The incidence of nasopharyngeal carcinoma (NPC) is high in Southeast China, including Hong Kong. Current therapy on NPC relies largely on radiotherapy and chemotherapy, but treatment failures remain the major challenge for advanced stage and recurrent/metastatic NPC. Previous studies indicated that after completion of primary treatment, the tumor recurrent rate for NPC was between 15% and 58%. Recent researches suggest the existence of cancer stem cells (CSCs). CSC refers to a sub-population of cells within the bulk tumor. CSCs exhibit the stem cell property of self-renew and differentiation, and are responsible for sustaining tumorigenesis and establishing the heterogeneity in the tumor. CSCs are generally more resistant to conventional treatment methods and might be responsible for tumor recurrent after treatment. Therapies that can eliminate cells with CSCs-characteristics might provide a more durable response and better prognosis. Sulforaphane (SFN) is a natural compound present in Cruciferous vegetables. SFN has been shown to inhibit the in vitro and in vivo growth of various types of tumor cells through the (i) induction of cell cycle arrest and apoptosis, (ii) inhibition of angiogenesis and metastasis, and (iii) suppression of cancer stem cells (CSCs). However, the effects of SFN on NPC have not been examined in detail. The present study aims to study the anti-tumor activities of SFN on NPC. In the first part of this study, the effects of SFN on the in vitro growth of NPC cells were examined. The growth of both EBV-negative HONE-1 and EBV-positive C666-1 cells was found to be inhibited by SFN. The growth inhibition was associated with the induction of G2/M cell cycle arrest and apoptosis. The effects of SFN on the growth of NPC cells with CSCs characteristics were also examined by the tumor spheres formation assay. SFN was found to reduce the capacity of both HONE-1 and C666-1 cells to form CSCs-enriched tumor spheres. The population of cells with high expression of NPC CSCs-associated markers (Sox2 and ALDH) was found to be reduced after SFN treatment. Under the culture conditions for CSCs, ALDH inhibitor was found to reduce the capacity of NPC cells to form tumor spheres. Similarly, the capability of Sox2 siRNA-treated NPC cells to form tumor spheres was also reduced in the spheroids assay. Results from these studies indicated that the growth of NPC cells with CSCs characteristics could be reduced by SFN. After the in vitro study of SFN on the growth of NPC cells, mechanisms that are associated with the SFN-induced growth inhibition on NPC cells were examined. MIF is a NPC biomarker that is highly expressed in NPC patients. Previous study has shown that SFN could interact with MIF and affect the biological function of MIF. In the present study, SFN was found to down-regulate the expression of MIF in NPC cells. One of the receptors of MIF, CXCR2, was found to be down-regulated after the SFN treatment. The downstream Akt signaling was also inhibited. Results from the second part of this study indicated that SFN-mediated inhibition of MIF/CXCR2/Akt signaling was involved in the growth inhibitory effects of SFN on NPC cells. In NPC, many genes were found to be down-regulated through hypermethylation and such down-regulation contributed to NPC development. In the present study, DNMT1 was found to be down-regulated after SFN treatment, and the effect was accompanied with the restored expression of WIF1 and Rassf1a. Further mechanistic study showd that siRNA-mediated DNMT1 knock-down could reduce the capacity of tumor spheres formation of NPC cells. Interestingly, the expression of the tumor suppressor genes WIF1 and Rassf1a was restored. In addition, exogenously added WIF1 could reduce the formation of tumor spheres. These findings suggested that SFN-mediated down-regulation of DNMT1 was associated with the growth inhibitory effects of SFN on NPC cells. Finally, the in vivo efficacy of SFN alone or SFN in combination with cisplatin on the growth of NPC xenograft was examined. SFN was found to inhibit the growth of C666-1 xenograft and enhance the anti-tumor effects of cisplatin on the C666-1 xenograft. Taken together, results from this study demonstrated the anti-tumor effects of SFN in NPC and suggested that SFN could be a potential therapeutic drug for NPC.

No description available.

No description available.

Background: ICG-001, an antagonist of CBP (CREB-binding protein), has been demonstrated to exert anti-tumor activity via the modulation of the Wnt signalling pathway. It has previously been demonstrated that miRNAs play an important role in ICG-001-mediated tumor suppression. In the present study, the role of miRNA-134 and 1-integrin in ICG-001-mediated anti-tumor activity in nasopharyngeal carcinoma (NPC) was examined. Methods: NPC cell lines including C666-1, HONE-1 and HK-1 were used in this study. RT-PCR and Western blot were used to study the expression of miRNA-134 and the protein expression of the target proteins, respectively. Confocal microscopy was used to analyse the subcellular localization of 1-integrin. In the functional studies, in vitro endothelial adhesion assay and in vivo nude mice model were used to evaluate the adhesion and migration of ICG-001-treated NPC cells in animals, respectively. Results: ICG-001 was found to up-regulate the expression of miRNA-134 and down-regulate 1-integrin in NPC cells. The effect was accompanied with the inhibition of the adhesion of NPC cells to lung endothelial cells. In addition, over-expression of miRNA-134 would down-regulate the expression of 1-integrin. Results from 1-integrin 3'UTR Renilla luciferase reporter assay confirmed that 1-integrin is a target of miRNA-134 in NPC cells. In the animal study, the ability of ICG-001-pretreated NPC cells or stable miRNA-134 expressing NPC cells to migrate to the mouse lung was greatly reduced. Conclusion: The CBP antagonist ICG-001 may further be developed as an anti-tumor agent for the treatment of nasopharyngeal carcinoma

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Chinese Medicine / Master / Master of Philosophy

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg 2017. / Background: Nasopharyngeal colonisation is a pre-requisite for developing bacterial respiratory and invasive disease. Immunisation of children with the pneumococcal conjugate vaccine (PCV) impacts upon colonising pneumococcal serotypes, which in turn could also affect the biome of the nasopharynx in relation to colonisation by other bacteria. Due to limitations in standard culture methods, the association between PCV-immunisation and bacterial carriage density is still unclear, including among HIV-infected children. In this study we aimed to evaluate the effect of infant vaccination with the 7-valent PCV (PCV7) on vaccine-serogroup colonisation in order to determine whether the increase in non-vaccine serotype (NVT) colonisation was due to unmasking of previously low density colonising serotypes or increase in acquisition of NVT. Also, we evaluated the association between PCV7 immunisation and HIV-infection on the prevalence density of nasopharyngeal colonisation by other common potentially pathogenic bacteria. Methods: A multiplex real-time qPCR assay was set up to detect 44 common pneumococcal serotypes and 5 bacterial pathogens including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Streptococcus pyogenes. All assays were optimised according to MIQE guidelines and their ability to detect multiple pneumococcal serotype/group and bacteria in archived nasopharyngeal swabs were evaluated. The multiplex qPCR assays were then used to evaluate vaccine-serotype, non-vaccine serotype and bacterial nasopharyngeal colonisation in achieved swabs of PCV7-vaccinated (at 6, 10 and 14 weeks of age) and PCV-unvaccinated African children at 9 and 15-16 months of age, prior to routine vaccination of children with PCV through the public immunisation program. In order to address the limitations of the qPCR assays, a nanofluidic real-time PCR assay was developed to simultaneously detect 53 pneumococcal serotypes, 6 serotypes of H. influenzae and 11 bacterial pathogens. Further, all assays were optimised and evaluated according to the MIQE guidelines and findings from Fluidigm and traditional qPCR assays were compared. Lastly, Fluidigm was used to evaluate the association of HIV-infection on the prevalence and density of nasopharyngeal colonisation at 9 and 16 months of age by common, potentially pathogenic bacteria including PCV7 pneumococcal serotypes, non-PCV7 serotypes, Haemophilus influenzae, non-typable Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Streptococcus pyogenes, Neisseria meningitidis, Neisseria lactamica, Bordetella pertussis, Bordetella parapertusis, Bordetella bronchiseptica and Bordetella holmesii in achieved nasophartngeal swabs collected from PCV7-vacciniated HIV-infected and HIV-uninfected children. Results: Molecular qPCR was more sensitive than culture in detecting multiple concurrent colonising pneumococcal serotypes as well as other common nasopharyngeal colonisers, with the majority of additional isolates detected by qPCR having a low carriage density (<104 CFU/ml). Further, qPCR identified a lower prevalence of PCV7-serotype colonisation among PCV7-vaccinated compared to PCV-unvaccinated children at 9 and 16 months of age [adjusted Odds Ratio (aOR): 0.37; 95% CI; 0.19-0.7 and 0.41; 95% CI; 0.26-0.63, respectively]; and an increase in NVT-serotype [aOR: 1.88; 95% CI; 1.02-3.48 and 2.2; 95% CI; 1.18-4.1] colonisation respectively. The increase in NVT carriage among PCV7-vaccinees was driven by serotype 19A, which increased by 53.4% (p=0.021) and 70.7% (p<0.001) at 9 and 16 months of age respectively. Further, 19A had a higher density of colonisation in PCV7-vaccinated groups compared to PCV-unvaccinated groups and was more likely to be identified as a primary than non-primary isolate in PCV7-vaccinated children alone. PCV immunisation was also associated with an increased prevalence of H. influenzae at 9 months (55.8% vs. 66.3%, p<0.001) and 16 months (72% vs. 62%, p=0.017) of age, while a temporary increase in the carriage prevalence of S. aureus was found in PCV7-vaccinated (18.9%) compared to PCV-unvaccinated children (11.1%, aOR 2.1; 95% CI 1.0-1.4; p=0.049) at 9 months of age only. The density of pneumococcus (4.68 vs. 4.28 CFU/ml; p=0.007), H. influenzae (3.86 vs. 4.34 CFU/ml; p=0.008), M. catarrhalis (2.98 vs. 3.52 CFU/ml; p<0.001) and S. aureus (3.06 vs. 4.02 CFU/ml; p=0.02) were also higher among PCV7-vaccinated compared to PCV-unvaccinated children at 9 months age, although this difference diminished with increasing age. There was excellent concordance between the qPCR and Fluidigm for carriage prevalence and density of the majority of assays, with Fluidigm identifying an additional 7 pneumococcal serotypes and 11 bacterial species above those detected by qPCR. Further, discordant results between the two PCR methods were strongly associated with a low carriage density (<102 CFU/ml). Using molecular Fluidigm, a lower carriage prevalence of overall pneumococci (58.6% vs. 69.9%; p=0.02), non-vaccine serotypes (27.8% vs. 40%; p=0.047) and H. influenzae (64.2% vs. 42.3%; p=0.01) was identified in HIV-infected children compared to HIV-uninfected children who were immunised with PCV7 at 9 months of age. No difference in the carriage prevalence of overall pneumococci was however found at 16 months of age (p=0.20), although the carriage prevalence of non-vaccine serotypes (50.9% vs. 60.4%; p=0.049) and H. influenzae (56% vs. 73.4%; p=0.02) was lower in HIV-infected children at 16 months of age. In addition, the density of overall pneumococcus was found to be higher in HIV-infected children (4.81 vs. 4.44 CFU/ml; p=0.014), despite the lower carriage prevalence at 9 months of age, which was driven by a higher density of vaccine serotypes/serogroups (4.21 vs. 3.72 CFU/ml; p=0.04). By 16 months of age, there was no difference in density of pneumococcal colonisation between the HIV-infected and HIV-uninfected children (p=0.89). No difference in the density of H. influenzae was found between HIV-infected and HIV-uninfected infants at 9 months of age (p=0.08); however, by 16 months of age, HIV-uninfected children had a higher density of overall H. influenzae colonisation (4.95 vs. 4.32 CFU/ml; p<0.001), which was largely due to the higher carriage density of NThinf in HIV-uninfected children (5.0 vs. 4.23 CFU/ml; p<0.001). Conclusion: Molecular qPCR assays were shown to be a promising alternative to WHO recommended culture in that multiple pneumococcal serotypes and other bacterial pathogens could be simultaneously detected as well as the bacterial load of each colonising bacteria quantified. The mechanism behind the vaccine effect was shown to be a combination of both serotype replacement and unmasking; however, the reduction in PCV7-serotype colonisation impacted on colonisation prevalence and density of other bacterial species of the nasopharynx and the clinical relevance of this needs further exploration in relation to mucosal and invasive disease outcomes, as well as for higher valence vaccines. While the higher carriage density of overall pneumococcus in HIV-infected children, despite the lower carriage prevalence might explain the higher invasive disease burden in HIV-infected compared to HIV-uninfected children even in the era of antiretroviral therapy treatment and PCV immunisation, future studies are required to provide clarity. Nevertheless, the findings from this thesis highlight the importance of continued surveillance of the circulation of pneumococcal serotypes as well as other bacterial pathogens especially in a population with a high burden of HIV-1 infection. / MT2017

Objetivo: Determinar os valores normativos da área de secção transversa mínima nasofaríngea de indivíduos sem anomalias craniofaciais e em diferentes faixas etárias. Método: Participaram do estudo 96 indivíduos sem anomalias craniofaciais, de ambos os sexos, subdivididos em 4 grupos etários: crianças com idade entre 6 e 10 anos (G1), adolescentes de 11 a 17 anos (G2), adultos jovens entre 18 e 39 anos (G3) e adultos de meia-idade entre 40 e 59 anos (G4). Verificou-se o índice de massa corpórea (IMC), a partir das medidas de peso e altura, e a circunferência cervical (CC), por meio de fita métrica pediátrica. A área de secção transversa mínima nasofaríngea (área nasofaríngea ANF) foi determinada por meio de rinomanometria anterior modificada (técnica fluxo-pressão), utilizando o sistema PERCI-SARS (versão 3.50 Microtronics Corp.). A significância da diferença entre as médias dos quatro grupos etários foi verificada por meio do teste Kruskal-Wallis, para amostras não pareadas. A correlação entre ANF e IMC, e ANF e CC, em cada grupo estudado, foi verificada por meio do coeficiente de correlação de Spearman. Foram aceitos como significantes os valores de p<0,05. Resultados: Todos os indivíduos apresentaram IMC indicativo de peso normal, exceto um único adulto jovem que apresentou índice de sobrepeso. A CC média de mulheres e homens adultos (G3 e G4) apresentou-se dentro da normalidade, sugerindo ausência de risco para obesidade. Os valores médios±DP da ANF foram de 1,025±0,054cm2, 1,055±0,081cm2, 1,050±0,083cm2 e 1,054±0,081cm2, respectivamente, para G1, G2, G3 e G4, não havendo diferença entre as 4 faixas etárias. Não houve correlação entre a ANF e o IMC e a ANF e a CC, em nenhum dos grupos estudados. Conclusão: Os valores normativos de ANF foram determinados para indivíduos sem anomalias craniofaciais de diferentes faixas etarias e servirão como referência em estudos envolvendo obstrução nasofaríngea. / Objective: To establish normative values of minimum cross-sectional nasopharyngeal area in individuals without craniofacial anomalies at different age ranges. Method: Ninety-six individuals of both genders, without craniofacial anomalies were evaluated. Participants were divided into 4 age groups: children, aged 6 to 10 years (G1); adolescents, aged 11 to 17 years (G2); young adults, 18 to 39 years (G3) and middle-aged adults, 40 to 59 years (G4). The body mass index (BMI) was calculated based on weight and height, and neck circumference (NC) was measured with a pediatric measuring tape. Minimum cross-sectional nasopharyngeal area (nasopharyngeal area NPA) was assessed by means of modified anterior rhinomanometry (pressure-flow technique) using a PERCI-SARS system (version 3.50 Microtronics Corp.). The difference between the mean values of the 4 age groups were verified by the Kruskal-Wallis test, for unpaired samples. Correlations between NPA and BMI, and NPA and NC were verified for each group by the Spearmans correlation coefficient. Differences were analyzed at a significance level of 5%. Results: All individuals presented normal BMI, except one young adult, which presented BMI suggestive of overweight. Mean NC values from adult women and men (G3 and G4) were within the normal range, suggesting no risk for obesity. Mean±SD values of NPA were 1.025±0.054cm2, 1.055±0.081cm2, 1.050±0.083cm2 and 1.054±0.081cm2 respectively, for groups G1, G2, G3 and G4, showing that there were no differences between the four age ranges. There was no correlation between NPA and BMI, and NPA and NC, in none of the studied groups. Conclusion: Normative data of NPA were established for individuals without craniofacial anomalies from different age ranges and may be used as reference values for future studies concerning nasopharyngeal obstruction.