Protein kinase B (PKB), a serine threonine kinase, is highly involved in the regulation of cellular proliferation and survival. To characterize PKB’s function in lymphocyte development and activation, transgenic (tg) mice that express a membrane targeted constitutively active form of PKBa (myr PKB) in T and B cells were analysed. Thymocytes from myr PKB tg mice showed enhanced proliferation after T cell receptor (TCR) engagement compared to wild type (wt) mice. Astonishingly, myr PKB tg thymocytes were capable to proliferate in response to PMA only and were also less sensitive to inhibition by the calcineurin inhibitors CsA or FK506, which indicates the proliferative response of myr PKB tg T cells is relatively independent of calcium mobilisation and calcineurin activity. In addition, when TCR signalling was inhibited by the MEKinase inhibitor PD98059 or the Srckinase inhibitor PP1 myr PKB tg thymocytes again were more resistant to inhibition. Western blot analysis revealed myr PKB enhances activation of the kinases Lck, Raf and Erk after TCR/CD3 stimulation. Thus, myr PKB renders proliferative responses of thymocytes more sensitive to TCR signals by positive regulation of the Lck-Raf-MEK-Erk signalling pathway. Studies on the cellular location of the tg protein showed myr PKB is located in membrane socalled “lipid rafts”. Furthermore, we found that after TCR/CD3 ligation endogenous cytoplasmic PKB moves into “lipid rafts”, which highlights PKB as a crucial mediator of TCR proximal signalling events. Analysing three different TCR tg model systems for positive and negative selection of immature precursors in the thymus, we found myr PKB promotes positive selection of CD4+ but not CD8+ T cells. This most likely results from PKB’s positive cross-talk on Lck-Raf-Erk signalling, which is known to influence thymocyte selection and CD4/CD8-lineage choice. Furthermore, myr PKB enhances phosphorylation of glycogen synthase kinase 3 (GSK3), a negative regulator of the transcription factor NFAT (nuclear factor of activated T cells) and T cell activation, and of the adapter protein c-Cbl. Concerning negative selection, myr PKB enhanced (OT1 mice), reduced (HY mice) or had no influence (OT2 mice) on negative selection. Thus, myr PKB’s effect on negative selection strongly depends on the model system analysed and this most likely results from differences in TCR affinity/avidity and TCR specificity for MHC. 106 Peripheral CD4+ T cells from myr PKB tg mice showed enhanced production of both Th1 and Th2 cytokines. Furthermore, after TCR/CD3 stimulation in the presence of TGF-b1, wt CD4+ T cells showed a drastic inhibition of proliferation, whereas myr PKB tg CD4+ T cells proliferated even better, i.e. they were resistant to the inhibitory TGF-b1 signals. Expression of myr PKB in B cells leads to reduced Ca2+ flux and proliferation after BCR stimulation, but activation of Lyn, SLP-65, c-Cbl and GSK-3 were enhanced. When we analysed B cell subsets in myr PKB tg mice, a decrease in immature and mature B cells became obvious, whereas cell numbers for marginal zone (MZ) B cells were normal. In aged myr PKB tg mice we detected a very strong reduction of pro/pre and immature B cell populations in the bone marrow, indicating PKB is very important for maintenance of B cell development. Furthermore, myr PKB also lead to a strong reduction of peritoneal B-1 cells. However, expression of NFATc1, which is required for B-1 cell development, was comparable between wt and myr PKB tg B-1 cells. To analyse the effect of myr PKB on immunoglobulin production, mice were immunized with thymus dependent (TD) and independent (TI) antigens. In both cases, B cell responses were strongly elevated in myr PKB tg mice. Finally, RT-PCR analyses of in vitro expanded B cells revealed increased Blimp-1 and Notch3 expression in myr PKB tg B cells, which might be primary candidates involved in their enhanced effector function. In summary, this study clearly shows an important cross-talk between PKB and various critical signalling molecules downstream of the TCR and BCR. Thereby active PKB modulates and regulates the thresholds for thymocyte selection and T cell activation as well as for B cell development and function. / Proteinkinase B (PKB), eine Serin-Threonin Kinase, spielt bei der Regulation der Proliferation und des Überlebens vieler Zelltypen eine wichtige Rolle ein. Um die Funktion von PKB bei der Reifung und Aktivierung von Lymphozyten zu verstehen, wurden transgene (tg) Mäuse analysiert, die eine konstitutiv-aktive, myristoylierte Form der PKBa (myr PKB) in der T- und B-Zelllinie exprimieren. Thymozyten von myr PKB tg Mäusen zeigten im Vergleich zu wildtypischen (wt) Mäusen nach T-Zell-Rezeptor (TZR)-Stimulation eine deutlich erhöhte Proliferation. Myr PKB tg Thymozyten konnten zudem nur durch PMA zur Proliferation angeregt werden und waren auch weniger sensitiv gegenüber Inhibition durch die Calcineurin-Inhibitoren CsA und FK506. Dies weist darauf hin, dass die Aktivierung von T Zellen der myr PKB tg Mäuse relativ unabhängig von der Calcium-Mobilisierung und der Calcineurin-Aktivität ist. Wurde die TZR-Signalübertragung durch MEKinase-Inhibitor PD98059 oder den Src-Kinase- Inhibitor PP1 blockiert, so waren myr PKB tg Thymozyten wiederum sehr viel schlechter inhibierbar als wt Thymoyzten. Western-Blot-Analysen zeigten sodann, dass myr PKB nach TZR/CD3-Stimulation die Aktivierung der Kinasen Lck, Raf und Erk verstärkt. Somit führt aktive PKB über die positive Regulation des Lck-Raf-Mek-Erk Signalwegs zu einer erhöhten TZR-Sensitivität. Weiterhin verstärkt myr PKB die Phosporylierung der Glykogen Synthase Kinase 3 (GSK3), ein negativer Regulator des Transkriptionsfaktors NFAT (Nucleärer Faktor Aktivierter TZellen) und der T-Zell-Aktivierung sowie des Adaptorproteins c-Cbl. Unsere Untersuchungen zur zellulären Lokalisation von myr PKB ergaben, dass myr PKB in den sog. „lipid rafts“ der Membran lokalisiert ist. Weiterhin konnten wir zeigen, dass endogene cytoplasmatische PKB nach TZR/CD3-Stimulation in diese „lipid rafts“ wandert. Diese Daten weisen auf eine profunde Rolle von aktiver PKB bei der frühen und proximalen TZR-Signalübertragung hin. Die Analysen drei verschiedener TZR-tg Modellsysteme zur Selektion von unreifen TZellvorläufern im Thymus zeigten, dass myr PKB die positive Selektion von CD4+ aber nicht von CD8+ T-Zellen fördert. Dies resultiert sehr wahrscheinlich aus der positiven Regulation des Lck-Raf-Erk Signalweges, welcher ein zentraler Regulator der Thymozytenselektion und CD4/CD8-Linienentscheidung ist. Was den Einfluss von myr PKB auf die negative Selektion 108 betrifft, so verstärkte (OT1-Mäuse), verminderte (HY-Mäuse) oder hatte diese keinen Effekt (OT2-Mäuse). Die Effekte von myr PKB auf die negative Selektion sind daher stark abhängig vom Modellsystem, d.h. von der TZR-Affinität/Avidität und der Spezifität der TZRs für MHC-Moleküle. Periphere CD4+ T-Zellen von myr PKB tg Mäusen wiesen eine erhöhte Produktion von sowohl Th1- als auch Th2-Cytokinen auf. Erstaunlicherweise führte TZR/CD3-Stimulation in Anwesenheit von inhibitorischen TGF-b1-Signalen, ganz im Gegensatz zu wt T-Zellen, in myr PKB tg CD4+ T Zellen zu keiner Inhibition der Expansion, sondern sie proliferierten sogar stärker. Dies könnte mit der beobachteten erhöhten Zytokinproduktion von IL-2 und IFNg und/oder der erhöhten Phosphorylierung der Smad2/3 Proteine in myr PKB tg CD4+ T Zellen in Verbindung stehen. Die Expression von myr PKB in B-Zellen führte zu reduziertem Calcium-Flux und reduzierter Proliferation, wobei jedoch eine verstärkte Aktivierung von Lyn, SLP-65, c-Cbl und GSK-3 nachgewiesen werden konnte. Die Analyse der B-Zell-Populationen der myr PKB tg Mäuse zeigte eine Abnahme der unreifen und reifen B-Zellen in der Milz, jedoch war die Anzahl der Marginalzonen (MZ)-B-Zellen normal. Interessanterweise führte myr PKB zu einer sehr starken Reduktion peritonealer B-1-Zellen. Die Expression von NFATc1, welcher für die Entwicklung von B-1-Zellen benötig wird, war jedoch in den B-1-Zellpopulationen von wt und myr PKB tg Mäusen durchaus vergleichbar. Ältere myr PKB tg Mäuse zeigten einen starken Verlust der pro-/prä- und unreifen B-Zellen des Knochenmarks. Dies weist stark darauf hin, dass PKB für die Aufrechterhaltung der BZellentwicklung entscheidend ist. Darüber hinaus war, obgleich reduzierter B-Zellen, die Immunantwort auf Thymus-abhängige (TD) und –unabhängige (TI) Antigene in myr PKB tg Mäusen verstärkt. In RT-PCR Analysen von in vitro expandierten B-Zellen wurde sodann eine erhöhte Expression von Blimp-1 und Notch3 beobachtet, die zu der erhöhten Immunglobulin-Produktion der myrPKB tg B-Zellen beisteuern könnte. Zusammenfassend zeigen diese Arbeiten, dass aktive PKB die Expression/Aktivität zahlreicher wichtiger Signalmoleküle der TZR- und BZR-induzierten Signalleitung reguliert und somit die Schwellenwerte für die Selektion und Aktivierung von T-Zellen sowie für die Entwicklung und Funktion von B-Zellen entscheidend moduliert.

ABSTRACT Infection with the hepatitis B virus (HBV) is still a major global health problem with an estimated 6% of the world’s population chronically infected with the virus. Chronic infection with HBV subgenotype A1, which is hyperendemic to southern Africa, is associated with a particularly high incidence of liver cancer and cirrhosis. Understanding HBV replication and developing effective HBV treatment to prevent liver cancer remain important medical priorities. Although there is a preventative vaccine for HBV, efficacy of currently available treatment of established infection is limited. Exploiting the RNA interference (RNAi) pathway through the use of small interfering (siRNA) and short hairpin RNA (shRNA) is an attractive new approach for the development gene therapies against HBV infection. Our laboratory has designed and demonstrated the efficacy both in vitro and in vivo of several shRNAs designed to target the X open reading frame (ORF) of HBV. Thus, the objective of this study was to construct a replication competent plasmid vector of the A1 subgenotype, a reporter plasmid vector of HBV and to assess the efficacy of RNAi effecters against these vectors both in vitro and in vivo. The first HBV replication competent vector, pCR-HBVA1 1.3x, containing the sequence of an HBV subgenotype A1 isolate, was successfully constructed by generating a greater than genome length sequence of HBV, that starts just upstream of endogenous HBV basic core promoter (BCP) and ends just downstream of the unique HBV polyadenylation (pA) site. Human hepatoma (Huh7) cells transfected with this plasmid secreted HBV surface antigen (HBsAg) into Abstract viii culture supernatants. In the murine hydrodynamic injection model of HBV replication, serum HBsAg, hepatitis B e antigen (HBeAg) and viral particle levels as well as relative surface and core mRNA levels were shown to be significantly elevated as compared to mock-injected mice. The second HBV vector, pCH-FLuc, was successfully generated by replacing the surface ORF with the sequence encoding Firefly Luciferase. The ability of pCH-FLuc to express Firefly Luciferase was demonstrated in a liver cell line (Huh7 cells). Co-transfection of the reporter plasmid, pCH-FLuc, with shRNAs targeted to HBV caused a significant reduction in Luciferase expression. Co-transfection/injection of the pCR-HBVa1 1.3x with shRNAs caused significant inhibition in the level of viral antigens (HBsAg, HBeAg and hepatitis B core antigen (HBcAg) as well as relative surface and core mRNA levels. This was observed both in vitro and in vivo. Our results demonstrate the potential this model allows for the study of HBV replication as well as the assessment of potential therapeutic strategies in a regionally significant subgenotype of HBV.

Hepatitis B virus (HBV) is hyperendemic to southern Africa and parts of Asia where it is a major cause of serious liver disease. Licensed antivirals for chronically infected individuals are only partially effective and approximately one million deaths occur annually as a result ofpersistent infection with the virus. Although RNA interference (RNAi) based gene silencingof HBV has been successfully demonstrated, difficulties with delivery of anti-HBV RNAieffectors remains an obstacle to their clinical use. Recombinant adenoviruses (Ads), amongst the most efficient hepatotropic gene vectors following systemic administration, have been successfully used to deliver expressed anti-HBV RNAi sequences. However, a drawback of Ad vectors is diminished efficacy and toxicity that results from stimulation of innate andadaptive immunity.To attenuate these effects we used polyethylene glycol (PEG) to modify first generation recombinant Ad (FG Ad) vectors that express an anti-HBV short hairpin (shRNA) sequence. Efficient hepatocyte transduction occurred and expressed shRNAs were processed to generate intended HBV-targeting guides. Inhibition of HBV replication was achieved after intravenous administration of PEGylated or native recombinant first generation Ads (FG Ads) to HBV transgenic mice. Circulating HBV viral particle equivalents (VPEs) remained low for 3 weeks and began to increase after 5 weeks. A second dose of PEGylated anti-HBV Ad caused a less sustained decrease in circulating VPEs, but no silencing after a second dose was observed in animals treated with unmodified vector. Release of inflammatory cytokines was elevated in animals receiving unmodified vectors and only a modest increase in monocyte chemotactic protein-1 (MCP-1) was observed in mice that received a second dose of PEG Abstract Ads. Also, polymer-conjugated vectors induced a weaker adaptive immune response and were less hepatotoxic than their unmodified counterparts. To address concerns about the transient nature of transgene expression by FG Ads resulting from immunostimulation, third generation helper-dependent (HD Ad) were utilised to delivered anti-HBV RNAi effectors. Seven days after intravenous administration of infectious HD Ads to HBV transgenic mice, 80-90% of hepatocytes were transduced and markers of HBV replication were decreased by approximately 95% which was sustained for 8 weeks. HD Ad-induced release of proinflammatory cytokines was minimal in preparations that were enriched with infectious particles. PEGylated HD Ad vectors caused similar anti- HBV effects and may be useful to evade interaction with vector-sequestrating receptors and further attenuate immunostimulation. Collectively these observations indicate that PEG modification of Ads and the use of HD Ads may have utility for delivery of therapeutic HBVsilencing sequences. Future work will focus on improving strategies to avoid immune detection and utilisation of HD Ad vectors for other HBV targeting sequences.

Student Number : 9708297R - MSc (Med) dissertation - School of Medicine - Faculty of Health Sciences / B chronic lymphocytic leukaemia (B-CLL) is a monoclonal haematopoietic disorder with expansion of small lymphocytes of B-cells. B-CLL cells accumulate in blood, bone marrow, lymph nodes and spleen, resulting in enlargement of these organs and decreased bone marrow function. B-CLL is the most common leukaemia, with an annual incidence of 1.8 to 3.0 per 100 000 population in the United States. It is characterised by the accumulation of long-lived monoclonal CD5+ B lymphocytes. In vivo normal B-lymphocytes derive growth factors through interactions with T-cells and monocytes. In culture however, survival and growth of activated B-cells depends on the availability of external factors such as interleukins. B-CLL cells populations are unable to survive in culture long enough to respond to the addition of growth factors. Such factors are important for the proliferation and survival of many cell types and in the absence of cytokines, these cells die as a result of apoptosis. Chronic lymphocytic leukaemia cells are influenced in vitro by a number of exogenously added cytokines that include IFN- α, IFN-γ, IL-2, IL-4, IL-10, IL-13, IL-15, TGF- β and TNF- α. The aim of this study was to investigate the effect of cytokines e.g., IFN, IL-2, IL-6, IL7 and IL-10 on the proliferation and survival of B-CLL cells and furthermore to compare the induction and reduction of spontaneous and induced apoptosis in vitro. Patients with B-CLL were recruited from three centres. Thirty blood samples were collected, separated using Ficoll Hypaque Gradient and purified by rosetting with AET treated SRBC. The proliferation and survival of B-CLL cells were studied in vitro in response to GM-CSF, IFN, IL-2, IL-6, IL7 and IL-10,. The survival and apoptosis of B-CLL cells in cultures with or without interleukins and other growth factors were studied under microscopic examinations and DNA agarose gel electrophoresis. It was observed in B-CLL cells cultures that IFN and IL-2 enhanced proliferation significantly. IL6, IL-7 and GM-CSF also enhanced proliferation of B-CLL cells but not to the greater extent than IL2 and IFN. IL-10 inhibited proliferation of B-CLL cells when compared to controls. In a long-term (5-day) culture, survival of B-CLL cells was greatly enhanced by IFN and followed by IL-2. Therefore it appeared that IFN and IL-2 are the two most potent growth factors tested in this study to promote B-CLL cells proliferation and survival. The combination of these mitogens did not further enhanced proliferation. IL-6 and GM-CSF enhanced proliferation and survival of B-CLL cells. IL-7 promoted proliferation but had no effect on survival of B-CLL cells in-vitro. IL-10 enhanced apoptosis and did not promote survival of B-CLL cells in-vitro. IFN and IL2 are survival and promoting growth factors for B-CLL cells in culture. In contrast, IL-10 has demonstrated to induce apoptotic cell death of B-CLL cells. In conclusion B-CLL cells proliferated equally well with IFN and IL-2. IL-6, IL-7 and GM-CSF had a much lower proliferation and survival effect with noticeable antiapototic activity when compared to IFN and IL-2. IL-7 was found not to promote survival of B-CLL cells and IL-10 enhanced cell death by apoptosis.

Thesis (Ph.D.)--University of the Witwatersrand, Faculty of Health Sciences, 2013.

Positive social interaction between members of opposing social groups (intergroup contact) is an effective method of prejudice reduction (Allport, 1954; Pettigrew & Tropp, 2006). This thesis explores how intergroup contact theory can be applied to age groups to reduce ageism towards older adults. Chapters 1 to 3 form the theoretical chapters of the thesis. Chapter 1 defines psychological processes underpinning ageism, provides details of its prevalence, outlines its consequences in society, and gives a justification for its reduction. Chapter 2 introduces the psychological processes underpinning intergroup contact theory and its different formations. This is followed by a literature review of intergenerational contact research, which identifies research gaps in the field and research questions addressed in this thesis. Four empirical chapters then present findings from eight studies. In Chapter 4, Study 1 provides initial correlational evidence of the relationships between direct intergenerational contact, ageism and related psychological processes. Chapter 5 addresses the research question of whether extended contact can be successfully applied to age groups. Studies 2, 3 and 4 provide novel evidence that extended intergenerational contact reduces ageism and is effective via reduced intergroup anxiety, ageing anxiety and ingroup norms. These studies also support prior research demonstrating that direct contact reduces ageism via intergroup and ageing anxieties. Chapter 6 presents two studies that extend the focus of the thesis to include age stereotypes. Secondary analysis of national survey data in Study 5 explores the perception of older adults' competence across the lifespan and friendships with older adults. The degree to which young and middle-aged adults perceive that competence declines with age is attenuated by having as little as one older friend. Building on these findings, Study 6 explores the relationships between direct and extended intergenerational contact, ageist attitudes and warmth and competence stereotypes. Corroborating Chapter 4, both direct and extended contact predicted reduced ageism and are effective via increased competence stereotypes and increased warmth stereotypes. In the final empirical chapter in the thesis Chapter 7 presents two studies that explore intergenerational contact theory in applied contexts. Using an experimental design, Study 7 evaluated an intergenerational programme in which students had conversations with older adults about their technology use. Compared to a control group, the experimental group rated older adults as warmer yet more incompetent. However, only warmth and not incompetence stereotypes formed indirect pathways to subsequent attitudes towards older adults more widely. Study 8 examined care workers positively and negatively experienced intergenerational contact with care home residents. Although care workers experienced more positive than negative contact, negative (but not positive) contact was associated with their attitudes towards care home residents and it generalised to older adults more widely. This indirect effect of negative contact to older adults was effective only for subtle and not blatant ageist attitudes. Overall, the thesis provides a range of evidence suggesting that intergroup contact theory can be successfully applied to the reduction of ageism. It presents a detailed overview of current knowledge, corroborates existing evidence and presents novel findings for extended contact and mediators of both direct and indirect intergenerational contact.

O uso de peptídeos na terapêutica apresenta inúmeras limitações, desde características físico-químicas até perfil farmacocinético inadequado para absorção oral. Entretanto, novas estratégias sintéticas para redução de custos e aumento da estabilidade química e metabólica de peptídeos, associada a vias alternativas de administração foram desenvolvidas e permitiram o acesso a essas moléculas no mercado farmacêutico. Diante disso, peptídeos naturais bioativos, subexplorados do ponto de vista químico medicinal, são considerados bons protótipos para o desenvolvimento de biblioteca de análogos não-naturais, gerando informações relevantes para a compreensão da relação entre a estrutura química e a atividade biológica destes compostos. No presente estudo, os peptídeos cíclicos rufomicina B (tuberculostático), chaiyaphumine A (anti-malárico), ciclozantoxilano A e desotamide B (antibiótico) foram estudados do ponto de vista sintético sob diferentes metodologias. Para tal, os aminoácidos Fmoc-N-Metil-L-Leucina e Fmoc-L-crotil-glicina foram sintetizados com rendimentos satisfatórios para síntese do antibiótico rufomicina B. O aminoácido Fenil-acetil-L-treonina foi obtido em bons rendimentos e a síntese de chaiyaphumine A e derivado foi testada em fase sólida, em solução e empregando combinação dos dois métodos. O peptídeo desotamide B e ciclozantoxilano A foram sintetizados, mesmo que em baixo rendimentos, em fase sólida com ciclização solução. Ainda, a síntese de desotamide B também foi possível através da metodologia \"safety-catch\". Por fim, realizamos análise sistemática das propriedades físico-químicas e estruturais dos peptídeos no mercado e em fase clínica e comparamos os resultados obtidos com os alvos desse estudo / The use of peptides in therapy presents several limitations, from physicochemical characteristics to inadequate pharmacokinetic profile for oral absorption. However, new synthetic strategies for cost reduction and increase of the chemical and metabolic stability of peptides associated with alternative routes of administration were developed and allowed access to these molecules in the pharmaceutical market. Therefore, natural bioactive peptides, is an underexploited class from the medicinal chemist\'s point of view, are considered good prototypes for the development of similar series, generating information relevant to understanding the relationship between chemical structure and biological activity of these compounds. In the present study, cyclic peptides rufomycin B (antituberculosis activity), chaiyaphumine A (anti-malarial), cyclozanthoxylane A and desotamide were studied from the synthetic point of view, through different metodologies. In this sense, the amino acids Fmoc-N-Methyl-L-Leu and Fmoc-L-crotyl-Gly were synthetized in good yields for the total synthesis of rufomycin B. The amino acid Phenyl-acetyl-L-threonine was obtained in high yield and the synthetic studies on the chaiyaphumine A and analogue has been performed in solution and solid phase peptide synthesis and also through the combination of the 2 methods. The peptides desotamide B and cyclozanthoxylane A were synthetized, in low yields, in solid phase and in solution cyclization. Desotamide B could also be synthetized through safety-catch solid phase method. At end, we performed a systematic review of the physical chemical properties of all market and on clinical trial peptide drugs and we compared the results with the target molecules in this study.

Plural Logic is an extension of First-Order Logic with plural terms and quantifiers. When its plural terms are interpreted as denoting more than one object at once, Plural Logic is usually taken to be ontologically innocent: plural quantifiers do not require a domain of their own, but range plurally over the first-order domain of quantification. Given that Plural Logic is equi-interpretable with Monadic Second-Order Logic, it gives us its expressive power at the low ontological cost of a first-order language. This makes it a valuable tool in various areas of philosophy. Some authors believe that Plural Logic can be extended into an even more expressive logic, Higher-Level Plural Logic, by adding higher-level plural terms and quantifiers to it. The basic idea is that second-level plurals stand to plurals like plurals stand to singulars (analogously for higher levels). Allegedly, Higher-Level Plural Logic enjoys the expressive power of type theory while, again, committing us only to the austere ontology of a first-order language. Were this really the case, Higher-Level Plural Logic would be a very useful tool, extending and strengthening some of the applications of Plural Logic. However, while the notions of plural reference and quantification enjoy widespread acceptance today, their higher-level counterparts have been received with scepticism. The main objection raised against them is that higher-level plural reference is unintelligible. This has been argued, among others, on the grounds that there are no higher-level plurals in natural language and that, if there were any, they could be eliminated. In this thesis, after introducing the debate on plurals in Chapters 1 and 2, I turn to defending the legitimacy of the notion of higher-level plural reference. To this end, in Chapter 3, I present and elucidate the notion. Next, in Chapter 4, I show that some natural languages clearly contain these expressions and that they do so in an ineliminable manner. Finally, in Chapters 5 and 6, I develop a semantics for higher-level plurals that employs only devices previously well-understood by English speakers. To finish, in Chapter 7, I describe an application of Higher-level Plural Logic: a strengthening of the neo-Fregean programme. After describing my proposal, I turn to the issue of the logical status of this formalism and defend an optimistic take on the matter.

Law Man Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 123-132). / Abstracts in English and Chinese; appendix in Chinese. / Precis --- p.vi / 摘要 --- p.viii / Captions for Tables --- p.x / Caption for Figure --- p.xii / Abbreviations --- p.xiii / Chapter Part 1 --- Literature Review --- p.0 / Chapter Chapter 1 --- Aims and Hypothesis --- p.1 / Chapter 1.1 --- Aims --- p.3 / Chapter 1.2 --- Hypothesis --- p.4 / Chapter Chapter 2 --- Epidemiology of HBV Infection --- p.5 / Chapter 2.1 --- Global and Local Epidemiology --- p.6 / Chapter 2.2 --- Modes of Transmission --- p.8 / Chapter 2.2.1 --- Perinatal Tansmission --- p.8 / Chapter 2.2.2 --- Percutaneous Transmission --- p.8 / Chapter 2.2.3 --- Sexual Transmission --- p.8 / Chapter 2.2.4 --- Healthcare Setting and Transplantation --- p.8 / Chapter 2.2.5 --- Transfusion --- p.9 / Chapter 2.2.6 --- Other --- p.9 / Chapter Chapter 3 --- Popularity of TCM --- p.10 / Chapter 3.1 --- Traditional Chinese Medicine Use --- p.11 / Chapter 2.2 --- Regulation on TCM Practice --- p.12 / Chapter Chapter 4 --- Philosophy of TCM on HBV Infection --- p.13 / Chapter 4.1 --- Basic Principles --- p.14 / Chapter 4.1.1 --- Yin-Yang Theory --- p.14 / Chapter 4.1.2 --- Five Elements Theory --- p.14 / Chapter 4.1.3 --- Zang Fu Theory --- p.14 / Chapter 4.2 --- Pathogenesis --- p.16 / Chapter 4.3 --- Diagnosis --- p.17 / Chapter 4.4 --- Treatment --- p.20 / Chapter Chapter 5 --- Western Medicine --- p.23 / Chapter 5.1 --- Natural History of HBV Infection --- p.24 / Chapter 5.1.1 --- Replicative Phase: Immune Tolerance --- p.24 / Chapter 5.1.2 --- Replicative Phase: Immune Clearance --- p.24 / Chapter 5.1.3 --- Noreplicating (Low-Replication) Phase --- p.25 / Chapter 5.2 --- Diagnostic Tests for HBV Infection --- p.26 / Chapter 5.2.1 --- Serologic Assays --- p.26 / Chapter 5.2.2 --- Serum Enzymes --- p.26 / Chapter 5.2.3 --- HBV DNA Assays --- p.27 / Chapter Chapter 6 --- Health-related Quality of Life --- p.29 / Chapter 6.1 --- Principle and Definition --- p.30 / Chapter 6.2 --- Assessment --- p.31 / Chapter 6.2.1 --- Generic Instrument --- p.31 / Chapter 6.2.2 --- Disease-Specific Instrument --- p.32 / Chapter Part 2 --- Studies & Results --- p.33 / Chapter Chapter 7 --- Research Methodology --- p.34 / Chapter 7.1 --- Study 1: Hospital-Based Surveys on Chronic Hepatitis B Patients: TCM Use --- p.35 / Chapter 7.1.1 --- Patients --- p.35 / Chapter 7.1.2 --- Survey Instrument & Logistic --- p.35 / Chapter 7.1.4 --- Statistical Analysis --- p.36 / Chapter 7.1.4 --- Sample Size Justification --- p.36 / Chapter 7.2 --- Study 2: Hospital-Based Survey on Chronic Hepatitis B Patients: HRQoL & Psychiatric Involvement --- p.38 / Chapter 7.2.1 --- Patients --- p.38 / Chapter 7.2.2 --- Survey Instrument and Logistic --- p.38 / Chapter 7.2.3 --- Statistical Analysis --- p.39 / Chapter 7.3 --- Study 3: Population-Based Survey on Chinese Medicine Practitioners (CMPs): Practice Behavior & Knowledge Assessment --- p.41 / Chapter 7.3.1 --- Study Population --- p.41 / Chapter 7.3.2 --- Survey Instrument --- p.41 / Chapter 7.3.3 --- Statistical Analysis & Knowledge Assessment --- p.42 / Chapter 7.4 --- Study 4: TCM Consultation Agreement --- p.44 / Chapter 7.4.1 --- Patients --- p.44 / Chapter 7.4.2 --- Chinese Medicine Practitioners --- p.44 / Chapter 7.4.3 --- Questionnaire --- p.44 / Chapter 7.4.4 --- Study Design --- p.45 / Chapter 7.4.5 --- Sample Size Estimation --- p.45 / Chapter 7.4.6 --- Data Analysis --- p.45 / Chapter 7.5 --- "Study 5: TCM Interpretation of Laboratory, Imaging & HRQoL Assessment" --- p.47 / Chapter 7.5.1 --- Patients --- p.47 / Chapter 7.5.2 --- HRQoL Assessment --- p.47 / Chapter 7.5.3 --- Statistical Analysis --- p.47 / Chapter Chapter 8 --- TCM Use on Chronic Hepatitis B Patients --- p.48 / Chapter 8.1 --- Patient Characteristics --- p.49 / Chapter 8.2 --- Health-Seeking Behavior of TCM Users --- p.51 / Chapter 8.3 --- Determinants of TCM Use --- p.53 / Chapter 8.4 --- Common Herbal Ingredients Used --- p.56 / Chapter Chapter 9 --- Impacts of HBV Infection on Patients --- p.58 / Chapter 9.1 --- Patient Socio-Demographic and Clinical Characteristics --- p.59 / Chapter 9.2 --- Patient Symptoms and Anxiety /Depression --- p.61 / Chapter 9.3 --- HRQoL & its Determinants --- p.63 / Chapter 9.3.1 --- Physical Aspect of HRQoL --- p.63 / Chapter 9.3.2 --- Mental Aspect of HRQoL --- p.67 / Chapter Chapter 10 --- Chinese Medicine Practitioners' (CMP) Practice --- p.71 / Chapter 10.1 --- CMPs Demographics and Training --- p.72 / Chapter 10.2 --- Practice Behavior --- p.75 / Chapter 10.3 --- Diagnostic and Therapeutic Approach --- p.76 / Chapter 10.4 --- Professional Knowledge Assessment --- p.79 / Chapter 10.5 --- Determinants of Diagnostic Approach --- p.82 / Chapter Chapter 11 --- Agreement of TCM Diagnosis among Different CMPs --- p.85 / Chapter 11.1 --- Patients Characteristics & Disease Severity --- p.86 / Chapter 11.2 --- Agreement of TCM Diagnosis & Treatment --- p.88 / Chapter Chapter 12 --- Interpretation of TCM diagnosis --- p.93 / Chapter 12.1 --- Patients --- p.94 / Chapter 12.2 --- Clinical Characteristics --- p.96 / Chapter 12.3 --- HRQoL Assessment --- p.98 / Chapter Part 3 --- Discussion & Conclusions --- p.100 / Chapter Chapter 13 --- Discussion --- p.101 / Chapter 13.1 --- TCM Popularity and its Practice --- p.102 / Chapter 13.2 --- Impact of TCM Symptomatology & Anxiety /Depression on HRQoL --- p.107 / Chapter 13.3 --- TCM Diagnosis Consistency --- p.111 / Chapter 13.4 --- Association of TCM Diagnosis with Western Medicine --- p.114 / Chapter 13.5 --- Limitations of the Study --- p.117 / Chapter Chapter 14 --- Conclusions --- p.120 / Reference --- p.122 / List of Publications of My Work Used in This Thesis --- p.133 / Acknowledgement --- p.136 / Appendix --- p.138

The pyrimidine biosynthetic pathway is essential and similar in all bacteria. The pathway from Pseudomonas is regulated by nucleotides which bind to the upstream region of the pyrBC’ gene complex. Work in our lab mapped the genes and showed that the pyrB and pyrC’ were part of an overlap complex. The Pseudomonas aeruginosa has one circular chromosome. A former Pseudomonas now called Burkholderia cepacia is similar to P. aeruginosa except that it contains three circular chromosomes (CI, CII, CIII) and one large plasmid. The primary chromosome named CI contains the pyrBC’. To our knowledge there has been no report of the activity of ATCase in Pseudomonas and contrasted with that of Burkholderia. Here, we compare the activity of ATCase in P. aeruginosa and B .cepacia. Cells of both organisms were grown in Pseudomonas minimal medium and in Enriched medium. The ATCase was extracted and partially purified from each sample. It is hypothesized that the B. cepacia has greater activity for ATCase than do the Pseudomonas.