Tesi sul tema "Genetic analyses"

Methods that enable interrogation of multiple genomic regions in parallel are very useful for efficient detection of genetic variation. Two different types of probes are described in this thesis that can be used for direct analysis or for sample preparation upstream of Next Generation Sequencing.  In addition to the development of molecular probing systems it also reports on the progress of two assay formats for biological experiments. The Selector probe enrich for genomic regions of interest by probe mediated specific circularization of target fragments. Amplification based enrichment of circles can be carried out using polymerase chain reaction, rolling-circle amplification or multiple displacement amplification. Enrichment of all exons in 28 genes known to be mutated in lung and/or colon cancer is demonstrated.  Selection and analysis by SOLiD Sequencing was performed on fresh frozen and formalin fixed paraffin embedded (FFPE) samples, and mutations previously detected by Sanger sequencing were detected.  The extractor probe is another probe variant that can be used for multiplex enrichment of DNA. It targets genomic fragments by using both ligation and sequence specific elongation for discrimination between on and off target sequences. A microfluidic platform fabricated by compact disc injection molding that can be used for biological assays is described.  Microchannel structures in thermoplastic material are coated with silicon dioxide by electron beam evaporation which facilitates closing of the structures by PDMS- glass bonding by ozone plasma. The platform’s utility for biological experiments is demonstrated by for detection of amplified single molecules (ASM), cell culturing and on-chip peristaltic pumping. The thesis also includes an exploratory study for the purpose of using a non-optical system for detection of ASM’s.  Optimizations were performed of the conditions needed in order to detect an increase in hydrodynamic size of magnetic particles, using a superconducting quantum interference device (SQUID), as they form complex with ASM’s.

Complex phenotypes are traits which are influenced by many factors, and not just a single gene, as for classical Mendelian traits. The brain, and its resultant behaviour, gives us a large subset of complex phenotypes to examine. Variation in these traits is affected by a range of different influences, both genetic and environmental, including social interactions and the effects of parents. Systems-genetics provides us with a framework in which to examine these complex traits, seeking to connect genetic variants to the phenotypes they cause, through intermediate phenotypes, such as gene expression and protein levels. This approach has been developed to exploit and analyse massive data sets generated for example in genomics and transcriptomics. In the first half of this thesis, I combine genetic linkage data from the BXD recombinant inbred mouse panel with genome-wide association data from humans to identify novel candidate genes, and use online gene annotations and functional descriptions to support these candidates. Firstly, I discovered MGST3 as a novel regulator of hippocampus size, which may be linked to neurodegenerative disorders. Secondly, I identified that CMYA5, MCTP1, TNR and RXRG are associated with mouse anxiety-like phenotypes and human bipolar disorder, and provide evidence that MCTP1, TNR and RXRG may be acting via inter-cellular signalling in the striatum. The second half of this thesis uses different cross-fostering designs between genetically variable BXD lines and the genetically uniform C57BL/6J strain to identify indirect genetic effects and the loci underlying them. With this, I have found novel loci expressed in mothers that alter offspring behaviour, novel loci expressed in offspring affecting the level of maternal care, and novel loci expressed in offspring, which alter the behaviour of their nestmates, as well as the level of maternal care they receive. Further I provide evidence of co-adaptation between maternal and offspring genotypes, and a positive indirect genetic effect of offspring on their nestmates, supportive of a role for kin selection. Finally, I demonstrate that the BXD lines can be used to investigate genes with parent-of-origin dependent expression, which have an indirect genetic effect on maternal care. In conclusion, this thesis identifies a number of novel loci, and in some cases genes, associated with complex traits. Not only are these techniques applicable to other phenotypes and other questions, but the candidates I identify can now be examined further in vitro or in vivo.

The identification of factors underlying complex trait variation is a major goal in the field of genetics. For normally distributed, fully observed trait data there are many well established statistical methods for partitioning phenotypic variation and for mapping quantitative trait loci (QTL). Survival or time-to-event traits often follow non-normal distributions and frequently contain partially-known (or censored) trait data. If standard statistical methods are used to analyse age at onset data a bias can be introduced through a failure to account for the non-normal distribution of the data and the presence of censoring. Complex statistical methods have been developed to partition trait variation and map QTL for age at onset or survival traits. In this thesis, the use of these survival analysis methods is compared to more established statistical methods for the analysis of age-at-onset data. A brief introduction to the analysis of human variation and the issues associated with the analysis of age at onset data is given. The methods currently used to partition trait variation and map QTL for survival traits are discussed (Chapter 1). Age-specific penetrances can be used to model the age-at-onset of disease in unaffected individuals. This parametric method is used to identify loci underlying susceptibility to a novel co-morbid psychiatric phenotype (depression and unexplained swelling). The method is compared to a non-parametric variance component (VC) QTL mapping method that does not account for the age at onset of the disease. Parametric linkage analysis identified two suggestive loci, neither of which were supported by the standard variance component analysis. VC analysis identified a suggestive linkage region on chromosome 14 which decreased upon fine mapping (Chapter 2). Many of the current methods used to analyse survival data in human genetics are based on methods originally derived by animal geneticists. The analysis of survival traits in some experimental populations is simplified by the presence of fully inbred lines. However, for complex traits the methods are both computationally intensive and not widely available. A grouped linear regression method is proposed for the analysis of continuous survival data in fully inbred lines. Using simulation the method is compared to both the Cox and Weibull proportional hazards models and a standard linear regression method that ignores censoring. The grouped linear regression method is of equivalent power to both the Cox and Weibull proportional hazards methods, is significantly better than the standard linear regression method when censored observations are present and is computationally simple (Chapter 3). A sample of 446 monozygotic (MZ) twins, 633 dizygotic (DZ) twins and 223 siblings was used to partition the inter-individual variance in age at menarche. The analysis was carried out using both a standard method which failed to account for the censored nature of the data and a mixed effects Cox model which fits a frailty model to the random effects. The standard methodology suggested that an additive genetic model best described the data. The most parsimonious model when using the frailty method included additive genetic and common environmental effects (ACE). The difference between the two models was caused by the different ascertainment of the siblings. The frailty model estimated the heritability of age at menarche to be 0.57 (Chapter 4). In Chapter 5, a sample of 2,685 pseudo-independent sib-pairs is used in a genomewide linkage scan for QTL underlying variation in age-at-menarche. The sample comprises of the adolescent sample discussed in chapter 4, and three adult cohorts. The proportion of censoring in the sample is 1.20% so a standard QTL mapping method is used. Two QTL of suggestive significance are identified on chromosomes 11p and 3p. The candidate genes WT1 and FSHB are located within the linkage peak on 11p. After the removal of bivariate outliers a locus on chromosome 12q was identified. No significant QTL were detected which suggests age-at-menarche is influenced by multiple genes of small effect. The thesis concludes with a general discussion (Chapter 6).

A proper understanding of biological processes requires an understanding of genetics and evolutionary mechanisms. The vast amounts of genetical information that can routinely be extracted with modern technology have so far not been accompanied by an equally extended understanding of the corresponding processes. The relationship between a single gene and the resulting properties, phenotype of an individual is rarely clear. This thesis addresses several computational challenges regarding identifying and assessing the effects of quantitative trait loci (QTL), genomic positions where variation is affecting a trait. The genetic information available for each individual is rarely complete, meaning that the unknown variable of the genotype in the loci modelled also needs to be addressed. This thesis contains the presentation of new tools for employing the information that is available in a way that maximizes the information used, by using hidden Markov models (HMMs), resulting in a change in algorithm runtime complexity from exponential to log-linear, in terms of the number of markers. It also proposes the introduction of inferred haplotypes to further increase the power to assess these unknown variables for pedigrees of related genetically diverse individuals. Modelling consequences of partial genetic information are also treated. Furthermore, genes are not directly affecting traits, but are rather expressed in the environment of and in concordance with other genes. Therefore, significant interactions can be expected within genes, where some combination of genetic variation gives a pronounced, or even opposite, effect, compared to when occurring separately. This thesis addresses how to perform efficient scans for multiple interacting loci, as well as how to derive highly accurate empirical significance tests in these settings. This is done by analyzing the mathematical properties of the objective function describing the quality of model fits, and reformulating it through a simple transformation. Combined with the presented prototype of a problem-solving environment, these developments can make multi-dimensional searches for QTL routine, allowing the pursuit of new biological insight.
eSSENCE

The aim of this thesis is to understand the genetic basis of the variation of human quantitative traits using data from twins and (to some extent) their families. Traits investigated include behavioural traits (intelligence), clinical traits (the metabolic syndrome) and anthropometric measures (height). The importance of human twins for understanding genetic variation in human quantitative traits is reviewed. The use of a novel finite mixture distribution model to partition phenotypic co(variance) of a trait into underlying genetic and environmental factors from twins of unknown zygosity is presented. The Scottish Mental Surveys of 1^st June 1932 and 4^th June 1947, respectively, administered the same validated verbal reasoning test (the Moray House Test) to almost everyone born in 1921 or 1936 and attending school in Scotland. Information on zygosity was unavailable. A novel application of a finite mixture distribution model estimated a large and consistent heritability of cognitive ability of about ~0/7. This study is the first to estimate genetic and environmental components of cognitive ability in entire school-attending populations and implies that large (national) data collections can provide sufficient information on twin pairs to estimate genetic parameters, even without known zygosity. The precision and bias of the finite mixture distribution model were assessed using computer simulations and application to IQ measures from a large sample of known zygosity twins (i.e. twins from the UK Twins’ Early Development Studies). It is shown that the mixture distribution is unbiased provided that the twins’ trait values are (bivariate) normally distributed and the sample size is large. However, it the bivariate normality assumption is violated, then the mixture distribution provides biased estimates.  The extension of the model to multivariate analysis is discussed. The simulations show that multivariate analysis decreases the standard error of the variance component estimates. Another statistical model, a mixed linear model is used to partition the phenotypic (co)variances of traits into genetic and environmental factors from twins of known zygosity (twins from the Danish Twin Registry). Its application to understand underlying genetic and environmental aetiology showed that endophenotypes associated with the metabolic syndrome do not appear to share a substantial common genetic or familial environmental background. Finally, a genome-wide linkage analysis to identify gene/chromosomal regions associated with adult height reveals several chromosomal regions that showed a modest linkage to adult height. This analysis provides further evidence for the polygenic nature of body height.

In the present study, within and among breed genetic diversity in thirteen Turkish sheep breeds (Sakiz, Karagü
l, Hemsin, Ç
ine Ç
apari, Norduz, Herik, Akkaraman, Dagliç
, Gö
kç
eada, Ivesi, Karayaka, Kivircik and Morkaraman
in total represented by 628 individuals) were analyzed based on 20 microsatellite loci. Loci were amplified by Polymerase Chain Reactions and products were electronically recorded and converted into [628 x 20] matrix representing genotypes of individuals. Reliability of the genotyping and genetic diversity analyses were done by means of various bioinformatics tools. For the analyses, various statistical methods (Fisher'
s Exact Test, Neighbor-Joining tree construction, Factorial Correspondence Analysis (FCA), Analysis of Molecular Variation, Structure Analysis and Delaunay Analysis) were used. Since, inputs of some software were not compatible with the outputs of other software some Java classes were written whenever necessary. Analyses revealed that among the major breeds Dagliç
, Karayaka and Morkaraman breeds are highly admixed but Kivircik, Akkaraman and Ivesi are relatively distinct. Among the minor breeds, distinctness of Hemsin, Sakiz, Ç
ine Ç
apari, Gö
kç
eada and Karagü
l are more pronounced compared to all of the examined breeds. Since highly admixed individuals can be identified by Structure and FCA tests, results of the present study, which is part of a national project with the acronym TURKHAYGEN-I (www.turkhaygen.gov.tr), were found to be promising in establishing and managing relatively pure conservation flocks for the Turkish native sheep breeds which are believed to be the reservoirs of genetic variability.

Chloroplasts are photosynthetic organelles in plant and algal cells that capture sunlight energy to form energy-rich molecules that are the basis for almost all life. Chloroplast development requires more than 3000 different proteins, most of which are encoded by nuclear DNA. Thus, chloroplasts must import most of their proteins from the cytosol. They are surrounded by a double membrane called the envelope. Embedded in the envelope are the TOC and TIC complexes (translocon at the outer and inner envelope membrane of the chloroplast, respectively), which mediate protein import into the organelle. Several components of the TOC and TIC complexes have been identified. One example is the receptor Toc159, which in the model plant Arabidopsis thaliana has four isoforms: atToc159, atToc132, atToc120 and atToc90. It is known that atToc159 supports accumulation of photosynthetic proteins, while atToc132 and atToc120 support the import of non-photosynthetic, housekeeping proteins. However, the role of atToc90 remains uncertain. I investigated the function of atToc90 genetically by studying a series of Arabidopsis toc90 double and triple mutants, and by overexpressing atToc90 in mutants lacking other receptor isoforms. This work suggested limited functional redundancy between atToc90 and other TOC receptors (most notably, atToc159). By tagging TOC receptors known to act in each of the photosynthetic and non-photosynthetic import pathways, I was able to purify different TOC complexes from transgenic plants using tandem affinity purification (TAP). This indicated that atToc90 is present promiscuously in both atToc159- and atToc132/120-containing TOC complexes. Publicly available Affymetrix microarray data suggested a role for atToc90 during senescence. Thus, I investigated whether toc90 knockout mutants display any differences from wild type regarding leaf senescence. Indeed, some defects were observed, suggesting a role for atToc90 in the biochemical changes that occur in chloroplasts during leaf senescence.

Thesis (MScAgric (Animal Sciences))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: Fluctuations and a general decline in the ratio between wool and meat prices resulted in marked changes in the South African sheep industry. Commercial producers now exploit other mechanisms such as terminal crossbreeding of Merino-type with meat type breeds or dual-purpose breeds to attain short-term benefits resulting from price fluctuations between wool and mutton without compromising the wool-producing capacities of ewe flocks. Most components of lamb production have low heritability. However, heterosis can be achieved by mating wool-type breeds with specialist meat breed rams. Genetic improvement of livestock depends on defining breeding objectives, estimation of genetic parameters and accurately identifying the right animals to be used for future breeding. Genetic parameters for traits of economic importance in terminal sire sheep breeds that could be used on Merino-type ewes in commercial operations in South Africa had not been published for the national flock apart from a preliminary study having been conducted by Olivier et al. (2004). Selection objectives were poorly defined due to lack of parameter estimates for variance and covariance components. Against this background, this study obtained pedigree information and live weight data from the National Small Stock Improvement Scheme for the Dormer, Ile de France and Merino Landsheep and estimated non-genetic factors and genetic parameters influencing early growth traits. Genetic and phenotypic trends for early growth traits were constructed for the three breeds to monitor genetic progress. Non-genetic factors influencing early growth traits in the Dormer, Ile de France and Merino Landsheep were estimated using data obtained from the National Small Stock Improvement Scheme of South Africa. The original data sets for the Dormer, Ile de France and Merino Landsheep consisted of the following number of records respectively: 52 202, 35 553 and 7 772. However, pre-weaning weights were available for the Ile de France and Merino Landsheep breeds only and post-weaning weights were available only for the Dormer breed. The data sets were complicated to such an extent that smaller data sets had to be generated to analyse for fixed effects. The traits that were analysed were birth weight, pre-weaning weight, weaning weight and post-weaning weight. The fixed effects, identified as having a significant effect (P < 0.01) on early growth traits were sex of lamb, birth type, age of dam, contemporary groups, age at which the trait was recorded and month of birth and year of birth in the Merino Landsheep breed. Although some significant interactions were found, they were subsequently ignored owing to their very small effects. In all three breeds, male lambs were significantly (P < 0.001) heavier than female lambs and single-borne lambs were significantly heavier at birth than multiple borne lambs. The age of dam had a significant curvilinear regression on all early growth traits in all three terminal sire sheep breeds. It was concluded from the study that the influence of non-genetic factors on early growth traits should be adjusted for or eliminated statistically in genetic evaluations to get accurate genetic parameter estimations. (Co)variance estimates for birth weight, weaning weight and post-weaning weight were obtained for the Dormer breed using restricted maximum likelihood procedures (REML). Direct heritabilities (h2) in single-trait analyses were 0.21 ± 0.03, 0.23 ± 0.02 and 0.29 ± 0.05 for birth weight, weaning weight and post-weaning weight, respectively. Direct heritabilities of 0.28 ± 0.04, 0.55 ± 0.06 and 0.32 ± 0.02 for birth weight, weaning weight and post-weaning weight respectively were obtained using three-trait analysis. Direct maternal genetic effects (m2) were excluded from the analyses because of the failure to partition maternal effects into maternal genetic and maternal permanent environmental effects (m2 and c2). This culminated as a consequence of poor data and population structures emanating from the loss of genetic links across flocks due to the random entrance and exit of flocks from the recording scheme. Maternal permanent environment was estimated at 0.15 ± 0.02, 0.13 ± 0.02 and 0.20 ± 0.03 for birth weight, weaning weight and post-weaning weight respectively using single-trait analysis. The correlation between direct effects and maternal effects (ram) was excluded from the analyses due the structure of the data. Genetic, phenotypic and environmental correlations between early growth traits were low to moderate. The medium to high heritability estimates for early growth traits obtained in the study led to the conclusion that Dormer sheep can successfully be used in terminal crossbreeding programs to improve meat production characteristics. Direct heritability estimates were 0.31 ± 0.14, 0.09 ± 0.02 and 0.14 ± 0.003 for birth weight, pre-weaning weight and weaning weight respectively using single-trait analysis for the Ile de France breed. Maternal effects were significant for all the traits studied despite the failure to properly partition them into their components due to the loss of genetic linkages across generations emanating from poor data structure. Genetic, phenotypic and environmental correlations were estimated using three-trait analysis and were found to be low to moderate for early growth traits. Direct genetic and maternal permanent environmental ratios were also computed and they did not differ much from the results obtained using single-trait analyses. The reasonable genetic parameter estimates obtained in the study led to the conclusion that the Ile de France can be selected to use as sires in crossbreeding programs. Genetic parameters were estimated for early growth traits in the Merino Landsheep breed. REML estimates of birth weight, pre-weaning weight and weaning weight were obtained using animal models in single-trait analyses. The direct heritability estimate for birth weight was 0.23 ± 0.13 using an animal model with additive direct genetic effects and dam permanent environmental effects as the only random factors. The dam permanent environmental effect for birth weight amounted to 0.10 ± 0.07. Direct heritability for pre-weaning weight was 0.36 ± 0.05 and the dam permanent environmental effect 0.56 ± 0.03. Weaning weight was estimated using an animal model that contained direct additive effects and dam permanent environmental effects. The direct heritability estimate for weaning weight was 0.17 ± 0.03. Maternal genetic effects were estimated to be 0.02 ± 0.01. Genetic and phenotypic trends were constructed for early growth traits in the Dormer, Ile de France and Merino Landsheep breeds. The traits that were considered were birth weight, pre-weaning weight, weaning weight and post-weaning weight. However, pre-weaning weights were available for the Ile de France and Merino Landsheep breeds only and post-weaning weights were available only for the Dormer breed. The Dormer exhibited significant improvement in the phenotypic and genetic aspects of early growth traits during the 17 years of evaluation (1990-2007). The average predicted direct breeding values of birth weight decreased by 0.055 % during the evaluation period. The predicted direct breeding value for weaning weight increased by 0.12 % during the 17 year period. Post-weaning weight improved by 0.32 % per annum. The Ile de France registered an increase in the predicted breeding value of birth weight which amounted to 0.025 % per annum. Averaged direct breeding values for pre-weaning weight increased at an annual rate of 0.23 %. and that of weaning weight increased by 1.21 %. In the Merino Landsheep the predicted direct breeding value for birth weights decreased by 0.04 % per annum and pre-weaning and weaning weights increased by 0.36 % and 0.10 % respectively. The trends were obviously biased due to inconsistencies in data structure and very few records available for analysis in this breed. In conclusion, it was evident that the additive genetic variation was available for all the early growth traits in all the three breeds. Although adequate genetic variation for substantial genetic progress was available, only modest rates of progress were attained for all the traits in all three breeds. The only possible exception was weaning weight in the Ile de France breed, which improved at > 1 % per annum. At least all changes were in the desired direction. Breeders should be encouraged to record data consistently, as one of the major shortcomings in the data for all breeds were a lack of continuity in the submission of data to the NSIS. More informative analyses ought to be feasible if this requisite could be met.
AFRIKAANSE OPSOMMING: Die wisselende en algemene afname in die prysverhouding van wol tot vleis het merkbare veranderinge in die Suid-Afrikaanse skaapbedryf teweeggebring. Kommersïele produsente maak nou gebruik van ander metodes soos terminale kruisteling van Merino-tipe ooie met vleis tipe vaars of dubbel-doel rasse om korttermynvoordele uit die wisselende wol en vleis pryse te behaal, sonder om die wol-produksie potensiaal van die ooi-kudde te benadeel. Die meeste van die lamproduksie eienskappe het ‘n lae oorerflikheid. Nietemin, kan heterose wel behaal word deur die kruisteling van wol-tipe rasse met spesialis vleisramme. Genetiese verbetering van vee is afhanklik van die beskrywing van die teeltdoelwitte, die akkurate beraming van genetiese parameters en die noukeurige identifikasie van die geskikste diere vir toekomstige teling. Genetiese parameters vir ekonomies belangrike eienskappe van terminale ramrasse wat gebruik kan word op Merino-tipe ooie in die kommersiële skaapbedryf in Suid-Afrika is nog nie gepubliseer vir die nasionale kudde nie, behalwe vir ‘n voorlopige studie wat gedoen is deur Olivier et al. (2004). Seleksiedoelwitte is nie duidelik beskryf nie a.g.v ‘n tekort aan akkurate parameterberamings vir (ko)variansie komponente. Hierdie studie het dus stamboominligting en lewende gewig data verkry vanaf die Nasionele Kleinveeverbeteringsskema vir die Dormer-, Ile de France- en die Merino landskaaprasse en nie-genetiese faktore sowel genetiese parameters vir vroeë lamgewigte beraam. Genetiese en fenotipiese tendense vir vroeë lamgewigte is vervolgens opgestel vir drie rasse om genetiese vordering te evalueer. Die oorspronklike datastelle vir die Dormer, Ile de France en die Merino Landskaap het uit die volgende aantal rekords bestaan, onderskeidelik: 52 202, 35 553 en 7 772. Voor-speen gewigte was net beskikbaar vir die Ile de France- en die Merino Landskaaprasse, en na-speen gewigte was net beskikbaar vir die Dormerras. Die beperkings in die datastelle het genoodsaak dat dat kleiner datastelle ontwikkel moes word om die vaste effekte te analiseer. Die eienskappe wat ge-analiseer was, was geboortegewig, voor-speengewig, speengewig en naspeengewig. Die vaste effekte wat vroeë lamgewigte betekenisvol (P < 0.01) beïnvloed het, was geslag van die lam, geboortestatus, ouderdom van die ooi, kontemporêre groep, die ouderdom waarop die eienskap aangeteken is en (in sommige gevalle) die maand en jaar van geboorte. Alhoewel daar sommige betekenisvolle interaksies was, is dit nie in die finale modelle ingesluit nie, omdat dit min tot die verklaarde variasie bygedra het. In al die rasse het ramlammers swaarder (P < 0.001) geweeg as ooilammers. Enkelinge was ook swaarder (P<0.001) as meerlinge. Die ouderdom van die moer van die lam het ‘n beduidende kromlynige invloed op alle vroeë lamgewigte by al drie terminale ramrasse gehad. Die gevolgtrekking van hierdie studie is dat die invloed van nie-genetiese faktore op vroeë lamgewigte in ag geneem moet word, of dat dit moet statisies elimineer word in die genetiese evaluasie om akkurate genetiese beramings te verkry. (Ko)variansie beramings vir geboortegewig, speengewig en na-speengewig is deur gebruik te maak van die “restricted maximum likelihood procedures” (REML) vir die Dormerras verkry. Die direkte oorerflikheid (h2) wat verkry was deur die mees geskikste diere model in ‘n enkel-eienskap analise te gebruik was onderskeidelik 0.21 ±0.02, 0.23 ±0.02 en 0.29± 0.05 vir geboortegewig, speengewig en na-speengewig. Direkte ooreenstemende oorerflikheid wat uit die drie-eienskap analise was 0.28±0.04, 0.55±0.06 en 0.32±0.02 onderskeidelik vir geboortegewig, speengewig en na-speengewig. Direkte maternale genetiese effekte (m2) is uitgesluit vanaf die analise weens die onvermoë om die maternale effekte te verdeel in maternale genetiese effekte en maternale permanente omgewings effekte (m2 en c2). Dit was a.g.v onvolledige data en populasiestrukture wat gelei het tot die gebrek in genetiese bande oor kuddes, wat ontstaan het weens kuddes wat slegs tydelik data tot die skema bygedra het. Maternale permanente omgewingeffekte is geskat op onderskeidelik 0.15±0.02, 0.13±0.02 en 0.20±0.03 vir geboortegewig, speengewig en na-speengewig met die gebruik van die enkel-eienskap analise. Die korrelasie tussen direkte effekte en maternale effekte (ram) is uitgesluit a.g.v die gebrekkige struktuur van die data. Genetiese-, fenotipiese- en omgewingskorrelasies tussen die vroeë lamgewigte was laag tot matig. Die matige tot hoë oorerflikheidberamings vir vroeë lamgewigte uit hierdie studie het gelei tot die gevolgtrekking dat Dormer skape suksesvol gebruik kan word in terminale kruisteel programme om vleisproduksie te verbeter. Direkte oorerflikheid skattings was 0.31±0.14, 0.09±0.02 en 0.14±0.003 vir die geboorte gewig, voor-speen gewig en speen gewig onderskeidelik met die gebruik van ‘n enkel-faktor analise vir dir Ile de France skaap ras. Maternale effekte was beduidend vir al die eienskappe wat bestudeer was , ten spyte van die onvermoë om dit behoorlik te verdeel in hul komponente weens die verlies van genetiese bande dwarsoor die generasies wat uitvloei vanaf ‘n swak data struktuur. Genetiese, fenotipiese en omgewings korrelasies was geskat deur gebruik te maak van ‘n drie-faktor analise en was gevind om laag tot matig te wees vir die vroeë groei eienskappe. Direkte genetiese en maternale permanente omgewings ratios was bereken en dit het nie veel verskil van die resultate verkry deur die enkel-faktor analise. Die aanvaarbare genetiese parameter skattings verkry in hierdie studie het gelei tot die gevolgtrekking dat die Ile de France geselekteer kan word as teelramme in kruisteel programme. Genetiese parameters was geskat vir vroeë groei eienskappe in die Merino Landskaa ras. REML skattings van geboorte gewig, voor-speen gewig en speen gewig was verkry deur diere modelle in enkel-faktor analises. Die direkte oorerflikheid skatting vir geboorte gewig was 0.23±0.13 met die gebruik van die diere model met additiewe direkte genetiese effekte en ooi permanente omgewings faktore as die enigste ewekansige faktore. Die ooi permanente omewings effek vir geboorte gewig was 0.10±0.07. Direkte oorerflikheid vir voor-speen gewig was 0.36±0.05 en die ooi permanente omgewings effek 0.56±0.03. Speen gewig was geskat deur die gebruik van ‘n diere model wat die direkte additiewe effekte en die ooi permanente omgewings effekte bevat het. Die direkte oorerflikheids skatting vir speen gewig was 0.17±0.03. Maternale genetiese effekte was geskat as 0.02±0.01. Genetiese en fenotipiese tendense is verkry vir vroeë lamgewigte in die Dormer-, Ile de France- en Merino Landskaaprasse. Die eienskappe wat oorweeg is, was geboortegewig, voor-speengewig, speengewig en naspeengewig. Voor-speengewigte was net beskikbaar was vir die Ile de France- en die Merino Landskaap rasse en die na-speense gewigte net vir die Dormerras. Die Dormer het beduidende verbetering vertoon in die fenotipiese en genetiese aspekte vir vroeë lamgewigte gedurende die 17 jaar van evaluasie (1990-2007). Die gemiddelde voorspelde direkte teeltwaarde van speen gewig het met 0.12% per jaar gestyg gedurende die 17- jaar periode. Na-speen gewig het met 0.32% per jaar verbeter. By die Ile de France het ‘n toename in die voorspelde teelwaarde van geboortegewig (0.025% per jaar) voorgekom. Gemiddelde direkte teelwaardes vir voor-speengewig het toegeneem teen ‘n jaarlikse tempo van 0.23% en speengewig het met 1.21% per jaar toegeneem. In die Merino Landskaapras het die voorspelde direkte teelwaarde vir geboortegewig met 0.04% per jaar gedaal, terwyl voor-speen- en speengewigte met 0.36% en 0.10% onderskeidelik toegeneem het. Die tendense was ooglopend gekompromiteer weens probleme met die data struktuur, en a.g.v van die relatief min rekords wat beskikbaar was vir die analise in die ras. Dit was duidelik dat die additiewe genetiese variasie beskikbaar was vir al die vroeë groei eienskappe in al die drie rasse. Alhoewel voldoende genetiese variasie vir wesentlike genetiese vordering beskikbaar was, is daar slegs matige vordering verkry vir al die eienskappe in al drie rasse. Die enigste moontlike uitsondering was speengewig in die Ile de France ras, wat met 1.21 % per jaar gestyg het. Alle veranderinge was minstens in die gewensde rigting. Telers word versoek om data deurlopend en akkuraat aan te teken , aangesien een van die groot tekortkominge met die data van al die rasse ‘n tekort aan deurlopendheid in die indiening van die data aan die NISS was. ‘n Meer verteenwoordigende analise sal uitvoerbaar wees, as daar aan al die bogenoemde aanbeveling voldoen kan word.

When an organism migrates from one area to another it comes into contact with many boundaries to its survival and fitness. The fruitfly Drosophila melanogaster has migrated from Africa, into Europe and colonised much of the rest of the world. The subject of this thesis is to better understand how Drosophila has adapted to survive the temperate climates in Europe. The variability of temperature and light from one season to the next makes adaptation of the circadian clock and life history strategy all the more important. Drosophila appear to have adjusted to the new conditions by exhibiting diapause in low temperatures when the nights are long and by altering several other characteristics of its circadian clock that may be related to diapause. One of these is a novel European single nucleotide polymorphism in the timeless gene that allows flies to maintain a more robust diapause than flies carrying the ancestral allele. This variant exists in all European populations and winter simulation experiments reveal that it maintains its diapause for longer than the ancestral variant. These experiments also supported the possibility that Drosophila diapause can be maintained for much longer periods than previous studies have indicated. Experiments with ancestral African D. melanogaster lines alongside several closely related species indicates that diapause may not be a recent adaptation, but an ancient response to stressful conditions that has adapted in Europe to be more sensitive to low temperatures and short photoperiods. I also discover that a splice variant of the period gene has a dramatic, further cementing the controversial relationship between clock genes and diapause. In addition I have performed a study of putative functional polymorphisms in this untranslated region around this splice site from European populations. Finally, a study of putative clock genes reported in Chapter 3 provides a cautionary tale as to the dangers of using RNAi.

[eng] Wall lizards from the Podarcis genus, which have diversified and evolved in the Mediterranean basin, present wide morphological, ecological, and genetic variability. This diversity plays a key role in the maintenance of the evolutionary and adaptative potential of all the Podarcis populations and, consequently, in their conservation management. The main point of this thesis was to investigate adaptive processes in the Podarcis genus, examining how evolutionary mechanisms have shaped genomic and phenotypic divergence in different Podarcis populations. Specifically, it focuses on the Podarcis species complex that inhabit the Iberian Peninsula, especially the southeast (SE) region and the Columbretes archipelago, and on one endemic species from the Balearic Islands (Podarcis lilfordi). This objective was addressed through different approaches based on specific genetic markers, ecology, morphology, and/or genome-wide analyses. Regarding the Podarcis hispanicus species complex, a multilocus methodology showed that three different clades presenting an overlapping distribution inhabit the SE region. Phylogenetic relationships and geological history enabled a new species to be defined in this region, Podarcis galerai sp. nov. and the nominal taxon (Podarcis hispanicus sensu stricto) to be renamed. The latter was identified with Albacete/Murcia lineage since it is situated at the closest point to the restricted-type locality of Monteagudo and presents the morphological traits that are most similar to those indicated for the nominal taxon. The taxonomic status of the third group, P. hispanicus (Valencia lineage), could not be fully defined due to the lack of sampling in the global distribution. The evolutionary origin of the Podarcis form from the Columbretes archipelago was corroborated to be conspecific with the mainland form Podarcis liolepis, specifically from the region of Peñagolosa, (Castellón, Spain). The divergence time between the insular and mainland forms was dated at 1.77 Ma coinciding with a sea level fluctuation period, when the two regions could have been connected. Current results disclose low genetic diversity in insular populations, which seems to have suffered diverse events of expansion and/or decrease in diversity (bottlenecks) throughout their evolutionary history. The extraordinary intra-specific variability present in the endemic lizard from the Balearic archipelago (P. lilfordi) was explored at the genetic, morphological, ecological, and behavioural level. The discordance found between phylogenetic, morphological, and ecological results indicates that the use of Evolutionarily Significant Units (ESUs) as taxonomic classification is better to ensure the evolutionary future of these populations and their consideration in conservation policies. Genome-wide analyses using double digest restriction-site associated DNA sequencing (ddRADseq) made it possible to detect more than 70,000 genome-wide single nucleotide polymorphisms (SNPs) that corroborated the uniqueness of these insular populations and highlighted the combined role of genetic drift and natural selection in driving divergence. Tests of selection identified approximately 2% of loci putatively under selection (outlier loci). Correlation analyses with different environmental variables found predation and human pressure as the most explanatory variables in shaping this adaptive divergence. The genetic base of the phenotypical trait of melanism present in several P. lilfordi populations was not found in either genome-wide analyses or MC1R candidate gene expression analyses.
[spa] Las lagartijas del género Podarcis, que han divergido y evolucionado en la cuenca Mediterránea, presentan una amplia variabilidad morfológica, ecológica y genética. Esta diversidad juega un papel clave en el mantenimiento del potencial evolutivo y adaptativo de todas las poblaciones de Podarcis y consecuentemente, en la gestión de su conservación. El principal objetivo de esta tesis fue investigar los procesos adaptativos en el género Podarcis, examinando como los mecanismos evolutivos han moldeado la divergencia genómica y fenotípica en las diferentes poblaciones de Podarcis. Específicamente, se centró en el complejo de especies Podarcis que habita la Península Ibérica, en concreto la región sureste (SE) y el archipiélago de Columbretes, así como una de las especies endémicas de las Islas Baleares (Podarcis lilfordi). Este objetivo se ha abordado desde diferentes enfoques basados en marcadores genéticos específicos, ecología, morfología, y/o análisis genómicos. En cuanto al complejo de especies Podarcis hispanicus, la metodología multilocus mostró que en la región SE habitan tres clados diferenciados que presentan una distribución solapada. Las relaciones filogenéticas y la historia geológica permitieron definir una nueva especie en esta región, Podarcis galerai sp. nov. y redefinir el taxón nominal (Podarcis hispanicus sensu stricto). Este último se identificó con el linaje Albacete/Murcia ya que está situado en el punto más próximo de la localidad tipo de Monteagudo y presenta las características morfológicas más similares a las indicadas para el taxón nominal. El estatus taxonómico del tercer grupo, P. hispanicus (linaje Valencia), no se pudo definir totalmente debido a la falta de muestreo de toda su distribución. Se corroboró que el origen evolutivo de las Podarcis del archipiélago de Columbretes es el mismo que el de la forma continental P. liolepis, específicamente de la región de Peñagolosa (Castellón, España). El tiempo de divergencia entre la forma insular y continental fue datada hace 1.77 Ma, coincidiendo con un período de cambios en el nivel del mar, durante el cual ambas regiones pudieron estar conectadas. Los resultados de este estudio revelan una baja diversidad genética en la población insular, que parece haber sufrido diversos eventos de expansión y/o disminución de la variabilidad (cuellos de botella) a lo largo de su historia evolutiva. La extraordinaria variabilidad intraespecífica presente en las lagartijas endémicas del archipiélago balear (P. lilfordi) fue explorada a nivel genético, morfológico, ecológico y de comportamiento. La discordancia encontrada entre los resultados filogenéticos, morfológicos y ecológicos indicó que el uso de las Unidades Evolutivamente Significativas (UES) para la clasificación taxonómica es mejor para asegurar el futuro evolutivo de estas poblaciones y su consideración en las políticas de conservación. Los análisis genómicos usando secuenciación de DNA asociada a sitios de restricción mediante doble digestión (ddRADseq) hicieron posible la detección de más de 70,000 polimorfismos de nucleótido único (SNPs) genómicos que corroboraron la singularidad de estas poblaciones insulares y destacaron el papel compartido de la deriva genética y la selección natural en el impulso de la divergencia. Los test de selección identificaron aproximadamente un 2% de loci supuestamente bajo selección (loci atípicos). Los análisis de correlación con diferentes variables ambientales encontraron que la depredación y la presión humana son las variables más influyentes en la conformación de esta divergencia adaptativa. La base genética del carácter fenotípico del melanismo manifestado en varias poblaciones de P. lilfordi no se encontró ni en los análisis genómicos ni en los análisis de expresión génica del gen candidato MC1R.
[cat] Les sargantanes del gènere Podarcis, que s’han diversificat i evolucionat a la conca del Mediterrani, presenten una àmplia variabilitat morfològica, ecològica i genètica. Aquesta diversitat juga un paper clau en el manteniment del potencial evolutiu i adaptatiu de totes les poblacions de Podarcis i en conseqüència, en la gestió de la seva conservació. L’ objectiu principal d’aquesta tesi va ser investigar els processos adaptatius en el gènere Podarcis examinant com els mecanismes evolutius han modelat la divergència genòmica i fenotípica a les diferents poblacions de Podarcis. Específicament, es va centrar en el complex d’espècies Podarcis que habita la Península Ibèrica, concretament la regió sud-est (SE) i l’arxipèlag de Columbretes, així com una de les espècies endèmiques de les Illes Balears (Podarcis lilfordi). Aquest objectiu s’ha abordat des de diferents enfocaments basats en marcadors genètics específics, ecologia, morfologia i/o anàlisi genòmica. En quant al complex d’espècies Podarcis hispanicus, la metodologia multilocus va mostrar que en la regió SE habiten tres clades diferenciats que presenten una distribució superposada. Les relacions filogenètiques i la història geològica permeteren definir una nova espècie en aquesta regió, Podarcis galerai sp. nov i redefinir el tàxon nominal (Podarcis hispanicus sensu stricto). Aquest últim es va identificar amb el llinatge Albacete/Murcia ja que es troba situat al punt més pròxim a la localitat tipo de Monteagudo i presenta les característiques morfològiques més similars a les indicades pel tàxon nominal. L’ estatus taxonòmic del tercer grup, P. hispanicus (llinatge Valencia), no es va poder definir totalment a causa de la manca de mostreig a tota la seva distribució. Es va corroborar que l’origen evolutiu de les Podarcis de l’arxipèlag de Columbretes és el mateix que el de la forma continental P. liolepis, concretament de la regió de Peñagolosa (Castelló, Espanya). El temps de divergència entre la forma insular i la continental es va datar fa 1.77 Ma, coincidint amb un període de canvis en el nivell del mar, durant el qual ambdues regions van poder estar connectades. Els resultats d’aquest estudi van revelar una baixa diversitat genètica en la població insular, que sembla haver sofert diversos esdeveniments d’expansió i/o disminució de la variabilitat (colls d’ampolla) al llarg de la seva història evolutiva. L’ extraordinària variabilitat intraespecífica que presenten les sargantanes endèmiques de l’arxipèlag balear (P. lilfordi) va ser explorada a nivell genètic, morfològic, ecològic i de comportament. La discordança trobada entre els resultats filogenètics, morfològics i ecològics va indicar que l’ús de les Unitats Evolutivament Significatives (UES) per a la classificació taxonòmica és més adequat per assegurar el futur evolutiu d’aquestes poblacions i la seva consideració en les polítiques de conservació. L’anàlisi genòmica emprant seqüenciació de DNA associada a llocs de restricció mitjançant doble digestió (ddRADseq) va fer possible la detecció de més de 70,000 polimorfismes de nucleòtids simples (SNPs) genòmics que van corroborar la singularitat d’aquestes poblacions insulars i van destacar el paper combinat de la deriva genètica i la selecció natural en l’impuls de la divergència. Els tests de selecció van identificar aproximadament un 2% de loci suposadament sota selecció (loci atípics). Les anàlisis de correlació amb diferents variables ambientals van identificar la depredació i la pressió humana com les variables més explicatives en la formació d’aquesta divergència adaptativa. La base genètica del caràcter fenotípic del melanisme manifestat a diverses poblacions de P. lilfordi no es va trobar ni a l’ anàlisi genòmica ni a les anàlisis d’expressió gènica del gen candidat MC1R.

Kangal dogs are the most popular dogs of Turkey due to their strength, intelligence, loyalty, endurance to extreme temperatures and their lack of predatory behavior towards livestock. In this study to provide genetic information about the distinctness of Kangal and Akbash dogs and hence to provide a basis to conserve them separately, 585 base pair of the mitochondrial DNA (mtDNA) control region sequence was analysed in 105 Kangal and 9 Akbash dog samples. All the results indicated that Kangal and Akbash dogs were different from each other. Comparison of the Turkish data with those from other dogs revealed that Kangal dogs harbour a rare haplogroup which is only seen in Scandinavian (36%), Portuguese (20%), Turkish (20%) dogs and only one Spanish dog, but not in Akbash, Middle Eastern, other European, Eastern Asian and Indian dogs. Furthermore, comparison of the Kangal and Akbash dogs with the dogs from different geographic regions indicated that Kangal dogs are genetically closer to Scandinavian and South West Asian dogs whereas Akbash dogs are more similar to European and East Asian dogs, based on the mtDNA control region sequences Today, the sizes of Kangal and especially Akbash populations are decreasing. An urgent program for their conservation is needed. In order to conserve them separately, it must be understood that these two dogs are genetically distinct. That is why, the main purpose of the present study is to provide genetic information about the distinctness of Kangal and Akbash dogs.

Padlock probes are useful in a variety of genetic applications, some of which require that the probes are amplified in order to generate detectable signals. Two general padlock amplification methods, RCA and PCR, are discussed in this thesis.

The isothermal rolling circle amplification (RCA) mechanism is described in detail as well as how a target strand affects primer extension. A mechanism to resolve the topological constraint imposed by the target strand, to which a padlock probe has been linked, is also discussed. We also present a more powerful amplification technique, termed serial circle amplification, which provides a highly precise tool for nucleic acid studies. Rolling circle products are digested to unit lengths, and each monomer converted to new circular oligonucleotides that can serve as templates in consecutive rounds of RCA. The final products are single-stranded DNA molecules, readily available for hybridization-based detection, for instance using molecular beacons or array hybridization.

Padlock probes have the potential to be combined in large numbers for parallel gene analysis. A significant improvement of the level of multiplexed genotyping is presented using padlock probes and a molecular inversion strategy. Padlock probes containing common primer sequences along with locus-specific tag sequences were combined in multiplexed ligation reactions. After exonucleolytic selection for circular molecules, the probes were cleaved at uracil residues situated between the primer sequences, which facilitated release from the genomic DNA. A single PCR primer pair amplified all molecularly inverted probes, and the products were finally sorted on microarrays for simultaneous readout. Up to 1,500 genotypes could be detected in parallel, with sufficient signal strength for further scale-up. Finally, an application of the same parallel genotyping strategy is described where a set of padlock probes was used to study tumor induced immune responses. The distribution of TCR Vβ transcripts in tumor infiltrating T-cells and in normal control tissues were investigated in a microarray format.

Natural killer (NK) cells, lymphocytes of the innate immune system, can recognize a wide array of cells via several receptors families such as Ly49 and NKR-P1. The Nkrp1 gene family encode for C-type lectin-like receptors which can recognize their ligands, Clr, on target cells. Nkrp1 and Clr genes are intertwined in the NK gene complex and are thus inherited together. The Nkrp1-Clr genes in 129S6 and BALB/c mouse strains show significant sequence polymorphism compared to those of C57BL/6 mice while the overall gene organization and gene number are conserved. RT-PCR was utilized to study the expression of individual Nkrp1-Clr genes. In situ hybridization was performed to validate expression results from RT-PCR, as well as to verify the cell types in which Nkrp1-Clr genes are expressed. Surprisingly, our expression studies reveal an interesting pattern of expression of Nkrp1 and Clr genes not only in lymphoid tissues but also in the epithelial cells of the intestine, kidney, eye and lung, the myocytes of the heart and skeletal muscle, and possibly some endothelial cells, indicating novel functions of NK cells in these tissues.

Through a binding partner the CARD15 gene activates NF-kB, a molecule with a role in the initiation of the inflammatory immune response. The gene is highly conserved in both structure and function in human and mouse and has recently been implicated as a disease resistance gene in Crohn's disease and Blau Syndrome in human. The gene's relationship to disease and its conservation between species suggests that it may also have a conserved role in bovine disease resistance. To elucidate the potential role of bovine CARD15 in disease resistance, the gene was characterized in cattle. Bovine CARD15 is located 4.2 cR5000 telomeric to ADCY7 on chromosome 18. It spans ~30 kb and is comprised of 12 exons, 11 of which are coding. Bovine CARD15 is expressed in many tissues, but is most abundant in peripheral blood leukocytes. An extensive comparative analysis between the bovine, mouse and human CARD15 genes revealed high levels of inter-species conservation in sequence, genomic structure and protein domains. Conserved putative regulatory motifs were identified in the three species comparison of the 5'UTR, 3'UTR and the intronic sequences flanking exons. Additionally, diverse regulatory motifs were identified in each of the species indicating an evolutionary divergence in the mechanisms of regulation of gene expression. To assess the extent of genetic diversity within bovine CARD15, 41 individuals from nine breeds representing two subspecies were sequenced and screened for polymorphisms. Thirty-six single nucleotide polymorphisms (SNPs) were identified including 26 within the gene transcript. Haplotypes were estimated for each individual and parsimonious SNP sets were identified with which the multi-locus Bos taurus and Bos indicus haplotypes may be reconstructed. There was a significantly higher rate of substitutions within Bos indicus than in Bos taurus. A significantly higher rate of nonsynonymous to synonymous substitutions was found in Bos taurus indicating that positive Darwinian selection is acting on the gene within this subspecies. Association analyses were performed between these SNP loci and haplotypes with Johne's disease. No overwhelming evidence for a simple causal relationship was detected. Assays are provided to screen populations of cattle for variation in the CARD15 gene.

Scaffolding proteins are transiently associated with morphogenetic intermediates but are not found in the mature viral particle. These proteins promote the efficiency and fidelity of particle formation by ensuring proper interactions between viral proteins, promoting the nucleation of assembly, and aiding in determining appropriate capsid size. The goal of the proposed research is to understand how scaffolding proteins recognize and interact with viral precursors thus enabling them to obtain an assembly active form and defining the requirements for, and constrains on, these interactions. Microviridae morphogenesis is dependent upon two scaffolding proteins, an internal and external species. The genes encoding three Microviridae (ØX174, G4 and α3) internal scaffolding proteins (B proteins) have been cloned, expressed in vivo and assayed for the ability to complement null mutations of different Microviridae species. Despite divergence as great as 70% in amino acid sequence over the aligned length, cross-complementation was observed, indicating that these proteins are capable of directing the assembly of foreign structural proteins into infectious particles. These results suggest that the Microviridae internal scaffolding proteins may be inherently flexible. There was one condition in which a B protein could not cross-function. Substitutions conferring utilization map to the viral coat. The more efficient substitution is located in a region where coat-scaffolding interactions have been observed in the atomic structure and may emphasize the importance of interactions in this region. This is supported by chimeric analyses where efficient complementation was observed only when the viral coat protein and COOH-terminus of internal scaffolding were of the same origin. Despite 70% homology on the amino acid level, over-expression of a foreign Microviridae external scaffolding protein is a potent cross-species inhibitor of morphogenesis. To define the requirements for and constraints on scaffolding protein interactions, chimeric external scaffolding proteins have been constructed and analyzed for effects on in vivo assembly. The results of these experiments suggest that at least two cross-species inhibitory domains exist within these proteins. One domain most likely blocks procapsid formation and the other domain allows procapsid assembly but blocks DNA packaging. These results demonstrate how closely-related proteins could be developed into antiviral agents that specifically target virion morphogenesis.

Thesis (MScAgric (Animal Sciences))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: The aim of this study was to evaluate the Simbra breed of cattle for certain non-genetic as well as genetic parameters influencing live weight traits in the breed. Live weight traits included birth weight (BW), weaning weight at 200 days of age (WW), yearling weight at 400 days of age (YW) and 600 day weight. The Simmental and Simbra Breeders’ Society of Southern Africa availed 148751 records for analysis from the year 1987 till 2009. Due to deficiencies of various kinds in the data and the restrictions imposed for the purposes of the analysis, 56.44% of the records were discarded for BW, 76.55% for WW, 91.54% for YW and 96.32% for 600-day weight. Non-genetic parameters affecting BW, WW, YW and 600-day weight were analysed using the General Linear Models procedure of the Statistical Analysis System (SAS, 2004) software. During this procedure sex of calf, breed composition of calf, breeder of calf, month of birth, year of birth and dam age were fitted in the models. BW, WW, YW and Mature Cow Weight (MCW) were fitted as covariates where possible. It was determined that the fixed effects of sex, dam age, breeder, year and month had a significant (P < 0.05) effect of BW and WW while dam age was not significant (P > 0.05) for YW or 600-day weight. Breed was found non significant for YW. Breeder of the calf accounted for the most variation in BW, WW, YW as well as 600-day weight with a contribution of 17.55%, 25.77%, 18.35% and 10.71% respectively. Tukey’s multiple range tests were performed for testing differences between least square means. Results indicated male calves to be significantly heavier than females for all four traits measured. Breed composition differences were found significant until WW. Calves with higher Brahman percentage weighted more at birth while calves with higher Simmental percentage weighed more at weaning. Middle-aged dams were found to account for heavier calves at both BW and WW while very young dams and very old dams produced lighter calves for the two live weight traits. A number of years showed a significant difference from each other for all the traits measured as well as month of birth. (Co) variance components and the resulting genetic parameters were estimated using single-traits and three-traits analysis by means of Restricted Maximum Likelihood procedures (Gilmour et al., 2002). Appropriate models were selected by means of Log likelihood ratios tests and implemented to estimate genetic parameters for each of the traits studied. Direct additive heritabilities for BW, WW, YW and 600-day weight in the Simbra were respectively 0.56 ± 0.08, 0.67 ± 0.09, 0.70 ± 0.11 and 0.10 ± 0.03 when the most suitable animal model was fitted in single-trait analyses for each trait. Single traits analysis also included maternal additive as well as the correlation between direct additive and maternal additive for BW, WW and YW. Maternal additive heritability estimates of 0.24 ± 0.07, 0.33 ± 0.06 and 0.38 ± 0.07 was obtained for BW, WW and YW. Correlation estimates between direct additive and maternal additive were -0.75 ± 0.07, -0.93 ± 0.07 and -0.85 ± 0.08 for BW, WW and YW respectively. Furthermore, dam permanent environment was included as an additional random effect that increased the log likelihood value significantly. A value of 0.04 ± 0.05 was obtained for dam permanent environment estimate for WW. When a three traits analysis was done for the same traits, but using a significantly smaller data set, direct additive heritabilities of 0.24 ± 0.07 for BW, 0.33 ± 0.06 for WW and 0.38 ± 0.07 for YW were obtained. Genetic and environmental correlation estimates of 0.18 ± 0.16 and 0.09 ± 0.06 between BW and WW; 0.27 ± 0.16 and 0.07 ± 0.06 between BW and YW; as well as 0.52 ± 0.10 and 0.45 ± 0.05 between WW and YW were obtained during the three-trait analysis. The magnitude of the heritabilities obtained in this study indicates that the opportunity exists to make genetic progress through proper selection objectives.
AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die Simbra bees ras te evalueer op grond van sekere niegenetiese so wel as genetiese parameters wat lewende gewig beïnvloed. Gereelde en akkurate opnames van lewende gewig, is ‘n goeie indikasie van groei potensiaal en is ‘n minimim vereiste vir meeste beesras telings genootskappe. Lewende gewigs eienskappe sluit in geboorte gewig (BW), speen gewig gemeet op 200 dae (WW), jaaroue gewig gemeet op 400 dae (YW) en finale gewig gemeet op 600-dag gewig. Die Simmentaler en Simbra genootskap van Suid Afrika het 148751 rekords beskikbaar gestel vir evaluasie vanaf die jaar 1987 tot 2009. Daar was egter groot tekort komings aan die gewewe data en dus is daar 56.44% van die rekords vir BW nie gebruik nie, 76.55% vir WW, 91.54% vir YW en 96.32% vir 600-dag gewig. Nie-genetiese parameters wat die onderskeie lewende gewigte beïnvloed het, is geanaliseer deur Algemene Lineêre Modelle met behulp van die Statistiese Analitiese Sisteem (SAS, 2004) sagteware. Gedurende die analise is geslag van die kalf, ras samestelling, teler van die kalf, maand van geboorte, jaar van geboorte asook moeder ouderdom gepas in die modelle vir die onderskeie gewigte. Geboorte gewig, speen gewig, jaaroue gewig asook volwasse koei gewig is gepas in elk van die modelle as ko-variate. Volgens die resutate is daar vasgestel dat geslag van die kalf, moeder ouderdom, teler, jaar, maand en volwasse koei gewig almal ‘n betekenisvolle (P < 0.05) invloed gehad het op BW en WW. Die moederouderdom was nie betekenisvol (P > 0.05) vir YW of 600-dag gewig nie. Die ras samestelling was ook nie betekenisvol gevind vir YW. Teler van die kalf was verantwoordelik vir die meeste variasie in BW, WW, YW asook 600-dag gewig met ‘n bydrae van 17.55%, 25.77%, 18.35% en 10.71% onderskeidelik. Tukey se veelvuldige vergelykings toets is gebruik om onderskeid te tref tussen “least square means”. Resultate het aangedui dat manlike diere swaarder weeg as vroulike diere tot en met finale gewig. Ras samestelling vir BW en WW was betekenisvol verskillend vir die diere. Kalwers met ‘n hoër Brahmaan persentasie het swaarder BW opgelewer as dié met ‘n hoër Simmentaler persentasie, terwyl kalwers met ‘n hoër Simmentaler persentasie swaarder geweeg het met speen en dus ideal is vir speen kalwer produksie stelsels. Middel-jarige moeders het swaarder kalwers geproduseer met geboorte en speen as baie jong en - ou moeders. Sommige jare waarin van die kalwers gebore is, het ook betekenisvol van mekaar verskil asook die maand waarin die kalf gebore is. Ko) variansie faktore en opeenvolgende genetiese parameters is bepaal met behulp van enkeleienskap analises asook meervuldige-eienskap analises deur middel van die “Restricted Maximum Likelihood” prosedure (Gilmour et al., 2002). Modelle is opgestel vir elk van die gewigte deur die geskikte genetiese terme toe te voeg en te toets met behulp van “Log likelihood tests” om sodoende die onderskeie genetiese parameters te bepaal. Direkte genetiese oorerflikhede bepaal deur enkeleienskap analises vir die Simbra ras was as volg, 0.56 ± 0.08 vir BW, 0.67 ± 0.09 vir WW, 0.70 ± 0.11 vir YW en 0.10 ± 0.03 vir 600-dag gewig. Die direkte maternale genetiese oorerflikhede tydens dieselfde enkel-eienskap analise vir die onderkeie gewigte was 0.24 ± 0.07 vir BW, 0.33 ± 0.06 vir WW en 0.38 ± 0.07 vir YW. Korrelasies tussen direkt genetiese en direk maternale eienskappe was sterk negatief. ‘n Waarde van -0.75 ± 0.07 is bepaal vir BW, -0.93 ± 0.07 vir WW en -0.85 ± 0.08 vir YW. ‘n Adisionele faktor was ook ingelsuit vir WW, naamlik die permanente omgewing van die moeder, wat ‘n waarde opgelewer het van 0.04 ± 0.05. Tydens die veelvuldige-eienskap analise het die oorerflikhede merkwaardig verminder vir die betrokke gewigte en kan ook waargeneem word as die meer korrekte genetiese weergawe. Direkte genetiese oorerflikhede van 0.24 ± 0.07 vir BW, 0.33 ± 0.06 vir WW en 0.38 ± 0.07 vir YW was bepaal. Hierdie matig tot hoë parameters dui op genetiese vordering deur middel van korrekte seleksie prosedures. Genetiese- en omgewing korrelasies is ook bepaal tydens die analise en het positiewe waardes opgelewer. ‘n Genetiese korrelasie waarde van 0.18 ± 0.16 tussen BW en WW is bepaal asook ‘n waarde van 0.27 ± 0.16 tussen BW en YW en ‘n waarde van 0.52 ± 0.10 tussen WW en YW. Hierdie korrelasies dui daarop dat na-speengewigte vermeerder kan word deur te selekteer vir verhoogde WW sonder om BW dramties te vermeerder. Omgewings korrelasie waardes van 0.09 ± 0.06 tussen BW en WW, 0.07 ± 0.06 tussen BW en YW asook ‘n waarde van 0.45 ± 0.05 tussen WW en YW is gevind. Genetiese neigings is bepaal vir die onderskeie gewigte deur die gemiddelde voorspelde teelwaardes aan te teken teenoor elke jaar wat bereken was tydens die enkel-eienskap analises vir die onderskeie gewigte. Groot variasie asook negatiewe tendense vir WW en YW is ondervind van jaar tot jaar en dui daarop dat die seleksie doelwitte vir lewendige gewig nie in plek gestel is nie en is dit nodig om te her evalueer.

Sexual development begins with the process by which the bipotential gonads of the embryonic urogenital ridge develop into either testes or ovaries. In the mouse, sex determination occurs at around 11.5 dpc and depends on the presence or absence of the Y chromosome and the associated activity of the testis-determining gene, Sry, in supporting cell precursors. The mutually antagonistic male and female developmental pathways are regulated by many cellular and molecular processes, disruption of which can lead to disorders of sex development (DSDs). However, many of the molecular mechanisms regulating the differentiation of the two gonads are still unknown. The boygirl (byg) mutant was identified in an ENU-based forward genetic screen for embryos with gonadal abnormalities. On the C57BL/6J background, XY byg/byg homozygotes exhibited complete embryonic gonadal sex reversal. The defective gene in byg, Map3k4, is a component of the mitogen-activated protein (MAP) kinase signalling pathway and provides the first evidence for a function of this pathway in sex determination. This thesis describes experiments aimed at investigating the cellular and molecular basis of the sex reversal phenotype associated with the XY Map3k^4byg/byg mutant. Cellular characterisations revealed a defect in male-specific proliferation at 11.5 dpc, which was attributed to a defect in Sry up-regulation. Elucidation of the downstream kinases activated by MAP3K4 during sex determination was attempted, with particular focus on identifying a role for p38α MAP kinase (MAPK). Using a conditional knockout approach, the function of p38α in Steroidogenic factor-1 (Sf1)-positive somatic cells was assessed. However, specific inactivation in these cells did not affect gonad development. Conditional inactivation of Map3k4 itself in these Sf1¬-positive cells also did not disrupt gonad development, suggesting that this pathway is either initiated in a different cell lineage or at an earlier stage than deletion driven by Sf1-Cre can disrupt. Conditional inactivation of p38α in the Müllerian duct mesenchyme and ovarian granulosa cells using Amhr2-Cre did reveal a function for p38α in female fertility, but did not disrupt embryonic sexual development. Gene knockdown in organ culture was attempted to determine a role for multiple p38 MAPKs in all cell types of the gonad. Therefore, this thesis details further characterisations of a novel signalling pathway important for the expression of Sry, focussing on the role of the p38 MAPKs.

Preliminary experiments, to find phage or bacteriocin activity which might be plasmid-encoded amongst available strains of the Rhodopirillaceae were unsuccessful, and no phage were isolated from the natural environment. The technique for phototrophic plate growth of Rm. vannielii was found to be unsuitable for basic genetic experiments and a method using microaerophilic growth was developed. The latter was found to yield better results in both mutagenesis and conjugation experiments despite an increased incubation time relative to phototrophic growth (i.e. 14 instead of 3-5 days to obtain colonies). The growth rate under microaerophilic conditions was increased by adding more yeast extract to the media and workable colonies were obtained after 10 days. NTG and UV-mutagenesis were successfully used to isolate a variety of mutant types including pigment mutants, motility mutants and temperature sensitive motility mutants. The pigment mutants were classified into four groups according to colour and spectroscopic analysis. A number of them were selected for further analysis. PAGE showed that in general these mutants were very similar to pigment mutants of other purple non-sulphur bacteria. Transposon mutagenesis was successfully carried out using pSUP2021 and pJB4JI. Tn5-insertion pigment mutants, were isolated and Southern hybridization demonstrated that the transposon was inserted at single non-specific sites on the chromosome. This was verified by analysis of revertants obtained using a * light-lethal’ selection technique on a photosynthetically incompetent mutant. An EcoRI fragment containing Tn5 plus flanking sequences was subsequently cloned from this mutant into pBR322. Conjugation experiments showed that broad-host-range plasmids from incompatability groups P, Q and W could be transferred to and maintained in Rm.vannielii.

Triatomine bugs (Hemiptera: Reduviidae: Triatominae) are the vectors of Chagas disease in South and Central America. Chagas disease predominantly affects poor rural communities with simply constructed housing susceptible to infestation by triatomines. Chagas disease is restricted to the Americas largely due to the limited distribution of triatomine bugs. The global diversity of triatomines is -130 species, of which only -10% are known to occur outside the Americas, one species (Triatoma rubrofasciata) is tropicopolitan, and the others are concentrated on the Indian subcontinent (Linshcosteus spp. ) and adjacent south east Asian island groups (Triatoma spp. ). The main objectives of this PhD programme were to: a) assess the facility of morphometric approaches (measurement and robust statistical analysis of morphological variation) in the study of population structure of vector species with proximal domestic and silvatic distributions to detect population structure and give information on the risk of reinvasion, b) study interspecific and higher taxonomic level relationships of New World and Old World triatomine bugs. To these ends geometric morphometric analyses were conducted in concert with molecular genetic analyses of mitochondrial and nuclear DNA sequences. The principal question being: Does the relatively low cost method of morphometrics reveal patterns consistent with population structure, as otherwise determined by more expensive molecular genotyping methods? Or are such patterns disrupted by environmental effects and intraspecific convergent/divergent morphological evolution? Combined morphometrics and molecular genetics were used to study vector populations in three of the countries that continue to be most affected by Chagas disease. In Venezuela and Ecuador Rhodnius species (R. prolixus and R. ecuadoriensis respectively) were studied, in areas where they occur in both domestic and silvatic environments, and in Paraguay T. infestans from a domestic and a putative silvatic focus. Head and wing morphometrics were compared to mitochondrial DNA sequence data to assess the population structure and disparity among domestic and silvatic samples in each case. The results presented suggest that head shape variation is subject to morphological plasticity and/or selective pressure and functional constraint and does not correlate well with the 11 Abstract phylogeny. However, in all examples, wing shape was found to be congruent with the phylogenetic patterns inferred from sequence analysis. Consequently, it is recommended that wing shape and not head shape be used in morphometric assessments of population dynamics. It is also asserted here that if population structure is suggested by morphometrics, it should be followed by robust population genetic analysis. As such, morphometrics could be used as a tool for broad surveillance to identify areas of concern. A further objective was to elucidate the broader phylogeny of Triatominae and their relationships with other reduviid subfamilies. To investigate the debated polyphyletic origin of the Triatominae molecular approaches were used. Combined head and wing morphometric and molecular genetic analyses of New World and Old World Triatominae have revealed patterns of convergent morphological evolution (among New World and Old World Triatoma) and striking examples of strongly divergent morphological evolution (between Old World Triatoma and Linshcosteus). Applying a molecular clock based on the rate of sequence divergence for a fragment of ribosomal DNA (D2-28S), calibrated to the fossil record and vicariant events (the divergence of ancestral lineages due to separation by topographical or ecological barriers) it has been possible to reconstruct a likely evolutionary history for the Triatominae and the Reduviidae as a whole. The weight of evidence presented supports a polyphylectic origin for blood-feeding for the Triatominae. The apparent independent development of blood feeding among the main lineages of the Triatominae represented by the genera Triatoma and Rhodnius highlights a fundamental biological difference among important vector species. This difference is likely to become evident in the eventual post genomic era in studies of vector/parasite interactions and it highlights the importance of sequencing genomes from different vector genera.

Leptin is a 16-kDa protein that is primarily secreted by adipose tissue. It affects body mass regulation by constituting part of the adipostat, that acts to alert the brain of the body's stored energy levels. Additional roles in immune function, reproduction and inflammation are known. Genetic studies of single nucleotide polymorphisms (SNPs), that exist in the extracellular domain of the leptin receptor gene, were undertaken in a population of European Caucasian postmenopausal women to investigate associations with indicators of adiposity. Homozygosity of the G allele of the LYS109ARG SNP was associated with lower mean fat mass levels and BMI. Furthermore, linkage disequilibrium was detected between this SNP and GLN223ARG, which in previous studies was also associated with indicators of adiposity. No associations were found between the LYS656ASN SNP and the tested phenotypes. To complement genetic studies of the leptin receptor, cDNA constructs representing different combinations of the alleles for SNPs in the leptin receptor were generated and subsequent expression of protein variants was conducted in COS-7 cells. Using a radioactive ligand-binding assay, labelled leptin was shown to specifically bind to the LYS109ARG223 and GLN223ARG protein variants, thereby testing the effect of the GLN223ARG mutation on LBA Preliminary data, suggest that the ARG allele appeared to bind less leptin than the GLN.Genetic studies were carried out on polymorphisms in related candidate genes. A promoter polymorphism (G-2548 A) in the leptin gene was associated with lower mean BMI and leptin levels in a cohort of European Caucasian postmenopausal womenIndividuals who lacked the 2 repeat allele of the variable number tandem repeat (VNTR) polymorphism present in intron two of the interleukin 1 receptor antagonist gene had an association with lower leptin levels but not BMI or fat mass. This suggests a potential feedback and/or cross-talk mechanism between leptin and members of the IL-1 family of cytokines in processes other than adiposity. Immunity and inflammation are processes where the interleukin one receptor antagonist protein has a prominent role and in which the function of leptin is increasingly being investigated, therefore an interaction between the two cytokines may be specific for these conditions. The TNF alpha (G-308 A) SNP was also investigated but no associations were observed between this SNP and the phenotypes in the postmenopausal cohort. To investigate the influence of the leptin receptor gene in conditions at the opposite end of the body weight spectrum to obesity, a case-control association study was undertaken to compare allele frequencies of the LYS109 ARG, GLN223ARG and LYS656ASN leptin receptor SNPs between anorexic women and controls. No significant differences were observed in allele or haplotype frequencies.

Thesis (M.Sc. (Molecular and Life Sciences)) --University of Limpopo, 2007
Colophospermum mopane (sensu lato) is currently recognised on morphological and physiological characteristics. To add to the suite of taxonomic characters, the genetic variability of C. mopane (sensu lato) was investigated using the RAPD technique. DNA was extracted from young seedlings and mature leaves using the CTAB method. Initially, the DNA extraction was problematic due to the presence of polysaccharides, making PCR nearly impossible. An additional phenol precipitation step was introduced to purify the DNA used to perform RAPD analyses. Twenty random primers were tested for their suitability and reproducibility to reveal polymorphism in C. mopane (sensu lato). Nine of the primers tested amplified the genomic DNA. Subsequently, three primers (OPA 03, OPA 08 and OPA 09) were selected based on their reproducibility and demonstration of polymorphism. OPA 03 amplified most of the samples tested whereas OPA 08 and OPA 09 amplified 50% of the samples. RAPD bands ranged from 180 bp to 2000 bp. RAPD profiles of C. mopane (sensu lato) with three random primers showed few polymorphisms. Individual trees of different ecotypes show similar RAPD banding pattern, instances were found where trees of the same ecotype showed different bands. The total character difference based on presence and absence of bands revealed both variability and similarity of C. mopane (sensu lato). Phylogenetic trees from individual primers and combined primers were constructed using Neighbour Joining and Parsimony analysis. The phylogenetic tree from the combined primers of bootstrap parsimony generated three clades with low and high parsimony bootstrap values. The first clade receives weak support (61%) while the second and third clades receive support of 90% and 70%, respectively. The other remaining entities collapsed resulting in basal polytomy. The third clade shows some members of Alba (Alba 11 Phala, Alba 1 Phala and Alba 7 Musina) grouped together. The overall results of C. mopane (sensu lato) show high (84.1%) genetic similarity. No ecotypic marker was obtained. Most of the ecotypes have not diverged genetically far from one another or from the parental material (Mopane – sensu stricto). The genetic results partially support the perceived morphological differences. In this study the RAPD technique has established its value as an additional tool to express the genetic variability in C. mopane (sensu lato).
The National Research Foundation

Precise and complete replication of the genome is essential for a cell. Chromosome replication follows a defined temporal order, depending on the efficiency and timing of the replication origins. However, the mechanism regulating origin activity has not been properly explained to date. Yeast replication origins are very well characterized and well studied. Genome wide replication in yeast was detailed through deep sequencing in various studies. In yeast, there are multiple replication origins for the complete replication of the genome. In Saccharomyces cerevisiae, there are ~400 replication origins, which are also referred to as Autonomously Replicating Sequences. Replication origins have varied levels of activity and varying times of activation. There are many lines of evidences, which suggest that the origins function differently in mitotic and meiotic cell cycles. It was thought that same origins function both during mitosis and meiosis. However, there is a difference in the replication timings of both the cell cycles, the reason for which is not known. Meiotic cell cycle is longer than the mitotic cell cycle. By using the plasmid-based assays, specific origins were selected and origin activity was analyzed during mitosis and meiosis to see if individual origins show any differences in origin activity. For all the origins tested, the meiotic activity was found to be less than the mitotic activity, which provides a possible explanation for a longer pre-meiotic S phase. Most of the confirmed yeast replication origins are present in the intergenic regions of the chromosome. Due to the presence of majority of replication origins in the intergenic regions and not on the genes, it was thought that the gene transcription might be detrimental to origin activity, hence not supporting the existence of an origin on a gene. Careful analysis of genome wide replication data along with plasmid based ARS assays confirmed that a few replication origins are present within genes. Assays were preformed to study the relation between transcription and origin activity both during mitosis and meiosis. Mitotic origin activity was shown to have no known affect from gene transcription. However, due to some unknown technical faults or other reasons, assays to find out transcription and meiotic activity were not successful.

Diminishing worldwide fossil fuel reserves coupled with the negative impact of their use on the environment has led to increased research and development of renewable energy sources. Renewable liquid biofuels are in demand for the transport sector, particularly if they can be used directly in existing internal combustion engines. Jatropha curcas L. is a perennial plant which belongs to the Euphorbiaceae family. J. curcas seeds contain about 30% oil which is suitable for biodiesel production, and therefore it has received global interest as a source of biofuel. However, to date J. curcas has not been put through any stringent breeding program for traits improvement and thus has not reached its full potential. Improving seed yield, seed oil quality and quantity is necessary for large scale biodiesel production, and developing other by products will add to the economic value of this crop. Apart from oil, J. curcas seeds also contain a high percentage of proteins, which makes the seed meal potentially useful as animal feed. However, seeds from most of the current global J. curcas plantations in Asia and Africa are characterized as non-edible, due to the existence of a few toxins or antinutrients. Phorbol esters have been considered as the main toxic agent. Edible provenances exist in Mexico which are devoid of phorbol esters. Among the other toxins, curcin (a type I ribosome inactivating protein) levels have not been reported for the edible and non-edible varieties. To improve our understanding of J. curcas natural variation and biochemical composition, seed samples were collected from a variety of locations in Madagascar, Mexico, and purchased from five other countries. Seed oil content, fatty acid composition and phorbol esters content were measured to establish the diversity in these traits. Seed oil content and fatty acid composition was found to vary in seeds collected from different sites, and oleate and linoleate composition were found to correlate strongly with cultivation site temperature indicating the importance of environmental conditions for the production of an optimal feedstock for biodiesel. Phorbol esters were found to be present in all seed samples originating from outside Mexico, and in the Mexican provenance Rosario Chiapas. All other Mexican samples lacked phorbol esters. This suggests that the presence of phorbol esters is a qualitative trait. AFLP analysis revealed that most genetic variation was present in Mexican samples, with all material originating from outside Mexico showing very limited genetic diversity. Edible samples and non-edible samples were found to be genetically distinct, with the edible samples forming a single cluster. The large amount of variation in oil quantity and quality in Madagascan samples, together with the limited genetic diversity in these samples, implies that J. curcas seed oil is largely influenced by environmental factors. Seed curcin levels were determined in the edible and non-edible varieties. The results showed that curcin levels are equally abundant in both varieties. This demonstrates that curcin is not playing any significant role in determining seed edibility and is consistent with the predominant role understood to be played by phorbol esters in determining this trait. The spatial and temporal expression of different curcin genes was further examined. Four curcin genes showed different patterns of expression with seed and leaf specific patterns of expression being identified. Further analyses revealed that CURCIN2 is induced in mature leaves in response to various abiotic stresses. Furthermore it appears that the induction of CURCIN2 in response to wounding is regulated via the JA (jasmonic acid) signalling pathway. Together these results represent a valuable addition to the knowledge base underpinning the development of J. curcas as an industrial crop through molecular breeding.

Nance-Horan Syndrome (NHS) is an X-linked developmental syndrome characterised by congenital cataract, dental anomalies, and dysmorphological features often associated with mental retardation. The NHS locus on Xp22.13 is encompassed by the disease locus for X- linked congenital cataract (CXN). Analysis of microsatellites within the CXN family resulted in refinement of the CXN disease interval, reducing the region of overlap between the CXN and NHS disease loci. Candidate genes in the overlapping intervals were identified bioinformatically and their genomic structures evaluated. Patient DNA was screened by direct sequencing, resulting in the identification of mutations within a novel gene in four British families with NHS, but not the CXN family. This novel gene, named NHS, is encoded by at least 10 exons transcribed into at least five mRNA isoforms A, B, C, D, and E (encoding a putative 1,630 a.a., 1,335 a.a., 1,474 a.a., 1,453 a.a., and 1,473 a.a. protein, respectively). All mutations identified are truncating and three mutations have been identified in exon 1, which are only expressed in isoform A. This implies that mutations in isoform A are sufficient to cause disease in families with NHS. Functional clues for the NHS protein were investigated resulting in identification of three new genes with significant homology to NHS {NHS-Like 1 (NHSL1), NHSL2 and NHSL3). All four genes share a conserved genomic structure. Fetal expression analysis of NHS, NHSL1 and NHSL2 suggests that NHSL1 and NHSL2 are more ubiquitous than NHS. Analysis of the NHS family of proteins revealed significant homology to members of the WASP family, which consists of WASP, N-WASP and WAVE 1-3. The WASP protein family play a crucial role in regulating actin dynamics, directly linking small GTPase signalling to actin polymerisation through activation of the Arp2/3 complex. An anti-peptide antibody to the C-terminus of NHS, completely conserved across species, was raised and characterised. A major NHS isoform (approximately 170 kDa) was detected in several cell lines. Subcellular localisation studies in MTLn3 cells showed localization of endogenous NHS to the leading edge of lamellipodia, a localisation pattern reminiscent of the Arp2/3 complex. Endogenous NHS also localised to some actin stress fibres. Homology to the WASP protein family and localisation of endogenous NHS to the leading edge of lamellipods strongly supports a role for NHS in actin cytoskeletal dynamics during development.

Complex diseases, unlike Mendialian diseases, are often characterized by genetic heterogeneity and multifactorial inheritance, involving defects in genes from the same or multiple alternative pathways. Many congenital diseases and psychiatric disorders are complex diseases, and incur heavy health care burden on the society. With the advancement in high-throughput genotyping technologies and the availability of the human single nucleotide polymorphism (SNP) catalogue, genome-wide association study (GWAS) has been widely used to investigate the genetic component of complex diseases. Copy number variations (CNV) can also be identified using the data from the same SNP array. Aiming to identify more disease susceptibility loci for complex diseases, separate GWAS using a case-control design were conducted on anorectal malformations (ARMs) and schizophrenia. ARMs are rare congenital diseases with heterogeneous phenotypes which could probably be explained by the genetic heterogeneity among patients, while schizophrenia is a common psychiatric disorder that is well known for its multigenic inheritance. The GWAS studies on ARM and schizophrenia included 4,369 (patients: N=363; controls: N=4,006) and 1,231 Han Chinese (patients: N=381; controls: N=850) respectively. The two studies were mainly focused on investigating the contribution of rare CNVs to the diseases, involving analyses on global CNV burden, rare CNV association, protein-protein interaction (PPI) network, pathway and chromosomal aberrations. The associations of SNPs with ARMs were also examined. Apart from elucidating the genetic components in these two diseases, a systematic analysis on four CNV detection programs (CNV partition, PennCNV, QuantiSNP and iPattern) was also undertaken. In the study of schizophrenia, a new approach in CNV filtering which was based on latent class analysis was adopted to gather information from multiple CNV prediction programs. The study of ARMs revealed 79 genes which were disrupted by CNVs in patients only. In particular, a de novo duplication of DKK4 (an antagonist of WNT signaling) was identified, and addition of Dkk4 protein was demonstrated to cause ARMs in mice. Another 10 genes uniquely disrupted in ARMs patients are also related to WNT signaling. Interestingly, this pathway was also significantly inferred by CNV in patients with schizophrenia. A different set of genes related to WNT signaling was disrupted in ARMs patients and patients with schizophrenia. WNT signaling is crucial for the development of multiple parts in the embryo. The contribution of different WNT signaling pathways at different development stages may vary. Apart from the WNT signaling pathway, other genes with biological relevance were also implicated in the two studies through gene-network and pathway analyses. The results from these two GWAS studies support our existing understanding of complex diseases that defects in various interacting genes could contribute to the same disease. In summary, the CNV results from the two studies have demonstrated the genetic heterogeneity nature of these two complex diseases. The findings also uncovered a set of putative disease candidate genes, which can be used as reference materials for future genetic research for ARMs and schizophrenia.
published_or_final_version
Psychiatry
Doctoral
Doctor of Philosophy

Murine cytomegalovirus (MCMV) is used as a biological model for human cytomegalovirus (HCMV). Latency, persistence and reactivation are same of the important aspects of the murine model that share analogies with human CMV infections. In order to elucidate the molecular mechanisms leading to these events, in-depth analyses of the murine model are required at the transcriptional level. During the MCMV replication cycle, there is a sequential expression of different regions of the viral genome, hence the transcripts are divided into three kinetic classes; the immediate early (IE), early (E) and late (L). This study presents the analyses of MCMV (Smith strain) transcripts of the major IE and E transcriptional units, and a more detail analysis of one of the major E regions, E10. The IE and E transcripts were studied by probing them with Ctoitplementary DNAs (cDNAs). The cDNAs were prepared from mRNA isolated from the IE and E phases of the viral replication cycle and cloned into the bacteriophage Lambda gt10. Ten E cDNAs were mapped to specific locations of the virus genome, and these represented transcripts from the major E regions in Hindlll fragments A, B, E, F, and I-J. Five E cDNAs, each representing a different major E region, and two IE cDNAs representing the major IE region, were applied as probes in one of the studies to determine the relative transcript levels during the course of infection of 3T3L1 fibroblast cells with MCMV. The major E transcriptional units were investigated further in a study where Northern blots of RNAs, isolated from different phases of the viral replication cycle, were probed with the five E cDNAs. This study revealed transcripts that were temporally regulated since they were present only during the E and usually L phases of the viral replication cycle. In addition, the quantities of these transcripts varied depending on the phase. However, all five cDNAs detected more than one transcript which indicates complex splicing events, overlapping genes, multiple initiation sites and/or the presence of gene(s) in the complementary DNA strand. One of the E cDNAs, E10, corresponding to a transcript from a major E region of Hindlll fragment I-J, was selected for further analysis. The E10 cDNA detected four transcripts of 9.5, 6.9, 4.7 and 2.1 kb in size, which were found to be transcribed from the same DNA strand. The DNA sequence of this E10 cDNA was determined and shown to contain 3223 nucleotides, however it lacked a polyadenylation signal and a poly A tract at the 3' end. The missing 3' terminus, designated as E10-A, was isolated using the polymerase chain reaction (PCJR) method and its DNA sequence of 1422 nucleotides was also determined. The combined sequence of E10 and E10-A (total of 4606 nucleotides) was designated as E10-C and is presented in this thesis. The E10-C cDNA (4.6kbp) most likely represents the 4.7 kb transcript. The E10-C cDNA sequence has one minor and one major open reading frame (ORF). The minor ORF is initiated by the first ATG triplet (nucleotide position 114) while the major ORF is initiated by the second triplet (nucleotide position 155). Since the sequence preceeding the second ATG triplet is in "good context" with regard to the translation initiation consensus sequence, it is most likely that the major ORF is translated. The major ORF (3600 bases) encodes a 1200 amino acid polypeptide, the putative E10 protein of approximately 135 kd in size. A protein close to that size was detected in one of the experiments in which RNAs, that were hybrid-selected by the E10 cDNA and eluted, were translated in vitro. The putative E10 protein lacks homology with any other protein in the data banks (SWISSPRT and GENPEPT). Portions of the viral genomic fragments Hindlll I and J were also sequenced to reveal the orientation of the gene coding for the E10 cDNA and its related transcripts.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

Raman spectroscopy offers much better spectral selectivity but its usage has been limited by its poor sensitivity. The discovery of surface-enhanced Raman scattering (SERS) effect, which results in increased sensitivities of up to 108-fold for some compounds, has eliminated this drawback. A new SERS active substrate was developed in this study. Silver nanoparticle-doped polyvinyl alcohol (PVA) coated SERS substrate prepared through chemical and electrochemical reduction of silver particles dispersed in the polymer matrix. Performances of the substrates were evaluated with some biologically important compounds. The specific detection of DNA has gained significance in recent years since increasingly DNA sequences of different organisms are being assigned. Such sequence knowledge can be employed for identification of the genes of microorganisms or diseases. In this study, specific proteasome gene sequences were detected both label free spectrophotometric detection and SERS detection. In label free spectrophotometic detection, proteasome gene probe and complementary target gene sequence were attached to the gold nanoparticles separately. Then, the target and probe oligonucleotide-modified gold solutions were mixed for hybridization and the shift in the surface plasmon absorption band of gold nanoparticles were followed. SERS detection of specific nucleic acid sequences are mainly based on hybridization of DNA targets to complementary probe sequences, which are labelled with SERS active dyes. In this study, to show correlation between circulating proteasome levels and disease state we suggest a Raman spectroscopic technique that uses SERGen probes. This novel approach deals with specific detection of elevated or decreased levels of proteasome genes&rsquo
transcription in patients as an alternative to available enzyme activity measurement methods. First, SERGen probes were prepared using SERS active labels and specific proteasome gene sequences. Then DNA targets to complementary SERGen probe sequences were hybridized and SERS active label peak was followed.