The human cell is complex, with countless processes ongoing in parallel in specialized compartments, the organelles. Cells can be studied in vitro by using immortalized cell lines that represent cells in vivo to a varying degree. Gene expression varies between cell types and an average cell line expresses around 10,000-12,000 genes, as measured with RNA sequencing. These genes encode the cell’s proteome; the full set of proteins that perform functions in the cell. In paper I we show that RNA sequencing is a necessary tool for studying the proteome of the human cell. By studying the proteome, and proteins’ localization in the cell, information can be assembled on how the cell functions. Image-based methods allow for detailed spatial resolution of protein localization as well as enable the study of temporal events. Visualization of a protein can be accomplished by using either a cell line that is transfected to express the protein with a fluorescent tag, or by targeting the protein with an affinity reagent such as an antibody. In paper II we present subcellular data for a majority of the human proteins, showing that there is a high degree of complexity in regard to where proteins localize in the cell. Cellular energy is generated in the mitochondria, an important organelle that is also active in many other different functions. Today approximately only a third of the estimated mitochondrial proteome has been validated experimentally, indicating that there is much more to understand with regard to the functions of the mitochondria. In paper III we explore the mitochondrial proteome, based on the results of paper II. We also present a method for sublocalizing proteins to subcompartments that can be performed in a high-throughput manner. To conclude, this thesis shows that transcriptomics is a useful tool for proteome-wide subcellular localization, and presents high-resolution spatial distribution data for the human cell with a deeper analysis of the mitochondrial proteome. / <p>QC 20170512</p>

Chromogranin A (CgA) and its cleavage product Catestatin (CST) are two very interesting proteins, especially due to their possible involvement in the development of type 1 diabetes. There are currently only a few primary antibodies against CgA and CST, and the ones that do exist are often only recommended for use on one method. Recently though, two new multifunctional antibodies were released, which are said to function on multiple methods. The aim of this project was to validate these two antibodies, one being the Chromogranin A Polyclonal Antibody (PA5-35071) from Thermo Fischer Scientific and the other being the Catestatin Polyclonal Antibody (289-MM-0288) from MediMabs. The methods of validation for these antibodies were western blot, flow cytometry, and immunofluorescent staining of frozen tissue. All study material were from mice and the tissues were chosen due to their established expression of CgA and CST. The results show that the antibodies are highly appropriate for use on flow cytometry and immunofluorescent staining of frozen tissue. The most appropriate concentration for the CgA-antibody was deemed as 1:25 for both flow cytometry and immunofluorescent staining. The most appropriate concentrations for the CST-antibody were deemed as 1:800 for flow cytometry and 1:1000 for immunofluorescent staining. However, the antibodies could not be validated for use on western blot. Further investigations are needed to determine if the antibodies are functional in western blot analysis or not. The validation performed here is an important first step in studying the involvement of these proteins in type 1 diabetes onset.

Active and passive transport of substances between the microcirculation in the brain and the central nervous system is regulated by the Blood-Brain Barrier (BBB). This barrier allows for chronic and acute modulation of the CNS microenvironment, and protects the brain from potentially noxious compounds carried in the circulatory system. In-vitro modeling of the BBB has become the target of much research over the past decade, as there are many unanswered questions regarding modulations in the permeability of this barrier. Additionally, the development of a practical and inexpensive model of the BBB would facilitate a much more efficient drug development process. The goal of this project is to investigate the formation of the BBB through assessment of tight junction formation and endothelial cell monolayer permeability. Accomplishment of this goal will include completion of the two primary aims of this thesis, which are 1) development of an immunohistochemical staining protocol for the labeling of tight junctional proteins, and 2) characterization of permeability across a porous membrane co-cultured with bovine aortic endothelial cells (BAECs) and C6 glioma cells. Both of these aims were met, as a reliable IF protocol for tight junctional staining was developed, and permeability values across a permeable membrane seeded with BAECs and C6s were collected. The completion of these aims has helped to accomplish the goal of investigating the formation of tight junctions in an in-vitro model of the BBB. The IF protocol that has been developed, along with the collected permeability data will aid the development of a more dynamic in-vitro model of the BBB to aid in research surrounding acute modulation of the BBB, along with facilitating a timelier drug development process.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

High-grade serous ovarian cancer (HGSOC) is the most aggressive subtype of ovarian cancer, and its heterogeneity poses a challenge for the discovery of reliable diagnostic biomarkers, therapeutic targets, and predicting treatment response, particularly to immunotherapy. The current standard diagnostic and treatment options are inadequate, resulting in late diagnosis and poor prognosis. To improve our understanding of the immunophenotype of tumours, potentially enhancing diagnostic and treatment capabilities, the aim of the present study was to develop a stringent workflow for studying the immune microenvironment of HGSOC tumours. We utilized publicly available single-cell RNA sequencing data and literature to identify genes enriched in certain cell types of HGSOC tumours, followed by validation using immunofluorescent-based multiplex protein profiling. A 9-plex immunofluorescence workflow was developed using the Opal™ system, and quantitative image analysis was performed to evaluate the expression of PD-L1, CD8A, FoxP3, CD163, KRT7, PDGFRB, and CD79A in large tissue sections of ovarian cancer. Each of these markers are specific to different cell types, and by staining the multiplex marker panel together with new markers with little or no literature linked to HGSOC we can gain novel insights on the immune microenvironment of HGSOC. In this project, for a proof of concept, we focused on two proteins; GZMK and SLAMF7. The optimized multiplex panel developed as part of this project will be used to identify cell-type-specific markers that may play a crucial role in the immune microenvironment of HGSOC, which could lead to better immunophenotype stratification of patients and a more optimal immunotherapy response. Moreover, the panel could also be used to study markers of less well-known immune cell types, further improving our understanding of HGSOC. Overall, this project has the potential to significantly contribute to the development of reliable diagnostic biomarkers and therapeutic targets for HGSOC, ultimately improving patient outcomes.

Cryptosporidium parvum is an important diarrhoea-associated protozoan, which is difficult to propagate in vitro. In 2017, a report described a continuous culture of C. parvum Moredun strain, in the oesophageal squamous cell carcinoma cell line COLO-680N, as an easy-to-use system for C. parvum propagation and continuous production of oocysts. Here, we report that—using the Köllitsch strain of C. parvum—even though COLO-680N cells, indeed, allowed parasite invasion and early asexual parasite replication, C. parvum proliferation decreased after the second day post infection. Considering recurring studies, reporting on successful production of newly generated Cryptosporidium oocysts in the past, and the subsequent replication failure by other research groups, the current data stand as a reminder of the importance of reproducibility of in vitro systems in cryptosporidiosis research. This is of special importance since it will only be possible to develop promising strategies to fight cryptosporidiosis and its ominous consequences for both human and animal health by a continuous and reliable methodological progress.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative tauopathy associated with exposure to repetitive head impacts (RHI) in contact sports. No treatments are currently available. Much of the focus in CTE has been on the microtubule-binding protein tau, which tends to accumulate within neurons and glia around blood vessels at the depths of cortical sulci. The mechanisms of tau accumulation and propagation in CTE are still unknown. The predilection for the perivascular region suggests inherent structural and/or cellular vulnerabilities in this area. Astrocytes are glial cells in this perivascular region that help form the blood brain barrier (BBB) and the neurovascular unit (NVU). Their endfeet envelop blood vessels and help transport nutrients from the blood into the brain, as well as clear harmful waste products out of the brain. Astrocytes are also vital players in many of the brain’s other normal physiological functions, including providing structural and metabolic support to neurons and maintenance of ion and water homeostasis. In response to injury or disease, astrocytes undergo a series of structural and functional changes in a process known as reactive astrogliosis. Astrogliosis is widely considered a hallmark of brain pathology, however, only recently have we begun to understand its functional implications. Astrocytes can respond heterogeneously to CNS insults, including either loss or increase of homeostatic functions, or gain of new, possibly toxic functions. These different astrocytic responses can either assist in recovery or further exacerbate injury. Our current understanding of how astrocytes are altered in RHI and CTE is limited. A degenerative phenotype has been identified in older donors with later stage CTE, but its presence in younger donors with earlier stage disease is unknown. The hypothesis of this study is that exposure to repetitive head trauma causes astrocytes to become reactive and adopt altered phenotypes, including loss of homeostatic functions, in brain areas known to be biomechanically susceptible to the shearing forces of head trauma, such as the perivascular region and interface of the grey and white matter at the depth of the cortical sulcus. These altered phenotypes are expected to be found in athletes with and without pathological tau deposition, highlighting astrocytes as potential therapeutic targets in the post-traumatic injury cascade. Specifically, I seek to characterize reactive astrocyte phenotypes and assess changes in their perivascular function in the brains of former American football players with and without a neuropathological diagnosis of CTE.

The normal aging process in humans is characterized by a number of hallmark changes including decreased white matter volume in the brain and accompanying cognitive decline. This is in contrast to neurodegenerative aging processes which involve acute pathology which results in neuronal cell death. Studying non-degenerative normal aging in humans can be difficult because of the high prevalence of neurodegenerative diseases in the population and other potentially confounding effects. Rhesus monkeys are an excellent model organism for the study of normal aging, as their aging process has been demonstrated to involve diminished white matter volume, but they do not suffer from neurodegenerative diseases such as Alzheimer's. In this study we seek to quantify levels of myelin degradation using confocal microscopy in regions of interest where it has been previously demonstrated that loss of white matter integrity results in lower levels of cognitive function across different treatment groups including aging monkeys, calorie restricted monkeys, and controls for calorie restricted monkeys. These areas include prefrontal white matter which is vital to executive function, the hippocampus which is integral to memory consolidation and the learning process, and finally the anterior, middle, and posterior cingulum bundle. The cingulum bundle contains a diverse variety of projections between cortical and subcortical regions, including but not limited to projections to and from the cingulate cortex which has been demonstrated to be vital for emotional processing, the limbic system, and a wide spectrum of other functions. We aim to quantify white matter degradation in these regions by using immunofluorescent tagging for healthy myelin basic protein (MBP) and degraded myelin basic protein (dMBP) and by measuring the colocalization between the two. For prefrontal white matter and hippocampus, we did not find significant differences in myelin degradation across treatment groups. In the cingulum bundle, however, we did find a significant effect of treatment on overall myelin damage throughout the bundle, and in particular we determined that there was a significant difference in colocalization in the anterior cingulum bundle between aging monkeys and control calorie restricted monkeys. Analysis of behavioral testing data yielded surprising results as we were unable to find a strong correlation between our measure for myelin degradation, and level of cognitive impairment. Our results indicate that there are likely differences in regional vulnerability to age related myelin damage across different white matter regions of the brain, however we would like to expand on this study to gain a more accurate understanding of how loss of white matter volume is distributed through the brain and the impact that has on cognitive outcomes.

No description available.