Thesis (Ph. D.)--Oregon Graduate Institute, 2000.

The confluence of next generation DNA sequencing and synthesis when combined with the application of concepts such as standardization and modular design has led to the genesis of a new discipline. The nascent field of Synthetic Biology concerns the rational design and construction of genetic circuits, pathways, machines and eventually whole organisms. The immaturity of this field dictates that early research efforts, including this Thesis, describe foundational work towards the creation of tools which make biology more amenable to being engineered. The first part of this Thesis describes an attempt to standardize the measurement of transcriptional promoter activity in E. coli. A method to measure in vivo promoter activity was developed for E. coli and tested in a multi-institution trial. Comparable results were achieved with less than a two-fold range for the measured promoters across eight laboratories. A standardized measurement kit was created and distributed for use by the teams participating in the 2008 international Genetically Engineered Machines competition. Techniques learned measuring the activity of E. coli promoters were applied to a collection of S. cerevisiae strains. Several promoters were measured in synthetic dextrose media and ADH1 was measured in multiple media conditions. The outcome of these experiments is to consider proposing ADH1as the reference promoter in S. cerevisiae. The second aspect of this Thesis describes the construction of artificial organelles in S. cerevisiae. Artificial organelles hold the prospect of being able to insulate synthetic genetic pathways from the cell. Two proteins are essential for the biogenesis of the peroxisome organelle in humans and yeast, Pex3p and Pex19p. Pex3p functions as a peroxisomal membrane receptor for Pex19p, while Pex19p shuttles other peroxisomal proteins to the membrane, including Pex3p, creating a feedback loop. Human Pex19p has previously been shown to dock to yeast Pex3p and a version of yeast Pex19p has been shown to work with human Pex3p as a high degree of evolutionary conservation exists between these proteins. Because of these inter-species protein docking characteristics, there exists the possibility of creating bimodality: the ambition of the work was therefore to create a cell strain which possessed both synthetic “humanized” and natural yeast peroxisomes. An S. cerevisiae BY4741a derivative strain was engineered with fluorophore tagged versions of human (CFP) and yeast (YFP) Pex3p and untagged yeast and human Pex19p proteins. The results indicated the creation of a single population of peroxisomes when a measure of fluorescently imaged CFP and YFP peroxisomes were plotted on a scatter plot. A log of the ratio of CFP to YFP peroxisomes was plotted on a histogram and a normal distribution was found to best fit the curve, indicating a lack of bimodality. Finally, microscopy images of this strain were reviewed and by visual inspection, showed no evidence of distinct human or yeast peroxisomes. This experiment therefore produced no evidence of bimodality when examining the interactions of human and yeast Pex3p and Pex19p proteins. However, the four proteins were shown to interact closely to produce a single population of chimeric human-yeast peroxisomes. The peroxisome-deficient mutant phonotype strain was rescued using human Pex3p and Pex19p.

Thesis (Ph.D.) -- McMaster University, 1999. / (Alpha) in title is a Greek letter. Includes bibliographical references (leaves 203-228). Also available via World Wide Web.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Cell Biology. Title from pdf file main screen (viewed on July 25, 2010). Includes bibliographical references.

Colorectal cancer (CRC) is a common disease with significant morbidity and mortality. In spite of significant advances in understanding the molecular signaling in this disorder, unanswered questions remain. Cyclooxygenase-2 (COX-2) and β-catenin have established roles in colorectal carcinogenesis, with both being upregulated early in the disease course. The role of peroxisome proliferator-activated receptor γ (PPARγ) is less clear, but has been shown to be downregulated in colon cancer models. Butyrate, a short chain fatty acid, produced by colon microbiota and transported into the colonocyte by transporter proteins, appears to be important in early carcinogenesis. The butyrate concentration is reduced in CRC and so are its transporters. Interleukin-17 (IL-17) plays a role in colitis-associated colorectal cancer (CAC), but its function in sporadic CRC is less clear. Similarly, Protein kinase C (PKC) has proven involvement in many solid tumours, including CRC, but its exact mechanistic role is still speculative. AIM: To investigate the role and possible signaling pathways of the major role players, β-catenin, COX-2 and PPARγ in early CRC. Further, to elucidate the mechanistic pathways of butyrate and its transporters, IL-17 and PKC in CRC. METHOD: Informed consent was obtained for all patients. Patients were recruited in various disease categories, including normal, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) and CRC. Colon biopsy specimens were obtained during colonoscopy and used for immunohistochemistry (IHC) and gene expression analysis of the above genes by quantitative polymerase chain reaction (qPCR). RESULTS: β-catenin mRNA and protein expression was increased in CRC and the IBD groups compared to the normal group, while it was reduced in the IBS groups. COX-2 mRNA expression showed a steady increase from normal, through IBS, IBD and CRC groups to a statistically significant degree. The COX-2 protein expression, however, did not match the mRNA expression with increased COX-2 protein expression in normal and IBS groups and reduced expression in IBD and CRC groups. PPARγ mRNA expression was unchanged in IBD and CRC groups, but significantly increased in the IBS group compared to normal. Butyrate transporter, SLC16A1 mRNA was significantly reduced in CRC, but also in the IBS groups, which was unexpected. In the IBD group, SLC16A1 mRNA was unchanged in Crohn’s disease (CD) but significantly reduced in ulcerative colitis (UC). Similarly, SLC5A8 mRNA expression was significantly reduced in the CRC as well as the IBS groups. In the IBD groups, SLC5A8 was unchanged in UC but significantly increased in CD. IL-17 mRNA expression was significantly reduced in CRC and IBS groups, but unchanged in the IBD groups. PKCε mRNA was significantly increased in CRC as expected. In the IBD groups, PKCε mRNA was unchanged in CD but significantly increased in UC. In the IBS groups, PKCε mRNA in constipation –IBS (C-IBS) was significantly reduced, but unchanged in diarrhoea – IBS (D-IBS). CONCLUSIONS: β-catenin mRNA and protein expression was increased in CRC and the CRC promoting IBD groups. COX-2 protein expression was incongruent with the COX-2 mRNA expression and this may reflect homeostatic control mechanisms. High COX-2 mRNA expression in CRC and CRC promoting IBD groups may be a secondary phenomenon reflecting the inflammatory milieu, rather than a true carcinogenesis-related event. PPARγ does not appear to play a central role in early colon carcinogenesis, in spite of available literature suggesting otherwise. Butyrate transporters showed inconsistent results and for now no firm conclusions can be drawn from this. IL-17 may play a role in CAC as confirmed in this and other studies, but its role in sporadic CRC is tenuous and requires further investigation. Likewise for PKCε, upregulation is associated with increased tumourigenecity as shown in this study, however, the mechanistic pathway(s) involved is still speculative and requires further study.

The presented thesis includes three related projects, that are linked by a common interest in the evolution of eukaryotic organelles and machineries that import proteins into these compartments. The first project considers the possibility of peroxisomes (eukaryotic organelles known in aerobic organisms) being conserved in two related anaerobic protists: a free-living amoeba Mastigamoeba balamuthi and a parasite Entamoeba histolytica. The most important hint for the presence of peroxisomes was the discovery of proteins that are homologous to known components of the peroxisomal protein import machinery. The second project aims to characterize the unknown protein translocase of the inner membrane (TIM) in the mitosomes (extremely reduced mitochondria) of an anaerobic protozoan Giardia intestinalis. We have discovered an important subunit of the mitosomal translocase (Tim44), which usually tethers the Hsp70/PAM (presequence translocase-associated motor) complex to the TIM translocon. The last project shows that the protein translocase of the outer mitochondrial membrane in trypanosomatids is related to a typical eukaryotic channel Tom40. This finding is important because the absence of Tom40 was previously considered an ancestral feature of trypanosomatids.

Chan, Pui Ting. / Thesis submitted in: May 2007. / On t.p. "alpha" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 110-118). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Abbreviations --- p.xi / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Peroxisome proliferater-activated receptors (PPARs) --- p.1 / Chapter 1.1.1 --- What are PPARs? --- p.1 / Chapter 1.1.2 --- PPAR isoforms --- p.1 / Chapter 1.1.3 --- PPARα ligands --- p.2 / Chapter 1.2 --- Biological role of PPARα --- p.3 / Chapter 1.2.1 --- Lipid metabolism --- p.3 / Chapter 1.2.2 --- Glucose metabolism --- p.5 / Chapter 1.2.3 --- Oxidative stress and carcinogenesis --- p.6 / Chapter 1.3 --- Discovery of PPARα-regulated and starvation-induced gene (PPSIG) --- p.7 / Chapter 1.4 --- Objectives of the present study --- p.9 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.10 / Chapter 2.1 --- Cloning of PPSIG cDNA into a pCMV-Tag epitope tagging mammalian expression vector --- p.10 / Chapter 2.1.1 --- Materials --- p.10 / Chapter 2.1.2 --- Methods --- p.10 / Chapter 2.2 --- Transient transfection of PPSIG cDNA into CHO-K1 and AML-12 cells --- p.16 / Chapter 2.2.1 --- Cell culture and transfection --- p.16 / Chapter 2.2.1.1 --- Materials --- p.16 / Chapter 2.2.1.2 --- Methods --- p.19 / Chapter 2.2.2 --- Western blot analysis --- p.20 / Chapter 2.2.2.1 --- Materials --- p.20 / Chapter 2.2.2.2 --- Methods --- p.20 / Chapter 2.3 --- Stable transfection of PPSIG cDNA into CHO-K1 and AML-12 cells --- p.22 / Chapter 2.3.1 --- Linearization of the pCMVT4B-PPSIG construct --- p.22 / Chapter 2.3.1.1 --- Materials --- p.22 / Chapter 2.3.1.2 --- Methods --- p.22 / Chapter 2.3.2 --- Cell culture and stable transfection --- p.23 / Chapter 2.3.2.1 --- Materials --- p.23 / Chapter 2.3.2.2 --- Methods --- p.23 / Chapter 2.3.3 --- Selection of the G418-resistant clones --- p.26 / Chapter 2.3.3.1 --- Materials --- p.26 / Chapter 2.3.3.2 --- Methods --- p.29 / Chapter 2.3.4 --- Picking and expanding the G418-resistant clones --- p.30 / Chapter 2.3.4.1 --- Materials --- p.30 / Chapter 2.3.4.2 --- Methods --- p.30 / Chapter 2.3.5 --- Screening and confirmation of the stable transfectants --- p.31 / Chapter 2.3.5.1 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.31 / Chapter 2.3.5.1.1 --- Materials --- p.31 / Chapter 2.3.5.1.2 --- Methods --- p.31 / Chapter 2.3.5.2 --- Northern blot analysis --- p.35 / Chapter 2.3.5.2.1 --- Materials --- p.35 / Chapter 2.3.5.2.2 --- Methods --- p.35 / Chapter 2.3.5.3 --- Western blot analysis --- p.37 / Chapter 2.3.5.3.1 --- Materials --- p.37 / Chapter 2.3.5.3.2 --- Methods --- p.37 / Chapter 2.3.5.4 --- Immunoprecipitation --- p.37 / Chapter 2.3.5.4.1 --- Materials --- p.37 / Chapter 2.3.5.4.2 --- Methods --- p.38 / Chapter 2.3.5.5 --- Matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry analysis --- p.39 / Chapter 2.3.5.5.1 --- Materials --- p.39 / Chapter 2.3.5.5.2 --- Methods --- p.39 / Chapter 2.4 --- "Analysis of the all-trans-13,14-dihydroretinol saturase (RetSat) activity by high-performance liquid chromatography (HPLC) analysis" --- p.41 / Chapter 2.4.1 --- Materials --- p.41 / Chapter 2.4.2 --- Methods --- p.42 / Chapter 2.4.2.1 --- Preparation of all-trans-retinol --- p.42 / Chapter 2.4.2.2 --- Treatment of PPSIG-transfected cells with all-trans-retinol --- p.42 / Chapter 2.4.2.3 --- Retinoid analysis --- p.43 / Chapter 2.5 --- Analysis of fatty acid compositions by gas chromatography-mass spectrometry (GC-MS) --- p.43 / Chapter 2.5.1 --- Materials --- p.43 / Chapter 2.5.2 --- Methods --- p.44 / Chapter 2.5.2.1 --- Preparation of fatty acid-BSA complex --- p.44 / Chapter 2.5.2.2 --- Treatment of PPSIG-transfected cells with fatty acid-BSA complex --- p.44 / Chapter 2.5.2.3 --- Extraction of fatty acids --- p.45 / Chapter 2.5.2.4 --- Methylation of the fatty acids --- p.45 / Chapter 2.5.2.5 --- GC-MS analysis --- p.46 / Chapter 2.5.2.6 --- Statistical analysis --- p.47 / Chapter CHAPTER 3 --- RESULTS --- p.48 / Chapter 3.1 --- The PPSIG cDNA was subcloned into a pCMV-Tag epitope tagging mammalian expression vector --- p.48 / Chapter 3.2 --- The pCMVT4B-PPSIG expression construct was transiently transfected into CHO-K1 and AML-12 cells --- p.54 / Chapter 3.3 --- Stable transfection of the pCMVT4B-PPSIG expression construct into CHO-K1 and AML-12 cells --- p.54 / Chapter 3.3.1 --- PPSIG-transfected CHO-K1 and AML-12 cells were obtained after G418 selection --- p.54 / Chapter 3.3.2 --- PPSIG-transfected CHO-K1 and AML-12 cells had high PPSIG mRNA expression --- p.58 / Chapter 3.3.3 --- PPSIG-FLAG fusion protein was over-expressed in the PPSIG- transfected CHO-K1 and AML-12 cells --- p.61 / Chapter 3.3.4 --- The stable transfectants were immunoprecipitated and identified as PPSIG protein by the mass spectrometry analysis --- p.64 / Chapter 3.4 --- PPSIG protein posseses saturase activity towards all-trans-retinol --- p.66 / Chapter 3.5 --- PPSIG protein is not a fatty acid transporter --- p.78 / Chapter CHAPTER 4 --- DISCUSSION --- p.101 / FUTURE STUDIES --- p.107 / REFERENCES --- p.110 / Appendix A: Prediction of the molecular weight of pCMVT4B- PPSIG protein --- p.119 / Appendix B: Theoretical tryptic peptides of PPSIG --- p.120 / Appendix C: Protein-peptide mass reports --- p.122 / Chapter C1. --- Peptide mass summary of trypsin-digested PPSIG immunoprecipitated protein from clone L2H4B18 --- p.122 / Chapter C2. --- Peptide mass summary of trypsin-digested PPSIG immunoprecipitated protein from clone AL2L7 --- p.123 / Appendix D: HPLC spectrum of the RetSat activity towards all- trans retinol --- p.124 / Chapter D1. --- RetSat activity towards all-trans retinol according to the Moise's group study ((Moise et al. 2004) --- p.124

Ho Wing Man. / On t.p. "gamma" appears as the Greek letter. / Thesis submitted in: December 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 95-106). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.III / ACKNOWLEDGMENTS --- p.V / ABBREVIATIONS --- p.VI / LIST OF FIGURES --- p.IX / LIST OF TABLES --- p.X / CONTENTS --- p.XII / Chapter CHAPTER ONÉؤ --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Background --- p.2 / Chapter 1.1.1 --- Thyroid cancer --- p.2 / Chapter 1.1.2 --- Apoptosis and thyroid cancer --- p.4 / Chapter 1.2 --- Estrogen receptors and apoptosis --- p.5 / Chapter 1.2.1 --- Estrogen receptor-α (ERα) and estrogen receptor-β (ERβ) --- p.5 / Chapter 1.2.2 --- Differential roles of estrogen receptor-α(ERα) and estrogen receptor-β (ERβ) in apoptosis --- p.6 / Chapter 1.2.3 --- Bcl-2 family --- p.8 / Chapter 1.3 --- Peroxisome proliferator-activated receptor-γ (PPARγ) --- p.9 / Chapter 1.3.1 --- Molecular aspects of PPAR --- p.9 / Chapter 1.3.2 --- PPAR/RXR complex --- p.13 / Chapter 1.3.3 --- PPARγ ligands --- p.16 / Chapter 1.3.4 --- PPARγ and apoptosis in thyroid cancer --- p.19 / Chapter 1.3.5 --- PPARγ ligands-mediated apoptosis pathway --- p.21 / Chapter 1.4 --- Previous results from our laboratory --- p.25 / Chapter 1.5 --- Summary of previous studies --- p.27 / Chapter 1.6 --- Perspectives --- p.28 / Chapter 1.7 --- Objectives of this project --- p.29 / Chapter CHAPTER TWÓؤ --- GENERAL MATERIALS AND METHODS --- p.30 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.1.1 --- Cell lines --- p.31 / Chapter 2.1.2 --- Plasmid vectors used in this study --- p.31 / Chapter 2.1.3 --- Antibodies --- p.32 / Chapter 2.1.4 --- Culture media and transfection reagents --- p.32 / Chapter 2.1.5 --- Materials for protein manipulation --- p.33 / Chapter 2.1.6 --- Drugs for treatment --- p.34 / Chapter 2.1.7 --- Kits --- p.35 / Chapter 2.1.8 --- Instruments --- p.35 / Chapter 2.2 --- Methods --- p.36 / Chapter 2.2.1 --- Cell culture --- p.36 / Chapter 2.2.2 --- Cell viability analysis --- p.36 / Chapter 2.2.3 --- Preparation of protein extract --- p.37 / Chapter 2.2.4 --- Determination of the concentration of target protein --- p.37 / Chapter 2.2.5 --- Gel electrophoresis and protein transfer --- p.38 / Chapter 2.2.6 --- Immunoblotting --- p.39 / Chapter 2.2.7 --- Apoptosis detected by Cell Death ELISAplus --- p.41 / Chapter 2.2.8 --- PPARγ-ligand Enzyme Immunoassay --- p.45 / Chapter 2.2.8.1 --- 15d-PGJ3 Enzyme Immunoassay --- p.45 / Chapter 2.2.8.2 --- 15(S)-HETE Enzyme Immunoassay --- p.46 / Chapter 2.2.8.3 --- 13(S)-HODE Enzyme Immunoassay --- p.46 / Chapter 2.2.9 --- Transient tranfection and luciferase activity assay --- p.47 / Chapter 2.2.10 --- Statistical Analysis --- p.52 / Chapter CHAPTER THREÉؤ --- ESTROGEN RECEPTORa (ERa) AND ESTROGEN RECEPTORP(ERP) MEDIATE THE PROLIFERATION AND APOPTOSIS OF HUMAN THYROID PAPILLARY CARCINOMA CELLS --- p.53 / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Cell culture and treatment --- p.56 / Chapter 3.2.2 --- Western Blot --- p.56 / Chapter 3.2.3 --- Cell proliferation determined by MTT assay --- p.57 / Chapter 3.2.4 --- Apoptosis detected by Cell Death ELISAplus assay --- p.58 / Chapter 3.3 --- Results --- p.59 / Chapter 3.3.1 --- "The expression of ERα, ERβ and PPARγ in NPA, FRO, ARO and WRO thyroid cancer cell lines" --- p.59 / Chapter 3.2.2 --- Effects of PPT and DPN on cell viability --- p.61 / Chapter 3.3.3 --- Apoptotic cells quantification by Cell Death ELISAplus assay --- p.64 / Chapter 3.4 --- Discussion --- p.67 / Chapter CHAPTER FOUŔؤ --- THE RELATIONSHIP BETWEEN PPARγ AND ESTROGEN RECEPTOR AND THE REGULATION OF THE APOPTOSIS IN THYROID CANCER CELL LINES --- p.70 / Chapter 4.1 --- Introduction --- p.71 / Chapter 4.2 --- Material and Methods --- p.74 / Chapter 4.2.1 --- Transient transfection --- p.74 / Chapter 4.2.2 --- Luciferase assay --- p.74 / Chapter 4.2.3 --- 15d-PGJ2 ELISA assay --- p.75 / Chapter 4.2.4 --- 15S-HETE ELISA assay --- p.76 / Chapter 4.2.5 --- 13S-HODE ELISA assay --- p.77 / Chapter 4.3 --- Results --- p.78 / Chapter 4.3.1 --- "PPT, ERα-agonist, increased thyroid cancer cell proliferation and caused the decrease the level of PPARγ ligands" --- p.78 / Chapter 4.3.2 --- "DPN, ERβ-agonist, inhibited thyroid cancer cell proliferation, induced apoptosis and caused the increase the level of PPARγ ligands" --- p.83 / Chapter 4.3.3 --- PPT did not alter the transcriptional activity of PPARγ --- p.88 / Chapter 4.4 --- Discussion --- p.90 / Chapter CHAPTER FIVÉؤ --- CONCLUSIONS AND FUTURE PROSPECT --- p.92 / Chapter 5.1 --- Summary of results --- p.93 / Chapter 5.2 --- Conclusion --- p.94 / Chapter 5.3 --- Future prospects --- p.94 / REFERENCE LIST --- p.95

Copies of author's previously published articles inserted. / Bibliography: leaves 112-166. / 179, [38] leaves, [29] leaves of plates : ilol. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to contribute to the understanding of the mechanisms of peroxisomal assembly in human hepatocytes by investigating the nature of the ultrastructural changes in peroxisomes in those patients where peroxisomal biogenesis or function is impaired. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1994

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.