Haemophilia A (HA) is a an X-linked bleeding disorder that manifests due to a mutation in the F8 gene encoding the coagulation factor VIII (FVIII) protein. Therapeutic management of HA involves intravenous FVIII infusions which are either plasma derived or recombinant concentrates that are administered to prevent or manage bleeding episodes promptly. A critical complication of repeated FVIII replacement therapy is the production of FVIII neutralising inhibitors which affect the coagulation potential of the replacement therapy, thus compromising the ability to manage bleeding episodes. The genetic and environmental factors predisposing to inhibitor development remain uncertain, and require improved understanding to provide optimal patient care and surveillance. The study firstly aimed to characterise a cohort of South African HA patients in terms of clinical severity, ethnicity, int22 mutation status and inhibitor development; secondly, to explore whether the genetic factors (clinical severity, ethnicity, int22 mutation status, F8 gene haplotype) influence inhibitor development. A total of 229 probands who had diagnostic HA testing at the Molecular Genetics Laboratory, Division of Human Genetics, of the National Health Laboratory Services (NHLS) and School of Pathology, University of the Witwatersrand, Johannesburg, were included in the study. The majority of patients (91%) in the cohort had severe HA. There were a similar proportion of black and white patients in the cohort. There was a 13% incidence of inhibitor development in the cohort of which 72% were black and 28% were white patients. To investigate the influence of genetic factors on inhibitor development only the probands with known inhibitor status were included (n=216). It was established that 36% (77/216) of patients were int22 positive of which 20% (15/77) were reported to be inhibitor positive while 10% (14/139) of the int22 negative patients (n=139) were shown to be inhibitor positive. Therefore, the int22 positive patients had a two-fold higher incidence of inhibitor development than int22 negative patients. F8 gene haplotype analysis revealed that the H1 and H2 haplotypes were the most common in the cohort while the H3 and H5 haplotypes were only reported in black patients. Black patients were shown to have a higher prevalence of inhibitor development within each haplotype, thus suggesting that factors other than F8 gene haplotype are important in inhibitor development. Overall, black int22 positive probands had a significantly higher prevalence (p=0.04) of inhibitor development than white int22 positive and negative patients in the cohort which is suggestive that ethnicity and F8 gene mutation may play a more major role in inhibitor development compared to F8 gene haplotype. Hence, there is a need to identify other genetic factors that may predispose HA patients to inhibitor development.

Thesis (D.PT.)--Sage Colleges, 2010. / "May 2010." "A Capstone project for PTY 768 presented to the faculty of The Department of Physical Therapy Sage Graduate School in partial fulfillment of the requirements for the degree of Doctor of Physical Therapy." Includes bibliographical references.

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The coagulation and fibrinolytic systems are linked by the thrombin-thrombomodulin complex which regulates each system through activation of protein C and TAFI, respectively. We have used novel assays and techniques to study the enzymology and biochemistry of TAFI and TAFIa, to measure TAFI activation in hemophilia A and protein C deficiency and to determine if enhancing TAFI activation can improve hemostasis in hemophilic plasma and whole blood. We show that TAFIa not TAFI attenuates fibrinolysis in vitro and this is supported by a relatively high catalytic efficiency (16.41μM-1s-1) of plasminogen binding site removal from fibrin degradation products (FDPs) by TAFIa. Since the catalytic efficiency of TAFIa in removing these sites is ~60-fold higher than that for inflammatory mediators such as bradykinin it is likely that FDPs are a physiological substrate of TAFIa. The high catalytic efficiency is primarily a result of a low Km which can be explained by a novel mechanism where TAFIa forms a binary complex with plasminogen and is recruited to the surface of FDPs. The low Km also suggests that TAFIa would effectively cleave lysines from FDPs during the early stages of fibrinolysis (i.e. at low concentrations of FDPs). Since individuals with hemophilia suffer from premature fibrinolysis as a result of insufficient TAFI activation we quantified TAFI activation in whole blood from hemophilic subjects. Both the rate of activation and the area under the TAFI activation time course (termed TAFIa potential) was determined to be reduced in hemophilia A and the TAFIa potential was significantly and inversely correlated with the clinical bleeding iii phenotype. Using a novel therapeutic strategy, we used soluble thrombomodulin to increase TAFI activation which improved the clot lysis time in factor VIII deficient human plasma and hemophilic dog plasma as well as hemophilic dog blood. Finally, we briefly show in a biochemical case study that TAFI activation is enhanced in protein C deficiency and when afflicted individuals are placed on Warfarin anticoagulant therapy, TAFI activation is reduced. Since TAFIa stabilizes blood clots, this suggests that reducing TAFI activation or inhibiting TAFIa may help restore blood flow in vessels with pathological thrombosis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2010-09-23 10:12:18.892

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Dissertation (Ph.D.) -- The Institute for Clinical Social Work, 1997. / A dissertation submitted to the faculty of the Institute of Clinical Social Work in candidacy for the degree of Doctor of Philosophy.

Thesis (M.S.)--University of Wisconsin--Madison, 1966. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 51-52).

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, South Africa in fulfilment of the requirements for the degree of Master of Science, 2019 / The study of flower colour is a particularly valuable approach to investigating fundamental evolutionary questions such as the maintenance of variation within species, the role of natural selection and genetic drift in maintaining polymorphisms, and how such polymorphisms contribute to biodiversity. Flower colour is a phenotype that is easily measured and it provides a strong indicator of the outcomes of selection pressures. It is both driven and maintained by either non- pollinator mediated selection agents, pollinator selection agents, or a combination of both. The overall aim of this study was to investigate the maintenance of flower colour using Rhodohypoxis baurii (Baker) Nel. var. confecta Hilliard and Burtt as a study system. This involved understanding the genetic mechanisms that regulate flower colour and examining the environmental variables that drive this trait variation across populations. Molecular and ecological methods were used in combination to understand flower colour by investigating the genes responsible, as well as the environmental selection pressures acting on this phenotype. Rhodoypoxis baurii var. confecta is a Drakensberg near-endemic species that has naturally occurring populations of magenta, pink, and white flower colour morphs. These flower colour morphs are discrete variants that co-exist in single populations and are thus considered to be true polymorphisms. Three populations of R. baurii var. confecta that occur in Sentinel Peak in the Royal Natal National Park, South Africa were studied. Non-pollinator mediated selection agents were investigated as potential drivers of flower variation in R. baurii var. confecta. The frequency of unpigmented (white) and pigmented (pink/magenta) flower colour morphs was measured over the flowering season. No pollinator visitation was observed at populations of R. baurii var. confecta and therefore pollinator-mediated selection was excluded. The extent to which non pollinator selection agents drive this intra-population variation was investigated by measuring the change in soil moisture, temperature, and precipitation over the flowering season. One population shifted from a greater proportion of unpigmented morphs at the beginning of the flowering season to a relatively greater proportion of pigmented morphs at the end of the season. In this population, a positive correlation was found between the proportion of pigmented morphs and soil moisture. This suggests that the accumulation of water in the soil promotes the production of pigmented flowers. The anthocyanin biosynthetic pathway (ABP) is responsible for the production of pigment in R. baurii var. confecta. The ABP is pleiotropic and in addition to pigment production is responsible for traits related to plant physiology and environmental stress response. Consequently, the ABP is cited as being well conserved among angiosperm species. The presence of expression in four ABP genes (CHI, F3H, F3’H, and F3’5’H) was tested for and compared between unpigmented and pigmented R. baurii var. confecta flowers. As there is no sequence data for Rhodohypoxis or any Hypoxidaceae species, primers for this molecular work were designed using closely related monocotyledon ABP gene sequences. Sequencing results showed that the amplified PCR products were not the targeted ABP genes. These results suggested that the designed primers were non-specific. The similarity of the four ABP genes within angiosperm species was investigated. All available complete sequences of the four regions of interest were aligned and sequence similarity was quantified. The results from this alignment indicate that the four investigated ABP gene sequences are polymorphic and are likely not well conserved within angiosperm species as a whole and, to some extent, within monocotyledon and dicotyledon species respectively. This suggests that the basic structure of the ABP is well conserved amongst angiosperm species however, the gene sequences that make up the pathway are polymorphic and less well conserved. The study highlights the importance of non-pollinator mediated selection on the presence and maintenance of flower colour polymorphisms. In addition, it provides some insight on the ABP and its conservation amongst angiosperm species. It also contributes to understanding how flower colour polymorphisms are maintained in natural populations both on a molecular and ecological level and is the foundational work in understanding the polymorphisms of R. baurii var. confecta. / TL (2020)

Hemophilia A (HA) is a hemorrhagic disorder caused by a decrease/absence of coagulation Factor VIII (FVIII) in circulation. Management involves administration of FVIII to prevent bleeding episodes. The most serious complication of this replacement therapy is the development of inhibitory anti-FVIII antibodies which neutralize the infused FVIII. Low zone tolerance (LZT) is a state in which the immune system is unresponsive to an antigen induced by repeated prior exposure to low doses of said antigen. Previous animal studies exploring LZT demonstrated successful T-cell tolerance induction by this mechanism. This study investigated whether the administration of low-dose FVIII could induce immune tolerance to FVIII in a HA mouse model. HA mice received intravenous FVIII at doses ranging from 0.01 IU/kg - 5 IU/kg to determine the most promising doses (0.25 IU/kg, 2.5 IU/Kg) to further investigate. Naïve mice were treated with 0.25 IU/kg or 2.5 IU/kg weekly for 6 weeks, then immunized with 25 IU/kg FVIII weekly for 4 weeks. Following a two-week rest period (week 12), all mice received a 25 IU/Kg booster shot. Blood was collected on weeks 7, 11 and 13 and anti-FVIII antibody concentrations were measured by ELISA. Control mice received phosphate buffered saline (PBS) during weeks 1-6 of the experiment, followed by identical immunizations as stated above. Within the 2.5 IU/Kg FVIII treatment group, 11% of the mice developed tolerance to the treatment, indicated by undetectable anti-FVIII IgG titer by ELISA. The remaining 89% of these mice developed high titer antibodies, therefore they were not tolerized to FVIII. In the PBS treatment group, 62% of the mice developed high anti-FVIII antibodies. Conversely, 50% of the mice treated with 0.25 IU/kg were tolerized to FVIII and the remaining mice had significantly reduced antibody titers when compared to the controls. Moreover, upon booster dose injection, 100% of 0.25 IU/kg and 2.5 IU/Kg treated mice that were previously tolerized retained tolerance, suggesting that tolerance through low-dose injections is maintained upon FVIII re-exposure. The LZT experiment conducted here shines light on a new approach to preventing FVIII inhibitors. This study suggests that frequently administering low doses of FVIII effectively induces tolerance to FVIII in HA mice. Moreover, treatment through LZT induction may confer long lasting protection against inhibitor development as indicated by the retention of tolerance in mice subjected to a rest period and post-treatment booster shot. / Thesis / Master of Science (MSc)