Fibroblast growth factor 2 (FGF-2) is known to be released from cartilage upon injury and is able to influence chondrocyte gene expression in vitro. In cartilage, FGF-2 regulates E11/podoplanin expression in murine joints following surgical destabilisation (DMM model of osteoarthritis (OA)), and in cartilage explant injury models. In bone, E11 is critical for the early stages of osteocytogenesis and is responsible for the acquisition of the osteocyte dendritic phenotype. This dendritic phenotype is dysregulated in OA and given the known role of the osteocyte in controlling bone remodelling, this may contribute to the subchondral bone thickening observed in OA. Hence, the aim of this study was to elucidate the nature of FGF-2- mediated E11 expression and osteocytogenesis in skeletal health and disease. This thesis has shown that FGF-2 dose-dependently increased E11 mRNA expression in MC3T3 cells, primary osteoblasts and in primary calvaria organ cultures, which was confirmed by E11 protein western blotting data. The FGF-2 induced changes in E11 expression were accompanied by significant increases in the mRNA expression of the osteocyte markers Phex and Dmp1, and significant decreases in the mRNA expression of the osteoblast markers Col1a1, Postn, Bglap and Alpl expression. This thus supports the hypothesis that FGF-2 drives osteocytogenesis. The acquisition of osteocyte phenotype involves the re-organisation of the cytoskeleton, such as F-actin. This step is important for the transition of cuboidal-shaped osteoblasts to the stellate-shaped osteocyte phenotype. FGF-2 stimulation of MC3T3 cells and primary osteoblasts revealed more numerous and longer dendrites, as visualised by phalloidin staining for F-actin and indicative of the acquisition of the osteocyte phenotype. In contrast, control cells had a typical rounded morphology with fewer and shorter dendrites. Furthermore, immunofluorescence labelling for E11 in control cells revealed uniform distribution throughout the cytoplasm, especially in the perinuclear region. In contrast, FGF-2 treated cells showed a modified distribution where E11 was negligible in the cytoplasm, but concentrated in the dendrites. The use of siRNA knockdown of E11 achieved a 70% reduction of basal E11 mRNA expression. This knockdown also effectively abrogated FGF-2-related changes in E11 expression and dendrite formation as disclosed by mRNA and protein expression, immunofluorescence and F-actin staining with phalloidin. Despite these FGF-2 driven increases in E11 and osteocyte dendrite formation in vitro, immunohistochemical labelling revealed no differences in E11 expression in subchondral, trabecular and cortical osteocytes from naïve Fgf-2 deficient mice in comparison to wild-type mice. Similar results were observed upon sclerostin immunolabelling. FGF-2 stimulation of MC3T3 cells elicited activation of ERK1/2, Akt and p38 MAPK. However, inhibition of the aforementioned pathways failed to reduce FGF-2- mediated E11 expression and as such, the specific signalling pathway responsible remains unclear. Upstream, the expression of Fgfr1 was increased (>10-fold) over 24 h time point, while a reduction was seen in Fgfr2/3 expression over same time point especially in the FGF-2 treated cultures. This suggests that increased E11 expression and the acquisition of the osteocyte phenotype may be speculatively though upregulation of Fgfr1. The expression of E11 and sclerostin in OA pathology in mice, human and dogs were investigated. Initially sequence homology using the Clustal Omega alignment program showed both proteins to be homologous in the domestic animals under study. A comparative study using canine subchondral bone osteocytes revealed increased E11 expression in the OA samples relative to the control. This feature may be related to newly embedded osteocytes during sclerosis. However, E11 and sclerostin were unchanged in both murine (DMM) and human OA subchondral bone osteocytes in comparison to controls. In mice, this may be due to limited OA development; whilst in humans the sample size, age, stage of the disease and sourcing from same diseased joint may be important in the interpretation of the results. The expression of E11 and sclerostin during OA pathology was also investigated in Fgf-2 deficient mice in which OA was induced using the DMM model. There was no difference in E11 expression between the OA and control (sham-operated) samples, suggesting that compensation of E11 expression may be mediated by growth factors from the FGF family. Surprisingly, increased E11 expression was observed in the control Fgf-2 deficient mice, in comparison to the wild-type control mice. This suggests a potential adjustment to loading by the contralateral knee, as this was not observed in naïve mice from both groups. Together, these data show that FGF-2 promotes the osteocyte phenotype, and that this is mediated by increased E11 expression. These results may help explain (1) the altered osteocyte phenotype and (2) increased subchondral bone thickening observed in OA. This knowledge will be of interest in the search for disease modifying therapeutics for skeletal health, including OA and osteoporosis.

The protein Poly was first discovered in Drosophila melanogaster, during a screening for third chromosome lethal mutations. Drosophila poly mutant larvae exhibit a slower rate of development. However, they reach the third instar larval stage and remain at that stage for 21 days before they die without reaching pupation. This phenotype is attributed to developmental impairment of the imaginal discs, therefore suggesting defects in cell growth and/or proliferation. During that stage, the mutant larvae develop melanotic masses. Poly is conserved and its homolog, Elp6, is one of the small subunits of the Elongator Complex, a complex involved in many cellular functions including transcription and translation. Drosophila larvae mutated at the Elp3 gene, the gene encoding the catalytic subunit of the Elongator complex, develop melanotic masses, a phenotype very similar to that of poly. The Heck laboratory published that Poly is a positive mediator of the Insulin Receptor/TOR (InR/TOR) pathway, which leads to protein, glycogen and fatty acid synthesis, regulates cell growth and apoptosis. It was shown that Poly interacts with InR, at least in some cases, therefore promoting cell growth and metabolism (Bolukbasi et al., 2012). The Drosophila genome shares 60% similarity to the human, with 77% of the genes attributed to a human disease having a Drosophila counterpart (Chien et al., 2002). In 2011, an intriguing study by Musselman et al. reported that feeding wild type Drosophila larvae with excessive amounts of sucrose led to the development of an insulin resistance phenotype similar to that of Type 2 Diabetes (T2D), thus rendering Drosophila an easily accessible T2D model. The phenotype included impaired metabolism, slower rate of development, and excessive accumulation of triglycerides (TAG) in the larval fat body. In my thesis research, I examined the involvement of Poly in insulin resistance - T2D using Drosophila as a model. The understanding of the connection between the protein and the disease came upon the discovery of a new form of Poly, Poly14. Poly14 is enriched in the Drosophila fat body, the equivalent of the human fat tissue and liver and its protein levels are significantly decreased when larvae are fed a high sucrose diet, compared to other types of diets – potentially linking the protein to the onset of T2D. Poly mRNA levels were also lower. To examine whether the overexpression of poly might be able to rescue the insulin resistance phenotype, two new Drosophila transgenes were generated with the ability to express the gene in a tissue of interest. In these two new transgenes, Poly is tagged with tRFP (Red Fluorescence Protein) at the N’- (UAS_N’RFPpoly) or the C’-terminus (UAS_polyC’RFP). Overexpression of Poly rescued the insulin resistance phenotype, therefore implicating Poly as a possible important regulator in the development of the insulin resistance phenotype. All of the above findings suggest a vital role of Poly in metabolism and the development of the insulin resistance/diabetic phenotype in Drosophila, providing us the opportunity for new tools in this very medically-relevant field of research.

Objetivos: Avaliar a variabilidade glicêmica, respostas metabólicas e cardiovasculares após uma sessão de exercício aeróbico em pacientes com diabetes em tratamento com vildagliptina ou glibenclamida. Métodos: Foi realizado ensaio clínico aberto e paralelo que incluiu 13 pacientes com diabetes tipo 2 tratados com vildagliptina (50mg bid) ou glibenclamida (5mg bid) por 12 semanas. Antes e após a intervenção, a variabilidade glicêmica (glicose média, variância da glicose, coeficiente de variação e média da amplitude das oscilações glicêmicas), respostas metabólicas (HbA1c, glicose, insulina e 8-iso prostaglandina F2α) e cardiovasculares (débito cardíaco, variabilidade da frequência cardíaca e componentes do controle autonômico) foram avaliadas no repouso, durante e após uma sessão de exercício aeróbico de 30 minutos. A variabilidade da pressão arterial foi aferida nas 24 horas após o exercício. Resultados: Doze semanas de tratamento resultou em redução da glicemia de 18% com vildagliptina e 35% com glibenclamida (p grupo=0.007). A HbA1c reduziu significativamente (1.24 % e 1.52%) nos grupos vildagliptina e glibenclamida, respectivamente.A variabilidade glicêmica não se alterou após o tratamento com glibenclamida ou vildagliptina (MAGE=55.8 ±5.3 mg/dL e 69.9 ± 13.3 mg/dL, respectivamente; p grupo=0.091; p tempo=0.234). Foi encontrada uma diminuição da glicose avaliada por monitoramento contínuo durante as 3 horas de recuperação do exercício, com AUC (de 6h) menor no grupo glibenclamida vs vildagliptina (p=0.04). A glibenclamida induziu maiores concentrações de insulina na recuperação do exercício. O grupo tratado com vildagliptina obteve 6.3mmHg de redução da pressão arterial sistólica, enquanto a glibenclamide reduziu 3.6mmHg, sem diferença entre os grupos. Os pacientes tratados com vildagliptina apresentaram menor variabilidade da pressão arterial sistólica (0.0445 ±0.05 mm/Hg), medida por time rate, comparados aos tratados com glibenclamida (0.601 ±0.12 mm/Hg), p=0.012. Conclusão: Este é o primeiro estudo conduzido em pacientes com DM2 que fornece dados sobre a influência da terapia medicamentosa padrão (metformina e glibenclamida) vs outra classe disponível (metformina e vildagliptina) em respostas a uma sessão de exercício aeróbico, um dos tratamentos recomendados para pacientes com DM2. Além de melhora no controle glicêmico e redução da pressão arterial sistólica obtidas por ambos tratamentos, foi observada menor variabilidade da pressão arterial nos 9 pacientes submetidos ao tratamento com vildagliptina. Não foi encontrada menor variabilidade da glicemia após o exercício nos pacientes tratados com vildagliptina comparados aos tratados com glibenclamida, refutando a hipótese de estudo.

Tese de mestrado integrado. Engenharia Civil (Construções Civis). Universidade do Porto. Faculdade de Engenharia. 2011

Tese de mestrado integrado. Engenharia Mecânica. Faculdade de Engenharia. Universidade do Porto. 2010

Tese de mestrado integrado. Engenharia Civil (especialização em Estruturas). Faculdade de Engenharia. Universidade do Porto. 2008

Head and neck cancer accounts for approximately 3 percent of all cancers in the United States, and over 90% of them are head and neck squamous cell carcinoma (HNSCC). Chemotherapeutic drugs that treat HNSCC can activate BCL-2 family dependent apoptosis. Pro-apoptotic NOXA induced by adenovirus (Ad-NOXA) or fenretinide inactivates anti-apoptotic MCL-1, while ABT-263 can inactivate other anti-apoptotic BCL-2 family members such as BCL-2 and BCL-XL. We used p53 inactive HN8 and HN12, p53 wild-type UMSCC1, and HPV-positive UMSCC47 human HNSCC cell lines and five mouse HNSCC cell lines. Cells were treated with Ad-NOXA, ABT-263, and fenretinide alone or in combinations. Combinational treatment of ABT-263 with Ad-NOXA or fenretinide enhanced cell death among all cell lines we tested regardless of p53 status. These findings support the hypothesis that combinational treatment of Ad-NOXA or fenretinide with ABT-263 increases cell death by simultaneously inhibiting all anti-apoptotic BCL-2 family proteins in HNSCC cells.

Thesis (M Med (Family Medicine))--University of Limpopo (Medunsa Campus), 2010. / Introduction: Diabetes Mellitus still remains an important non- communicable disease globally. The burden of the disease continues to rise even in the sub-Saharan Africa. Aim: The study aimed to assess and describe the dietary practices and the prevalence of obesity among type 2 diabetic patients seen at Scott Hospital. Methods: A descriptive cross- sectional study was conducted among type 2 diabetic patients at Scott Hospital, Morija, Lesotho. A simple random sample of 50 participants was selected and each completed an interviewer administered questionnaire. The administration of the questionnaire was done by the researcher and a research assistant. Socio- demographic characteristics of the participants were obtained, anthropometric measurements were height and weight of each participant with subsequent determination of the BMI. Dietary intake was assessed using questions from a modified food frequency questionnaire of 16 food groups that reflect the commonest food items available. Results: The results showed that 86% (43/50) of the participants were females and 14% (7/50) were males with a female: male ratio of 6:1. The majority (72%) were above age 50 years and with regards to educational status, 62% had primary school education as the highest level attained. Four percent (4%) had no formal education. Fifty percent (50%) of the participants were unemployed. The mean weight of the participants was 84.22 ± 13.51 kg and the mean height was 157.76 ± 6.10 cm. The mean BMI was 33.97 ± 4.99 kg/m². Seventy-eight percent (38/50) of the participants were obese, and 14% (7/50) of the participants who were females, were morbidly obese with BMIs ≥ 40kg/m². The prevalence of obesity when stratified by gender showed that 57% (4/7) of male participants and 81% (35/43) of female participants were obese. According to the dietary assessments, food groups with the highest reported consumption per unit per month were; maize meals, breakfast-cereals and bread respectively. Conclusion: The study showed a predominantly female and elderly population of type 2 diabetics at Scott Hospital, Morija, Lesotho. The prevalence of obesity was high (78%) among the participants and this further supports the view that obesity is a major risk factor for type 2 diabetes. Reported dietary intake showed a higher consumption of carbohydrates and a very low intake of dietary fibre as represented by legumes. Keywords: Type 2 diabetes mellitus, dietary habits and obesity.

Thesis (M Med (Family Medicine))--University of Limpopo, 2010. / The number of diabetic patients will continue to rise even in rural settings and the burden of this disease will continue to take its effect on the limited resources of these communities. The effect of such burden will be more pronounced if we are to add the various complications associated with substandard management of diabetes mellitus. The first step in assessing the level of care we give to this category of patients is to measure their level of adherence, in an effort to expose the pitfalls on both the side of the patients and on the side of the health care provider. The aim of the study is to assess the level of adherence to treatment among type2 diabetic patients in Matlala district hospital; Limpopo Province. This cross-sectional study used the convenience method of sampling with the aid of a tested, structured questionnaire, to obtain data from respondents between December 2009 and March 2010, a period of 4 months. The excel computer program was used for data capturing. Percentages and numbers were used for interpretation and cross tabulation was used to determine association. The result of the study indicated that 137 {70%} of the respondents adhere to diabetes treatment. There were two demographical characteristics that are significantly associated with non adherence: age {p=0.028} and employment status {p=0.018}. Of those respondents that keep their appointments, 98% are adherent to treatment. When considering reasons for poor adherence; 29% of respondents stated that the clinic did not have their pills, 16% stated that they forgot to take their medication and 14% stated that they travelled to visit ix and did not take enough pills with them. On the reasons for poor adherence to lifestyle: 29% of the respondents said that they were too old, 22% stated no specific reason, 13% struggled to motivate themselves and 10% simply forgot what to do. Most, 68%, of the respondents that adhere to the recommended use of medication agreed that they take it at meal time, 14% set a reminder, 8% employed the assistance of a treatment supporter and other respondents used other means to remember. The study revealed an above average level of adherence in my setting and it will be logical to assess whether this corresponds to the metabolic control expected of good adherence. More is needed to be done on the reasons why our patients do not adhere to both medication and lifestyle changes and each stake holder needs to address their short comings.

Thesis (M Med(Family Medicine))--University of Limpopo (Medunsa Campus) 2010. / Aim & Objectives Aim: To explore the lay beliefs of type 2 diabetic patients seen at Prince Mshiyeni Memorial Hospital. Objectives: To understand lay beliefs of effective life long management of type 2 diabetes patients and consequently to make recommendations of improving management of diabetes in conjunction with the findings of the study. Methodology Study Design: An exploratory study from a qualitative perspective using free attitude interviews as a data collection technique. Setting: Prince Mshiyeni Memorial Hospital a district hospital located in Umlazi in the outskirts of Durban, South Africa. Study Population: All type 2 diabetic patients above the age of 40 on treatment for 18 months or more. Ten respondents were purposively selected using maximum variation sampling strategy. Participants were asked individually to give an account of their beliefs and experience in the management of diabetes. All interviews were tape recorded, transcribed verbatim, and analyzed to identify emerging themes. Results The interpretative thematic analysis generated the following main themes: 1) Combination therapy 2) Modern versus Traditional 3) The bitter stuff 4) Traditional healers and alternative remedies viii 5) Stress: Physical, financial, emotional, psychosocial strain 6) Spiritual believes: Religious and Traditional 7) Eating right food and loose weight Conclusions This study has described most lay beliefs about the management of type 2 diabetes in this setting. The research has identified that type 2 diabetic patients seen at Prince Mshiyeni Memorial Hospital in Umlazi South of Durban held different diabetes management beliefs based on their respective initial symptom perception and beliefs, their illness origin and healing beliefs, their spiritual and traditional beliefs and values and to a significant extent, the beliefs of spouses or life partner in couples and of family members. These findings have challenged the functionality of the health care in its capacity to respond to the population expectations based on their ethnic, spiritual and cultural background.