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Investigating the Clinical Validity of CUB and Zona-Pellucida-Like Domain-Containing Protein 1 (CUZD1) in Malignant and Non- Malignant Human Diseases

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Investigating the Clinical Validity of CUB and Zona-Pellucida-Like Domain-Containing Protein 1 (CUZD1) in Malignant and Non- Malignant Human Diseases Investigating the Clinical Validity of CUB and Zona Pellucida- like Domain-Containing Protein 1 (CUZD1) in Malignant and Non-Malignant Human Diseases by Sofia Farkona A thesis submitted in conformity with the requirements for the degree of Doctorate of Philosophy Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Sofia Farkona 2018 Investigating the Clinical Validity of CUB and Zona-Pellucida-like Domain-Containing Protein 1 (CUZD1) in Malignant and Non- Malignant Human Diseases Sofia Farkona Doctor of Philosophy Laboratory Medicine and Pathobiology University of Toronto 2018 Abstract CUB and zona pellucida-like domain-containing protein 1 (CUZD1) has been previously shown to be specifically expressed in normal pancreas and was proposed as a candidate biomarker for pancreatic related disorders. Due to the lack of specific reagents and techniques, its levels in tissues and biological fluids have not been extensively examined. We generated mouse monoclonal antibodies against recombinant CUZD1 and used them for the development of an enzyme-linked immunosorbent assay (ELISA). Analysis of various human extracts showed that CUZD1 is measured in high levels in pancreas and at much lower (but detectable) levels in several other tissues. Analysis of biological fluids showed that CUZD1 is detected exclusively in pancreatic juice. CUZD1 has been previously linked to diseases (such as pancreatitis, ovarian cancer and IBD) but it is currently unknown if the expression levels of this antigen are elevated in any of the aforementioned or other disorders. Analysis of a large number of serum samples from patients with various malignant and benign disorders showed that CUZD1 levels were elevated in patients with ovarian cysts but not ovarian cancer. ii CUZD1 is a pancreas-specific protein but it is unclear if its expression is elevated in malignant conditions of the pancreas. IHC staining of pancreatic ductal adenocarcinoma (PDAC) and acinar cell carcinoma (ACC) tissue sections revealed that CUZD1 protein was highly expressed in ACC but not in PDAC. CUZD1 is one of the targets of pancreatic autoantibodies (PABs) which have been emerged as possible biomarkers for Inflammatory Bowel Disease (IBD). Data assessing the diagnostic significance of CUZD1 autoantibodies in patients with IBD are scarce, mainly due to the lack of high throughput techniques for their detection. We developed an ELISA targeting CUZD1 autoantibodies and used it to analyze 200 serum samples from IBD patients and 129 patients assessed for various autoimmune diseases (vADs). CUZD1 autoantibodies were detected in 16% of CrD patients in 9% of UC patients and in less than 5% of patients being tested for vADs. In conclusion, this thesis encompasses the development and validation of analytical techniques targeting CUZD1 antigen and CUZD1 autoantibodies. These tools can facilitate future investigations aiming to delineate the role of CUZD1 in physiology and pathobiology. iii Acknowledgments First and foremost, I would like to thank my supervisors Dr. Eleftherios Diamandis and Dr. Ivan Blasutig for their continuous support and guidance throughout my PhD. I cannot thank you both enough for giving me the opportunity and freedom to pursue my scientific interests and for the patience and mentorship throughout my PhD. I am most thankful to colleagues who contributed to this work and to all members of ACDC laboratory for supporting me and making my time here enjoyable. I will never forget the time we shared together. I would also like to extend my gratitude and thanks to the members of my PhD advisory committee, Dr. Yousef and Dr. Schmitt Ulms for volunteering their time and for providing invaluable advice and feedback that led to completion of this exciting work. Additionally, I am thankfully to our collaborators Dr. Bogdanos, Dr. Ruckert and Dr. Serra who shared my enthusiasm and who graciously provided me with samples and resources. I would also like to thank the examiners of this thesis Dr. Charames, Dr. Chandran and Dr. Kavsak for reading it and giving useful feedback during the final exam. I should thank the members of the Department of Laboratory Medicine and Pathobiology, including Dr. Harry Elsholtz, Sue Sarju and Rama Ponda for administrative help in helping scheduling the defense. I would like to acknowledge the funding I received from the LMP department. Last but not least, none of this work would have been possible without the love and support of my family. Thank you to my brother, Peter, and more importantly my parents, Markos and Eutychia, for always believing me and encouraging me to follow my dreams. I know that I would not be where I am today if it wasn’t for you. I am eternally grateful, and this thesis is dedicated to you. iv Table of Contents Acknowledgments.......................................................................................................................... iv Table of Contents .............................................................................................................................v List of Abbreviations ..................................................................................................................... ix List of Tables ............................................................................................................................... xiv List of Figures ................................................................................................................................xv Chapter 1 | Introduction ................................................................................................................1 1.1 Pancreas ...............................................................................................................................2 1.1.1 General information .................................................................................................2 1.1.2 History of the pancreas ............................................................................................2 1.1.3 Structure of the pancreas ..........................................................................................2 1.1.4 Development ............................................................................................................4 1.1.5 Development and Molecules....................................................................................5 1.1.6 Physiology of the pancreas ......................................................................................8 1.1.7 Islet-acinar axis/interaction ....................................................................................14 1.1.8 Common diseases...................................................................................................16 1.2 Inflammatory Bowel Disease .............................................................................................22 1.2.1 General information (classification, symptoms and prevalence) ...........................22 1.2.2 Causes ....................................................................................................................23 1.2.3 Diagnosis................................................................................................................24 1.3 CUZD1 ...............................................................................................................................27 v 1.3.1 Genomic location and structure .............................................................................27 1.3.2 CUZD1 endogenous expression ............................................................................31 1.3.3 Cellular localization of CUZD1 .............................................................................34 1.3.4 Proteins similar to CUZD1 ....................................................................................36 1.3.5 Function of CUZD1 (based on experiments) .........................................................38 1.3.6 CUZD1 autoantibodies in patients with IBD .........................................................40 1.4 Rationale and Objectives ...................................................................................................42 1.4.1 Rationale ................................................................................................................42 1.4.2 Hypothesis..............................................................................................................42 1.4.3 Objectives ..............................................................................................................42 2 Chapter 2 | Novel immunoassays for detection of CUZD1 autoantibodies in serum of patients with inflammatory bowel diseases...............................................................................44 2.1 Introduction ........................................................................................................................45 2.2 Materials and Methods .......................................................................................................47 2.2.1 Patients ...................................................................................................................47 2.2.2 Production of recombinant CUZD1 .......................................................................47 2.2.3 Selected Reaction Monitoring (SRM) ...................................................................50 2.2.4 Chromatographic and MS conditions ....................................................................51 2.2.5 Data analysis
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