Advanced-stage nasopharyngeal carcinoma (NPC) presents very difficult scenarios for radiation therapy (RT) planning. The infiltration of tumor to the skull base and beyond means that the tumor is very close to critical normal organs (organs at risk, OARs). Despite the advent of intensity-modulated radiotherapy (IMRT) treatment technique---the state-of-art RT technique, conflicting requirements between organ protection and target dose conformity is still problematic. The objectives of the present research are (1) to investigate the dosimetry properties of IMRT treatment in advanced-stage NPC in respect of its dosimetric limitations and planning problems, (2) to develop new methods and tools to resolve such problems, in particular to improve the quality of treatment plans and efficiency of the dose planning and optimization process. A series of four inter-linked studies were conducted to address these issues. / In conclusion, the solutions to several major problems in IMRT planning for advanced-stage NPC were investigated and established. It has been demonstrated in this research that, by applying these methods and tools, significant improvement in the dosimetry and efficiency of IMRT treatment planning can be accomplished as compared with conventional IMRT planning techniques. It is expected that such would translate into an improvement in treatment throughput, better tumor control and reduction in normal tissues complications. The methods developed have potential to be applied to all stages of NPC and to other tumor sites. / The first study was to improve the efficacy in target coverage and organs sparing using an "organ-splitting" approach. The OARs which overlapped with targets were split into target-overlapping and non-overlapping segments and each segment was assigned with different constraints parameters to increase the degree of flexibility during optimization. As a result, a steep gradient in the dose distribution at the regions of interface between the targets and normal critical organs could be achieved and treatment quality was improved. In the second study, a thorough dosimetric comparison between conventional 2-dimensional (2D) RT and IMRT plans was conducted to determine, with reference to outcome of 2D treatments, the extended tolerance dose limits for the critical organs, especially that of the brainstem and spinal cord, and their planning organ at risk volume. Such data could then serve as reference in IMRT planning when the dose of critical organs need be exceeded in order to allow adequate dose to a very close by target. In the third study, the feasibility of using interpolated contours for segmentation of targets and OARs in IMRT planning was investigated. The result indicated that the use of interpolated contours in IMRT planning could significantly reduce the contouring time by about 50% without degrading the target coverage and OARS sparing. In the final study, an array of dose constraint templates that could accommodate different degrees of overlap between the targets and OARs, together with a template selection program, were developed to improve the efficiency of IMRT planning. By applying the methods and tools developed, IMRT treatment planning of advanced NPC could become more efficient and less dependent on planner's experience. / Chau, Ming Chun. / Adviser: Anthony Chan Tak Cheung. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0948. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 118-128). / 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. / Abstract also in Chinese. / School code: 1307.

A comprehensive immunohistochemical study was carried out to investigate the phenotypes and prevalence of intraepithelial lymphocytes in NPC samples semi-quantitatively. CD25+/FOXP3+ T-cells were highly prevalent in primary NPCs, suggesting the presence of the immunosuppressive Tregs in tumor microenvironments. The low abundance of CD4+ T-cells, and the positive correlation between FOXP3 and CD8 staining in NPC samples imply that CD8+FOXP3+ Tregs may be present and play role in the suppression of anti-tumor immune response in NPC patients. The involvement of chemokine in the migration of tumor-infiltrating lymphocytes was studied. Chemokine ligand 20 (CCL20) was overexpressed in all EBV-positive NPC cell lines and xenografts compared to EBV-negative NPC, and immortalized normal nasopharynx epithelial cell lines. The presence of CCL20 was also found in primary tumors but not in normal epithelium. Furthermore, the ability of LMP1 to upregulate CCL20 expression in epithelial cells indicates that EBV may induce the production of chemokine involved in lymphocyte migration. / Epstein-Barr virus (EBV) is invariably associated with the development of nasopharyngeal carcinoma (NPC). Although the association of EBV and cancer has been reported for about four decades, it is still not clear how EBV latent infection contributes to the transformation of nasopharyngeal epithelial cells. The aims of this study are to identify EBV-regulated cellular genes and pathways and to determine the potential role of EBV in the modulation of anti-tumor immune responses in NPC. / In summary, EBV plays critical roles in the development of NPC by regulation of multiple cellular genes and pathways such as the Notch signaling cascade, and modulation of anti-tumor immune responses through the induction of chemokine important in migration of immune cells. / Notch signaling pathway functions in diverse cellular processes such as proliferation, apoptosis, adhesion, and epithelial to mesenchymal transition. In the current study, aberrant expression of activated Notch1 receptor (NICD), Notch ligand (Jagged1), negative regulator of Notch ( NUMB) and Notch downstream effector (HEY1) was detected in NPC cell lines and xenografts. Overexpression of NICD, Jagged1 and HEY1 proteins was also commonly found in primary tumors of NPC. / Transfection of Jagged1 to normal nasopharynx epithelial cells resulted in increased cell proliferation. Moreover, EBERs, which is abundantly expressed in EBV-positive NPC tumors, was capable of inducing the expression of Jagged1 in epithelial cells. The current data shows that Notch signaling pathway is aberrantly activated by the deregulated expression of multiple Notch components in NPC. The induction of Jagged1 by EBERs also implies the potential role of EBV in the activation of Notch signaling cascade in NPC. / Using high-density oligonucleotide microarray, expression profiles of EBV-infected NPC cell lines, HK1+EBV and HONE1+EBV, and their uninfected counterparts, HK1 and HONE, were generated. From the microarray results, six EBV-upregulated (JDP2, IL8, ATP6V0E2L, PLAP, PIK3C2B and AKR1B10 ) and three EBV-downregulated genes (BACE2, PADI3 and MMP1) were identified in both HK1 and HONE1 cells upon EBV latent infection. One hundred and thirty-eight and seventy-six genes were also found to be differentially modulated by EBV in HK1 and HONE1 cells, respectively. This study shows that cellular genes involved in wide range of biological processes and cellular functions are differentially regulated by EBV, which suggest that EBV modulates multiple pathways and processes during NPC tumorigenesis. / Hui, Wai Ying. / Adviser: Kw Lo. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0806. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 166-204). / 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. / Abstract also in Chinese. / School code: 1307.

Objetivo: Analisar as áreas seccionais, distâncias e volumes de segmentos específicos da cavidade nasal de crianças com fissura de lábio e palato unilateral, comparativamente a crianças sem fissura, por rinometria acústica. Modelo: Estudo prospectivo. Local de Execução: Laboratório de Fisiologia, HRAC-USP. Participantes: Trinta e nove crianças com fissura transforame unilateral e 34 crianças sem fissura, de ambos os sexos, com idade de 6 a 9 anos. Intervenções/Variáveis: Foram avaliadas as áreas seccionais correspondentes ao 2º, 3º e 4º entalhes do rinograma (AST1, AST2 e AST3), suas respectivas distâncias (d1, d2, d3) e os volumes nos segmentos correspondentes a 1-3,2cm (V1), 3,3-6,4cm (V2) e 7-12cm (V3), relativamente à narina, antes e após a aplicação tópica de vasoconstritor nasal (VC), utilizando um Rinômetro Acústico Eccovision-Hood Laboratories. Resultados: No lado fissurado (LF) as médias (±DP) de AST1, AST2, AST3 (em cm2), d1, d2 e d3 (em cm) e V1, V2 e V3 (em cm3) foram: 0,17±0,12, 0,29±0,20, 0,40±0,28, 2,02±0,40, 3,74±0,51, 5,50±0,44, 0,54±0,30, 1,22±0,86 e 4,52±3,37, respectivamente. No lado não-fissurado (LNF) as médias corresponderam a 0,33±0,11, 0,65±0,28, 0,90±0,43, 1,69±0,48, 3,67±0,53, 5,60±0,70, 1,04±0,37, 2,57±1,20, 8,77±4,85, respectivamente. As médias de AST e V em LF foram estatisticamente menores e a média de d1 foi maior que as observadas em LNF e no grupo controle, antes e após o uso do VC (p<0,05). A vasoconstrição provocou aumento dos valores de AST e V em ambos os grupos. Conclusão: Os resultados obtidos mostram significativo comprometimento da permeabilidade nasal em crianças com fissura labiopalatina unilateral. / Objective: To evaluate cross-sectional areas, distances and volumes of specific segments of the nasal cavity of children with unilateral cleft lip and palate, compared to children without cleft, by acoustic rhinometry. Design: Prospective analysis. Setting: Laboratory of Physiology, HRAC-USP. Participants: Thirty-nine children with unilateral cleft lip and palate and 34 controls without cleft, of both genders, aged 6 to 9 years. Interventions/Variables: Cross-sectional areas measured at the 2nd, 3rd and 4th notches in the rhinogram (CSA1, CSA2 and CSA3), their distances from the nostril (d1, d2 and d3) and volumes measured at 1-3.2cm (V1), 3.3-6.4cm (V2) and 7-12cm (V3) from the nostril were evaluated by means of an Eccovision Acoustic Rhinometer-Hood Laboratories, before and after nasal decongestion. Results: At the cleft side (CS), the mean values (±SD) of CSA1, CSA2, CSA3 (in cm2), d1, d2 and d3 (in cm) and V1, V2 e V3 (in cm3) were: 0.17±0.12, 0.29±0.20, 0.40±0.28, 2.02±0.40, 3.74±0.51, 5.50±0.44, 0.54±0.30, 1.22±0.86 and 4.52±3.37, respectively. At the non-cleft side (NCS) mean values corresponded to 0.33±0.11, 0.65±0.28, 0.90±0.43, 1.69±0.48, 3.67±0.53, 5.60±0.70, 1.04±0.37, 2.57±1.20, and 8.77±4.85, respectively. CSA and V means at CS were significantly lower and d1 mean was higher than NCS and control means, before and after decongestion (p<0.05). Increased CSA and V mean values were seen after decongestion in both groups. Conclusion: The results show a significant impairment of nasal patency in children with unilateral cleft lip and palate.

As alterações volumétricas da cavidade oral, nasal e espaço faríngeo após a cirurgia ortognática, têm sido objetivo de muitos estudos, uma vez que, essas alterações dependem da direção e magnitude da movimentação dos segmentos ósseos, porém, poucos são os trabalhos que combinam os diferentes métodos para a avaliação da nasofaringe. O objetivo deste estudo foi correlacionar o espaço aéreo faríngeo em pacientes com fissura labiopalatina, submetidos à cirurgia ortognática com avanço de maxila e/ou recuo de mandíbula, por meio da análise do volume e área seccional mínima utilizando imagens de tomografia computadorizada de feixe cônico e pela rinomanometria anterior modificada (técnica fluxo-pressão) no pré e pós-operatório de um ano da cirurgia ortognática. Desse modo, a amostra foi composta por 41 indivíduos, que foram avaliados no pré-operatório e no pós-operatório, na qual, avaliou-se a área seccional mínima pela rinomanometria anterior modificada expressos em mm2 e pelas imagens de tomografia de feixe cônico, que foram importadas em DICOM e avaliadas pelo software Dolphin Imaging 11.0, obtendo os valores numéricos de volume (V), expressos em cm3, bem como a área seccional mínima, expressa em mm2. Notou-se que, em todas as variáveis, houve aumento médio dos valores no pós-operatório em relação ao pré-operatório. Além disso, notouse uma diferença estatisticamente significante ao comparar os resultados dos volumes e da área seccional mínima da área nasofaríngea no pré e pós-operatório pelo software Dolphin Imaging 11.0 ao aplicar o Teste de Wilcoxon. Observou-se também, o aumento discreto da área nasofaríngea avaliada pela rinomanometria, no préoperatório de 105,9 mm2 para 107,1 mm2 no pós-operatório, mas sem diferença estatisticamente significante com p=0,493 pelo Teste de Wilcoxon. E ao comparar a ASM pela TCFC (ASMD) e pela rinomanometria (notou-se diferença estatística (p= 0,033) pelo Teste de Wilcoxon. Por conseguinte, concluiu-se que, existe diferença estatisticamente significante entre a área seccional mínima obtida da TCFC com a rinomanometria pela técnica de fluxo-pressão. / The volumetric changes of the oral cavity, nasal cavity and pharyngeal space after orthognathic surgery have been the objective of many studies, since these alterations depend on the direction and magnitude of the movement of the bone segments, however, there are few studies that combine the different methods for evaluating the nasopharynx. The objective of this study was to correlate the pharyngeal air space in patients with cleft lip and palate submitted to orthognathic surgery with maxillary advancement and / or mandible retreatment, by means of volume analysis and minimum sectional area using conical beam computed tomography by modified anterior rhinomanometry (flow-pressure technique) in the pre- and postoperative year of orthognathic surgery. Thus, the sample consisted in 41 individuals, which were evaluated preoperatively and postoperatively, in which the minimum sectional area was determined by modified anterior rhinomanometry expressed in mm2 and by conical beam tomography images, which were imported into DICOM and evaluated by Dolphin Imaging 11.0 software, obtaining the numerical values of volume (V), expressed in cm3, as well as the minimum sectional area, expressed in mm2. It was observed that, in all variables, there was an average increase in postoperative values in relation to the preoperative period. In addition, a statistically significant difference was observed when comparing the results of the volumes and the minimum sectional area of the nasopharyngeal area in the pre and postoperative period by the Dolphin Imaging 11.0 software when applying the Wilcoxon Test. It was also observed a discrete increase in the nasopharyngeal area evaluated by rhinomanometry, in the preoperative period from 105.9 mm2 to 107.1 mm2 postoperatively, but without a statistically significant difference with p = 0.493 by the Wilcoxon test. When comparing ASM by CBCT (ASMD) and rhinomanometry (a statistical difference (p = 0.033) was noted by the Wilcoxon test. Therefore, it was concluded that there is a statistically significant difference between the minimum sectional area obtained from the CBCT with rhinomanometry.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Medical Sciences

Medical imaging informatics is one of the important research areas in radiology that studies how information available on medical images is retrieved, analyzed, and enhanced. Recent development in medical imaging informatics has resulted in improvement of diagnostic accuracy based on imaging examinations, as well as efficiency in clinical workflow. Computer aided diagnosis (CAD) and electronic patient record system (ePR) are both topics in medical imaging informatics that have matured from research concepts into commercially available computerized systems in clinical environment. The current challenges are to further broaden their scope of applications. In this thesis project, I developed a CAD system for interpreting PET/CT examinations and an ePR system for patient data integration in neurosurgery suites. Specifically, the CAD system in this project was designed to automatically diagnose nasopharyngeal carcinoma (NPC) on Positron emission tomography/computed tomography (PET/CT) examinations, which aimed to detect and classify both the primary NPC and its nodal metastasis. The regions of interests (ROIs) were segmented from the PET images and registered onto the CT in order to combine the imaging features from both modalities and the a priori anatomical knowledge of the suspicious lesion. These combined features were then classified by a support vector machine (SVM) to generate the final diagnosis result. The system was validated with 25 PET/CT examinations from 10 patients suffering from NPC, and the result produced by the system was compared to the gold standard of lesions manually contoured by experienced radiologists. The results confirmed that the system successfully distinguished all 53 genuine lesions from the mimickers due to normal physiological uptake and artifacts that also produced potentially confusing signals. The second part of the project involved development of an electronic patient record system (ePR) that integrated all the myriad of images and different types of clinical information before, during, and after neurosurgery operations, in order to enhance efficiency of work flow in this unique clinical environment. The system comprises of pre-, intra-, and post-operation modules which correspond to the different stages of the neurosurgery. The pre-op module was developed to store and categorize all images and data before the procedure to assist the surgeons in planning operation. The intra-op module integrates all the input signals, waveforms, images and videos that are produced by different imaging and physiological monitoring devices in the operation room during the surgery, and displays all the relevant information in a single large screen in real time to ease monitoring of the procedure. The post-op module helps surgeons to review all the data acquired from all the prior stages for follow-up of the treatment outcome. One-tumor case was utilized to test the pre-op module, and the signals and waveforms simulators were used to evaluate the performance of the intra-op module. In summary, two different medical informatics systems, a CAD and an ePR system were developed. Both showed promising results in laboratory tests. Future work would involve performance enhancement and feedback of the systems, and ultimately evaluation of these systems in the clinical environment. / published_or_final_version / Diagnostic Radiology / Master / Master of Philosophy

Epstein-Barr virus (EBV) is one of the most successful human pathogens in the world and establishes a lifelong persistent infection in 95% of adult population worldwide. It is associated with a number of malignancies including Burkett’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma(NPC) and gastric carcinoma. EBV was the first virus reported to produce microRNAs (miRNAs) and it encodes 44 mature miRNAs from 2 viral transcripts, BART and BHRF1. The BART transcript is abundantly expressed in all latently infected cells, particularly in epithelial cells. The BART miRNAs (miR-BARTs) were shown to be involved in apoptosis inhibition, immune evasion, metastasis, viral and cellular transcripts regulation. The high expression profile and the diverse functions of miR-BARTs suggest that they may play a critical role in the development of EBV-associated NPC. In order to understand the importance of miR-BARTs in NPC development, in this thesis, I conducted a study on the miR-BARTs function in nasopharyngeal carcinogenesis. In the first part, I characterized the cellular target and function of an abundantly expressed miR-BART in NPC. In the second part, I established a novel recombinant EBV construction system for genetic studies of miR-BARTs in nasopharyngeal epithelial (NP) cells. In the first part of my study, I characterized the cellular target and function of miR-BART3* in NPC. As predicted by bioinformatics, tumor suppressor protein DICE1 was a cellular target of miR-BART3*. The specific targeting between miR-BART3* and DICE1 3’UTR was validated by luciferase assays and the downregulation of both endogenous DICE1 protein and mRNA was observed in EBV+epithelial cells and miR-BART3* expressing cells. In addition, restoration of DICE1 protein expression by inhibition of miR-BART3* was also demonstrated in EBV+epithelial cells. Moreover, miR-BART3* was shown to promote cell proliferation via suppression of DICE1. Analysis of22 human nasopharyngeal(NP)biopsy samples demonstrated the inverse correlation between miR-BART3* and DICE1 expression. Taken together, miR-BART3* downregulates the tumor suppressor DICE1 protein to promote cell proliferation and transformation in NPC. Besides the candidate approach, genetic studies can provide a systematic view of the functions of all miR-BARTsand shed light on the importance of miR-BARTs in NPC under a more physiological condition. At present, bacterial artificial chromosome (BAC) technology is commonly used for recombinant EBV construction. However, the intrinsic disadvantages of BAC prevent its use in NP epithelial cells. Therefore in the second part of my study, I established a novel CRISPR/Cas9-mediated recombinant EBV construction system and constructed a miR-BART deleted recombinant EBV. The CRISPR/Cas9 system was demonstrated to be effective in EBV genome editing and Akata cells were infected by the recovered recombinant mutant virus. Infected Akata cells served as the source for NP cell infection through co-culture. The new CRISPR/Cas9 system have many advantages over the conventional EBV BAC method. My work reported in this thesis not only illustrated the importance of miR-BARTs in NPC, but also provide a new technology platform for further study of miR-BARTs in NP epithelial cells. (An / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The etiology of nasopharyngeal carcinoma (NPC) is based on intricate interactions among environmental factors, genetic susceptibility and Epstein-Barr virus (EBV) infection. Information concerning the role of EBV infection, particularly during the early stage of NPC development is poorly understood. Our laboratory has shown that stable infection of EBV could be achieved in immortalized epithelial cell lines which harbor genetic alterations and altered cell signaling pathway. In this study, these cell models were used to elucidate early events involved in EBV infection in premalignant nasopharyngeal epithelial cell models and their implications on development and progression of nasopharyngeal carcinoma. The response of EBV-infected cells to a stromal inflammatory cytokine, interleukin-6 (IL-6), was examined. EBV infection and long-term propagation of EBV-infected nasopharyngeal epithelial cells confer enhanced sensitivity to STAT3 activation induced by IL-6. IL-6-induced STAT3 activation reinforced their malignant properties in nasopharyngeal epithelial cells and may play a role in the development of nasopharyngeal carcinoma. Furthermore, constitutive STAT3 activation was demonstrated to facilitate malignant transformation of EBV-infected premalignant nasopharyngeal epithelial cells to cancer cells, suggesting that EBV infection and STAT3 activation might synergistically promote the development of NPC. This study also provides support for the existence of a positive feedback loop of IL-6/STAT3/LMP in NP460hTert-EBV cells, which enhanced STAT3 activation in EBV-infected cells. Elevated levels of IL-6Rα expression were observed in EBV-infected NP460hTert cells compared with uninfected cells and were largely responsible for the enhanced sensitivity of IL-6-induced STAT3 activation in these cells. High expression level of IL-6Rα could amplify IL-6 signaling in nasopharyngeal epithelial cells to promote growth proliferation in NP460hTert cells and increase the growth rate of xenografted NPC cells in immune-suppressed animals, suggesting that IL-6Rα overexpression may play a role of contributing to the development of nasopharyngeal carcinoma. The serum concentrations of both IL-6 and sIL-6R were also higher in NPC patients than healthy individuals and may have prognostic values to predict clinical outcome and disease progression in NPC patients. In conclusion, these data support the hypothesis that EBV infection under inflammatory environment may activate aberrant gene expressions and cell signaling to facilitate malignant transformation. The inflammatory cytokine, IL-6, may mediate the role of EBV infection in the development of NPC. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy