The Papanicolaou Smear is a screening test which detects premalignant lesions of the uterine cervix. By treating these lesions, cervical cancer can be evaded. In 1988, a cytological diagnosis which communicated a state of uncertainty in the atypicality of cervical cells was first created in the Bethesda Cytology Classification scheme. This diagnosis is now known as atypical squamous cells of undetermined significance (ASC-US) and still little is known about its natural history. / This paper analyzes the results of a longitudinal study incorporating repeated regular measurements of viral and cytological endpoints as well as lifestyle and behavioural aspects, to understand the natural history of an ASC-US Pap smear and identify determinants of ASC-US acquisition and clearance. / Overall, the median duration of ASC-US is short, and is dependent on the definition of clearance since most lesions regress to normal. The factors most predictive of ASC-US acquisition but not clearance relate to HPV infection.

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Chan, Ho Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 263-283). / Abstracts in English and Chinese. / Examination Committee List --- p.i / Acknowledgements --- p.ii / Abstract --- p.iv / 摘要 --- p.vi / Table of Contents --- p.viii / List of Abbreviations --- p.xvii / List of Figures --- p.xx / List of Tables --- p.xxv / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- SUMO (Small Ubiquitin-like Modifier) and SUMOylation --- p.1 / Chapter 1.1.1 --- "Ubiquitin, Ubiquitin-like proteins and SUMO isoforms" --- p.2 / Chapter 1.1.2 --- SUMO cycle --- p.5 / Chapter 1.1.2.1 --- SUMO conjugation consensus sequence --- p.5 / Chapter 1.1.2.2 --- SUMO maturation --- p.6 / Chapter 1.1.2.3 --- SUMO conjugation cascade --- p.7 / Chapter 1.1.2.4 --- SUMO deconjugation --- p.9 / Chapter 1.1.3 --- Mode of SUMO action --- p.12 / Chapter 1.1.4 --- Biological functions of SUMO --- p.13 / Chapter 1.1.4.1 --- SUMO in cancer --- p.14 / Chapter 1.2 --- Human cervical cancer and human papillomavirus (HPV) --- p.17 / Chapter 1.2.1 --- Infectious cycle of HPV-16 --- p.18 / Chapter 1.2.1.1 --- Viral entry --- p.18 / Chapter 1.2.1.2 --- Maintenance --- p.18 / Chapter 1.2.1.3 --- Deregulation of cell cycle --- p.19 / Chapter 1.2.1.4 --- Amplification and virion release --- p.20 / Chapter 1.2.2 --- Viral cancer induction --- p.22 / Chapter 1.2.2.1 --- Integration into the host genome --- p.22 / Chapter 1.2.2.2 --- Viral oncoproteins E6 and E7 --- p.23 / Chapter 1.2.3 --- SUMOylation and HPV --- p.24 / Chapter 1.2.3.1 --- Known examples of virus-host SUMOylation system interaction --- p.24 / Chapter 1.2.3.2 --- Other possible mode of virus-SUMO interaction --- p.26 / Chapter 1.3 --- A novel labeling method: biarsenical-tetracysteine labeling in SUMO study --- p.28 / Chapter 1.3.1 --- Potential use of 2As-4Cys system in SUMO studies --- p.31 / Chapter 1.3.2 --- Potential use of 2As-4Cys system in SUMO proteomics --- p.31 / Chapter 1.4 --- Objectives of the present study --- p.34 / Chapter Chapter II --- Proteomics investigation of SUMOylation in human cervical carcinoma cell line HeLa --- p.35 / INTRODUCTION --- p.35 / Chapter 2.1 --- MATERIALS --- p.37 / Chapter 2.1.1 --- Vectors for expression of SUMO and SUMOylation enzymes in E. coli --- p.37 / Chapter 2.1.2 --- E.coli cell strains --- p.38 / Chapter 2.1.3 --- Mammalian cell lines --- p.39 / Chapter 2.1.4 --- E.coli growth mediums --- p.40 / Chapter 2.1.5 --- Mammalian cell growth medium --- p.41 / Chapter 2.1.6 --- Reagents and buffers --- p.41 / Chapter 2.1.6.1 --- Reagents and buffers for molecular cloning --- p.41 / Chapter 2.1.6.2 --- Reagents and buffers for E.coli protein expression --- p.43 / Chapter 2.1.6.3 --- Reagents and buffers for mammalian cell culture --- p.44 / Chapter 2.1.6.4 --- Reagents and buffers for Western blot study --- p.45 / Chapter 2.1.7 --- Reagents and solutions for two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) sample preparation --- p.46 / Chapter 2.1.7.1 --- Reagents and solutions for 2-DE --- p.46 / Chapter i. --- 2-DE sample preparation --- p.46 / Chapter ii. --- First dimensional gel electrophoresis -isoelectric focusing (IEF) --- p.46 / Chapter iii. --- Second dimensional gel electrophoresis -SDS-PAGE --- p.47 / Chapter iv. --- Silver staining --- p.47 / Chapter 2.1.7.2 --- Reagents and solutions for mass spectrometry sample preparation --- p.48 / Chapter i. --- Destaining of silver stained gel spots --- p.48 / Chapter ii. --- Trypsin digestion --- p.48 / Chapter iii. --- Peptide extraction --- p.48 / Chapter iv. --- Desalting and concentration of peptide mixture --- p.49 / Chapter 2.2 --- METHODS --- p.50 / Chapter 2.2.1 --- Molecular cloning of SUMO-1 into pET-28m and pHM6 vectors --- p.50 / Chapter 2.2.1.1 --- Design of primers for the cloning of SUMO-1 --- p.50 / Chapter 2.2.1.2 --- DNA amplification by polymerase chain reaction (PCR) --- p.51 / Chapter 2.2.1.3 --- DNA extraction from agarose gels --- p.52 / Chapter 2.2.1.4 --- Restriction digestion of vectors and purified PCR products --- p.54 / Chapter 2.2.1.5 --- Ligation of SUMO cDNA into expression vector pET-28m and pHM6 --- p.55 / Chapter 2.2.1.6 --- Preparation of competent cells --- p.56 / Chapter 2.2.1.7 --- Transformation of ligated mixture into competent DH5a --- p.56 / Chapter 2.2.1.8 --- Preparation of plasmid DNA --- p.57 / Chapter 2.2.1.8.1 --- Mini-preparation of plasmid DNA --- p.57 / Chapter 2.2.1.8.2 --- Midi-preparation of plasmid DNA --- p.58 / Chapter 2.2.1.8.3 --- DNA quantification and quality measurement --- p.60 / Chapter 2.2.2 --- "Expression of His6-tagged SUMO, ubc9, TDG, GST-tagged El and MBP-tagged Prdx 1 with E.coli" --- p.60 / Chapter 2.2.3 --- "Purification of His6-tagged SUMO, ubc9, TDG, GST-tagged El and MBP-tagged Prdx 1" --- p.62 / Chapter 2.2.3.1 --- Affinity chromatography --- p.65 / Chapter 2.2.3.1.1 --- Ni-NTA affinity chromatography --- p.65 / Chapter 2.2.3.1.2 --- Heparin affinity chromatography --- p.66 / Chapter 2.2.3.1.3 --- Glutathione affinity chromatography --- p.66 / Chapter 2.2.3.1.4 --- Amylose affinity chromatography --- p.67 / Chapter 2.2.3.2 --- Ion exchange chromatography --- p.68 / Chapter 2.2.3.2.1 --- Anion exchange chromatography --- p.68 / Chapter 2.2.3.2.2 --- Cation exchange chromatography --- p.68 / Chapter 2.2.3.3 --- Size exclusion chromatography --- p.69 / Chapter 2.2.3.4 --- Purification strategies --- p.70 / Chapter 2.2.3.4.1 --- Purification of His6-tagged SUMO --- p.70 / Chapter 2.2.3.4.2 --- Purification of His6-tagged TDG --- p.71 / Chapter 2.2.3.4.3 --- Purification of His6-tagged ubc9 --- p.72 / Chapter 2.2.3.4.4 --- Purification of GST-tagged El --- p.73 / Chapter 2.2.3.4.5 --- Purification of MBP-tagged Prdx 1 --- p.74 / Chapter 2.2.4 --- HeLa and C-33A cell culturing and protein extraction --- p.75 / Chapter 2.2.4.1 --- HeLa and C-33A cell culturing --- p.75 / Chapter 2.2.4.2 --- Protein extraction for in vitro SUMOylation assay --- p.76 / Chapter 2.2.5 --- Protein quantification with Bradford assay --- p.76 / Chapter 2.2.6 --- In vitro SUMO conjugation assay --- p.77 / Chapter 2.2.6.1 --- In vitro SUMO conjugation system optimization --- p.77 / Chapter 2.2.6.2 --- In vitro SUMO conjugation of HeLa cell extract --- p.78 / Chapter 2.2.7 --- Transient transfection of pHM6-SUMO-l into HeLa cells and protein extraction from HeLa cells --- p.79 / Chapter 2.2.7.1 --- Transfection with lipofection method --- p.79 / Chapter 2.2.7.2 --- Determination of transfection efficiency --- p.80 / Chapter 2.2.7.3 --- Whole cell protein extraction of transfected cells --- p.81 / Chapter 2.2.8 --- Protein quantification with BCA assay --- p.81 / Chapter 2.2.9 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.83 / Chapter 2.2.10 --- Western blot analysis --- p.84 / Chapter 2.2.10.1 --- Electro-transfer blotting --- p.84 / Chapter 2.2.10.2 --- Immunoblotting with antibodies --- p.84 / Chapter 2.2.10.3 --- ECL detection --- p.85 / Chapter 2.2.10.4 --- Mild stripping for re-probing --- p.86 / Chapter 2.2.11 --- Two-dimensional gel electrophoresis (2-DE) --- p.86 / Chapter 2.2.11.1 --- Sample preparation --- p.86 / Chapter 2.2.11.2 --- First dimension gel electrophoresis -isoelectric focusing (IEF) --- p.87 / Chapter 2.2.11.3 --- Second dimension gel electrophoresis -SDS-PAGE --- p.88 / Chapter 2.2.11.3.1 --- Strip equilibration --- p.88 / Chapter 2.2.11.3.2 --- 16 x 18cm SDS-PAGE --- p.88 / Chapter 2.2.11.4 --- Visualization of proteins on SDS-polyacrylamide gel --- p.90 / Chapter 2.2.11.4.1 --- Silver staining --- p.90 / Chapter 2.2.11.4.2 --- Coomassie Blue® R250 staining --- p.91 / Chapter 2.2.12 --- Sample preparation for mass spectrometry analysis --- p.92 / Chapter 2.2.12.1 --- Destaining and trypsin digestion --- p.92 / Chapter 2.2.12.2 --- Extraction of peptide mixture --- p.93 / Chapter 2.2.12.3 --- Desalting and concentration of peptide mixture --- p.93 / Chapter 2.3 --- RESULTS --- p.95 / Chapter 2.3.1 --- Construction of recombinant pET-28m-SUMO-l and pHM6-SUMO-l --- p.95 / Chapter 2.3.2 --- "Purification of His6-tagged SUMO, ubc9, TDG and GST-tagged El" --- p.98 / Chapter 2.3.2.1 --- Purification of His6-SUMO --- p.98 / Chapter 2.3.2.2 --- Purification of His6-TDG --- p.101 / Chapter 2.3.2.3 --- Purification of His6-ubc9 --- p.104 / Chapter 2.3.2.4 --- Purification of GST-El --- p.106 / Chapter 2.3.3 --- In vitro SUMO conjugation assay --- p.108 / Chapter 2.3.3.1 --- Optimization of in vitro SUMO conjugation system --- p.108 / Chapter 2.3.3.2 --- In vitro SUMO conjugation of HeLa cell protein extract --- p.111 / Chapter 2.3.3.2.1 --- Protein extraction for in vitro sumoylation assay --- p.111 / Chapter 2.3.3.2.2 --- In vitro SUMOylation of HeLa cell lysate --- p.114 / Chapter 2.3.4 --- Differential proteomes of control and in vitro SUMOylated HeLa total cellular extract --- p.116 / Chapter 2.3.4.1 --- Mass spectrometric identification of differential protein candidates --- p.123 / Chapter 2.3.5 --- Overexpression of SUMO-1 in HeLa cells by transient transfection --- p.127 / Chapter 2.3.6 --- Differential proteomes of total cellular protein extract from control and SUMO-1 transfected HeLa cells --- p.128 / Chapter 2.3.6.1 --- Mass spectrometric identification of differential protein candidates --- p.132 / Chapter 2.4 --- Proteins identified in proteomic study with in vitro SUMOylation -Analysis of protein candidate --- p.133 / Chapter 2.4.1 --- Proteins identified from the in vitro investigation --- p.133 / Chapter 2.4.2 --- Verification of putative SUMO substrate Prdx 1 --- p.139 / Chapter 2.4.2.1 --- Purification of Prdx 1 --- p.139 / Chapter 2.4.2.2 --- In vitro SUMOylation of Prdx 1 --- p.142 / Chapter 2.4.3 --- Highlights of the proteins identified --- p.145 / Chapter 2.4.3.1 --- DJ-1 protein --- p.145 / Chapter 2.4.3.2 --- nm23A --- p.145 / Chapter 2.4.3.3 --- v-crk protein of CT10 --- p.146 / Chapter 2.4.3.4 --- Annexin I --- p.146 / Chapter 2.4.3.5 --- "Enolase 1, aldolase A, triosephosphate isomerase (TIM) and phosphoglycerate mutase 1" --- p.147 / Chapter 2.4.3.6 --- CyclophilinA(CypA) --- p.148 / Chapter 2.4.3.7 --- Stress induced phosphoprotein 1 (Stip 1) --- p.148 / Chapter 2.4.3.8 --- TSA and peroxiredoxin 1 (Prdx 1) --- p.149 / Chapter 2.5 --- Proteins identified in proteomic study with overexpression of SUMO-1 in HeLa cells -Analysis of protein candidate --- p.150 / Chapter 2.5.1 --- Proteins identified from the in vivo investigation --- p.150 / Chapter 2.5.2 --- Verification of upregulation of keratin 17 --- p.157 / Chapter 2.5.2.1 --- Immunoblotting against keratin 17 --- p.157 / Chapter 2.5.3 --- Highlights of the proteins identified --- p.159 / Chapter 2.5.3.1 --- "Heat shock proteins (Hsp 60, 70 and 27)" --- p.159 / Chapter 2.5.3.2 --- 14-3-3σ protein (SFN protein) --- p.161 / Chapter 2.5.3.3 --- PDZ-RGS3 --- p.162 / Chapter 2.5.3.4 --- "Keratins 8, 17" --- p.163 / Chapter 2.5.3.5 --- XIAP-1 --- p.164 / Chapter 2.5.3.6 --- ISG15 --- p.164 / Chapter 2.6 --- DISCUSSION --- p.166 / Chapter Chapter III --- Characterization of a novel fluorescent labeling method: Biarsencial-tetracysteine labeling in SUMO study --- p.182 / INTRODUCTION --- p.182 / Chapter 3.1 --- MATERIALS --- p.184 / Chapter 3.1.1 --- "Molecular cloning, protein expression and purification of pET-28m-4Cys 1 -SUMO-1 and pET-28m-4Cys2-SUMO-1" --- p.184 / Chapter 3.1.2 --- Mammalian cell culture and transient transfection of pHM6-4Cysl-SUMO-1 and pHM6-4Cys2-SUMO-l into HeLa cells --- p.184 / Chapter 3.1.3 --- Reagents and buffers --- p.184 / Chapter 3.1.3.1 --- Reagents and buffers for Lumio´ёØ in-gel labeling --- p.184 / Chapter 3.1.3.2 --- Reagents and buffers for Lumio´ёØ in cell labeling --- p.185 / Chapter 3.1.3.3 --- Reagents and buffers for immunostaining --- p.186 / Chapter 3.2 --- METHODS --- p.187 / Chapter 3.2.1 --- Molecular cloning of tetracysteine-tagged SUMO (4Cys-SUMO) into pET-28m and pHM6 vectors --- p.187 / Chapter 3.2.1.1 --- Design of primers and oligonucleotides encoding tetracysteine tag --- p.187 / Chapter 3.2.1.1.1 --- For 4Cysl-SUMO-1 --- p.187 / Chapter 3.2.1.1.2 --- For 4Cys2-SUMO-l --- p.188 / Chapter 3.2.1.2 --- DNA amplification of 4Cysl-SUMO-1 by Polymerase chain reaction (PCR) --- p.189 / Chapter 3.2.1.3 --- Restriction digestion of vectors and purified PCR products of 4Cysl-SUMO-1 --- p.191 / Chapter 3.2.1.4 --- Ligation of 4Cysl-SUMO into expression vector pET-28m and pHM6 --- p.191 / Chapter 3.2.1.5 --- Restriction digestion of pET-28m-SUMO and pHM6-SUMO for ligation with 4Cys2 oligos --- p.192 / Chapter 3.2.1.6 --- Ligation of 4Cys2 oligos to the digested pET-28m-SUMO and pHM6-SUMO plasmids --- p.193 / Chapter 3.2.1.6.1 --- Self-annealing of the 4Cys oligonucleotides --- p.193 / Chapter 3.2.1.6.2 --- Phosphorylation of ds 4Cys2 oligos and ligation to the plasmids --- p.193 / Chapter 3.2.2 --- Expression and purification of pET-28m-4Cys 1 -SUMO-1 and pET-28m-4Cys2-SUMO-1 in E.coli expression system --- p.195 / Chapter 3.2.3 --- Immunohistochemistry (IHC) staining of endogenous SUMO in HeLa cells --- p.196 / Chapter 3.2.4 --- In-cell labeling of 4Cysl/2-SUMO with Lumio´ёØ Reagent --- p.197 / Chapter 3.2.4.1 --- Preparation --- p.197 / Chapter 3.2.4.2 --- In-cell Lumio´ёØ labeling --- p.198 / Chapter 3.2.4.3 --- Detection and imaging of the labeled cells --- p.199 / Chapter 3.2.5 --- In-gel labeling of 4Cysl/2-SUMO with Lumio´ёØ Reagent --- p.199 / Chapter 3.2.5.1 --- Lumio´ёØ in-gel labeling --- p.199 / Chapter 3.2.5.2 --- Visualization and imaging of the labeled gel --- p.200 / Chapter a. --- UV illumination at 302 nm --- p.200 / Chapter b. --- Typhoon Trio TMLaser-scanning at 532 nm --- p.201 / Chapter 3.2.5.3 --- Detection limit of fluorescent 4Cys2-SUMO-l in SDS-PAGE --- p.201 / Chapter 3.2.5.4 --- In-gel labelling in two-dimensional electrophoresis (2-DE) --- p.202 / Chapter 3.2.5.4.1 --- Modification of equilibration buffer before SDS-PAGE --- p.202 / Chapter 3.3 --- RESULTS --- p.203 / Chapter 3.3.1 --- Adoption of old version of 4Cys-tag (4Cys 1) in SUMO study --- p.203 / Chapter 3.3.1.1 --- Construction of recombinant pET-28m-4Cys 1 -SUMO-1 and pHM6-4Cysl-SUMO-1 --- p.203 / Chapter 3.3.1.2 --- In vivo HA-4Cysl-SUMO-1 Lumio´ёØ labelling --- p.205 / Chapter 3.3.1.3 --- Immunohistochemistry (IHC) staining of endogenous SUMO in HeLa cells --- p.207 / Chapter 3.3.1.4 --- Expression and purification of His6-4Cysl-SUMO-1 --- p.208 / Chapter 3.3.1.5 --- Validation of 4Cys1-SUMO-1 conjugate by Lumio´ёØ in-gel labeling --- p.211 / Chapter 3.3.2 --- Adoption of a modified version of 4Cys-tag (4Cys2) in SUMO study --- p.213 / Chapter 3.3.2.1 --- Construction of recombinant pET-28m-4Cys2-SUMO-l and pHM6-4Cys2-SUMO-l --- p.213 / Chapter 3.3.2.2 --- In vivo HA-4Cys2-SUMO-l Lumio´ёØ labelling --- p.216 / Chapter 3.3.2.3 --- Expression and purification of His6-4Cys2-SUMO-1 --- p.219 / Chapter 3.3.2.4 --- Validation of 4Cys2-SUMO-l conjugate Lumio´ёØ in-gel labeling --- p.221 / Chapter 3.3.3 --- 2As-4Cys labeling in two-dimensional electrophoresis (2-DE) --- p.223 / Chapter 3.3.3.1 --- Detection limit of 4Cys2-SUMO-l in SDS-PAGE --- p.224 / Chapter 3.3.3.2 --- Lumio´ёØ labeling in 2-DE --- p.226 / Chapter 3.4 --- DISCUSSION --- p.232 / Chapter Chapter IV --- Conclusion and Future Perspectives --- p.242 / Chapter 4.1 --- Conclusion on proteomic study of SUMOylation --- p.242 / Chapter 4.2 --- Future perspectives of proteomic study of SUMOylation --- p.245 / Chapter 4.2.1 --- In vitro study --- p.245 / Chapter 4.2.2 --- In vivo study --- p.246 / Chapter 4.3 --- Conclusion of the investigation of biarsencial-tetracysteine (2As-4Cys) system application on SUMO study --- p.247 / Chapter 4.4 --- Future perspectives of the application of 2As-4Cys system application on SUMO study --- p.249 / Chapter 4.4.1 --- In cell study --- p.249 / Chapter 4.4.2 --- In gel study --- p.250 / Appendices --- p.251 / Chapter 1. --- Genotype of E.coli strains --- p.251 / Chapter 2. --- Vector maps --- p.252 / Chapter a. --- Vector map and MCS of pET-28a --- p.252 / Chapter b. --- Vector map and MCS of pHM6 --- p.253 / Chapter c. --- Vector information of pTwo-E --- p.254 / Chapter 3. --- Primers used in this study --- p.255 / Chapter 4. --- Nikon TE2000 filter sets spectrums --- p.257 / Chapter a. --- FITC/GFP filter set --- p.257 / Chapter b. --- RFP filter set --- p.257 / Chapter c. --- UV/DAPI/Hoechst filter set --- p.258 / Chapter 5. --- Akt signalling pathway diagram --- p.259 / Chapter 6. --- DNA sequence of SUMOs and 4Cys2 oligonucleotide --- p.260 / Chapter 7. --- Electrophoresis markers --- p.261 / References --- p.263

BACKGROUND: Human papilloma virus (HPV) in patients with esophageal carcinoma has previously been studied with an average detection rate of 15%, but the role of HPV in relation to survival is less clear. In cervical cancer, lung cancer and tonsil cancer HPV viral load is a predictive factor for survival and outcome of treatment. The primary aim was to study the spectrum of high-risk HPV types in esophageal tumors. Secondary, as a pilot study we investigated the association between HPV status and the survival rates. METHODS: We compared both the presence and the viral load of high-risk HPV types 16, 18, 31, 33, 39, 45, 52, 58, and 67 in relation to clinical data from patients with esophageal carcinoma. Survival data and tumor samples were retrieved from 100 patients receiving treatment at the Department of Oncology, Uppsala Hospital, Uppsala, Sweden. The tumor samples were investigated for HPV viral load using real-time PCR. RESULTS: HPV 16 was detected in 16% of the patients; no other HPV type was detected. HPV 16 infection had no significant effect on survival (p = 0.72). Also, HPV 16 did not improve survival after treatment (radiotherapy or chemotherapy). CONCLUSION: Only HPV 16 was detected among the patients. HPV 16 in esophageal carcinoma patients did not influence survival or improve therapy response. However, given the size of the study there is a need to examine a larger cohort in order to understand in more detail the effect of high risk HPV types in esophageal carcinoma. / <p>De två första författarna delar förstaförfattarskapet.</p>