Investigation into the early pathogenesis of lynch syndrome associated endometrial cancer

Endometrial cancer is the most common gynecologic malignancy in Canada with increasing rates of both incidence and mortality. Historically, histopathological subtyping of endometrial cancer was employed but was determined to be less than accurate at patient risk stratification. Recent advances in molecular subtyping have revealed mismatch repair deficient endometrial cancer as the second most deadly subtype. Mismatch repair deficient endometrial cancers may be acquired through somatic or germline aberrations to the four mismatch DNA repair genes: MLH1, PMS2, MSH2, and MSH6. Germline mutations to mismatch DNA repair genes results in Lynch Syndrome, an autosomal dominant hereditary cancer predisposition syndrome which increases the risk of endometrial cancer in affected individuals, however, the pathogenesis of Lynch Syndrome associated endometrial cancer has yet to be fully elucidated. To investigate the early pathogenic events following the introduction of MLH1 and PMS2 deficiencies, both in vivo and ex vivo models were utilized. A cohort (n = 6) of archival FFPE tissues, taken from prophylactic hysterectomies performed for Lynch Syndrome patients with germline MLH1 and PMS2 mutations, were immunohistochemically profiled for mismatch repair deficient endometrial glands. MLH1 deficient benign endometrial glands were identified in four cases with a novel report of zonal MLH1 deficient benign endometrial glands. In one case with zonal MLH1 deficient endometrial glands, multiplex immunohistochemical staining based panel was used to quantify endometrial epithelial cell differentiation by examination of secretory, ciliated, and proliferative cell types in both MLH1 proficient and deficient endometrial glands where no significant difference in cell type proportion was observed. MLH1 and PMS2 deficient endometrial organoid models were established using benign hysterectomy tissues and CRISPR-Cas9 mediated gene editing. Single cell RNA sequencing of MLH1 and PMS2 deficient endometrial organoids revealed no significant alterations in MLH1 deficient organoid cells and slight alterations to epithelial mesenchymal transition genes in PMS2 deficient organoids. Altogether, this data suggests that mismatch repair deficiency does not result in immediate changes to gene expression or cell differentiation in endometrial glands and that progression towards endometrial cancer is reliant on the accumulation of mutations.Medicine, Faculty ofGraduat

Investigation of myo-inositol Phosphates on Human Cells by Phenotypic Cell-Based Assays

Myo-inositol phosphates (IPs) are a large family of compounds that are ubiquitous in eukaryotes. They are involved in many critical cellular functions, from signal transduction to DNA repair in eukaryotes. Myo-inositol hexakisphosphate (InsP6), commonly known as phytic acid, is the most abundant cellular IP and previous studies have suggested that exogenous InsP6 has anti-proliferative effects on multiple cancer cell lines. By contrast, derivatives of InsP6 with lower phosphate number, such as myo-inositol (1,2,4,5,6)-pentakisphosphate (InsP5), have not been studied due to their costly preparation. Here we investigated the effects of InsP6 and InsP5 on the human bone osteosarcoma cell line, U2OS, by phenotypic assays. After 48 h after treatment with either InsP6 and InsP5 at 15 or 50 µM, cells display a striking vacuole formation, as observed by light microscopy. By staining with acridine orange and observing by fluorescence microscopy, the vacuolated cells showed acidic vesicle accumulation, indicative of increased autophagy. By staining with the non-permeant fluorescent dye Lucifer yellow and fluorescence microscopy we observed that vacuolated cells showed higher levels of the fluorescent dye, indicative of endocytosis, than non-treated cells. Vacuoles observed by phase contrast microscopy are frequently stained with Lucifer yellow and acridine orange. These findings suggest that InsP5 and InsP6 affect endocytosis, which subsequently leads to increased lysosomal degradation the engulfed material. Understanding the effects of IPs on human cells is important in understanding inositol signalling pathways and may lead to the discovery of novel anticancer compounds. *Indicates presente

Cable Transit De-icing Project

Holmes Solutions has presented two projects that require similar solutions - ice removal from the Switchback and the Whoosh, with the Switchback’s cables currently being the primary focus. The Frost Fighters have been tasked to find a way to stop ice formation on the Switchback’s cables so that the ride can operate safely in cold climates and during the colder months of the year. To be an adequate solution, the chosen de-icing method must operate in temperatures from -10°C to 40°C and be on standby in temperatures from -40°C to 60°C. In addition, it must prevent any falling debris that may harm people or cause damage to the environment underneath. Without a solution in place, ice on the cable reduces the coefficient of friction between the wheels of the trolley and the cable itself, which could prevent adequate braking prior to corners. Excessive ice and frost also have the potential to cause the cable to sag under the additional weight, which would create larger inclines in certain areas, thus creating a hazard for ride participants. Additionally, blocks of ice left frozen on the cable can pose the risk of derailment. With this in mind, the team has chosen to pursue a thermal approach to combat ice formation. While many thermal approaches were considered, the Frost Fighters decided to pursue a final solution that involves a heat cable being strapped to the underside of the ride’s main cable. The top of the heat cable is in direct contact with the underside of the main cable and insulation encapsulates all heat cable surfaces - the underside and sides - that would otherwise be exposed to the air. These components are bound together by attachments that are of adequate thickness for the Switchback’s wheels to pass over unhindered. This heat cable setup is connected directly to a wifi junction box which is supplied 240V by the electrical grid and protected by a 40A circuit breaker. The inclusion of a wifi junction box allows remote operation of the heated cable via an included phone app that can also be set on a regular schedule. With this setup in place, the Switchback will be able to safely operate in cold climates and during the colder months of the year. By staying operable year-round, Holmes Solutions’ Switchback profits will increase as the number of operable days will rise. In addition, Holmes Solutions will have an effective method to remove ice and prevent ice formation which may one day be utilized in Holmes’ other “Whoosh” project - which is essentially a larger scale Switchback - as well as other cable systems

WildMe - Scout

The Wild Me Scout team designed and developed a multi-platform mobile app with the goal of providing a more accessible way to report sightings of endangered animals in the wild. Both conservationists and the general public can send valuable sighting information to help combat extinction.https://pilotscholars.up.edu/egr_project/1076/thumbnail.jp