Tesi sul tema "Genetic"

PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO DE JANEIRO
COORDENAÇÃO DE APERFEIÇOAMENTO DO PESSOAL DE ENSINO SUPERIOR
PROGRAMA DE EXCELENCIA ACADEMICA
Sistemas Fuzzy-Genéticos compreendem uma área que une Sistemas de Inferência Fuzzy e Meta-Heurísticas prevalentes nos conceitos de seleção natural e recombinação genética. Esta é de grande interesse para a comunidade científica, pois propicia a descoberta de conhecimento em áreas onde a compreensão do fenômeno em estudo é exíguo, além de servir de apoio à decisão para gestores público-privados. O objetivo desta dissertação é desenvolver um novo Sistema Fuzzy-Genético Genérico, denominado Genetic Programming Fuzzy Inference System (GPFIS). O principal aspecto do modelo GPFIS são as componentes do seu processo de Inferência Fuzzy. Esta estrutura é composta em sua base pela Programação Genética Multigênica e pretende: (i ) possibilitar o uso de operadores de agregação, negação e modificadores linguísticos de forma simplificada; (ii ) empregar heurísticas de definição do consequente mais apropriado para uma parte antecedente; e (iii ) usar um procedimento de defuzzificação, que induzido pela forma de fuzzificação e sobre determinadas condições, pode proporcionar uma estimativa mais acurada. Todas estas são contribuições que podem ser estendidas a outros Sistemas Fuzzy-Genéticos. Para demonstrar o aspecto genérico, o desempenho e a importância de cada componente para o modelo proposto, são formuladas uma série de investigações empíricas. Cada investigação compreende um tipo de problema: Classificação, Previsão, Regressão e Controle. Para cada problema, a melhor configuração obtida durante as investigações é usada no modelo GPFIS e os resultados são comparados com os de outros Sistemas Fuzzy-Genéticos e modelos presentes na literatura. Por fim, para cada problema é apresentada uma aplicação detalhada do modelo GPFIS em um caso real.
Genetic Fuzzy Systems constitute an area that brings together Fuzzy Inference Systems and Meta-Heuristics that are often related to natural selection and genetic recombination. This area attracts great interest from the scientific community, due to the knowledge discovery capability in situations where the comprehension of the phenomenon under analysis is lacking. It can also provides support to decision makers. This dissertation aims at developing a new Generic Genetic Fuzzy System, called Genetic Programming Fuzzy Inference System (GPFIS). The main aspects of GPFIS model are the components which are part of its Fuzzy Inference procedure. This structure is basically composed of Multi-Gene Genetic Programming and intends to: (i ) apply aggregation operators, negation and linguistic hedges in a simple manner; (ii ) make use of heuristics to define the consequent term most appropriate to the antecedent part; (iii ) employ a defuzzification procedure that, driven by the fuzzification step and under some assumptions, can provide a most accurate estimate. All these features are contributions that can be extended to other Genetic Fuzzy Systems. In order to demonstrate the general aspect of GPFIS, its performance and the relevance of each of its components, several investigations have been performed. They deal with Classification, Forecasting, Regression and Control problems. By using the best configuration obtained for each of the four problems, results are compared to other Genetic Fuzzy Systems and models in the literature. Finally, applications of GPFIS actual cases in each category is reported.

Today genetic services including genetic counselling are widespread across the world. Although developing countries, like Colombia, have started to apply genetic knowledge to the health area, genetic counselling is usually integrated in the routine clinical genetic consultation, however, before this study the process of communication involved in it had not been explored. In collaboration with the Colombian Association of Medical Genetics, the Bogotá Health Service, and the University of Warwick (UK), I observed 25 genetic consultations in five Colombian genetic clinics. I undertook semi-structured interviews with patients / families before and after the consultation. Thematic analysis of the interview transcripts established mismatches between physician perception and patient comprehension. Efficient communication was affected by patient, relatives, practitioner and external factors. Among these environmental factors were excessive administrative procedures, interruptions during the encounter, patients‟ lack of interest to medical terminology, doctors using scientific language, excessive information given in one session, beliefs and education level of the patient and/or relatives, patient distress caused by bad news, unfulfilled expectations and no availability/accessibility of treatment. I also interviewed 20 medical practitioners working in genetics services. There was general agreement that genetic counselling in Colombia was challenging, and that more training in communication skills was required at Medical schools at undergraduate and postgraduate level. Many physicians did not believe that other health professionals should work as genetic counsellors. There was a general recognition of limited genetic knowledge, awareness and understanding in most medical specialities. These results have made a valuable contribution to describe the current situation with genetics consultation and counselling in Colombian genetic clinics, and have already influenced the future development of an effective and robust genetic counselling service in Colombia. They will also be used in the development of the academic curriculum related to basic and clinical genetics at Colombian Universities.

Paddlefish (Polyodon spathula) is a commercially and recreationally important species, with a native range that extends over 22 US states. This is a large, long-lived, highly mobile riverine species that has been negatively impacted by habitat fragmentation, historic overharvest, and hatchery supplementation. Dams are the primary cause of habitat fragmentation, blocking migration routes, flooding spawning grounds, and isolating populations. A common management action to mitigate the impacts of habitat fragmentation and maintain harvestable populations is hatchery propagation and stocking. Reduction in stock size, isolation of populations, and stocking can all negatively impact the genetic integrity of Paddlefish. I evaluated the impacts of isolation and hatchery supplementation on the effective population size (Ne) of Paddlefish as well as the range-wide genetic structure of Paddlefish.

Malaria, an infectious disease caused by Plasmodium parasites, is one of the major world-scale health problems. Despite the efforts aimed at finding an effective way to control the disease, the success has been thwarted by the emergence of parasite drug resistance and mosquito resistance to insecticides. This thesis focuses on the genetic analysis of resistance to murine malaria induced by the lethal Plasmodium berghei ANKA using a wild-derived-inbred strain (WDIS). The aim of this thesis was to exploit the genetic diversity represented among WDIS for identifying loci contributing to resistance/susceptibility to murine malaria. The work included a genome-wide polymorphism survey using microsatellite markers performed on 10 WDIS. Comparisons of these strains to laboratory inbred strains confirmed a higher rate of polymorphism among the WDIS. We conclude that these WDIS represent repositories of unique naturally occurring genetic variability that may prove to be invaluable for the study of complex phenotypes. Next, we used the WDIS to search for novel phenotypes related to malaria pathogenesis. Whereas most laboratory strains were susceptible to experimental cerebral malaria (ECM) after infection with P. berghei ANKA, several WDIS were found to be resistant. To study the genetic inheritance of resistant/susceptibility to P. berghei ANKA infection we analysed backcross and F2 cohorts derived from crossing the WLA wild-derived strain with a laboratory mouse strain (C57BL/6). A novel phenotype represented by the cure of infection, clearance of parasitaemia and establishment of immunological memory was observed in the F2 progeny. The backcross progeny was used to genetically map one locus on chromosome 1 (Berr1) and one locus on chromosome 11 (Berr2) that mediate control of resistance to ECM induced by P. berghei ANKA. Genetic mapping using the F2 progeny showed that a locus on chromosome 1 (Berr1) and a locus on chromosome 9 (Berr3) were contributing to control survival time after infection with lethal Plasmodium. Finally, we identified, a locus on chromosome 4 (Berr4) that appears to control time of death due to hyperparasitaemia. This thesis underlines the value of using WDIS to reveal genetic factors involved in the aetiology of disease phenotypes. The characterisation of the genetic factors represented by the malaria resistance loci identified here are expected to provide a better understanding of the malaria pathology.

"November 2001".
Thesis (PhD)--Macquarie University, Division of Environmental and Life Sciences, Department of Biological Sciences, 2002.
Bibliography: leaves 136-157.
General introduction -- Molecular markers for comparative and quantitative studies in macropods -- Genetic linkage map construction in the tammar wallaby (M. eugenii) -- Intraspecific variation, sex-biased dispersal and phylogeography of the eastern grey kangaroo (M. giganteus) -- General discussion.
The analysis of DNA using molecular techniques is an important tool for studies of evolutionary relationships, population genetics and genome organisation. The use of molecular markers within marsupials is primarily limited by their availability and success of amplification. Within this study, 77 macropodid type II microsatellite loci and two type I genetic markers were characterised within M. eugenii to evaluate polymorphic levels and cross-species amplification artifacts. Results indicated that 65 microsatellite loci amplified a single locus in M. eugenii with 44 exhibiting high levels of variability. The success of crossspecies amplification of microsatellite loci was inversely proportional to the evolutionary distance between the macropod species. It is revealed that the majority of species within the Macropodidae are capable of using many of the available heterologous microsatellites. When comparing the degree of variability between source-species and M. eugenii, most were significantly higher within source species (P < 0.05). These differences were most likely caused by ascertainment bias in microsatellite selection for both length and purity. -- The production of a marsupial genetic linkage map is perhaps one of the most important objectives in marsupial research. This study used a total of 353 informative meioses and 64 genetic markers to construct a framework genetic linkage map for M. eugenii. Nearly all markers (93.7%) formed a significant linkage (LOD > 3.0) with at least one other marker. More than 70% (828 cM) of the genome had been mapped when compared with chiasmata data. Nine linkage groups were identified, with all but one (LG7; X-linked) allocated to the autosomes. Theses groups ranged in size from 15.7 cM to 176.5 cM, and have an average distance of 16.2 cM between adjacent markers. Of the autosomal linkage groups, LG2 and LG3 were assigned to chromosome 1 and LG4 localised to chromosome 3 based on physical localisation of genes. Significant sex-specific distortions towards reduced female recombination rates were revealed in 22% of comparisons. Positive interference was observed within all the linkage groups analysed. When comparing the X-chromosome data to closely related species it is apparent that it is conserved both in synteny and gene order. -- The investigation of population dynamics of eastern grey kangaroos has been limited to a few ecological studies. The present investigation provides analysis of mtDNA and microsatellite data to infer both historical and contemporary patterns of population structuring and dispersal. The average level of genetic variation across sample locations was exceedingly high (h = 0.95, HE = 0.82), and is one of the highest observed for marsupials. Contrary to ecological studies, both genic and genotypic analyses reveal weak genetic structure of populations where high levels of dispersal may be inferred up to 230 km. The movement of individuals was predominantly male-biased (average N,m = 22.61, average N p = 2.73). However, neither sex showed significant isolation by distance. On a continental scale, there was strong genetic differentiation and phylogeographic distinction between southern (TAS, VIC and NSW) and northern (QLD) Australian populations, indicating a current and / or historical restriction of geneflow. In addition, it is evident that northern populations are historically more recent, and were derived from a small number of southern eastern grey kangaroo founders. Phylogenetic comparisons between M. g. giganteus and M. g. tasmaniensis, indicated that the current taxonomic status of these subspecies should be revised as there was a lack of genetic differentiation between the populations sampled.
Mode of access: World Wide Web.
xv, 182 leaves ill

Thesis (M.S.)--University of Cincinnati, 2006.
Advisor: Nancy Steinberg Warren. Title from electronic thesis title page (viewed July 17, 2009). Includes abstract. Keywords: Genetic advocacy, professional collaborations, genetic counseling, genetic; conditions, support groups. Includes bibliographical references.

The main goal of the master's thesis is to compose a study on cartesian genetic programming with focus on evolution of circuits and to design a concept for visualisation of this evolution. Another goal is to create a program to visualise the circuit evolution in cartesian genetic programming, its generations and chromosomes. The program is capable of visualising the changes between generations and chromosomes and comparing more chromosomes at once. Several user cases had been prepared for the resulting program.

Bibliography: leaves 127-151. The successful application of genetic engineering in wheat is dependent on the availability of suitable tissue culture and transformation methods. The primary object of this project was the development of these technologies using elite Australian wheat varieties.

Una de les forces selectives més fortes que han afectat a les poblacions humanes en la història més recent és el paràsit de la malària: Plasmodium falciparum, que és la causa de varis exemples d'adaptació induïda per patògens en els éssers humans. Una forma especial de malària és l'associada a l'embaràs, que es caracteritza per l'acumulació d'eritròcits infectats en la placenta, i que pot arribar a causar fins a 200.000 morts maternoinfantils cada any. L'objectiu d'aquest treball és descriure com aquesta forma peculiar de malària ha afectat la variació genètica humana. Amb aquesta finalitat, hem utilitzat mètodes tant de la genètica evolutiva com de l'epidemiologia molecular, resultant en la primera investigació a gran escala de la base genètica de la malària placentària. Els resultats ofereixen una nova visió sobre els gens que modulen el risc d'infecció, ,així com de la selecció natural actuant sobre les vies cel·lulars implicades en la patogènesi de la malaltia. Finalment, també aportem noves dades sobre l'estructura genètica de les poblacions sub-saharianes analitzades.
One of the strongest selective forces affecting human populations in recent history is the malaria parasite Plasmodium falciparum, which is the cause of a variety of well-established examples of pathogen-induced adaptation in humans. A special form of malaria is pregnancy-associated malaria, which is characterised by the accumulation of infected erythrocytes in the placenta, and causes up to 200,000 maternal and infant deaths every year. The aim of this work is to characterise how this particular form of malaria has shaped human genetic variation. To that end we use methods of both evolutionary genetics and molecular epidemiology, reporting the first large-scale investigation of the genetic basis of placental infection. Our results provide new insights into genes modulating the risk of infection, as well as natural selection acting on cellular pathways involved in the pathogenesis of the disease. Finally, we also provide new data on the genetic structure of affected populations in Sub-Saharan Africa.

Fighting ability is a well-known attitude in Valdostana cattle (i.e., Aosta Chestnut and Aosta Black Pied cattle), due to the strong belligerency that cows exhibit at pasture, when unfamiliar animals met and new hierarchies for the access to resource have to be established. This peculiar attitude revives during the traditional tournaments of Batailles de Reines, annually performed by cows in the Aosta Valley. Annual competition consists of 20 eliminatory tournaments and a final challenge, where only the two autochthonous breeds are allowed to take part in the battles. Using data coming from the battles, fighting ability has been investigated aiming to assess both the behavioural and genetic aspects. Four steps have been followed, aiming to look at fighting ability under different but complementary point of views and to shed light on different concerns. At first, data from 4 tournaments undertaken in 2009 have been video recorded and analysed (i.e., a total of 168 fights) in order to depict both the dynamics of agonistic interaction among cows and what factors may affect the shape and the outcome of a conflict. A suitable phenotypic score for fighting ability (i.e., Placement score) was developed in a second study using data from 6 years of battles (i.e., 2001-2006, approximately 16,000 records belonging to about 6,000 cows). This in order to build a genetic model able to investigate the variance components of fighting ability and to quantify genetic parameters. Additionally (i.e., third study), the analyses included the incidence of conspecifics in the genetic estimates of fighting ability, considering the introduction of the opponent either within the phenotype itself or directly in the model as indirect genetic effect (IGEs). As alternative analysis, the contribution of conspecifics has been retained directly into the phenotype, implementing the previous score into a Competitive Placement Score, thus applied into analogous genetic models. As further step, the effect of another force (i.e., inbreeding) and its relationship with the genetic values for fighting ability has been investigated in Aosta Chestnut and Aosta Black Pied cattle. Population data coming from the entire pedigree, as well as all available information on 9 years of battles (i.e., about 24,000 records of over 8,200 participants) permitted to assign individual inbreeding coefficients to cows. Thus, the incidence of inbreeding into genetic models for fighting ability was studied, as well the possible relation among genetic merits and inbreeding. Results obtained from this study indicate that fighting battles among cows seem to follow the typical dynamic of an escalated contest, with a cumulative assessment of the two contenders. Moreover, age, weight, and, most of all, prior experiences of the previous battles play a role in determining the outline and the intensity of the conflict. In addition, the genetic component of fighting ability results of main importance in affecting the outcome of the encounters. As well, weight, age, herd and tournament revealed as significant factor in the investigation of phenotypic variance, and, together with a direct additive and a permanent environmental, they have allowed the estimates of “non zero” genetic parameters. Heritability of fighting ability showed a level of about 0.08 when evaluated using a classical quantitative model, whereas the introduction of indirect genetic effects drove heritability estimates to levels of approximately 0.11. Moreover, including indirect genetic components showed a better general model fitting, whereas classical quantitative models taking into account of the opponent within the score exhibited the worst fitting. Reasoning on the different variance components that can be accounted into the model (i.e., either direct or indirect and due to conspecifics, as well as related to the permanent environment), models that included the opponent only as genetic effect have provided better estimates. Including the kinship as classes of inbreeding coefficient within the genetic models, heritability estimates undergo some small variations under classical quantitative models (i.e., +0.02) whereas models with IGEs did not experienced any shift due to inbreeding. However, the genetic values for fighting ability resulted as reduced in correspondence to increasing levels of inbreeding, as shown by the negative slope of the linear regression analysis performed on lineages of fighting cows with increasing inbreeding levels. As well as inbreeding shows a positive trend over years due to selection, it is interesting to note that, despite the incidence of inbreeding depression and a lack in planned selective programs, also fighting ability reveal a positive increase in mean breeding values (about 2-3% of gain/year). Thus, models including indirect genetic effects are the most appropriate tool in investigating social traits, and the study of fighting ability in Valdostana cattle may provide some interesting suggestions for the analysis of social traits in the area of animal breeding.
Le razze Valdostana Castana e Pezzata nera tradizionalmente si contraddistinguono per una spiccata belligeranza che emerge al momento del pascolo, quando gli animali provenienti da mandrie diverse si incontrano e combattono per definire nuove gerarchie sociali. Allo scopo di riproporre tale comportamento ad un pubblico più vasto, gli allevatori valdostani organizzano da secoli una caratteristica competizione che prende il nome di “Batailles de reines” e vede annualmente migliaia di esemplari contendersi il titolo di “Regina dell’anno”. Annualmente, la manifestazione consiste in 20 giornate di eliminatorie ed in un torneo finale a cui è consentito prendere parte soltanto alle bovine provenienti dalla Valle d’Aosta. I risultati dei combattimenti disputati nel corso degli anni sono divenuti materia di ricerca nel presente lavoro di tesi, allo scopo di studiare alcuni aspetti, sia genetici che comportamentali, dell’attitudine al combattimento nelle bovine valdostane. Il carattere in questione è stato studiato seguendo 4 passaggi successivi, condotti con metodologie diverse e volti a mettere in luce aspetti differenti del comportamento combattivo. La prima analisi si è focalizzata sulla dinamica delle interazioni agonistiche tra bovine combattenti, preoccupandosi di capire quali fattori possano incidere nel tipo di combattimento che viene espresso e sul suo eventuale esito. Allo scopo, sono stati considerati 168 combattimenti registrati mediante videocamera nel corso di quattro tornei svoltisi nella stagione 2009. Il dataset per le analisi è stato largamente ampliato nello studio successivo, allargato ai dati raccolti nel corso di sei anni di competizioni (dal 2001 al 2006, circa 16.000 record appartenenti a 6.000 esemplari), e volto a delineare un punteggio fenotipico (Placement Score) ben rappresentativo delle performance dei partecipanti. Tale punteggio è stato quindi utilizzato come variabile dipendente in un modello genetico volto a stimare le componenti di varianza ed i parametri genetici per l’attitudine al combattimento. Tale punteggio è stato quindi inserito come fenotipo in un modello genetico volto a stimare le componenti di varianza e i parametri genetici inerenti al carattere studiato. Un’ulteriore analisi (terzo passaggio), si è invece focalizzata sullo studio degli effetti genetici indiretti (IGEs) dovuti all’incidenza dei partner sociali nel fenotipo dell’individuo. Quale criterio di indagine, si è provveduto a confrontare modelli genetici privi dell’effetto dei conpecifici (i.e., membri della stessa specie), con modelli comprendenti invece l’avversario, alternativamente introdotto nel fenotipo (Competitive Placement Score) e nel modello genetico. Quale quarto e finale passaggio, è stato condotto uno studio di popolazione sul livello di inbreeding nelle due razze studiate, in grado di stimare coefficienti di parentela individuali. Tali coefficienti sono stati quindi inseriti nei modelli genetici (descritti in precedenza) allo scopo di determinare l’incidenza dell’inbreeding sulle stime dei parametri genetici per la combattività, nonché sul valore genetico degli individui consanguinei. Per quest’analisi si è reso disponibile un dataset più ampio, comprendente un ammontare di 24,000 record relativi ad oltre 8,200 esemplari. I risultati ottenuti a seguito di tutte le analisi condotte, dimostrano che i combattimenti tra bovine seguono le tipiche dinamiche della lotta scalata, costituita da valutazioni successive degli avversari con esibizioni ad intensità crescente. Quali risultano fattori chiave nello delineare l’esito dei conflitti e le dinamiche in cui essi si svolgono sono emersi l’età dei contendenti, il loro peso, e, soprattutto, le precedenti esperienze di combattimento. Analogamente, anche la componente genetica della combattività rivestire un ruolo significativo nell’influenzare l’esito dei conflitti. Le analisi statistiche e genetiche condotte su tale carattere hanno permesso di riconoscere come significativi fattori quali il peso, l’età, l’azienda e il torneo dell’esemplare, come pure le componenti genetica indiretta e ambientale. Le analisi condotte sui modelli quantitativi classici hanno permesso di stimare un’ereditabilità per l’attitudine al combattimento dell’8%, mentre le analisi effettuate sui modelli con effetti genetici indiretti hanno riportato valori di ereditabilità dell’11%. Confrontando le due tipologie di modelli, è emerso come l’inclusione degli effetti genetici indiretti porti a valori migliori nelle stime. Tra i vari modelli comprendenti le componenti indirette considerati negli studi, quello il più affidabile risulta includere gli effetti indiretti solo in termini di componente genetica additiva e non ambientale. L’introduzione del coefficiente di parentela nei modelli genetici, sia essi classici che comprendenti effetti indiretti, comporta delle variazioni soltanto lievi nelle stime dell’ereditabilità, dell’ordine del 2% modelli classici, e addirittura non percettibili negli altri. I valori genetici per la combattività sembrano comunque risentire negativamente dell’effetto dell’inbreeding, come suggerito dalla pendenza negativa della retta di regressione lineare ricavata analizzando genealogie di consanguinei con livelli di inbreeding crescenti. È infine interessante notare come, nonostante la mancanza di un’opera selettiva pianificata rivolta al miglioramento del carattere, l’attitudine al combattimento risulti comunque aumentare nel tempo, rivelando un incremento nei valori genetici del 2-3% annuo. Da questa, e dalle precedenti considerazioni effettuate per le precedenti analisi, è possibile concludere che l’attitudine al combattimento nella razza Valdostana può offrire al miglioramento genetico degli spunti di riflessione interessanti per l’analisi dei comportamenti e dei caratteri sociali.

The postglacial recolonization of northern Europe supposedly originated from Western Europe and the Russian Plain, however, recent molecular and macrofossil-based investigations suggest that the history may be more complex than previously thought. This study aims to investigate the genetic diversity and population structure of Scots pine from Scandinavia to Russia to re-evaluate its recolonization history, and to examine whether the pattern of spatial genetic diversity has any adaptive significance. Populations ranging from Norway to Russia were sampled and genotyped using genotyping-by-sequencing. The seedlings were freeze tested to provide an average degree of hardiness for every population. Eight hundred and thirty-two seedlings were analyzed, and 6,034 SNPs were recovered in these individuals after stringent filtering. Population structure was investigated using fastStructure and differentiation between populations was estimated with pairwise FST and analysis of molecular variance (AMOVA) to assess the genetic variability. Genetic diversity was measured as observed heterozygosity, H0, in populations, clusters and overall. Two genetic clusters were detected in the samples, one in Norway and Sweden and one in Russia. These clusters are weakly differentiated (FST = 0.01202) with only 0.66 % variation between them. Highest variation was found within populations (98.8 %) and the overall genetic diversity for all populations was high (Ho = 0.2573). The weak differentiation and high diversity are indicative of extensive gene flow between populations in this species. The composition of the clusters across the sampled area suggests a westward recolonization from the Russian Plain into Scandinavia, and a possible local origin of another polymorphism in Norway and Sweden. No clear relationship between cold hardiness and genetic variation was detected. The clinal variation in cold hardiness reflects local adaptation, and the difference between genetic and phenotypic variation is likely due to epigenetic regulation or polygenic inheritance. More extensive genome scan is needed to understand the genetic basis of local adaptation.

Thesis (MSc)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: The World Health Organisation (WHO) has defined preterm labour as the onset of labour before 37 completed weeks of gestation with an incidence ranging between 5-10%. Although patient care has improved, the rate of preterm birth has slowly been increasing and currently impacts significantly on maternal and fetal mortality and morbidity. The complex condition of preterm labour involves multiple etiologies and risk factors, which complicates the search for candidate markers and / or biomarkers. The aim of this prospective study was to investigate potential genetic associations with preterm labour. The study cohort consisted of consecutive first-time booking, low-risk primigravid pregnant women from a restricted geographical region. The study cohort comprised 421 [306 Coloured and 115 Black] pregnant women presenting at the Paarl Hospital Obstetric clinic. Subsequently, DNA was extracted from whole blood and investigated for a range of known polymorphisms in pro-inflammatory and anti-inflammatory cytokines, as well as the novel LGALS13 gene, for potential variants that may impact on pregnancy outcome. Screening techniques involve combinations of allele-specific PCR amplification, Multiphor SSCP/HD analysis, restriction enzyme analyses and DNA sequencing. A significant association was demonstrated between the IL-1RN*2-allele and adverse pregnancy outcome, mainly in the preterm labour and hypertension group. The presence TNFα-308 A-allele was associated with overall adverse pregnancy outcome and preterm labour. In addition to this, a novel IL-1RN allele was identified in the control group. Mutation screening and subsequent statistical methods revealed an association between a novel LGALS13 exonic variant, 221delT, and preterm labour in Coloured women. Two previouslydocumented intronic variants (IVS2-22A/G and IVS3+72T/A) demonstrated linkage disequilibrium, signifying evolutionary conservation of exon three. Additionally, two novel intronic variants, IVS2-36 G/A and IVS2-15 G/A, demonstrated no association with adverse pregnancy outcome. In this study we identified rare novel exonic variants; two non-synonymous variants in exon three (M44V, [N=2] and K87R, [N=1]) and a silent variant in exon four (P117P, [N=1]) - all identified in individuals from the control cohort. Within coding exon three, an interesting variant [“hotspot”] was identified, which represents six polymorphic bases within an 11bp stretch. No associations were demonstrated with these variants and pregnancy outcome. Furthermore, a previously documented 5' “‘promoter” variant, -98 A/C, was identified and demonstrated no association with adverse pregnancy outcome. However, subdivision of lateonset pre-eclamptic cases revealed a significant association with the A-allele and late-onset preeclampsia. Genotype-phenotype investigation demonstrated association between the IL-10 -1082 A/G, IL-4 C/T and 221delT loci and poor pregnancy progress which manifested as (i) delivery of infants weighing <2000g, (ii) before 37 weeks of gestation. The findings of this study will strengthen our understanding of the pathophysiology underlying pregnancy complications and facilitate the further development of effective treatment strategies to reduce maternal and fetal morbidity and mortality.
AFRIKAANSE OPSOMMING: Die Wêreld Gesondheid Organisasie (WHO) klassifiseer voortydse kraam as kontraksie voor 37 volledige weke, met ‘n insidensie tussen 5-10%. Alhoewel pasiënte-sorg verbeter het, neem die tempo van voortydse geboorte steeds toe, wat ‘n groot impak het op moederstrefte en fetale mortaliteit en morbiditeit. Die komplekse kondisie van voortydse kraam sluit veelvoudige oorsake en risiko faktore in, wat die navorsing van kandidaat en / of biologiese merkers kompliseer. Die doel van hierdie prospektiewe studie, was die potensiële navorsing van genetiese assosiasies met voortydse kraam. Die studie kohort bevat opeenvolgende eerste bespreking van lae risiko primigravida swanger vrouens vanaf ‘n beperkte geografiese omgewing. Die studie kohort beslaan 421 [306 Kleurling en 115 Swart] swanger vrouens teenwoordig by die Paarl Hospitaal Verloskunde kliniek. Vervolgens was DNS geëkstraeer van bloedmonsters en geondersoek vir ‘n verskeidenheid van bekende polimorfismes in pro-inflammatoriese en antiinflammatoriese sitokiene, insluitend die nuwe sifting van die LGALS13 geen potensiaal vir variante wat ‘n impak op swangerskap uitkomste sal hê. Die siftings tegnieke toegepas, sluit in ‘n kombinasie van alleel-spesifieke amplifikasie, Multiphor enkelstring konformasie polimorfisme / heterodupleks analise, restriksie ensiem verterings en volgorde bepalings tegnieke. ‘n Betekenisvolle assosiasie was gedemonstreer tussen die IL-1RN*2-alleel en nadelige swangerskap, beperk tot voortydse kraam en die hipertensie groep. Die teenwoordigheid van die TNFα-308 A-alleel was geassosieer met algehele nadelige uitkomste en voortydse kraam. Daarby, was ‘n nuwe IL-1RN alleel geïdentifiseer in die kontrole groep. Mutasie sifting en opeenvolgende statistiese metodes, het ‘n assosiasie getoon tussen ‘n nuwe LGALS13 koderende variant, 221delT, en voortydse kraam in Kleurling vrouens. Twee voorafbeskryfde introniese variante (IVS2-22 A/G en IVS3+72 T/A), het ‘n betekenisvolle bewys opgelewer dat daar koppelings-onewewig bestaan tussen hierdie variante, en toon evolusionêre konservasie van ekson drie. Addisioneel was twee nuwe introniese variante ontdek, IVS2-36 G/A en IVS2-15 G/A, wat geen assosiasie getoon nie. In hierdie studie het ons ‘n nuwe seldsame koderende variante geïdentifiseer in die kontrole groep, waarvan twee nie-sinonieme variante was in ekson drie (M44V, N=2 en K87R, N=1) en ‘n stil variasie in ekson vier (P117P, N=1). Geleë in die koderende area van ekson drie, was ’n interessante variant [“hotspot’] ontdek, waarvan ses basisse in ‘n 11 basis paar area polimorfies is. Geen assosiasie was getoon met hierdie variante en swangerskap uitkomste nie. Verder was ‘n voorafbeskryfde 5' ‘promotor’ variant, -98 A/C, geïdentifiseer wat geen assosiasie getoon met nadelige swangerskap uitkomste nie. Onderverdeling van laat-aanvangs preeklampsie, het getoon dat die A-alleel ‘n betekenisvolle assosiasie getoon het met die ontwikkeling van laat pre-eklampsie. Genotipe-fenotipe interaksies het ’n assosiasie getoon tussen die IL-10 -1082 A/G, IL-4 C/T en 221delT lokusse en nadelige swangerskap uitkomste, wat manifesteer as (i) kraam van suigelinge wat <2000g weeg, (ii) geboorte voor 37 weke. Die bevindings van hierdie studie sal ons basiese kennis verbeter oor die patologie beskrywend aan swangerskap komplikasies, asook die fasilitering en ontwikkeling van effektiewe behandelings strategieë, om moederstrefte en fetale mortaliteit en morbiditeit te verminder.

The present thesis covers two aspects of statistical analysis applied to the genetics of human diseases. First, the significance of LOD-score results for the confirmation of linkage is addressed, with special emphasis on small pedigrees. A new analytical approach is presented for the linkage analysis of heterogenetic traits, using hereditary spastic paraplegia as a model, a disease well suited for the analyses. The critical significance values for confirmation of linkage are evaluated using Bayesian statistics, and empirical P-values for LOD score results are calculated using computer simulation methods. The presented analytical approach resulted in conclusive linkage analyses on small to medium-size families, under the restrictions of genetic heterogeneity.
The second part addresses linkage-disequilibrium based fine mapping in the French Canadian population. The performance of five linkage-disequilibrium based fine-mapping methods is evaluated using French Canadian chromosomes with one of three diseases found in this population: oculopharyngeal muscular dystrophy (OPMD), hidrotic ectodermal dysplasia (HED), and sensorimotor polyneuropathy with or without agenesis of the corpus callosum (ACCPN). The gene for OPMD was recently mapped and cloned, allowing us to evaluate the performance of the methods with the OPMD results, and to make predictions about the ACCPN and HED putative gene positions. In addition, a new approach to linkage-disequilibrium based fine mapping is presented using FrenchCanadian ascending genealogies. The method involves two steps. First, the likely founding couple of a mutation-bearing chromosome is identified using a computerised randomisation statistic. Then, using a delete-d jackknife resampling scheme, the distribution of gene mapping estimates is calculated from the count of ancestral recombinants and ancestral meioses joining the identified founding couple to the disease gene carriers. Gene mapping estimates are calculated from each marker individually, and confidence intervals of the estimates are derived from the jackknife distributions. The method, when applied to French Canadian families with OPMD, successfully confirmed the localisation of PABP2 responsible for OPMD and performed better than other linkage disequilibrium-based mapping models.

The purpose of this study was two-fold. The first objective was to determine if Saccharomyces cerevisiae is a useful system for investigating the expression of T-DNA (it takes several months to obtain sufficient bacteria-free transformed plant tissue to investigate T-DNA transcription). A short fragment of T-DNA carrying T-DNA gene 7 was cloned into a yeast plasmid in an attempt to investigate the expression of gene 7 in yeast. The second objective was to determine the significance of a heat shock related sequence identified in the 5¹ region of T-DNA gene 7. Primer extension analysis, SI nuclease mapping, and Northern hybridizations indicate that transcription of T-DNA gene 7 in yeast is different from that of transcription of gene 7 in crown gall tumors. Transcription is different because the distance between the TATA box and the transcription initiation sites must be at least 40 nucleotides in yeast. Therefore, Saccharomyces cerevisiae does not appear to be a useful system for investigating the expression of T-DNA. Crown gall tumors were subjected to a number of stress agents, including heat shock, to determine the significance of the heat shock related sequence identified in gene 7. Primer extension analyses indicate that only cadmium and mercury have a significant effect on the expression of T-DNA gene 7. Although gene 7 responds to cadmium and mercury, the increase in transcription does not appear to be heat shock or metallothionein related, indicating that another mechanism is involved in the enhanced transcription of T-DNA gene 7 in crown gall tumors.
Medicine, Faculty of
Medical Genetics, Department of
Graduate

Diabetes mellitus represents a group of metabolic disorders caused by both environmental and genetic factors. The two most common forms of diabetes are type 2 diabetes (T2D) and type 1 diabetes (T1D). T2D is associated with obesity and the disease is caused by insulin resistance and pancreatic b-cell dysfunction. T1D is an autoimmune disease in which the insulin- producing b-cells in the pancreas are destroyed by infiltration of lymphocytes. The aim of this thesis was to identify genes conferring susceptibility to diabetes. This was approached using genetic methods, both linkage and association studies, within the population of northern Sweden. The northern Swedish population is well suited for genetic studies of familial forms of disease, since an internal expansion of the northern Swedish population, coupled with a low frequency of immigration and a high frequency of consanguineous marriages, has resulted in a relatively homogeneous gene pool. This simplified genetic background increases the probability of identifying genes contributing to disease. The family-based material used for the type 2 diabetes studies (papers I and II) consisted of 231 individuals from 59 families originating in northern Sweden. The type 2 diabetes case-control material (papers I and II) consisted of 872 cases and 857 matched controls, all from northern Sweden. In paper I we performed a genome-wide linkage scan, seeking T2D susceptibility loci. Linkage to the previously identified Calpain-10 region was found, however, association studies in the case-control material revealed no association to the CAPN10 gene. Using both the family-based and the case-control material, we were able to confirm the association of polymorphisms in the TCF7L2 gene to T2D in the population of northern Sweden (paper II). CTLA-4 is a negative regulator of T cell activity, belonging to the CD28 co-stimulatory receptor family. Numerous reports, including our own, have associated CTLA-4 variants with T1D as well as other autoimmune diseases, such as autoimmune thyroid disease (AITD). Allelic variation in the 3ÚTR of the CTLA-4 gene was associated to human T1D and this variant has also been suggested to affect the level of mRNA encoding the soluble form of the molecule (sCTLA-4). We confirmed the association of allelic variation in the 3ÚTR of the CTLA-4 gene in a T1D/AITD case-control material from northern Sweden, consisting of 104 individuals with ATID, 149 individuals with T1D and 865 matched controls. However, we were unable to identify any correlation between allelic variants in the 3ÚTR of the CTLA-4 gene and expression of sCTLA-4 (paper III). Based on recently published genome-wide association (GWA) scans, 33 single-nucleotide polymorphisms (SNPs) located within 16 genes were selected for an association analysis in T1D/AITD families from northern Sweden. The T1D/AITD family-based material consisted of 253 cases and 206 healthy individuals from 97 northern Swedish families. Analysis revealed association to T1D for SNPs in PTPN22, COL1A2, IL-2Ra and INS. In addition, SNPs in CTLA-4, IL-2 and C12orf30 were shown to be associated to AITD (paper IV). Together, these results underpin the notion that the population of northern Sweden is well suited for the detection of genes involved in complex diseases. The use of our more restricted patient material, compared to materials used in published GWA scans, enables the discovery of disease associated genes in a more cost effective manner and show that our population is capable of detecting general susceptibility genes.

In order to recover new mutant alleles of the Polycomb group gene Additional sex combs (Asx), mutagenized chromosomes were screened over the putative Asx allele XT129. Thirteen new mutant strains that fail to complement XT129 were recovered. Unexpectedly, the thirteen strains sorted into four complementation groups. Recombination mapping suggests that each complementation group represents a separate locus. The largest group fails to complement a deletion of Asx and maps in the vicinity of 2-72, the published location of Asx. All new mutant strains enhance the phenotype of Polycomb mutant flies and are not allelic to any previously discovered second chromosome Polycomb group genes. Therefore, the new mutants may be considered putative new members of the Polycomb group. This study suggests that Asx belongs to a sub-group of genes displaying intergenic non-complementation.
Science, Faculty of
Zoology, Department of
Graduate

Inledning: I Sverige har alla blivande föräldrar rätt till fri mödravård. I stort sett alla kvinnor utnyttjar den förmånen. Barnmorskor vid MVC är ofta den första kontakten som de blivande föräldrarna träffar under graviditeten, och har också huvudansvaret för graviditeten ända fram till barnets födelse. Under de senare åren har mödravården alltmer fokuserat på det ofödda barnets hälsa. Förutom de rutinmässiga undersökningarna och samtalen är barnmorskans uppgift också att ge information kring olika fosterdiagnostiska metoder och genetiska avvikelser som kan upptäckas. Utifrån informationen ska de blivande föräldrarna kunna fatta egna beslut kring fosterdiagnostik, och ofta ta ett snabbt beslut. Det är därför viktigt att den information som ges är tydlig och detaljerad. Barnmorskans information ska göra att man känner sig trygg med de beslut man tar. Den utökade kunskapen, medvetenheten och känsligare tekniken ökar den enskilda förälderns önskan om relevant information och därför ställs högre krav på barnmorskor i rollen som förmedlare av information kring genetik och fosterdiagnostik. Därför är det viktigt att undersöka hur barnmorskorna själva upplever sin kunskapsnivå inom dessa områden, om de själva anser sig ha fått tillräcklig utbildning för att ge denna information till de blivande föräldrarna. Syftet med studien var att undersöka hur barnmorskor upplever sin kunskapsnivå och utbildning inom fosterdiagnostik och genetik samt att undersöka vilka behov för utökade resurser inom dessa ämnen barnmorskorna ansågs föreligga. Material och metod: Totalt 200 barnmorskor på MVC, fördelade mellan olika regioner av Sverige, deltog i en enkätundersökning våren 2005. Svarsutfallet var 30 %. Frågeformuläret innehöll 49 frågor med både fasta och öppna svarsalternativ. Formuläret var bearbetat och utformat för denna studie. Resultat: Majoriteten av barnmorskorna ansåg sig ha bristfällig kunskap inom fosterdiagnostik och genetik. Många barnmorskor ansåg sig ha lite, men ej tillräcklig, kunskap kring ultraljud och moderkaks- och fostervattenprov. En fjärdedel av barnmorskorna ansåg sig inte ha någon kunskap alls kring NUPP eller genetik. Barnmorskorna ansåg också att de har fått för lite eller ingen utbildning alls inom dessa områden. Så stor andel som 67 % önskar ytterligare utbildning inom ovanstående ämnen. En liten andel (6 %) anser att det vore bra om några få barnmorskor fick ytterligare utbildning och på så sätt få en ökad kompetens. 11 % av barnmorskor ansåg att det vore bra med personer med specialkompetens, genetiska vägledare, knutna till mödravården, som de kan hänvisa blivande föräldrar till. Konklusion: Denna studie har resulterat i en sammanställning av hur de tillfrågade barnmorskorna vid MVC själva uppfattar sin kompetens och utbildning inom genetik och fosterdiagnostik. Då barnmorskorna själva ansåg sig ha bristfällig kunskap inom fosterdiagnostik och genetik samt ansåg att de fått för lite utbildning inom dessa ämnen bör tillvägagångssättet för genetisk vägledning vid svensk mödravård ses över. Resultaten från denna studie kan ses som ett viktigt underlag för hur genetisk vägledning för blivande föräldrar kan förbättras i Sverige.
Introduction: In Sweden, all pregnant women have the right and access to free antenatal care. A midwife is often the first professional contact the expecting parents will encounter, and the midwife is also the person with the key responsibility for the pregnancy. In recent years, antenatal care has increasingly focussed on the health of the unborn child. In addition to routine examinations and conversation, the midwives role is to inform about prenatal diagnosis, the risk figures, and the genetic divergence that can be detected. The parents should, based on this information, be able to make a decision about prenatal diagosis. It is therefore crucial that the information conveyed is clear and sufficient. As the knowledge and awareness in this area increases and as thetechniques gets more accurate, parents desire more relevant information, and the demand on midwives as informers in this area increases proportionally. Therefore it is important to investigate how the midwives experience their own knowledge about prenatal diagnosis and genetics, and if they consider that they have sufficient education in this area. Aims of the study: to investigate how midwives experience their own knowledge about prenatal diagnosis and genetics, to investigate if they consider that they have sufficient education in this area and to investigate what kind of needs for increased resources within this area the midwivesconsider to be important. Material and method: In total 200 antenatal care midwives from various towns of different sizes and from various regions throughout Sweden were invited to take part in a survey in the course of spring 2005. A questionnaire containing 49 questions was distributed by e- mail. About 30 % of the questionnaires were answered and returned. This questionairre was designed for this study. Results:Most midwives considered that they had insufficient knowledge about prenatal diagnosis and genetics. Many midwives said that they had some, but insufficient knowledge about ultrasound and chorionic villus sampling and amniocentesis. As many as 25% of the midwives thought that they had no knowledge about NUPP and genetics. The midwives also considered that they had none or only little education in prenatal diagnosis and genetics. Most midwives (67 %) answered that they would like additional education within prenatal diagnosis and genetics. A few (6 %) think it would be valuable if a few midwives could get extra education and thus increased competence. Others (11 %) consider it a good idea to employ genetic councellors within antenatal care. Conclusion: This study shows how midwives within antenatal care in Sweden assess their own knowledge and education within genetics and prenatal diagnosis. As the midwives consider that they have insufficient knowledge and education in this area, the way of giving this kind of information and genetic councelling within antenatal care in Sweden should be investigated.The results from this study can be regarded as a basis of how geneic councelling for expecting parents could be improved in Sweden.

Osteoarthritis is already a major cause of disability worldwide and with an ageing population the associated socio-economic burden is expected to soar. Complex interactions between genetic and environmental factors are responsible for the initiation of osteoarthritis. There is a clear genetic contribution to hip' osteoarthritis but to date no major susceptibility gene has been identified. A large collection of affected sib pairs with hip osteoarthritis had already been recruited from Northern Ireland (416 participants) and Nottingham (115 participants) . Additional unaffected (n=42) family members were recruited. As part of a genome wide screen 160 microsatellite markers from chromosomes 9, 10, 11, 14, 19, 20, 21 and 22 were typed in each family member (n=531)and controls (n=49). Candidate genes were identified in areas suggesting positive linkage. Single nucleotide polymorphism (SNP) typing or sequencing were used to investigate these genes further. The three regions most suggestive of linkage were found in chromosomes 9 (at 74.4mB), 11 (at 11.7mB) and 19 (at 8mB). Ten of the candidate genes identified were typed with SNPs in one member of each family (n=206) and controls (n=49). The ten exons of the other gene, osteoclast stimulating factor 1 (OSTFl), were sequenced in both cases (n=40) and controls (n= 8). The only candidate gene with significantly positive results was the collagen type V, alpha 3 gene (COL5A3) on chromosome 19. One haplotype block was significantly more common in the control population (p=O.Ol). Further analyses of the COL5A3 gene and its neighbouring genes are currently underway. Identification of a major susceptibility gene will improve our understanding of the underlying pathogenesis and potentially identify new targets for drug therapy. The global impact of finding a major susceptibility gene for hip osteoarthritis should encourage us to continue with our search to elucidate the genetics of familial hip osteoarthritis

This thesis investigates the novel idea of using a computer to create and optimise conceptual designs of a range of differently-shaped three-dimensional solid objects from scratch. An extensive literature review evaluates all related areas of research and reveals that no such system exists. The development of a generic evolutionary design system, using a genetic algorithm (GA) as its core, is then presented. The thesis describes a number of significant advances necessitated by the development of this system. Firstly, a new low-parameter spatial-partitioning representation of solid objects is introduced, which allows a wide range of solid objects to be appropriately defined and easily manipulated by a GA. Secondly, multiobjective optimisation is investigated to allow users to define design problems without fine-tuning large numbers of weights. As a result of this, the new concepts of acceptability, range-independence and importance are introduced and a new multiobjective ranking method is identified as being most appropriate. Thirdly, variable-length chromosomes in GAs are addressed, to allow the number of primitive shapes that define a design to be variable. This problem is overcome by the use of a new hierarchical crossover operator, which uses the new concept of a semantic hierarchy to reference chromosomes. Additionally, the thesis describes how the performance of the GA is improved by using an explicit mapping stage between genotypes and phenotypes, steady-state reproduction with preferential selection, and a new lifespan limiter. A library of modular evaluation software is also presented, which allows a user to define new design problems quickly and easily by picking combinations of modules to guide the evolution of designs. Finally, the feasibility of the generic evolutionary design of solid objects is demonstrated by presenting the successful evolution of both conventional and unconventional designs for fifteen different solid-object design tasks, e.g. tables, heatsinks, penta-prisms, boat hulls, aerodynamic cars.

Thesis (S.B. and M.Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 1999.
Includes bibliographical references (leaves 50-51).
by Jason C. Miller.
S.B.and M.Eng.

During the last two decades, genotyping technology has advanced rapidly, which enabled the tremendous success of genome-wide association studies (GWAS) in the search of disease susceptibility loci (DSLs). However, only a small fraction of the overall predicted heritability can be explained by the DSLs discovered. One possible explanation for this ”missing heritability” phenomenon is that many causal variants are rare. The recent development of high-throughput next-generation sequencing (NGS) technology provides the instrument to look closely at these rare variants with precision and efficiency. However, new approaches for both the storage and analysis of sequencing data are in imminent needs. In this thesis, we introduce three methods that could be utilized in the management and analysis of sequencing data. In Chapter 1, we propose a novel and simple algorithm for compressing sequencing data that leverages on the scarcity of rare variant data, which enables the storage and analysis of sequencing data efficiently in current hardware environment. We also provide a C++ implementation that supports direct and parallel loading of the compressed format without requiring extra time for decompression. Chapter 2 and 3 focus on the association analysis of sequencing data in population-based design. In Chapter 2, we present a statistical methodology that allows the identification of genetic outliers to obtain a genetically homogeneous subpopulation, which reduces the false positives due to population substructure. Our approach is computationally efficient that can be applied to all the genetic loci in the data and does not require pruning of variants in linkage disequilibrium (LD). In Chapter 3, we propose a general analysis framework in which thousands of genetic loci can be tested simultaneously for association with complex phenotypes. The approach is built on spatial-clustering methodology, assuming that genetic loci that are associated with the target phenotype cluster in certain genomic regions. In contrast to standard methodology for multi-loci analysis, which has focused on the dimension reduction of data, the proposed approach profits from the availability of large numbers of genetic loci. Thus it will be especially relevant for whole-genome sequencing studies which commonly record several thousand loci per gene.

Lactic acid bacteria (LAB), a group of generally recognized as safe (GRAS) organisms that metabolize sugars into primarily lactic acid, have traditionally been used for the fermentation and preservation of various foods and beverages. There is increasing interest in the genetic manipulation of LAB to improve existing characteristics or introduce novel, industrially pertinent phenotypes. However, because these bacteria have food-related applications, their genetic modification requires the use of food-grade genetic engineering tools. LAB plasmids, self-replicating extrachromosomal DNA molecules, can be used to derive food-grade cloning vectors. The rationale of this research was to develop a food-grade cloning vector using a lactobacilli cryptic plasmid and to investigate its cloning and expression properties. The main objectives were to (i) screen Lactobacillus spp. for plasmids, (ii) isolate and characterize a plasmid, and (iii) use the plasmid replicon to construct a cloning vector and express heterologous genes in various hosts. This is the first step in the development of a new family of food-grade cloning vectors for the genetic modification of lactobacilli.

Thesis (MSc)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: The genetic information of plants is found in the nucleus, the mitochondria, and the plastids. The DNA of plastids is comprised of multiple copies of a double-stranded, circular, prokaryoticallyderived genome of -150 kb. The genome equivalents of plastid organelles in higher plant cells are an attractive target for genetic engineering as high protein expression levels are readily obtained due to the high genome copy number per organelle. The resultant proteins are contained within the plastid organelle and the corresponding transgenes are inherited, in most crop plants, uniparentally, preventing pollen transmission of DNA. Plastid transformation involves the uniform modification of all the plastid genome copies, a process facilitated by homologous recombination and the non-Mendelian segregation of plastids upon cell division. The plastid genomes are in a continuous state of inter- and intra-molecular exchange due to their common genetic complement. This enables the site-specific integration of any piece of DNA flanked by plastid targeting sequences, via homologous recombination. The attainment of homoplasmy, where all genomes are transformed, requires the inclusion of a plastid-specific selectable marker. Selective pressure favouring the propagation of the transformed genome copies, as well as the random segregation of plastids upon cell division, make it feasible to acquire uniformity and hence genetic stability. From this, a complete transplastomie line is obtained where all plastid genome copies present are transgenic, having eliminated all wild-type genome copies. The prokaryotic nature of the chloroplast genetic system enables expression of multiple proteins from polycistronic mRNAs, allowing the introduction of entire operons in a single transformation. Expression cassettes in vectors thus include single regulatory elements of plastid origin, and harbour genes encoding selectable and screenable markers, as well as one or more genes of interest. Each coding region is preceded by an appropriate translation control region to ensure efficient translation from the polycistronic mRNA. The function of a plastid transformation vector is to enable transfer and stable integration of foreign genes into the chloroplast genomes of higher plants. The expression vector constructed in this research is specific for the transformation of the grape chloroplast genome. Vitis vinifera L., from the family, Vitaceae, is the choice species for the production of wine and therefore our target for plastid transformation. All chloroplast derived regulatory elements and sequences included in the vector thus originated from this species.
AFRIKAANSE OPSOMMING: Die genetiese inligting van plante word gevind in die kern, die mitochondria, en die plastiede. Die DNA van plastiede bestaan uit veelvuldige kopieë van 'n ~ 150 kb dubbelstring, sirkulêre genoom van prokariotiese oorsprong. Die genoomekwivalente van plastiede in hoër plante is 'n aantreklike teiken vir genetiese manipulering, aangesien die hoë genoom kopiegetal per organel dit moontlik maak om gereeld hoë vlakke van proteïenuitdrukking te verkry. Hierdie proteïene word tot die plastied beperk, en die ooreenstemmende transgene word in die meeste plante sitoplasmies oorgeërf, sonder die oordrag van DNA deur die stuifmeel. Plastied transformasie behels die uniforme modifikasie van al die plastied genoomkopieë, 'n proses wat deur homoloë rekombinasie en die nie-Mendeliese segregasie van plastiede tydens seldeling gefasiliteer word. As gevolg van die gemeenskaplike genetiese komplement, vind aanhoudende interen intra-molekulêre uitruiling van plastiedgenome plaas. Dit maak die setel-spesifieke integrasie, via homoloë rekombinasie, van enige stuk DNA wat deur plastied teikenvolgordes begrens word, moontlik. Vir die verkrying van homoplasmie, waar alle genome getransformeer is, word die insluiting van 'n plastiedspesifieke selekteerbare merker benodig. Seleksiedruk wat die vermeerdering van die getransformeerde genoomkopieë bevoordeel, en die lukrake segregasie van plastiede tydens seldeling, maak dit moontlik om genetiese stabiliteit en uniformiteit van die genoom te verkry. Dit kan op sy beurt tot die verkryging van 'n volledige transplastomiese lyn lei, waar alle aanwesige plastiedgenome transgenies is, en wilde tipe genoomkopieë geëlimineer is. Die prokariotiese aard van die chloroplas genetiese sisteem maak die uitdrukking van veelvuldige proteïene vanaf polisistroniese mRNAs moontlik, wat die toevoeging van volledige operons in 'n enkele transformasie toelaat. Uitdrukkingskassette in vektore bevat dus enkel regulatoriese elemente van plastied oorsprong, gene wat kodeer vir selekteerbare en sifbare merkers, asook een of meer gene van belang (teikengene). Voor elke koderingsstreek, is daar ook 'n toepaslike translasie beheerstreek om doeltreffende translasie vanaf die polisistroniese mRNA te verseker. Die funksie van 'n plastied transformasie vektor is om die oordrag en stabiele integrasie van transgene in chloroplasgenome van hoër plante moontlik te maak. Die uitdrukkingsvektor wat in hierdie studie gekonstrueer is, is spesifiek vir die transformasie van die druif chloroplasgenoom. Vitis vinifera L., van die familie Vitaceae, is die voorkeur species vir die produksie van wyn, en daarom die teiken vir plastied transformasie. Alle chloroplast-afgeleide regulatoriese elemente en volgordes wat in hierdie vektor ingesluit is, het huloorsprong vanaf VUis vinifera L.

Thesis (MSc (Genetics))—University of Stellenbosch, 2007.
In South Africa, pre-eclampsia is the second highest cause of maternal deaths. The incidence of this disease in the Western Cape alone is 6.8% and places a large burden of health care facilities. The placenta and implantation thereof is thought to play the most significant role in the onset of this disease. Among the many theories for its aetiology, is the acknowledged two - stage theory. This is based on evidence that pre-eclamptic placentas demonstrate altered remodelling and invasion into the uterine endometrium and myometrium. The sub-optimal endometrium invasion leads to less oxygenation of the placental environment causing transient hypoxia. Consequently, the placenta is thought to release unknown factors into the maternal circulation which then culminates in clinical features associated with pre-eclampsia. Neurokinin B is thought to be one of these placental factors and subsequently binds to the NKB receptor in the maternal system. Endothelium-derived nitric oxide synthase has recently been shown to activate this receptor. The aim of this study was to investigate the role of neurokinin B (TAC3) and the neurokinin B receptor (TACR3) genes in the predisposition of pre-eclampsia and their interaction with eNOS in the South African coloured population together with a matched control cohort.

Most common diseases have substantial heritable components but are characterized by complex inheritance patterns implicating numerous genetic and environmental factors. A longstanding goal of human genetics research is to delineate the genetic architecture of these traits - the number, frequencies, and effect sizes of disease-causing alleles - to inform mapping studies, elucidate mechanisms of disease, and guide development of targeted clinical therapies and diagnostics. Although vast empirical genetic data has now been collected for common diseases, different and contradictory hypotheses have been advocated about features of genetic architecture (e.g., the contribution of rare vs. common variants). Here, we present a framework which combines multiple empirical datasets and simulation studies to enable systematic testing of hypotheses about both global and locus-specific complex trait architecture. We apply this to type 2 diabetes (T2D).

The Roma (Gypsies) are a European people composed of a mosaic of culturally heterogeneous populations. Linguistic analyses point to their origins in the Indian subcontinent. Cultural diversity in extant Romani populations suggests that they are descended from a mixture of Indian populations. Previous population genetic studies of the Roma have supported this claim by demonstrating the genetic heterogeneity of Romani populations. More recently, medical genetic research has detected identical founder mutations in separated Romani populations, which provides evidence of their relatedness. In this thesis, the genetic heritage of the Roma and its significance for genetic disease and research is investigated. Male and female lineages were analysed in eight traditionally endogamous Romani populations. Asian specific Y chromosome haplogroup VI-68 and mitochondrial DNA (mtDNA) haplogroup M were detected in all populations and accounted for 39% and 25% of all lineages respectively. Diversity within haplogroups was assessed by genotyping Y chromosome short tandem repeats (YSTRs) and sequencing the mtDNA hypervariable segment 1 (HVSl). Lineages within haplogroups VI-68 and M were found to be closely related suggesting that Romani populations are predominantly descended from a single Indian ethnic population. The differing historical legacies of Romani populations and adherence to endogamous practices have resulted in genetic substructure and limited diversity within populations. Thus, the Roma are shown to comprise a conglomerate of related admixed population isolates. The unique genetic heritage of the Roma provides a powerful tool for the positional cloning of monogenic disease genes. This is demonstrated through the reduction of the critical chromosomal region for a novel genetic disorder, hereditary motor and sensory neuropathy type Lom (HMSNL). In the initial report, the HMSNL disease locus was defined as a 3cM region on chromosome 8q24. In this study, refined genetic mapping utilising historical and parental recombinations observed in Romani individuals from different populations reduced the HMSNL critical interval to 202kb. Sequence analysis of two genes contained within this genomic interval found all affected individuals to be homozygous for a CT mutation in codon 148 of N-myc downstream regulated gene 1 (NDRGJ), resulting in a truncating Rl48X mutation. Investigation of the population distribution of the R148X disease allele shows that it occurs in six of eight separated Romani populations. Another founder mutation, C283Y in the y-sarcoglycan gene (SGCG), which causes limb girdle muscular dystrophy type 2C (LGMD2C), was found in two of eight Romani populations. Profound founder effects are apparent within Romani populations with a carrier frequency of 19.5% determined for the R148X mutation in the Lom population, and 6.25% for the C283Y allele in the Turgovzi population. High carrier frequencies for autosomal recessive diseases can be expected to pose a significant health risk for these communities. Thus, community-wide carrier testing represents a potential means of addressing this health problem. A pilot community based carrier-testing program was implemented in a Romani community of north eastern Bulgaria and relevant attitudes assessed by means of a questionnaire. Community-based carrier screening was demonstrated to be an appropriate approach to improving health amongst the Roma.