological and pathological processes, and have identified novel aspects of ABCA 1 function in specific cell types.

The emerging field of personalized medicine and the prediction of side effects experienced due to pharmaceutical drugs is being studied intensively in the post genomic era. The molecular basis of inheritance and disease susceptibility is being unravelled, especially through the use of rapidly evolving new technologies. This in turn facilitates analyses of individual variations in the whole genome of both single subjects and large groups of subjects. Genetic variation is a common occurrence and although most genetic variations do not have any apparent effect on the gene product some do exhibit effects, such as an altered ability to detoxify xenobiotics. The human body has a highly effective detoxification system that detoxifies and excretes endogenous as well as exogenous toxins. Numerous studies have proved that specific genetic variations have an influence on the efficacy of the metabolism of pharmaceutical drugs and consequently the dosage administered. The primary aim of this project was the local implementation and assessment of two different genotyping approaches namely: the Applied Biosystems SNaPshot technique and Affymetrix DMET microarray. A secondary aim was to investigate if links could be found between the genetic data and the biochemical detoxification profile of participants. I investigated the approaches and gained insight into which method would be better for specific local applications, taking into consideration the robustness and ease of implementation as well as cost effectiveness in terms of data generated. The final study cohort comprised of 18 participants whose detoxification profiles were known. Genotyping was performed using the DMET microarray and SNaPshot techniques. The SNaPshot technique was used to genotype 11 SNPs relating to DNA repair and detoxification and was performed locally. Each DMET microarray delivers significantly more data in that it genotypes 1931 genetic markers relating to drug metabolism and transport. Due to the absence of a local service supplier, the DMET - microarrays were outsourced to DNALink in South Korea. DNALink generated raw data which was analysed locally. I experienced many problems with the implementation of the SNaPshot technique. Numerous avenues of troubleshooting were explored with varying degrees of success. I concluded that SNaPshot technology is not the best suited approach for genotyping. Data obtained from the DMET microarray was fed into the DMET console software to obtain genotypes and subsequently analysed with the help of the NWU statistical consultation services. Two approaches were followed: firstly, clustering the data and, secondly, a targeted gene approach. Neither of the two methods was able to establish a relationship between the DMET genotyping data and the detoxification profiling. For future studies to successfully correlate SNPs or SNP groups and a specific detoxification profile, two key issues should be addressed: i) The procedure for determining the detoxification profile following substrate loading should be further refined by more frequent sampling after substrate loading. ii) The number of participants should be increased to provide statistical power that will enable a true representation of the particular genetic markers in the specific population. The statistical analyses, such as latent class analyses to cluster the participants will also be of much more use for data analyses and interpretation if the study is not underpowered. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

The emerging field of personalized medicine and the prediction of side effects experienced due to pharmaceutical drugs is being studied intensively in the post genomic era. The molecular basis of inheritance and disease susceptibility is being unravelled, especially through the use of rapidly evolving new technologies. This in turn facilitates analyses of individual variations in the whole genome of both single subjects and large groups of subjects. Genetic variation is a common occurrence and although most genetic variations do not have any apparent effect on the gene product some do exhibit effects, such as an altered ability to detoxify xenobiotics. The human body has a highly effective detoxification system that detoxifies and excretes endogenous as well as exogenous toxins. Numerous studies have proved that specific genetic variations have an influence on the efficacy of the metabolism of pharmaceutical drugs and consequently the dosage administered. The primary aim of this project was the local implementation and assessment of two different genotyping approaches namely: the Applied Biosystems SNaPshot technique and Affymetrix DMET microarray. A secondary aim was to investigate if links could be found between the genetic data and the biochemical detoxification profile of participants. I investigated the approaches and gained insight into which method would be better for specific local applications, taking into consideration the robustness and ease of implementation as well as cost effectiveness in terms of data generated. The final study cohort comprised of 18 participants whose detoxification profiles were known. Genotyping was performed using the DMET microarray and SNaPshot techniques. The SNaPshot technique was used to genotype 11 SNPs relating to DNA repair and detoxification and was performed locally. Each DMET microarray delivers significantly more data in that it genotypes 1931 genetic markers relating to drug metabolism and transport. Due to the absence of a local service supplier, the DMET - microarrays were outsourced to DNALink in South Korea. DNALink generated raw data which was analysed locally. I experienced many problems with the implementation of the SNaPshot technique. Numerous avenues of troubleshooting were explored with varying degrees of success. I concluded that SNaPshot technology is not the best suited approach for genotyping. Data obtained from the DMET microarray was fed into the DMET console software to obtain genotypes and subsequently analysed with the help of the NWU statistical consultation services. Two approaches were followed: firstly, clustering the data and, secondly, a targeted gene approach. Neither of the two methods was able to establish a relationship between the DMET genotyping data and the detoxification profiling. For future studies to successfully correlate SNPs or SNP groups and a specific detoxification profile, two key issues should be addressed: i) The procedure for determining the detoxification profile following substrate loading should be further refined by more frequent sampling after substrate loading. ii) The number of participants should be increased to provide statistical power that will enable a true representation of the particular genetic markers in the specific population. The statistical analyses, such as latent class analyses to cluster the participants will also be of much more use for data analyses and interpretation if the study is not underpowered. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

Diss. (sammanfattning) Alnarp : Sveriges lantbruksuniversitet, 2006. / Härtill 5 uppsatser.

This thesis comprises of two published papers and one accepted manuscript, focused on various aspects of liverwort reproduction. Treated aspects include patterns of asexual reproduction, sex ratio and sex-specic pattern in vegetative growth, and patterns of genetic variation and spatial genetic structure of populations differing in availability of substrate on localities and the population connectivity, and consequently in size, density, and prevailing reproductive mode. These characteristics were studied on representatives of the family Scapaniaceae s.l., belonging to the largest liverwort order Jungermanniales. The results showed that asexual propagules were formed and present in course of the whole growing season and can be considered as a sufficient substitution for sexual reproduction. In contrast with the female-biased sex ratio observed earlier in most dioicous bryophytes, unexpectedly high male-biased sex ratio was observed in the aquatic liverwort, which was speculated to represent a strategy to overcome sperm dilution in aquatic environment. In addition, no size differences between female and male shoots were detected, although the evidence for higher cost of sexual reproduction in females was found. The study of population genetic structure has shown that even small and predominantly asexually reproducing populations are important sources of genetic variation. However, we were able to demonstrate notably low levels of gene flow among populations where habitat fragmentation poses a significant barrier to dispersal of diaspores. The fine scale study of spatial genetic structure revealed a strong aggregation of genotypes, particularly in smaller populations, and at the same time showed that asexual reproduction is an efficient mean of maintaining the populations at not only the short distances, given the spatial extent of clones spanning dozens of meters.

The ATP-binding cassette transporter, sub-family A, member 1 (ABCA1) mediates the major pathway for cholesterol exit from non-hepatic cells and thereby controls the rate-limiting step in the biogenesis of high density lipoprotein (HDL) particles. In humans,ABCA1 deficiency results in Tangier disease, characterized by low levels of HDL cholesterol, cellular cholesterol accumulation and increased risk for atherosclerosis. More than 100 coding variants have been described in the ABCA1 gene. We attempted to understand how both naturally occurring and engineered mutations in ABCA1 impact its role in cholesterol transport in a variety of in vitro and in vivo systems. We attempted to correlate specific genetic variants in ABCA 1 with phenotypes in patients carrying the sevariants, and used an evolutionary approach to predict which specific variants in ABCA1would impact its function. We then turned to the study of tissue-specific genetic deletion of ABCA1 in mice to study its role in HDL biogenesis, atherosclerosis and glucose metabolism. We found that intestinal ABCA1 is an important site of HDL biogenesis and that activation of intestinal ABCA1 raises HDL levels in vivo. Hepatic ABCA1, which is a major site of HDL biogenesis, was shown to significantly contribute to susceptibility to atherosclerosis. Finally, we show that ABCA1 plays an unsuspected role in B-cell function and insulin secretion. These studies have contributed to our understanding of the impact of genetic variation in ABCA1 on diverse biological and pathological processes, and have identified novel aspects of ABCA 1 function in specific cell types. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

A survey of the genetic variation in the ability to produce brown pupae was conducted using 23 broods reared from wild caught females. In addition a selection experiment was designed to investigate the genetic basis of the dimorphism. It was concluded that the genetic basis of the dimorphism is a threshold trait with quantitative genetic variation underlying the two phenotypes of green and brown pupal color. It was further concluded that the dimorphism is maintained in nature by weak stabilizing selection, thus explaining the occurrence of mismatches in pupal and background color. / Master of Science

Breed characterization is a primary step in designing appropriate management and conservation programs of livestock in developing countries. Since cattle represent a major food animal species in Zambia, its conservation is a major goal for both the government and non-governmental organizations. To support the conservation effort, the objective of this thesis research was to assess the phenotypic and molecular characteristics of indigenous Zambian cattle breeds including Angoni, Barotse, Tonga, and Baila based on body measurements and randomly amplified polymorphic DNA (RAPD) markers, respectively. A total of 100 animals, 25 from each of the four breeds associated with different tribes and region of Zambia, were used in the molecular analysis research. Additionally, 10 Holstein x Jersey crossbred animals were used as a reference and to test the extent of cross-breeding, if any, of the indigenous stock with exotic breeds. To further compare the Zambian indigenous breeds, morphometric measurements including body length, heart girth, and height at withers on 50 animals of each breed were measured. Blood was collected from animals at randomly selected farms and DNA isolated by standard protocols in Zambia. A total of 10 primers, of the 20 evaluated for informativeness, were used in the RAPD-PCR analyses. Differences among the four breeds for all the three morphometric measurements were significant with the Barotse significantly higher than the other three (P<0.05). The average number of bands per primer was 7.1 and the percentage of polymorphic bands per primer ranged from 40 to 71.4 with an average of 64.8%. Breed divergence was highest between the Tonga and the Barotse and lowest between the Tonga and Baila breeds. Both the morphometric measurements and RAPD-based distance estimates suggest that the Barotse may be different from the other indigenous breeds while the Tonga and Baila were more closely related. In addition, the genetic distance estimates imply that the Holstein x Jersey crosses are different from the four Zambian indigenous cattle breeds evaluated. This thesis research provides, for the first time, the basic genetic information necessary for conservation of Zambian cattle breeds and the use of these populations for effective crossbreeding. The data suggest that though there is isolated by geographic distance and cultural differences among the tribes, two of the breeds are significantly related. / Master of Science