abstract: Biomarkers find a wide variety of applications in oncology from risk assessment to diagnosis and predicting and monitoring recurrence and response to therapy. Developing clinically useful biomarkers for cancer is faced with several challenges, including cancer heterogeneity and factors related to assay development and biomarker performance. Circulating biomarkers offer a rapid, cost-effective, and minimally-invasive window to disease and are ideal for population-based screening. Circulating immune biomarkers are stable, measurable, and can betray the underlying antigen when present below detection levels or even no longer present. This dissertation aims to investigate potential circulating immune biomarkers with applications in cancer detection and novel therapies. Over 600,000 cancers each year are attributed to the human papillomavirus (HPV), including cervical, anogenital and oropharyngeal cancers. A key challenge in understanding HPV immunobiology and developing immune biomarkers is the diversity of HPV types and the need for multiplexed display of HPV antigens. In Project 1, nucleic acid programmable protein arrays displaying the proteomes of 12 HPV types were developed and used for serum immunoprofiling of women with cervical lesions or invasive cervical cancer. These arrays provide a valuable high-throughput tool for measuring the breadth, specificity, heterogeneity, and cross-reactivity of the serologic response to HPV. Project 2 investigates potential biomarkers of immunity to the bacterial CRISPR/Cas9 system that is currently in clinical trials for cancer. Pre-existing B cell and T cell immune responses to Cas9 were detected in humans and Cas9 was modified to eliminate immunodominant epitopes while preserving its function and specificity. This dissertation broadens our understanding of the immunobiology of cervical cancer and provides insights into the immune profiles that could serve as biomarkers of various applications in cancer. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2018

Luk, Chun Shui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 174-189). / Abstract also in Chinese. / Declaration --- p.I / Acknowledgements --- p.II / Funding Support --- p.IV / Abstract of thesis entitled --- p.V / 論文摘要 --- p.VII / Abbreviations --- p.IX / Table of Contents --- p.XIII / List of Figures --- p.XVIII / List of Tables --- p.XX / List of Appendix --- p.XXI / Chapter Chapter One - --- Literature Review --- p.1 / Chapter 1.1 --- History of Knowledge on Human Papillomavirus --- p.1 / Chapter 1.2 --- Virology of Human Papillomavirus --- p.2 / Chapter 1.2.1 --- Taxonomic Classification of Human Papillomavirus --- p.2 / Chapter 1.2.2 --- Morphology of Human Papillomavirus --- p.3 / Chapter 1.2.3 --- The Viral Genome --- p.3 / Chapter 1.2.4 --- The Viral Gene Products --- p.5 / Chapter 1.2.4.1 --- E1 and E2 Proteins --- p.5 / Chapter 1.2.4.2 --- E4 Protein --- p.6 / Chapter 1.2.4.3 --- "E5，E6, E7 Proteins" --- p.7 / Chapter 1.2.4.4 --- L1 and L2 Proteins --- p.8 / Chapter 1.3 --- Evolution of Human Papillomavirus --- p.9 / Chapter 1.3.1 --- Rates of Evolution --- p.11 / Chapter 1.3.2 --- Co-evolution Between Human Papillomavirus and Human --- p.11 / Chapter 1.4 --- Human Papillomavirus Infection and Disease --- p.13 / Chapter 1.4.1 --- Human Papillomavirus and Cervical Cancer --- p.13 / Chapter 1.4.1.1 --- Disease Burden of Cervical Cancer --- p.13 / Chapter 1.4.1.2 --- Epidemiology of Cervical Cancer --- p.14 / Chapter 1.4.1.3 --- Distribution of HPV types in Cervical Precancerous Lesions --- p.14 / Chapter 1.4.2 --- Human Papillomavirus and Non-cervical Diseases --- p.15 / Chapter 1.5 --- Human Papillomavirus Type 58 --- p.15 / Chapter 1.5.1 --- Biology of Human Papillomavirus Type 58 --- p.15 / Chapter 1.5.2 --- Epidemiology of Human Papillomavirus Type 58 Infections --- p.16 / Chapter Chapter Two - --- Background and Objectives of Study --- p.17 / Chapter 2.1 --- Background of study --- p.17 / Chapter 2.1.1 --- The Need for Research on HPV58 --- p.17 / Chapter 2.1.2 --- Intratypic Classification System for HPV --- p.17 / Chapter 2.2 --- Implication and Impact of Study --- p.19 / Chapter 2.2.1 --- Implication on HPV Virology --- p.19 / Chapter 2.2.2 --- HPV58 Classification --- p.19 / Chapter 2.2.3 --- Improvement on in the Detection of HPV58 --- p.20 / Chapter 2.2.4 --- Implication on Vaccine Development --- p.20 / Chapter 2.3 --- Objectives of Study --- p.21 / Chapter 2.3.1 --- To Generate a Database for Intratypic Variation of Different Gene Regions of HPV58 --- p.21 / Chapter 2.3.2 --- To Study the Variability of Seven Gene Regions of HPV58 --- p.21 / Chapter 2.3.3 --- To Study the Geographical Distribution of HPV58 Variants --- p.22 / Chapter 2.3.4 --- To Study the Phylogeny of HPV58 --- p.22 / Chapter 2.3.5 --- To Develop an Intratypic Classification System for HPV58 --- p.22 / Chapter 2.3.6 --- To Predict the Effectiveness of Commonly Used Primers on the Detection of HPV58 --- p.22 / Chapter Chapter Three - --- Materials and Methods --- p.24 / Chapter 3.1 --- Overall Study Design --- p.24 / Chapter 3.2 --- Study Population --- p.25 / Chapter 3.3 --- Sample Processing and Storage --- p.25 / Chapter 3.4 --- Primer Design --- p.26 / Chapter 3.5 --- Specimen Quality Assessment and Sample Selection --- p.30 / Chapter 3.6 --- Amplification of Gene Region --- p.30 / Chapter 3.7 --- Agarose Gel Electrophoresis --- p.34 / Chapter 3.8 --- Sequencing Reaction --- p.34 / Chapter 3.8.1 --- Purification of PCR Product --- p.34 / Chapter 3.8.2 --- Sequencing Reaction --- p.35 / Chapter 3.8.3 --- Purification of Fluorescence-labelled Product --- p.35 / Chapter 3.8.4 --- Sequence Identification --- p.35 / Chapter 3.9 --- Sequence Analysis --- p.36 / Chapter 3.9.1 --- Sequence Editing --- p.36 / Chapter 3.9.2 --- Criteria for Confirming the identity of HPV58 --- p.36 / Chapter 3.9.3 --- Identification of Variants --- p.38 / Chapter 3.9.4 --- Identification of Conserved and Variable Regions --- p.39 / Chapter 3.9.5 --- Phylogenetic Analysis --- p.40 / Chapter 3.9.5.1 --- Construction of Maximum Likelihood Tree --- p.40 / Chapter 3.9.5.2 --- Bootstrap Analysis --- p.41 / Chapter 3.9.5.3 --- Bayesian Phylogenetic Analysis --- p.42 / Chapter 3.9.5.4 --- Non-synonymous to Synonymous Substitution Rate Ratio (dN/dS) --- p.42 / Chapter 3.9.6 --- Evaluation of Performance of Commonly Used Primers --- p.43 / Chapter Chapter Four - --- Results --- p.44 / Chapter 4.1 --- Specimen Quality Assessment and HPV58 Confirmation --- p.44 / Chapter 4.2 --- HPV58 Genome Variability --- p.44 / Chapter 4.2.1 --- E6 Open Reading Frame --- p.45 / Chapter 4.2.2 --- E7 Open Reading Frame --- p.51 / Chapter 4.2.3 --- E2 Open Reading Frame --- p.56 / Chapter 4.2.4 --- E4 Open Reading Frame --- p.61 / Chapter 4.2.5 --- E5 Open Reading Frame --- p.66 / Chapter 4.2.6 --- L1 Open Reading Frame --- p.71 / Chapter 4.2.7 --- Long Control Region --- p.88 / Chapter 4.2.8 --- Whole HPV genome --- p.94 / Chapter 4.3 --- Evaluation of Commonly Used Primers --- p.99 / Chapter 4.3.1 --- PGMY09/11 Primers --- p.99 / Chapter 4.3.2 --- MY09/11 Primers --- p.99 / Chapter 4.3.3 --- GP5+/6+ Primers --- p.100 / Chapter 4.3.4 --- SPF Primers --- p.100 / Chapter 4.3.5 --- L1F/L1R Primers --- p.101 / Chapter Chapter Five - --- Discussion --- p.111 / Chapter 5.1 --- Overall Variation of HPV58 Genome --- p.111 / Chapter 5.2 --- Variability of Each Gene Region --- p.114 / Chapter 5.2.1 --- E6 Open Reading Frame --- p.115 / Chapter 5.2.2 --- E7 Open Reading Frame --- p.116 / Chapter 5.2.3 --- E2 Open Reading Frame --- p.117 / Chapter 5.2.4 --- E4 Open Reading Frame --- p.118 / Chapter 5.2.5 --- E5 Open Reading Frame --- p.119 / Chapter 5.2.6 --- L1 Open Reading Frame --- p.120 / Chapter 5.2.7 --- Long Control Region --- p.121 / Chapter 5.3 --- Phylogenetics of HPV58 --- p.122 / Chapter 5.3.1 --- Natural Selection Pressure --- p.122 / Chapter 5.3.2 --- HPV58 Lineage Using the L1 Gene --- p.124 / Chapter 5.3.3 --- Methods for Lineage Identification --- p.125 / Chapter 5.3.4 --- Geographical Distribution of the Four Lineages --- p.126 / Chapter 5.3.5 --- Recombination --- p.127 / Chapter 5.4 --- Evaluation of Commonly Used Primers --- p.128 / Chapter 5.5 --- Limitations of the Current Study --- p.129 / Chapter 5.6 --- Future Studies --- p.130 / Appendix --- p.133 / References --- p.174

O câncer de colo do útero é o segundo tumor mais frequente entre as mulheres brasileiras, os tratamentos disponíveis trazem complicações de curto a longo prazo e estudos que avaliam a qualidade de vida destas mulheres ainda são escassos no Brasil. Isto é justificado pela falta de instrumentos específicos e validados para esta finalidade em língua portuguesa (Brasil). Neste estudo objetivou-se testar as propriedades psicométricas dos questionários EORTC QLQ-CX24 e FACT-CX em uma amostra de mulheres brasileiras com câncer do colo do útero. O estudo é metodológico e foi realizado com 100 mulheres tratadas no Hospital de Câncer de Barretos, por meio de três avaliações (no início do estudo, 15 dias e 18 a 24 meses após o início). Foi testada a consistência interna (alfa de Cronbach), reprodutibilidade (coeficiente de correlação intraclasse), validade convergente (correlação com o questionário SF-36 e com o questionário EORTC QLQ-C30), a validade discriminante pelo estadiamento, tipo de tratamento e autoclassificação de saúde e a responsividade pelo método de distribuição dos dados, além da análise da facilidade de compreensão das questões e dos questionários. Confirmou-se a confiabilidade do questionário EORTC QLQ-CX24, a reprodutibilidade foi baixa nas escalas de preocupação sexual, atividade sexual e prazer sexual, bons resultados observou-se ao avaliar a validade convergente e discriminante. A análise da responsividade não confirmou todas as hipóteses. Ao avaliar o questionário FACT-CX, por meio da consistência interna, exceto pela escala de bem-estar emocional, as demais apresentaram valores de Alpha de Cronbach superiores a 0,70. A reprodutibilidade foi satisfatória nas cinco escalas que compõem o FACT- CX. Na análise da validade convergente, somente as escalas de bem-estar físico e bem-estar emocional mostrou boa correlação com as escalas do SF-36. Em geral, o questionário revelou boa validade discriminante, principalmente em relação a autoclassificação da saúde. Resultado satisfatório encontrou-se na análise da responsividade. Em geral observou-se um bom grau de entendimento das questões e dos questionário. Concluindo os questionários desenvolvidos para medir a QVRS em mulheres com câncer de colo do útero EORTC QLQ-CX24 e FACT-CX obtiveram resultados aceitáveis recomendando-se que outros estudos testem a responsividade por outros métodos / Cervical cancer is the second most common tumor among Brazilian women. Available treatments bring short and long-term complications, and studies assessing the quality of life of these women are still rare in Brazil. Such situation is warranted by the lack of specific and validated tools in (Brazilian) Portuguese for such a purpose. The goal of this study was to test the psychometric properties of EORTC QLQ-CX24 and FACT-CX questionnaires in a sample of Brazilian women affected by cervical cancer. This is a methodological study and it was conducted on 100 women treated at the Barretos Cancer Hospital by way of three assessments (at baseline, 15 days and 18 to 24 months after the onset). We tested the internal consistency (Cronbach\'s alpha), reproducibility (intraclass correlation coefficient), convergent validity (correlation with the SF-36 questionnaire and the EORTC QLQ-C30 questionnaire), the discriminant validity by way of staging, type of treatment and health self-classification and responsiveness by the method of data distribution, as well as the analysis of how easy it was their understanding of questions and questionnaires. The reliability of the EORTC QLQ-CX24 questionnaire was confirmed, reproducibility was low in the scales of sexual worry, sexual activity and sexual enjoyment, and good results were observed when evaluating the convergent and discriminant validity. The analysis of responsiveness did not confirm all hypotheses. Upon evaluation of the FACT-CX questionnaire through internal consistency, except for the scale of emotional well-being, the others showed Cronbach\'s alpha values above 0.70. Reproducibility was satisfactory in the five scales comprising FACT-CX. In convergent validity analysis, only the scales of physical well-being and emotional well-being showed good correlation with the scales of the SF-36. In general, the questionnaire demonstrated good discriminant validity, especially in relation to the self-ratings of health. Satisfactory result was found in the analysis of responsiveness. In general there was a good degree of understanding of questions and questionnaires. In conclusion, questionnaires developed to measure HRQOL in women with cervical cancer such as EORTC QLQ-CX24 and FACT-CX achieved acceptable results, and we do recommended that further studies are performed to assess responsiveness by other methods

Perception of cervical cancer screening among Immigrant African women residing in Houston, Texas Esther I. Orji M.Ed., University of Lagos, 2002 B. Ed, Abia State University, 1998 A.C. E. University of Benin, 1990 Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Public Health Walden University August 2016 Disparities in cervical cancer screening participation still exist especially among immigrant and minority women compared to non immigrant populations. Research investigators through multiple studies have explored reasons for non participation in cervical cancer screening among immigrant and minority women. However, there was limited research specifically on how immigrant women of African descent could be empowered to overcome barriers associated with cervical cancer screening in Houston Texas. The purpose of this study was to explore and understand the perceptions of factors which are barriers to perform cervical cancer screening tests among immigrant women of African descent, as well as their beliefs on preventive medicine, and how immigrant women could be empowered in order to overcome barriers associated with cervical cancer screening. The theoretical framework was based on the health belief model. The participants for the study were 20 immigrant women of African descent aged 21-65. A semi-structured interview schedule which comprised of open ended questions was used to collect data on the perspectives of immigrant women towards cervical cancer screening. Data was transcribed verbatim and analyzed thematically. The social change implications of the study can be that immigrant women could be more encouraged to seek access to the appropriate state of the art in cervical cancer screening which could contribute in reducing mortality, morbidity rate, and costs associated with cervical cancer.

Sexual minority women (SMW) demonstrate lower rates of cervical cancer screening than heterosexual women. This is concerning as lesbian and bisexual women tend to engage in higher rates of substance-related cancer risk behaviors, unprotected sex, and tend to have higher body mass indices, all of which are risk factors for cancer development. Another major risk factor, screening avoidance, places SMW at increased risk for the development of high grade cervical lesions in the absence of early detection practices, which is likely to impact overall cervical cancer morbidity in this population. The aim of the present study was to utilize the Health Equity Promotion Model in order to investigate the interplay of medical heterosexism, social and community, behavioral, biological, and social identity/position factors on cervical cancer screening rates in a sample of SMW from a large metro area in the southeastern United States. 145 women who identify as sexual minorities were recruited from local LGBT-friendly venues, events, community organizations, email LISTSERVs, and related social media accounts and were asked to complete a fifteen-minute survey. A series of bivariate correlation, t-test, and multivariate regression analyses were run. Findings from mediation analyses demonstrated that health communication factors mediated the relationship between perceived medical heterosexism and cervical cancer screening outcomes. Further, after accounting for demographic factors, greater provider communication quality, provider trust, eHealth literacy, and ever having an HIV test significantly and differentially predicted cervical cancer screening outcomes in the multivariate models (ps < .05). Findings suggest that health communication factors play an important role in facilitating cervical cancer screenings for SMW, and provider training interventions and policy that focus on reducing medical heterosexism may aid in improving patient-provider relationships in this population.

Cervical cancer, caused by oncogenic (high risk [hr]) human papillomavirus (HPV) subtypes, is the most common cancer in women in Guatemala and the most common cause of cancer mortality in women aged 15-44 years. Visual inspection with acetic acid (VIA) with onsite cryotherapy “test-and-treat” is recommended for underserved Guatemalan indigenous rural women. This research assessed: 1) hrHPV infection prevalence in women screened by VIA; 2) Sensitivity and specificity of VIA in detecting hrHPV infection and cytologically identified precancerous and cancerous lesions; and 3) Factors associated with hrHPV infection. Analysis of anonymous data collected during VIA clinics in 2013 (N = 205) and 2017 (N = 234) for indigenous women aged 21-65 years in six villages showed 22.6% (95% confidence interval [CI]=18.7%-27.2%) had hrHPV cervical infection. VIA results were abnormal in 5.9% (95%CI=3.8%-8.8%). Only nine VIA exams in 89 women with hrHPV were abnormal (Sensitivity=10.1%, 95%CI=4.7%-18.3%), although abnormal VIA was associated with hrHPV (Prevalence Ratio [PR])=1.8; 95%CI=1.1-3.1; P=.05). Of 221 women who had VIA, hrHPV nucleic acid testing and liquid preparation cytology (equivalent to Papanicolaou or “Pap”) testing, 10 (4.7% [95%CI=2.3%-8.5%]) had abnormal cytological results, including one cancer, four high- and five low-grade squamous intraepithelial lesions. VIA sensitivity and specificity for detection of precancerous cytological abnormalities and cancerous lesions were 20.0% (95%CI=2.5%-55.6%) and 96.0% (95%CI=92.3%-98.3%) respectively. In contrast, hrHPV sensitivity and specificity were 100% (95%CI=71.7%-100%) and 88.7% (95%CI: 83.9%-92.7%). In both years combined, women aged fewer than 29 years or reporting fewer than four pregnancies were more likely to have hrHPV cervical infection (36.8%, 27.3%, respectively) than those who were older or reported more pregnancies (18.7; P=.025, respectively); 60.0% reported some form of modern contraception. Progesterone injections or implant users were more likely to have hrHPV infection (31.9%) than women using other or no contraceptives (19.5%); PR=1.6; 95%CI=1.1-2.4; P=.01). These data suggest that VIA may not be sufficiently sensitive for use in cervical cancer screening. “Test-and-treat” screening using hrHPV real-time testing, as recommended by the World Health Organization may be preferable to VIA, and may be acceptable using self-collected specimens.

O câncer cervical é um dos tipos de câncer mais comuns entre as mulheres, e a infecção persistente pelos HPV-16 e HPV-18 é responsável por 70% dos casos. As vacinas profiláticas disponíveis possuem alta eficácia na prevenção da infecção pelos tipos mais prevalentes de HPV. No entanto, este tipo de abordagem não beneficia mulheres que já apresentam lesões precursoras ou tumores cervicais avançados, e a busca por abordagens terapêuticas para esse tipo de câncer é considerada uma necessidade. A qualidade do antígeno representa um aspecto fundamental para o sucesso de vacinas terapêuticas baseadas em proteínas recombinantes. Neste sentido, os sistemas de expressão em células eucarióticas, como leveduras e células de mamíferos são considerados adequados para a produção de proteínas com aplicação biotecnológica. O objetivo principal deste trabalho contemplou a expressão das proteínas de fusão gDE7E6 do HPV-16 e do HPV-18 e a oncoproteína E7 do HPV-16 em células da levedura Pichia pastoris e expressão da gDE7E6 do HPV-16 e do HPV-18 em células de mamífero HEK293T e CHODG-44 para obtenção de antígenos purificados com futura aplicação em vacinas terapêuticas contra tumores associados ao HPV-16 e HPV-18. Os genes que codificam as proteínas gDE7E6 dos HPV-16 e HPV-18 e da E7 do HPV-16 foram clonados no vetor pPIC9K, os quais foram linearizados por digestão enzimática e utilizados na transformação da P. pastoris. A expressão das proteínas foi analisada nos tempos de 24, 48, 72 e 96 horas, no entanto, não foi observada a produção das proteínas no sobrenadante e nem no lisado celular. Diante desta constatação, iniciamos a expressão das proteínas gDE7E6 do HPV-16 e gDE7E6 do HPV-18 em células de mamíferos HEK293T e CHODG-44. As sequências genéticas das proteínas gDE7E6 do HPV-16 e do HPV-18 foram clonadas no vetor de expressão pNU1 e analisadas por digestão enzimática. Análises de SDS-PAGE e western blot demonstraram a expressão das proteínas gDE7E6 do HPV-16 e do HPV-18 em até 96 horas em células HEK293T. Em paralelo, realizamos a transfecção estável dos plasmídeos contendo as sequencias da gDE7E6 do HPV-16 e gDE7E6 do HPV-18 em células CHO-DG44. Com o intuito de aumentar a expressão das proteínas de interesse na população mista de CHODG-44, realizamos amplificação genômica com metotrexato (MTX), sendo possível observar aumento da expressão das proteínas, conforme aumento gradativo nas concentrações de MTX. Posteriormente, foram feitas tentativas para isolar um clone produtor das proteínas gDE7E6 HPV-16 e HPV-18, através de clonagem por diluição limitante e sistema automatizado, sendo possível isolar um clone para cada construção através de matriz semisólida, confirmado por western blot e citometria de fluxo. Apesar de demonstrar a expressão das proteínas de interesse em sistema de expressão baseado em células de mamífero, o rendimento obtido após a purificação por afinidade ao níquel foi extremamente baixo, o que dificulta a obtenção dos antígenos para fins vacinais. / Cervical cancer is one of the most common cancers among women, and persistent infection with HPV-16 and HPV-18 accounts for 70% of the cases. Available prophylactic vaccines are highly effective in preventing infection by the most prevalent types of HPV. However, this type of approach does not benefit women who already have precursor lesions or advanced cervical tumors, and the search for therapeutic approaches to this type of cancer is considered a necessity. Antigen quality represents a key aspect for the success of therapeutic vaccines based on recombinant proteins. In this sense, expression systems based in eukaryotic cells such as yeast and mammalian cells are considered suitable for the production of proteins with biotechnological applications. The main objective of this work was to express the gDE7E6 fusion proteins HPV-16 and HPV-18 and the E7 oncoprotein HPV-16 in Pichia pastoris and expression of gDE7E6 HPV-16 and HPV-18 in mammalian cells HEK293T and CHODG-44 to obtain purified antigens with future applications in therapeutic vaccines against HPV-16 and HPV-18 associated tumors. The genes encoding the gDE7E6 proteins HPV-16 and HPV-18 and E7 HPV-16 were cloned into the pPIC9K vector, which were linearized by enzymatic digestion and used in the transformation of P. pastoris. Expression of the proteins was analyzed at 24, 48, 72 and 96 hours, however, the production of the proteins in the supernatant and in the cell lysate was not observed. In light of this finding, we initiated the expression of gDE7E6 proteins HPV-16 and HPV-18 in mammalian cells HEK293T and CHODG-44. The genetic sequences of gDE7E6 proteins HPV-16 and HPV-18 were cloned into the pNU1 expression vector and analyzed by enzymatic digestion. SDSPAGE and western blot analyzes demonstrated expression of gDE7E6 proteins HPV-16 and HPV-18 within 96 hours in HEK293T cells. In parallel, we performed stable transfection of plasmids containing gDE7E6 HPV-16 and HPV-18 sequences into CHODG44 cells. In order to increase the expression of the proteins in the mixed population of CHODG-44, we performed genomic amplification with methotrexate (MTX), and it was possible to observe an increase in protein expression, as a gradual increase in MTX concentrations. Therefore, attempts were made to isolate a clone producing gDE7E6 proteins HPV-16 and HPV-18 by limiting dilution and automated system, being possible to isolate one clone for each construct through a semisolid matrix, confirmed by western blot and flow cytometry. Despite observing protein expression in mammalian cell-based expression system, the yield obtained after nickel affinity purification was extremely low, which makes it difficult to obtain the antigens for vaccine purposes.

The research described in this thesis reviewed previous uses of Interactive Voice Response (IVR), developed appropriate software, and employed IVR to obtain self-report of sensitive issues in surveys and conduct brief public health interventions. Chapter 1 introduces IVR and describes a systematic critical review of the use of IVR. IVR is a telephone interviewing technique where the human speaker is replaced by a high quality recorded interactive script to which the respondent provides answers by pressing the keys of a touch-telephone (touchphone). IVR has numerous advantages, including: economy, autonomy, confidentiality, access to certain population groups, improved data quality, standardised interviewing, multi-lingual interfaces, and detailed longitudinal assessments. Despite this, there have been few survey applications of IVR compared to alternative methods such as Computer-Assisted Telephone Interview (CATI). There has not been any evaluation of the use of IVR for asking sensitive questions in surveys or as a tool for health promotion at the community level. A literature review, described in Chapter 2, was conducted to identify other methods of asking sensitive questions. The literature review identified 19 different methods. Those methods that were most successful were those that provided the greatest degree of anonymity to the respondent. It was suggested that IVR may be a suitable method for community surveys. As described in Chapter 3, a custom software called Generalized Electronic Interviewing System (GEIS) was developed. This provided both CATI and IVR interviewing modes. As described in Chapter 4, it was found that the response rate obtained using IVR was unacceptably low, and an alternative interviewing method, the Hybrid method was developed. In the Hybrid method the interview was initiated by the interviewer but completed using IVR with GEIS. As described in Chapter 5, the IVR, CATI and Hybrid methods were used to investigate self-reported rates of alcohol and drug consumption within a telephone household survey of 2880 households. The self-report rates were compared to the National Drug Strategy Household Survey (NDSHS). Response rates did not differ significantly between the CATI and Hybrid methods, however the response rate for IVR was significantly less than the other methods. The Hybrid and IVR methods obtained significantly higher self-report consumption rates for alcohol and marijuana, and significantly higher hazardous drinking scores using Alcohol Use Disorders Identification Test (AUDIT). In Chapter 6 a pilot of an IVR cervical screening brief advice interface is described. A total of 5000 households were contacted by the IVR system. The system randomly selected an eligible woman aged 18-69 per household and determined her cervical screening status. A total of 661 women listened to the IVR message. The IVR call was shown to be acceptable and inexpensive compared to a mail pamphlet intervention. In Chapter 7 a randomized controlled trial of an IVR cervical screening brief advice involving 17,008 households is described. Cervical screening rate data were obtained from the Health Insurance Commission (HIC) for a period spanning six months before and following the intervention. The cervical screening rate was increased in the intervention postcodes by 0.43% compared to the control postcodes, and the increase was greater for older women at 1.34%. This was a desirable outcome since this group is considered to be an at-risk group. The overall conclusion was that IVR technology could be feasibly used to contact women to deliver brief interventions aimed at increasing cervical screening rates, but the cost per screen was likely to be high. It is suggested that an IVR system could be linked to cervical screening registers to more directly and economically contact women, and provide an efficacious complementary approach to the existing letter reminder system. / PhD Doctorate

ABSTRACT Whiplash associated disorders (WAD) is one of the most debated musculoskeletal conditions. Sensory disturbances including hypersensitive responses to mechanical, thermal and electrical stimulation have been consistently shown to be a feature of both the acute and chronic stages of the whiplash condition. More importantly, such dysfunctions have also been found to be associated with higher risk of poor functional recovery. It is apparent that better understanding of the sensory disturbances in WAD is needed in order to elucidate mechanisms underlying the pain and disability of this recalcitrant condition and to facilitate the development of more effective management strategies. Comprehensive Quantitative Sensory Testing (QST) combining sensory detection and pain threshold measures is proving to be a valuable tool to advance the classification and illuminating the underlying mechanisms of an array of musculoskeletal pain disorders but such protocol has never been undertaken in a WAD cohort. In order to fill this gap in knowledge, the series of studies in the thesis aimed to utilize comprehensive QST to investigate the presence of somatosensory dysfunction in chronic WAD and to compare the somatosensory profile of WAD to cervical radiculopathy and idiopathic (non-traumatic) neck pain. Once a better understanding of the potential underlying mechanisms in chronic WAD was established, the research then focused on documenting the presence of such somatosensory disturbances soon after whiplash injury and its temporal development over a 6 months period. The results have provided a number of significant insights into some of the potential underlying mechanisms of the somatosensory dysfunction in WAD as well as other neck pain conditions of different aetiology. It is clear that generalised sensory hypoaesthesia coexisted with sensory hypersensitivity in chronic WAD and a combination of pain and detection measures best discriminated patients with WAD and controls. Similar sensory presentation was also found in patients with cervical radiculopathy but not in idiopathic neck pain patients. This finding indicates that different mechanisms underlie various musculoskeletal conditions with disordered central processes contributing to a greater degree in some conditions. Patients with whiplash and those with cervical radiculopthy may share similar underlying pain mechanisms involving the central nervous system and the discrepant findings in the idiopathic neck pain group may be due to the magnitude of nociceptive input required to induce/maintain altered central adaptive changes. Another important observation from the studies was that sensory hypoaesthesia was present in the majority of patients with whiplash injury in the acute stage. However, it persisted only in individuals who initially reported higher levels of pain and disability levels and sign of hypersensitivity. It was this group of individuals who predominantly developed persistent symptoms at six months post injury. The longitudinal findings indicate that such sensory impairments can be identified very early on and treatment interventions directed at these sensory disturbances (both sensory hypersensitivity and hypoaesthesia) should aim to reduce the nociceptive input and this may improve recovery post whiplash injury. The findings in this thesis demonstrated the clear association between sensory hypersensitivity and other sensory disturbances and their potential influence on recovery. Furthermore, the heterogeneity of the whiplash condition highlighted the importance of the early identification of “low-risk and “high-risk” patients in order to assist the clinicians to make clinical decisions on the best management approach. It cannot be overemphasised that the early assessment of whiplash injured patients should aim to identify features associated with poor recovery and a better classification system will be an important step. Implications for assessment and management of whiplash are vital in the acute stage of injury and may well go some way toward preventing the transition to chronicity, particularly in those with a more complex clinical presentation involving somatosensory disturbances. Further research directions have also been identified in order to improvement management of this complex clinical condition.

Dans cette thèse, nous avons analysé le comportement biomécanique In Vitro et In Vivo d'une prothèse discale cervicale à articulation sphérique. Les expérimentations In Vitro nous ont permis d'étudier la cinématique segmentaire dans les trois plans de l'espace au niveau instrumenté et aux niveaux adjacents. La mesure de la pression intradiscale a apporté des informations sur les variations de contraintes après arthroplastie au niveau des disques adjacents. Différentes configurations ont ainsi été évaluées : arthroplastie et arthrodèse à 1 et 2 niveaux, montage hybride. L'influence de la précharge sur le comportement biomécanique du rachis cervical a fait l'objet d'un travail spécifique en se basant sur les essais In Vitro et sur la simulation numérique. Les analyses In Vivo 2D ont été effectuées sur des clichés dynamiques en flexion-extension et ont permis le calcul des amplitudes de mobilité et de la position des centres moyens de rotation dans une population de patients opérés (n=32). Celles-ci ont intéressé le niveau opéré mais également les niveaux adjacents à court et moyen terme. Grâce au système stéréo-radiographique EOSTM, nous avons pu compléter l'analyse cinématique In Vivo par la mesure des amplitudes de mobilité des segments instrumentés dans les trois plans de l'espace.