Hemophilia BVancouver, is a moderately severe hereditary disorder in which the factor IX antigen is present in relatively normal amounts but the biological activity of factor IX is markedly reduced. Previous studies have demonstrated that although the patient has 62% of the normal factor IX antigen level in his plasma, he shows only 2.6% of normal factor IX procoagulant activity. In addition, radioimmunoassays have shown that epitopes on both the heavy and light chains of activated factor IX are present. These two results were taken as an indication that the molecular defect causing the hemophilia may be a point mutation involving an amino acid change in the protein. In order to identify the mutation involved, DNA was isolated from lymphocytes in a blood sample from the patient. This DNA was used to construct a genomic library in the λ vector EMBL3. One million of the resultant clones were screened with a labelled factor IX cDNA probe to identify those clones containing portions of the factor IX gene. DNA inserts from three λ clones, which together span the entire gene, were subcloned to facilitate sequence analysis of the exons and intron / exon junctions of the factor IX gene. The nucleotide sequences of the coding regions were found to match the published sequence of the normal gene, except for one nucleotide. A single mutation was found at nucleotide 31,311 of the factor IX gene (Yoshitake et al.,1985), corresponding to amino acid 397 of the mature protein. This alteration, which changes an isoleucine codon, ATA, to a threonine codon, ACA, is novel among the mutations which have been reported to cause hemophilia B. A three dimensional model of the protease domain of factor IXа, which was prepared on the basis of its homology to the pancreatic serine proteases, was examined in the vicinity of residue 397. The position of threonine 397 in this model suggests that this mutation could alter the hydrogen bonding between factor IXа and its substrate, factor X. Taken together, these data suggest that this mutation is the cause of the hemophilia in this patient. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The concept of (b, s)-continuous functions in topological spaces is introduced and studied. Some of their characteristic properties are considered. Also we investigate the relationships between these classes of functions and other classes of functions.

In a recent discussion of disenchantment and re-enchantment Charles Taylor suggests that it is possible to respond to the disenchanted view of the world, in which meaning and value are understood as subjective projections, by articulating a re-enchanted sense of nature or the universe from the perspective of human ‘agency-in-the-world’, in which meaning and value are objective. The question I address in this thesis is, what could it mean to articulate a re-enchantment from within our ‘agency-in-the-world’? In Chapter One I examine the work of John McDowell in order to explore the possibility that he gives sense to the idea of a re-enchantment from within our agency-in-the-world. I conclude that he provides one way of doing so. However I argue that McDowell’s naturalism of second nature can seem limited as it does not address the ‘proto-religious’ dimension to Taylor’s understanding of re-enchantment. In Chapter Two I turn to the work of Roger Scruton to consider whether he provides a re-enchantment from within our agency-in-the-world that does accommodate this proto-religious dimension. I conclude that he does, but raise concerns about how convincing Scruton’s re-enchantment is. I argue that, from a McDowellian point of view, a case can be made that Scruton implicitly accepts as true certain significant elements of the disenchanted view of the world. In Chapter Three I look to the later Heidegger for an alternative re-enchantment from within our agency-in-the-world that attempts to accommodate the proto-religious. I focus on two interpretations of the later Heidegger given by Julian Young and Charles Taylor. In response to a worry put forward by Young, I argue that Charles Taylor’s interpretation can accommodate a proto-religious dimension. In my Conclusion I argue that McDowell’s naturalism of second nature and the understanding of our agency-in-the-world as presented by Taylor’s Heidegger, form interestingly continuous re-enchantments. On this basis I argue that although McDowell himself does not extend his idea of second nature to accommodate the proto-religious, the example of later Heidegger shows that there is nothing inherently limited about the framework of second nature that means it cannot be extended to encompass important proto-religious responses to the world.

This thesis is focused on immunochemical determination of concentration of active and inactive form of cathepsin B in patients with bladder cancer in order to compare diagnostic efficiency of methods for their possible use for routine diagnosis. Cathepsin B and procathepsin B were measured in serum and urine in 82 patients with bladder cancer (47 men and 35 women), with the average age of 66.5 year. The control group contain of 72 healthy subjects (31 men and 41 women), with the average age of 58.5 year. The concentration of cathepsin B and procathepsin B in the urine were corrected to creatinine, which was determined by the enzymatic creatinase method. The concentrations of cathepsin B in urine were singnificantly elevated in patients than in control group (median = 3.5 µg/l vs. 0.9 µg/l, P = 0.01), similarly the results of the cathepsin B/creatinine ratio were elevated (median = 0.4 µg/mmol vs. 0.1 µg/mmol, P = 0.01). There were no significant difference in concentration in serum between patients and control group (median = 4.8 µg/l vs. 4.2 µg/l, P = 0.8). The concentration values of procathepsin B were significantly higher in patients compare to control group both in urine (median = 3.9 µg/l vs. 1.4 µg/l, P < 0.0001), in serum (median = 73.3 µg/l vs. 58.7 µg/l, P = 0.0005) and similarly in...

Factors regulating the induction and development of B cell–mediated autoimmunity are not well understood. Here, we report that targeted deletion in murine B cells of X-linked Cosmc, encoding the chaperone required for expression of core 1 O-glycans, causes the spontaneous development of autoimmune pathologies due to a breakdown of B cell tolerance. BC-CosmcKO mice display multiple phenotypic abnormalities, including severe weight loss, ocular manifestations, lymphadenopathy, and increased female-associated mortality. Disruption of B cell tolerance in BC-CosmcKO mice is manifested as elevated self-reactive IgM and IgG autoantibodies. Cosmc-deficient B cells exhibit enhanced basal activation and responsiveness to stimuli. There is also an elevated frequency of spontaneous germinal center B cells in BC-CosmcKO mice. Mechanistically, loss of Cosmc confers enhanced B cell receptor (BCR) signaling through diminished BCR internalization. The results demonstrate that Cosmc, through control of core 1 O-glycans, is a previously unidentified immune checkpoint gene in maintaining B cell tolerance.

Indiana University-Purdue University Indianapolis (IUPUI) / Vaccinia virus (VACV), the prototypical poxvirus, was used to eradicate smallpox worldwide and, in recent years, has received considerable attention as a vector for the development of vaccines against infectious diseases and oncolytic virus therapy. Studies have demonstrated that VACV exhibits an extremely strong bias for binding to and infection of primary human antigenpresenting cells (APCs) including monocytes, macrophages, and dendritic cells. However, very few studies have evaluated VACV binding to and infection of primary human B cells, a main type of professional APC. In this study, we evaluated the susceptibility of primary human peripheral B cells at different developmental stages to VACV binding, infection, and replication. We found that VACV exhibited strong binding but little entry into ex vivo B cells. Phenotypic analysis of B cells revealed that plasmablasts were the only subset resistant to VACV binding. Infection studies showed that plasma and mature-naïve B cells were resistant to VACV infection, while memory B cells were preferentially infected. Additionally, VACV infection was increased in larger and proliferative B cells suggesting a bias of VACV infection towards specific stages of differentiation and proliferative ability. VACV infection in B cells was abortive, and cessation of VACV infection was determined to occur at the stage of late viral gene expression. Interestingly, B cell function, measured by cytokine production, was not affected within 24 hours post-infection. In contrast to ex vivo B cells, stimulated B cells were permissive to productive VACV infection. These results demonstrate the value of B cells as a tool to aid in deciphering the intricacies of poxvirus infection in humans. Understanding VACV infection in primary human B cells at various stages of differentiation and maturation is important for the development of a safer smallpox vaccine and better vectors for vaccines against cancers and other infectious diseases.

The purpose of this study was to identify mutations in the basic core promoter and enhancer II region a* the hepatitis B virus (HBV) that might result in the hepatitis B virus e antigen (HBeAg)-negative phenotype and contribute to hepatocarcinogenesis in black African carriers of the virus. The basic core promoter/enhancer II overlaps the X gene. HBV DNAfrom serum of 47 asymptomatic carriers and 50 patients with hepatocellular carcinoma and from 29 tumorous and 10 nontumorous liver tissues was amplified and sequenced directly. That part of the enhancer II region not overlapping the basic core promoter was reasonably well conserved in all samples. Missense mutations at positions 1809 and 1812 were found in 80% of all sequences and may represent wiidtype sequence in southern African isolates. Nucleotide and amino acid divergences were higher in the basic core promoter of hepatocellular carcinoma patients than of asymptomatic carriers (p<0.0001). This applied particularly to the paired 1762 adenine to thymine (1762T) and 1764 guanine to adenine (1764*) missense mutations, the prevalence of which was 66% in patients with hepatocellular carcinoma compared with 11% in asymptomatic carriers (p<0.0001). There was no association between the presence of 1762T1764A and the rate of HBeAg negativity, although these mutations suppressed HBeAg titres in HBeAg-positive patients. Suppression of HBeAg expression as well as alteration of amino acid sequence of the X protein may be contributing factors in the development of hepatocarcinogenesis.

Thesis (M.Sc. (Med.) (Epidemiology and Biostatistics))--University of the Witwatersrand, Faculty of Health Sciences, 2012. / Introduction Cryptococcal disease (CD) is the most common cause of morbidity and mortality among patients living with HIV I AIDS in many parts of sub-Saharan Africa. Globally the highest number of HIV -associated CD cases occur in sub-Saharan Africa (720 000/957 900) and mortality rates among patients on antifungal treatment remain unacceptably high. This study aimed to estimate and compare in-hospital mortality of HIV-associated CD among patients who were treated with amphotericin B versus fluconazole versus mixed treatment with amphotericin B and fluconazole. Materials and Methods We performed an analytical, cross-sectional analysis of data from a national laboratory-based surveillance programme through a network of public sector laboratories in South Africa. The study period was 1 January 2005 to 31 December 2006. The analysis used a subset of data from laboratory confirmed cases with completed case report forms and available data on outcome. The exposure was measured in 3 levels defined as treatment during the induction phase of therapy for at least 7 days with either amphotericin B or fluconazole or mixed treatment (initiation of treatment with one regimen and switching on to the other within 7 days of treatment). Outcome was defined as: patients who died between 7 and 30 days in hospital. Chi-squared test was used to compare characteristics among the treatment groups and multiple logistic regression models were constructed to identify risk factors for in-hospital death. Results Sixty two percent of the patients (1,363/2,211) were treated for ?:.7 days and 38% (848/2,211) for <7 days. In the group treated for ?:.7 days, mortality was 359/1,363 (26%) and the median time to death was 12 days (IQR 9-18). There was no significant difference in case fatality among patients treated with: amphotericin B (29%), fluconazole (27%) or mixed treatment (24%), (p-value = 0.28). On multivariate analysis, factors significantly associated with in-hospital mortality were: patients between the age group of 40-59 years, province in which the patients resided and altered mental status. In the group treated for <7 days patients treated with fluconazole were 82% less likely to die than patients treated with amphotericin B. Discusslon and Conclusion In-hospital mortality was high and similar among all treatment regimens in the ?:.7 days group. However a significant reduction in mortality was noted in the <7 days group treated with fluconazole. The reason for the later findings is unclear. It may be that amphotericin B alone is not superior but equivalent to fluconazole in the first 7 days of treatment and 5-flucytocine may be the intervention required to improve outcome in the first 7 days. Or it may be that patients are reaching care too late for a significant impact in disease outcome to be observed and prevention of CD is required in the form of ART and primary prophylaxis. Selection bias in that patients in the fluconazole group are less sick than in the amphotericin B-group is the most likely reason for lower mortality. The study findings call for more standardization of optimum treatment as well as advocacy for the availability of 5-flucytocine. Factors associated with high mortality require further investigations for interventions to improve patient outcomes.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine Johannesburg, 2017 / Hepatitis B is a global health problem that kills about 600 000 people annually. It is an infectious disease caused by the Hepatitis B Virus (HBV), which infects the liver and leads to liver inflammation and secondary complications such as cirrhosis and Hepatocellular Carcinoma (HCC). The available therapies are only partially effective and are associated with adverse side effects and viral drug resistance. RNA interference (RNAi) pathway is a gene silencing pathway found in diverse living systems including mammals. Harnessing of this pathway to inhibit HBV replication has shown a lot of promise, with highly effective anti-HBV RNAi activator sequences designed. However, the lack of safe and efficient delivery and expression systems for these sequences is one of the obstacles that need to be overcome before RNAi effectors can reach clinical application. For easy assessment of transduction efficiency, Helper Dependent Adenoviral vectors (HDAd) expressing β-galactosidase (encoded by lac Z gene) are commonly used to deliver anti-viral RNAi activators. However, this reporter protein has been blamed for induction of innate immune response and concomitant clearance of the HDAds by the host. For the first time, this study explored the use of lac Z-deficient HDAds to deliver anti-HBV RNAi activators expressed under the control of a liver-specific phosphoenolpyruvate carboxykinase (PEPCK) promoter. HDAd expressing Firefly luciferase resulted in a significant luminescence detected in cell culture lysates and a sustained bioluminescence in mice. HDAds expressing anti-HBV sequences transduced the liver efficiently and did not induce a pronounced inflammatory response or liver toxicity in mice. However, this did not translate into maximal anti-HBV sequence expression and HBV replication inhibition in vitro and in vivo. This suggests that the PEPCK promoter is inadequate for RNAi activator expression. This study highlights the importance of careful RNAi activator regulatory elements selection and presents the therapeutic potential and utility of HDAd vectors for hepatotropic delivery of antiviral sequences with markedly attenuated immune response stimulation and toxicity. For improved anti-HBV RNAi activator expression and HBV knockdown, a different liver specific promoter mouse transthyretin receptor (MTTR) promoter is currently being investigated in our lab. / MT2017

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