Pneumocystis carinii is an opportunistic fungal pathogen that causes lifethreatening pneumonia in immunocompromised individuals. Infants appear to be particularly susceptible to Pneumocystis (PC) pulmonary infections. The higher incidence of PC as well as other pulmonary infections among infants is likely due to an immature immune system. The neonatal lung environment is deficient immunologically in preterm as well as term infants (1, 2). Decreased phagocytic capacity of macrophages in newborns may increase the risk of infection from inhaled pathogens (1, 2). We have previously demonstrated that there is approximately a 3-week delay in the clearance of PC organisms from pup mouse lungs compared to adults. Herein, we demonstrate that there is also a 1-week delay in the infiltration of AMs in pup compared to adult PC-infected mice. We go on to show that there is a delay in pup versus adult lung macrophage phenotypic expression and cytokine production in response to PC organisms. We demonstrated that pup AMs are competent to produce cytokine in response to LPS and that stimulation with zymosan generates cytokine production in pup AMs that is comparable to adult cytokine production. These data indicate that pup lung macrophages are specifically poorly responsive to PC organisms and likely require exogenous stimulation to mount a significant immune response and expedite clearance of the organism. We go on to show that heat-killed Escheriae coli improves cytokine response, cellular infiltration and reduces organism burden in PC-infected pup mice. The clinically relevant cytokine, GM-CSF, has been used to improve the clearance of several pulmonary infections, including PC in adult animal models. We show that monotherapy with GM-CSF is insufficient to improve PC clearance in pup mice; however, when combined with TMP/SMX it improves PC clearance and maintains a reduced PC burden following discontinuation of therapy. Furthermore, we have shown that GM-CSF improves the ability of human infant lung macrophages to phagocytose PC organsms without generating an increased inflammatory response. These data suggest that combination therapy with TMP/SMX and GM-CSF may be a viable treatment option for infants failing or intolerant to standard therapy.

Purpose - The purpose of this paper is to explore if there is any evidence that students actually can learn anything with the help of case studies. This study will answer the hypothesis “H” that are constructed as following: “H”. “The case study teaching method is a stimulating method for students to learn in.” Methodology - The method used in this paper is qualitative secondary research from databases. The databases consist of Diva, Google Scholar, Emerald, Web of Science and Scopus. Other secondary research is from research books and books about pedagogy. Implications/Findings - This research study shows that case studies have a positive influence on the student’s engagement and that learning gets more meaningful if the students are more engaged in the learning process. To be more engaged can move the student to a higher level of thinking. Furthermore, case studies enhanced the learning retention in students. Keywords - Student, Evidence, Learning, Case Study, Engagement, Stimulating, Motivation, Influence. Paper type – Research paper.

Macrophages are a natural target cell for HIV-1 infection, and they contribute to the development of disease as they are important for transmission, dissemination and persistence of the virus in an infected patient. Macrophages are less well-studied than T cells and cell lines in relation to HIV-1 infection, yet macrophages are highly specialised and key aspects of the HIV-1 life cycle in these cells are already known to differ compared to other cell types. HIV-1 entry into macrophages has been suggested to occur by macropinocytosis, however the entry route in these cells has not been fully characterised. In this thesis I have tested a panel of pharmacological inhibitors of cellular proteins and uptake pathways, in order to delineate the requirements for HIV-1 entry into macrophages and to determine the nature of the entry route. My findings suggest that the following host factors are important for entry; membrane cholesterol, actin rearrangements, dynamin, sodium-hydrogen exchange, Pak1, and Rac. Other factors including clathrin, PI-3 kinase, Rho kinase and some isoforms of PKC were found to be dispensable for infection or to inhibit infection. Macrophages are a heterogeneous group of cells, and tissue macrophages from different parts of the body differ in their morphology, phenotype and function. I have used the growth factors M-CSF and GM-CSF to direct monocytes to differentiate into distinct types of macrophage. This allowed me to determine that different macrophages differ in their susceptibility to infection and in their ability to support replication. This is likely to be due to variation in HIV-1 receptor expression and the levels of key HIV-1 transcription factors, respectively. Overall this thesis contributes to existing knowledge regarding HIV-1 infection of macrophages. These findings may assist with the design of entry inhibitors, and with therapies designed to eradicate HIV-1 from infected individuals.

Student Number : 9600048X - MSc (Med) research report - Faculty of Health Sciences / Disorders of the thyroid gland are amongst the most common endocrine disorders. The diagnosis of thyroid disease consists of a history and clinical examination, followed by specific confirmatory investigations. These investigations are an important diagnostic component in thyroid disease and are amongst the most common investigations ordered in clinical laboratories. Although these tests are relatively inexpensive individually, they account for a disproportionately large amount of health care expenditure for diagnostic testing. Appropriate laboratory investigation is critical to establish the diagnosis and cause of thyroid disease in the most costeffective way. Discovery Health released a set of evidence-based guidelines in order to educate the clinician with regard to the selection of thyroid function tests. According to these guidelines a TSH (Thyroid Stimulating Hormone) test is the investigation of choice in suspected thyroid disease. This study is a retrospective investigation that compares the difference in ordering patterns of laboratory investigations by clinicians before and after the publication of the guidelines. Two data sets were generated from the data bank of Discovery Health. The first data set (I) was based on records compiled before March 2003 whereas the second data set (II) was based on records compiled from April 2003. Following use of the exclusion and inclusion criteria the sample size totaled 73 850 cases. An analysis was made with regard to the requesting frequency of specific tests. This study will focus solely on the appropriateness of thyroid function tests ordered. It is beyond the scope of this study to attach a specific clinical diagnosis to the results. The thyroid function tests requested before the publication of the evidence-based guidelines were often requested without careful thought and consideration on the part of the clinician. Some of the combination tests ordered (Free T3 and Free T4) are not advocated as an initial investigation in the evaluation of thyroid function and waste funds in this instance. The ordering of inappropriate thyroid function tests often leads to the depletion of funds available to a patient within the financial year. The results revealed that after publication of the guidelines there was an increase in the requesting frequency of TSH as a first line investigation, as well as Free T4 while a decrease in requests for Free T3 was noted. The publication of evidence-based guidelines as a guide to requesting the correct thyroid function tests in order to diagnose suspected thyroid disease appears to have impacted in increasing awareness amongst clinicians with regard to the tests required to diagnose and monitor thyroid disease.

by Wong, Cheuk-fai. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 95-99). / List of figures --- p.iv / List of tables --- p.v / Abbreviations --- p.vi / Abstract --- p.vii / Chapter I. --- INTRODUCTION --- p.1 / Chapter 1. --- Inhibin a brief review --- p.1 / Chapter 1.1 --- "Definition and nomenclature, including related substances" --- p.1 / Chapter 1.2 --- Structure --- p.2 / Chapter 1.3 --- Historical background of the inhibin concept --- p.4 / Chapter 1.4 --- Actions of inhibin --- p.9 / Chapter 1.5 --- Control of inhibin production --- p.10 / Chapter 1.6 --- Measurement --- p.11 / Chapter 1.6.1 --- Immunoassay --- p.11 / Chapter 1.6.2 --- Bioassay --- p.13 / Chapter 1.6.2.1 --- In vivo methods --- p.13 / Chapter 1.6.2.2 --- In vitro methods --- p.14 / Chapter 1.7 --- Inhibin in clinical studies --- p.15 / Chapter 2. --- Project design --- p.17 / Chapter 2.1 --- Background --- p.17 / Chapter 2.2 --- Objectives --- p.20 / Chapter II. --- MATERIALS AND METHODS --- p.21 / Chapter 1. --- Materials --- p.21 / Chapter 1.1 --- Tracer preparation and purification --- p.21 / Chapter 1.2 --- Inhibin RIA --- p.21 / Chapter 1.3 --- Other immunoassays --- p.22 / Chapter 2. --- In-house inhibin RIA development --- p.22 / Chapter 2.1 --- The tracer preparation --- p.22 / Chapter 2.2 --- The radioimmunoassay --- p.25 / Chapter 2.3 --- Optimization of assay parameters --- p.26 / Chapter 2.3.1 --- Optimization of serum content and second antibody titre --- p.26 / Chapter 2.3.2 --- Verification of second antibodies' precipitating activity --- p.27 / Chapter 2. --- INHIBIN-EASIA --- p.27 / Chapter 3. --- Progesterone --- p.28 / Chapter 4. --- "Oestradiol, LH, and FSH" --- p.28 / Chapter III. --- RESULTS --- p.30 / Chapter Part I. --- In-house inhibin assay development --- p.30 / Chapter 1. --- Iodination and purification --- p.30 / Chapter 1.1 --- Step 1:Iodination followed by Sephadex column purification --- p.30 / Chapter 1.2 --- Step 2: Red A gel column purification --- p.33 / Chapter 1.3 --- Step 3: Sephadex column purification --- p.33 / Chapter 2. --- Inhibin RIA: Binding and antibody dilution curve experiment --- p.36 / Chapter 3.1 --- Verification of binding activity of the second antibody --- p.38 / Chapter 3.2 --- Optimization of serum content/ second antibody titre --- p.39 / Chapter 4. --- Discussion and conclusion --- p.41 / Chapter Part II. --- Hormone results of women undergoing in vitro fertilization --- p.42 / Chapter 1. --- Presentation of analytical results --- p.42 / Chapter 2. --- Comparison of hormone profiles of patients in the two GnRH agonist regimes --- p.43 / Chapter 2.1 --- Gonadotropins --- p.43 / Chapter 2.2 --- E2 --- p.56 / Chapter 2.3 --- Progesterone --- p.62 / Chapter 2.4 --- Inhibin --- p.68 / Chapter 3. --- Relationship between hormone --- p.74 / Chapter 3.1 --- Relationship between hormone changes --- p.74 / Chapter 3.2 --- Regression analysis --- p.87 / Chapter IV. --- DISCUSSSION --- p.89 / Chapter 1. --- Hormone profiles --- p.89 / Chapter 2. --- Hormone correlation --- p.91 / Chapter V. --- CONCLUSION --- p.94 / Chapter VI. --- REFERENCES --- p.95 / Chapter VIII. --- Appendix 1 Protocol for study on IVF and inhibin --- p.100 / Chapter IX. --- Appendix 2 Protocol for the management of IVF cycles --- p.101 / Chapter X. --- Appendix 3 Patients' results (table) --- p.103 / Chapter XI. --- Appendix 4 Patients ' results (graph) --- p.110

L’immunologie de la reproduction englobe les principes de l’immunologie générale et les aspects spécifiques de la reproduction et du développement. Les Colony Stimulating Factors (CSFs) sont une illustration de l'application médicale de ce domaine. Dans la famille des CSFs, le Granulocyte-Colony Stimulating Factor (G-CSF) apparaît aujourd'hui comme une thérapie innovante dans divers cas d'échec de la reproduction, bien que ses cibles et ses effets ne soient pas encore clairement établis. Dans ce travail, à travers une revue sur les CSFs dans la reproduction, une étude consacrée aux gènes cibles du G-CSF dans l'endomètre humain, et une étude consacrée aux effets de la supplémentation systémique en G-CSF sur l’implantation embryonnaire murine, nous avons essayé d'approcher certains mécanismes d'action possibles pour cette cytokine. Dans les modèles murins fertiles et pro-abortifs, la supplémentation systémique en G-CSF, ciblant spécifiquement l’endomètre préimplantatoire, modifie les taux d’implantation embryonnaire. Dans l’endomètre humain, certaines dérégulations préimplantatoires de gènes cibles du G-CSF ont également été observées chez les patients infertiles. L'influence du G-CSF sur ces gènes cibles a été également illustrée dans un modèle ex-vivo de culture endométriale. Ces cibles dont l’expression est influencée par le G-CSF sont décrites comme des molécules clés dans le processus implantatoire, intervenant sur l’adhésion embryonnaire, la migration cellulaire, le remodelage des tissus et l'angiogenèse locale. Ces données suggèrent des possibilités de diagnostic préventif et pré-conceptionnel de certains échecs de reproduction, considérés jusqu’à maintenant comme idiopathiques, et de thérapies innovantes orientées, afin d’optimiser la réceptivité du biosenseur endométrial afin de permettre une implantation embryonnaire harmonieuse et une grossesse évolutive. / Reproductive Immunology involves general immunology principles and special aspects of reproduction and development. Colony Stimulating Factors (CSFs) are an illustration of the medical application of this domain. In the CSF family, Granulocyte-Colony Stimulating Factor (G-CSF) appears today as a promising therapy in various cases of reproductive failure although its targets and effects are not clearly established. In this work, through a review on CSFs in reproduction, a study dedicated to human endometrial targets of G-CSF, and a study dedicated to systemic G-CSF supplementation effects on murine embryo implantation, we tried to approach some possible mechanisms of action of this cytokine. In the considered non-abortive and abortion-prone murine models, the timed systemic G-CSF supplementation, targeting specifically the pre implantation endometrium, influenced the embryo implantation process. Some pre conceptual human endometrial dysregulations of G-CSF target genes were also observed in infertile patients. The endometrial influence of G-CSF on these target genes was also illustrated in an ex-vivo model. These molecules under G-CSF influence are described as critically involved in embryo implantation process, by influencing embryo adhesion, cell migration, tissue remodelling and angiogenesis. These data suggest possible pre-conceptual preventive diagnosis of such reproductive failures and future orientated therapies to optimise the endometrial biosensor and the further embryo implantation and ongoing pregnancy.

Orientador: Flávio Ruas de Moraes / Banca: Claudinei da Cruz / Banca: Mauricio Laterça Martins / Resumo: Neste trabalho foi avaliada a cinética da evolução do processo cicatricial em Piaractus mesopotamicus alimentados com ração suplementada por cromo trivalente e/ou parede celular de Saccharomyces cerevisiae, distribuídos ao acaso em quatro grupos; T1= controle sem suplemento; T2= com 0,3% de parede celular; T3= com 0,3% parede celular +18 mg de cromo trivalente/kg de ração; T4= com 18 mg de cromo trivalente/kg de ração. Após o período de alimentação de 105 dias, foram realizadas incisões na pele para a remoção da epiderme e derme. A avaliação do processo cicatricial foi realizada macroscopicamente e microscopicamente após um, três, sete, 14, 21, 28 e 35 dias de induzir as lesões. Na avaliação macroscópica foi utilizada a área de retração da ferida representativa da média de cada grupo. Para avaliação microscópica foram retirados fragmentos de pele do bordo superior até o inferior incluindo a musculatura. Os dados obtidos foram submetidos à análise de variância, e quando significativos foram comparados pelo teste de Tukey a 5% de probabilidade. No exame macroscópico e na análise histológica, as lesões dos animais que receberam ração suplementada por parede celular de S. cerevisiae ou cromo trivalente os eventos do processo cicatricial foram antecipados em relação ao grupo controle. A área de retração das feridas apresentou diferença entre os dias de avaliação demonstrando um aumento progressivo da velocidade do processo cicatricial, entretanto sem diferenças entre os grupos. A espessura da epiderme e derme, o número de células caliciformes e de neovasos não diferiram entre os grupos, contudo demonstraram a evolução do processo cicatricial nos diferentes tempos avaliados. / Abstract: This study evaluated the kinetics of the evolution of the healing process in P. mesopotamicus fed with ration supplemented with trivalent chromium and/or cell wall of S. cerevisiae, distributed at random into four groups; T1 = control without supplement, T2 = with 0.3% of the cell wall, T3 = with 0.3% cell wall +18 mg of trivalent chromium/kg diet, T4 = with 18 mg of trivalent chromium/kg diet. After the feeding period of 105 days, incisions were made in the skin to remove the epidermis and dermis. The evaluation of the healing process was performed macroscopically and microscopically after one, three, seven, 14, 21, 28 and 35 days induction of injuries. For macroscopical evaluation was used the rate of retraction of the wound representative of the average of each group. For microscopical evaluation pieces of skin were removed from the upper to the lower including muscle. Data were submitted to analysis of variance and when significant were compared by Tukey test at 5% probability. On macroscopic examination and histological analysis, the lesions of animals fed with ration supplemented with cell wall of S. cerevisiae or trivalent chromium the events of the healing process were anticipated in the control group. The area of wound retraction was different between the days of valuation showing a progressive increase of speed the healing process, however without differences between groups. The thickness of the epidermis and dermis, the number of goblet cells and neovascularization didn't differ between groups, although, demonstrated the evolution of the healing process in different moments. / Mestre

Although much more work needs to be done to elucidate the functional roles of FSH and LH in fish reproduction, the preset study provides a relatively comprehensive study for us to understand the potential roles of FSH and LH during ovarian development in fish, especially the importance of FSH. / At the same time, functional studies were carried out to examine and compare bioactivities of the CHO-derived zfFSH and zfLH in zebrafish ovary, which is the major part of the present project. The following aspects were covered to investigate the actions of zfFSH and zfLH: steroidogenesis and folliculogenesis. / Both recombinant zfGTHs stimulated activin betaA expression but slightly suppressed activin betaB expression. During short-term treatment, zfFSH and zfLH exhibited similar stimulatory effects on activin betaA expression; the effect of zfLH became more prominent after 24 h treatment while zfFSH had little effect. / Previously, our laboratory had established two stable Chinese hamster ovary (CHO) cell lines expressing recombinant zebrafish FSH (zfFSH) and LH (zfLH). However, the production yields are very low. Therefore, the present study tried to adopt the yeast Pichia pastoris as another bioreactor to produce recombinant zfFSH and zfLH. Two different forms of expression vectors for a native form and a fusion form carrying a His-tag, respectively, were constructed for each hormone. Their bioactivities were monitored and confirmed by receptor-based reporter gene assays as well as ovarian fragment incubation. As expected, the native form exhibited much higher activities than the fusion form. / The pituitary gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are the key hormones controlling vertebrate reproduction. Although the two gonadotropins have been characterized in numerous teleost species, our understanding of their biological functions remains rather limited. This is largely due to the lack of pure form of homologous gonadotropins and inadequate understanding of gonadal physiology in most species studied as well as species variation of hormone actions. The present study aims at systematically investigating the functional roles of FSH and LH in the ovary using zebrafish as the model. Zebrafish is becoming more and more popular as the model of reproductive and developmental studies due to several advantages. First, though its body size is small, its ovary is relatively large and available all the year around. Second, zebrafish spawns everyday and its development is fast. Last but not least, its bioinformatics information is tremendous compared to other fish models. / We investigated the effects of zfFSH and zfLH on steroidogenesis by examining the regulation of aromatase by these two hormones. Aromatase catalyzes the conversion of androgens into estrogens during steroidogenesis. Both recombinant zfGTHs stimulated the aromatase expression during short-term treatment (8 h) in ovarian fragment culture, with zfFSH much more potent than zfLH. However, zfFSH continued to exhibit powerful effect on aromatase expression after 24 h treatment while zfLH had little effect at all. The stimulatory effect of zfFSH on aromatase expression was time-, dose- and stage-dependent and was also confirmed by in vivo study. Furthermore, it was also zfFSH but not zfLH that significantly stimulated StAR protein expression during short-term treatment. StAR protein is critical to steroidogenesis by facilitating the movement of cholesterol across the mitochondrial membrane. / zfLH was found to be able to induce GVBD in zebrafish, as demonstrated in other fish species. However, our preliminary data showed that zfFSH was also involved in this process. To our knowledge, this is the first time to demonstrate that homologous FSH induces GVBD in teleosts. / Yu, Xiaobin. / Adviser: Wei Ge. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis submitted in: December 2008. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 152-181). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Migration Stimulating Factor (MSF) is an oncofoetal protein which is constitutively produced by both epithelial and stromal cells during foetal development, not expressed by the majority of their normal adult counterparts, but re-expressed during pathological processes such as cancer and wound healing. Scotland has the highest occurrence of oral cancers in the UK; the incidence is still increasing, but patient survival remains very poor. The expression of MSF in oral tumours has not been previously reported. The aims of this study were: • To determine the effects of MSF on the migration of oral tumour cell lines and normal stromal cells in culture (chapter 3), • To ascertain the possible presence, diagnostic and prognostic value of MSF in oral squamous cell carcinoma (OSCC; chapter 4), and salivary gland tumours (SGT; chapter 5). • To identify the putative MSF receptors in oral tumour cell lines (chapter 6). For tissue culture studies, the effects of rhMSF (wild type and mutant proteins) were examined on human cell lines TYS, HSG, Endo 742 and FSF44. These cells were derived from OSCC, SGT, microvascular endothelial cells and skin fibroblasts, respectively. For ex-vivo studies, paraffin embedded archival specimens of OSCC and SGT were stained with specific MSF antibodies and the level of staining was assessed by consensus of 2-4 independent observers. The association between MSF expression and patient survival was determined by Kaplan-Meier and log-rank tests. Results presented in this thesis indicate that TYS and HSG cells secrete bioactive MSF in culture. rhMSF stimulated the migration of these tumour cells. The use of mutant proteins demonstrated marked differences among the cells examined: Five bioactive motifs (4x IGD and 1x HEEGH) were required for MSF bioactivity on TYS and HSG cells, whereas only one of these motifs was required for Endo 742 and two for FSF44. MSF+aa and MSF-aa showed the same migration-stimulating activity, but differ in their interaction with the MSF-inhibitor Neutrophil Gelatinase-Asscciated Lipocalin (NGAL). NGAL was shown to bind to and inhibit MSF+aa, but not MSF-aa. The bioactivity of MSF+aa and MSF-aa was inhibited by Insulin-like Growth Factor Binding Protein-7 (IGFBP7), MSF-function-neutralising antibody and antibody to the integrin avß3. This integrin was identified in the cell membrane material bound to MSF, suggesting that avß3 is a receptor for MSF.

Pre-clinical studies suggest that granulocyte-colony stimulating factor (GCSF) holds promise for the treatment of ischaemic heart disease (IHD). However, its safety and efficacy in this setting, and in particular in patients with chronic refractory 'no-option' IHD, is unclear. Therefore, a clinical study was initiated in 20 such 'no-option' patients, with the aim of assessing the safety and efficacy of both G-CSF administration, and also, that of an intracoronary infusion of G-CSF mobilised CD133+ cells. The study involved initial baseline cardiac ischaemia assessment (symptom based questionnaire, exercise stress test (EST), nuclear Sestamibi (MIBI) and dobutamine stress echocardiographic (DSE) imaging). Stable 'no-option' IHD patients then received open-label G-CSF commencing at 10μg/kg s/c for five days, with an EST on days four and six (to facilitate myocardial cytokine generation and stem cell trafficking). After three months, cardiac ischaemia assessment and the same regimen of G-CSF and ESTs were repeated, but in addition, leukapheresis and then a randomised double-blinded intracoronary infusion of CD133+ or unselected cells were performed. Final cardiac ischaemia assessment was three months thereafter. Eighteen male and two female subjects (mean age 62.4) were enrolled. Eight events occurred that fulfilled pre-specified 'adverse event' criteria: four ischaemic (troponin positive) episodes, two episodes of transient thrombocytopaenia (one profound), one episode of gout and one unscheduled hospitalisation for exhaustion. Troponin was positive on 17 further occasions (all CK-MB negative), however, at these instances angina severity was identical to baseline. Importantly, no adverse event(s) resulted in any detectable long-term adverse sequelae for any subject. From baseline to final follow-up, the administration of two cycles of G-CSF was associated with statistically significant improvements in a range of subjective outcomes, including anginal symptoms, quality of life and EST performance (all p < 0.005). However, the objective MIBI and DSE scans showed only trends towards improvement (all p > 0.1). Compared to unselected cells, an intracoronary infusion of CD133+ cells did not improve either subjective or objective outcomes. In conclusion, administering G-CSF to patients with refractory 'no-option' IHD warrants careful monitoring, but may be performed with safety. A larger, randomised double-blind placebo-controlled trial of G-CSF in these patients appears warranted.