VictorJuiz/thesis huggingface dataset:
The Roles of Presenilin and FKBP14 in Drosophila Development and Notch Signalling

The Roles of Presenilin and FKBP14 in Drosophila Development and Notch Signalling; Diana L. van de Hoef, Department of Molecular Genetics, University of Toronto, 2008. The multimolecular gamma-secretase complex cleaves type 1 transmembrane proteins such as Notch and one of the genes targeted in Alzheimer’s disease known as APP. This complex comprises four components, known as anterior pharynx defective 1, presenilin enhancer 2, nicastrin and presenilin. Presenilin is an aspartyl protease that comprises the catalytic core of gamma-secretase, and mutated forms of presenilin cause early-onset familial Alzheimer’s disease. To further define the role of Drosophila Presenilin (Psn), I performed a genetic modifier screen to identify Psn-interacting genes. One of the genes that was identified, known as FKBP14, encodes a peptidyl-prolyl isomerase that may be involved in protein folding in the ER. I demonstrate that an immunosuppressant drug known as FK506, which binds FKBPs and abrogates their function, reduced Psn, anterior pharynx defective 1 and presenilin enhancer 2 protein levels in vivo. I also show that FKBP14 colocalized with anterior pharynx defective 1 and Psn in the ER, suggesting a role in gamma-secretase stability. Consistent with this, I demonstrate that FKBP14 binds with Psn and mediates Psn stability and Notch signalling in vivo. To further characterize the role of FKBP14 in development, I analyzed its expression pattern and phenotypes of an FKBP14 null mutant. I show that FKBP14 localized to embryonic hemocytes and larval tissues, in addition to being expressed in developing egg chambers. FKBP14 function is required during development, since FKBP14 null mutants are recessive lethal. These mutants exhibited defects in larval disc development that resulted in eye, wing and notum phenotypes reminiscent of Psn dominant-negative and Notch-dependent phenotypes. Furthermore, FKBP14 mutants displayed enhanced apoptosis in larval tissues, suggesting a possible involvement in apoptosis regulation. I then examined the effects of FKBP14 overexpression, and observed enhanced Psn protein levels in vivo. Interestingly, co-expression of FKBP14 and Psn resulted in synergistic bristle phenotypes, suggesting a role for FKBP14 function in the Notch signalling pathway. Consistent with this, FKBP14 mutants enhanced Notch loss-of-function phenotypes in the wing. Altogether, my data demonstrate an essential role for FKBP14 during development, particularly in Psn protein maintenance and Notch signalling.

Keywords: Genetic Screen
Advisor: Boulianne, Gabrielle
Sequence and Structural Analysis of the BTB Domain

The BTB domain is a eukaryotic protein-protein interaction motif found in variety of proteins. This thesis describes an investigation into the general and specific properties of the sequence, structure and self-association properties of this domain. The work is divided by two complementary approaches. Chapter 2 describes compu-tational work in assembling a collection of BTB domain sequences from completely se-quenced eukaryotic genomes. This chapter describes analyses on this collection including the genomic distribution, domain architectures, identification of putative novel domains and predictions of interactions. Chapters 3, 4 and 5 are founded on experimental analyses on BTB domains from human BTB-ZF proteins. Chapter 3 describes the structure of the BTB domain from Leu-kemia/Lymphoma Related Factor (LRF). The structure closely resembles the previously determined structures of BTB domains. The structure showed a large number of sequence substitutions on the surface of the LRF BTB domain that is equivalent to the surface in-volved in an interaction between the BTB domain from B-Cell Lymphoma 6 (BCL6) and a peptide derived from the SMRT co-repressor (the SMRT-BBD). We show the LRF BTB domain does not interact with this peptide. Chapter 4 describes the structures of the BTB domains from FAZF and Miz-1. These proteins conserve most of the BTB fold but show some unexpected changes. The BTB domain from FAZF lacks domain swapping which is a novel feature. The BTB do-main from Miz-1 contains a naturally truncated N-terminus and a novel movement of 10 residues away from a conserved three-stranded β-sheet. We show these BTB domains are dimeric within a specific concentration range and that they do not interact with the SMRT-BBD. Chapter 5 describes the structure of the BTB domain from Kaiso. This structure showed interactions between Kaiso BTB domain dimers that extend through the crystal. We identified similar interactions between dimers in a number of other structures of other BTB domains which suggested a common mode of oligomerization.

Keywords: structural biology
biochemistry
biophysics
x-ray crystallography
bioinformatics
protein-protein interactions
Advisor: Privé, Gilbert G.
Elucidation of the Catalytic Mechanism of Golgi alpha-mannosidase II

The central dogma of molecular biology outlines the process of information transfer from a DNA sequence, to a protein chain. Beyond the step of protein synthesis, there are a variety of post-translational modiﬁcations that can take place, one of which is addition of carbohydrate chains to nascent proteins, known as glycosylation. The N-linked glycosylation pathway is responsible for the covalent attachment of multifunctional carbohydrate chains on asparagine residues of nascent proteins at Asn-X-Ser/Thr consensus sequences. These carbohydrate chains are thought to aid in cell signaling, immune recognition, and other processes. Golgi alpha-mannosidase II (GMII) is the enzyme in the N-glycosylation pathway that is responsible for cleaving two mannose linkages in the oligosaccharide GnMan5Gn2 (where Gn is N-acetylglucosamine and Man is mannose), thereby producing GnMan3Gn2 , which is the committed step in complex N-glycan synthesis. It has been speculated that GMII is an excellent therapeutic target for cancer treatment, as the unusual distribution of carbohydrates on the surface of tumour cells has been characterized in many cancers. In addition, swainsonine-—a strong, yet nonspecific inhibitor of GMII—-has been shown to block metastasis and improve the clinical outcome of patients with certain cancers, including those of the colon, breast and skin. This thesis examines Golgi alpha-mannosidase II from Drosophila melanogaster (dGMII) as a model for all GMII enzymes. First, a 1.80 Angstrom resolution crystal structure of a weak inhibitor, kifunensine, binding to dGMII provides mechanistic insights into the substrate distortion in the GMII reaction. It is hypothesized that the GMII reaction proceeds via a 1 Sinterintermedi-ate. Second, a 1.40 Angstrom resolution structure of a mutant dGMII bound to its natural substrate, GnMan5Gn, identiﬁes key substrate binding and catalytic residues, as well as expanding the deﬁnition of the GMII active site to include two distant sugar−binding subsites. Finally, the results are taken together, with knowledge of other related enzymes to synthesize a plausible itinerary for the GMII reaction.

Keywords: Glycosylation
Cancer
X-Ray Crystallography
Glycoside hydrolase
Catalytic mechanism
Drug Design
Advisor: Rose, David
Optimization of Imaging Performance and Conspicuity in Dual-Energy X-ray Radiography

Dual-energy (DE) x-ray imaging of the chest decomposes two radiographs acquired at low- and high x-ray energies into 'soft-tissue' and 'bone' images, reducing the influence of background anatomical noise and providing increased conspicuity of subtle underlying structures compared to conventional radiography. This thesis derives a quantitative theoretical model of imaging performance in DE x-ray imaging and employs the resulting framework to system optimization in thoracic imaging. Fourier domain metrics of signal and noise performance - including the noise-power spectrum (NPS), modulation transfer function (MTF), detective quantum efficiency (DQE), and noise-equivalent quanta (NEQ) - were computed using cascaded systems analysis extended to DE imaging and combined with a quantitative model of imaging task to yield estimates of detectability across a broad range of DE image acquisition and decomposition techniques. Specifically, the detectability index provided an objective function for optimizing the selection of kVp pair, added filtration, allocation of dose between low- and high- energy views, and choice of decomposition algorithm and parameters therein. Theoretical calculations were validated in comparison to measurements of NPS, MTF, DQE, and NEQ performed on an experimental DE imaging system and through human observer studies for a variety of imaging tasks. Overall, the detectability index was found to provide a reliable predictor of human observer performance. Results identified optimal DE image acquisition and decomposition techniques that boost detectability beyond that achieved by conventional radiography or other DE imaging approaches, in many cases boosting conspicuity of subtle lesions from barely visible to highly conspicuous at fixed dose to the patient. The results are particularly encouraging, as such performance was achieved with the DE imaging dose equivalent to that of a single chest radiograph. The theoretical framework provided a valuable guide to optimization of a clinical prototype for high-performance DE chest imaging and may be extended to other DE imaging approaches, such as DE mammography and DE computed tomography.

Keywords: Medical Imaging
Dual-energy X-ray Imaging
Advisor: Siewerdsen, Jeffrey H.
Distribution of Human Tissue Kallikrein-Related Peptidases in Tissues and Biological Fluids: Localization, Hormonal Regulation and Physiological Functions in the Female Reproductive System

Human tissue kallikrein-related peptidases (KLK) are fifteen genes located on chromosome 19q13.4, encoding hormonally regulated, secreted serine proteases with trypsin/chymotrypsin-like activity. I identified expression of many KLKs in tissues throughout the female reproductive system and in cervico-vaginal fluid (CVF). The female reproductive system is hormonally regulated during the menstrual cycle, suggesting KLKs may also be regulated by these hormones. Measurement of KLKs levels in CVF and saliva samples throughout the menstrual cycle revealed a peak in expression following ovulation in both fluids. Progesterone levels rise during this period suggesting KLK regulation by progesterone during the menstrual cycle. Using proteomic techniques, I resolved the CVF proteome to identify potential KLK substrates. Among 685 proteins identified, several cell-cell adhesion molecules, cervical mucins and defense-related proteins were found. KLKs play a role in the desquamation of skin corneocytes through cleavage of cell-cell adhesion proteins. The vaginal epithelium undergoes cyclical changes during the menstrual cycle involving desquamation of cells upon rising progesterone levels. The post-ovulatory peak in KLK expression suggests that KLKs may contribute to cell desquamation during the menstrual cycle. Cervical mucus acts to block the uterus from vaginal microorganisms. Around ovulation, cervical mucus loses viscosity to facilitate sperm passage through the cervix. Proteolytic enzymes are thought to aid in this mucus remodelling. Our immunohistochemical studies localized KLK expression to the mucus secreting cervical epithelium and I investigated KLK processing of cervical mucin proteins in vitro. KLKs 5 and 12 were found to cleave mucins, suggesting their potential involvement in cervical mucus remodelling. CVF plays a role in host defense. KLKs are known to process the antimicrobial cathelicidin protein in skin and I investigated whether KLKs may also process antimicrobial proteins found in CVF. KLK5 was found to cleave defensin-1 alpha, in vitro, suggesting KLKs may aid in defense of the female reproductive system. Here I provide evidence of potential physiological roles for KLKs in the female reproductive system: in desquamation of vaginal epithelial cells, remodelling of cervical mucus and processing of antimicrobial proteins. These findings suggest KLKs may function in female fertility, in pathological conditions such as vaginitis and in host defense.

Keywords: kallikrein
reproduction
serine protease
vaginal physiology
Advisor: Diamandis, Eleftherios P.
Proteomic Analysis of Prostate Cancer Cell Line Conditioned Media for the Discovery of Candidate BIomarkers for Prostate Cancer

Early detection of prostate cancer is problematic due to the lack of a marker that has high diagnostic sensitivity and specificity. The prostate specific antigen test, in combination with digital rectal examination, is the gold standard for prostate cancer diagnosis. However, this modality suffers from low specificity. Therefore, specific markers for clinically relevant prostate cancer are needed. Our objective was to proteomically characterize the conditioned media from human prostate cancer cell lines to identify secreted proteins that could serve as novel prostate cancer biomarkers. An initial proof of principle study of the PC3 prostate cancer cell line was conducted. From this study over 200 proteins were identified in the conditioned media. Through gene ontology analysis and literature searches Mac-2 binding protein was selected as a candidate biomarker for validation in the serum of prostate cancer patients. A preliminarily validation showed that Mac-2 binding protein has discriminatory ability in prostate cancer diagnosis. However, an extended validation did not confirm this. Based on our proof of principle study we optimized our workflow and extended our analysis by culturing three different prostate cell lines [PC3 (bone metastasis), LNCaP (lymph node metastasis), and 22Rv1 (localized to prostate)]. We conducted a bottom-up analysis of each cell line by 2-dimensional liquid chromatography and tandem mass spectrometry. Of the 2124 proteins identified, 12% (329) were classified as extracellular and 18% (504) as membrane-bound. Among the identified proteins were known prostate cancer biomarkers such as PSA and KLK2. To select the most promising candidates for further investigation, tissue specificity, biological function, disease association based on literature searches, and comparison of protein overlap with the proteome of seminal plasma and serum were examined. Based on these results, several candidates were selected for validation in serum of patients with and without prostate cancer. Of these four novel candidates: follistatin, chemokine (C-X-C motif) ligand 16, pentraxin 3 and spondin 2 showed discriminatory ability. Of the four candidates, follistatin was further studied in an extended validation in serum of patients with biopsy confirmed prostate cancer and tissues of prostate cancer patients of low and high grade tumours by immunohistochemistry. In addition, follistatin was also investigated in the tissue of colon and lung cancer where intense staining was observed in one specimen of lung squamous carcinoma.

Keywords: Prostate Cancer
Biomarker
Proteomics
Conditioned Media
Mass Spectrometry
Cell Culture
Advisor: Diamandis, Eleftherios P.
Kallikrein-related Peptidase Signalling via Proteinase-Activated Receptors

The family of human kallikrein-related peptidases (KLKs) numbers 15 serine proteinases implicated in tumour progression. Despite the wide tissue distribution of KLKs and the numerous reports of their differential expression in pathological settings, the signalling mechanism(s) whereby these enzymes regulate tissue function are not yet known. Further, knowledge of the levels of their activity, as well as of their potential endogenous targets, has only been extracted from in vitro studies and cell culture systems. We hypothesized that KLKs can trigger tumour signalling via proteinase-activated receptors (PARs), a family of G-protein-coupled receptors. To test our hypothesis, we evaluated the ability of KLKs 5, 6, and 14: to activate or prevent signalling via PARs 1, 2, and 4 in cells and tissues expressing these receptors. Further, we used a novel activity-based probe approach, coupled with conventional immunoassay (ELISA), to determine the abundance of active KLK6 relative to total immunoreactive KLK6 in cancer-related biological fluids. We concluded that KLKs can regulate multiple signalling pathways triggered by PARs 1, 2, and 4, resulting in calcium release, platelet aggregation and vascular relaxation, and they can cause murine inflammation. Further, our activity-based ELISA demonstrated the presence of active KLK6 in ovarian cancer ascites fluids and cancer cell supernatants. We, therefore, suggest that tumours can produce active KLKs, which can potentially control tumour behaviour by regulating PAR activity.

Keywords: kallikrein-related peptidases
proteinase-activated receptors
cancer
inflammation
Advisor: Diamandis, Eleftherios P.
Public Reason and Canadian Constitutional Law

Liberals claim that the exercise of state power must be justified on terms that all citizens can reasonably accept. They also support democracy. The challenge is to bring these two desideratum in line-- to ensure that democratic deliberations are somehow predicated on claims that all citizens can reasonably accept. Put differently, the challenge is to set the terms of public reason. Liberal philosophers advance grand theories of political justice towards this end. They claim that a reasonable argument in the political sphere is one that conforms to theory x. The difficulty is that there will be those who reasonably reject theory x, preferring theory y or z, or eschewing theory altogether. Pessimism at the prospect of agreement on higher-order theories of justice leads some to advocate simple majority rule. The thesis argues that convergence on higher order theory is not essential to public reason. The Supreme Court of Canada’s method of adjudication under the Canadian Charter of Rights and Freedoms is used as a model. Where basic rights are engaged, or are alleged to be engaged, the Court examines the reasonableness of law and policy using a series of open-ended tests. These tests discipline their deliberations by focusing attention on generally accepted facts and values (notably, the values expressed by the Charter). The thesis contends that the Court’s open-ended, contextual approach can serve as a model for broader public reasoning. The thesis then explores the role of religious arguments within this model. In a polity committed above all to Charter values, what is the place of religion in the justification of law? It is argued that religion is understood to be private and inscrutable under the Charter. This is what justifies the Court’s generous reading of the right to religious freedom. It also justifies our forbidding state coercion in the name of religion. With the preceding ideas in mind, the thesis examines Canadian law and public discourse on the issues of therapeutic cloning (ch.4) and same sex marriage (ch.5).

Keywords: Public Reason
Canadian Charter of Rights and Freedoms
Liberalism
Separation of Church and State
Constitutional Law
Rawls
Advisor: Moreau, Sophia
International Patent Law: Cooperation, Harmonization and An Institutional Analysis of WIPO and the WTO

This work considers international cooperation or harmonization in patent law and analyzes the two main international patent law governance institutions: the World Intellectual Property Organization (WIPO) and the World Trade Organization (WTO). A welfarist approach is adopted, proposing that international patent law should improve global welfare, subject to assumptions that the preferences of the world population are heterogeneous, that governments try to maximize the welfare of their citizens, and that international legal organization faces collective action problems. Normatively desirable patent law harmonization reconciles strong reasons for preserving diversity (including the static and dynamic satisfaction of local preferences and adapting to unpredictable change) with strong reasons for cooperation (reducing duplication in patent prosecution, and reconciling imbalanced national externalities, incentives to innovation and costs). The last reason leads to a system of national treatment and minimum standards. The risks presented by the skewed nature of invention are addressed in the international patent system through a form of regional insurance. These reasons for cooperation present two linked but separable collective action problems, supporting the existence of two international institutions to govern patent cooperation. WIPO is best positioned to address duplication in patent prosecution. The WTO is best positioned to address imbalanced national externalities, incentives and costs. However, both the WIPO and the WTO are needed to provide a comprehensive international governance system. Questions about the WTO dispute resolution system, the TRIPs Council, and the WTO’s legitimacy are addressed by advocating a trade stakeholders’ model. Whether international patent law should be seen as a multilateral obligation or a nexus of bilateral obligations is explored. Given diverse national preferences and high uncertainty surrounding the welfare effects of specific patent policies, the process of harmonization is inevitably a political process. This political aspect directly connects the topic of patent law harmonization with the institutional analysis of WIPO and the WTO. The implementation of welfare-enhancing patent law cooperation is best guarded by a process with a wide range of political inputs and transparency. Ultimately, only good international governance can deliver on the potential of the international patent system to promote international innovation, economic growth and world-wide prosperity.

Keywords: patent
internatioinal
WIPO
WTO
cooperation
harmonization
law
institutional analysis
Advisor: Trebilcock, Michael
Functions of Lunatic and Manic Fringe in Regulating the Strength and Specificity of Notch Receptor-ligand Interactions during Hematopoiesis

Notch signals are required to promote T lineage commitment and development and suppress alternative cell fates in the thymus. Although the Notch activating ligand(s) in the thymus is(are) not known, studies have shown that hematopoietic progenitors are sensitive to Delta-like (DL), but not Jagged (Jag)-type ligands. In Chapter 3, I show that DL-expressing bone marrow stromal cell lines exhibit Notch ligand-independent functional heterogeneity in their capacity to support T cell development in vitro. These findings thus suggest the existence of stromal cell-derived signals that work with Notch to support T cell development. In Chapters 4 and 5, I investigated the ability of Fringe proteins to modulate Notch ligand-receptor interactions and the developmental consequences of these interactions for hematopoetic progenitors. Fringe proteins are glycosyl-transferases that enhance Notch activation by DL ligands and inhibit Notch activation by Jag ligands. In Chapter 4 I show that Lunatic Fringe (Lfng) enhances the strength of DL-mediated Notch activation to drive proliferation and expansion of early thymocytes and that DL4 and DL1 display different potencies to induce Notch-dependent outcomes. In Chapter 5, I demonstrate for the first time in a mammalian system that Lfng and Manic Fringe (Mfng) co-operate to enhance DL-Notch interactions and inhibit Jag-Notch interactions in hematopoietic stem cells. Thus, Lfng and Mfng function together to induce T cell development and inhibit B cell, myeloid and NK cell development. Collectively, these data highlight the importance of Fringe proteins in modulating the strength and specificity of Notch signaling levels during hematopoieisis.

Keywords: Immunology
hematopoiesis
T cell development
Notch signaling
lymphocyte development
Advisor: Guidos, Cynthia J.