Medical Biology - Theses
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ItemMechanisms of tumorigenesis and therapy resistance in breast cancer( 2021)Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. Breast cancer treatments are subtype-based but eventually cancer cells become resistant, with tumour relapse remaining a significant challenge for patient management. This thesis addresses tumour suppressor genes that drive breast tumorigenesis and potential mechanisms of therapy resistance. The first section of the thesis describes a framework for the identification and validation of novel tumour suppressor genes that were identified in an in vivo genome-wide CRISPR/Cas9-screen in Trp53+/– heterozygous mice. The genes that encode for the scaffold protein Axin1 and the regulatory subunit from the protein kinase A complex, Prkar1a were identified as tumour suppressor genes that collaborate with loss of Trp53 in mammary tumorigenesis. Axin1- and Prkar1a-deleted organoids showed a morphological change with an increased proliferative capacity. RNAseq analysis revealed potential deregulation of cellular energetics in Axin1 mutant organoids. In addition, direct in vivo genome editing via intraductal injection of lentiviral vectors engineered to express dual short-guide RNAs validated Axin1 and Prkar1a as potent tumour suppressor genes that cooperate with loss of Trp53 to accelerate tumorigenesis each combination gave rise to hormone receptor negative tumours. In the second part of this thesis, mass cytometry was applied to breast cancer patient-derived xenografts (PDXs) to understand changes in cell survival and signal transduction pathways at the single cell level. Mass cytometry can detect up to 40 different protein markers via antibodies conjugated to heavy metals. Two primary questions were investigated: (a) what is the proportion of cells within a tumour that respond to treatment and (b) what molecular changes contribute to therapy resistance and tumour relapse? Short- and long-term dual therapy consisting of ABT-199 plus fulvestrant for ER+ PDX models or S63845 plus docetaxel for BRCA1-mutated triple negative PDX models were performed. Mass cytometry analysis revealed a PDX model-dependent response to short-term treatment, with the percentage of responsive tumour cells ranging from 1.5 - 33.5%. In four BRCA1-mutated and two ER+ PDX models tumour resistance was found to be accompanied by upregulation of a subset of proteins specific to each model. Recurrent upregulated proteins included EGFR, pAKT and BCL-xL. Inhibition of these targets in a triple combination treatment strategy may augment treatment response and prevent tumour relapse. Furthermore, tumour cells responsible for relapse are likely pre-existing since most PDX models showed similar number of cell clusters pre- and post-therapy.
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ItemMolecular mechanisms of liver infection by the human malaria parasite Plasmodium falciparum( 2021)Malaria is the disease caused by Plasmodium parasites. The parasite infects the red blood cells giving rise to symptoms but it musts first infect the liver to reach the blood. Blocking liver infection would prevent both malaria disease and onward transmission as well as stimulate immunity. However, little is known about parasite-host interactions during liver infection of Plasmodium falciparum, the species responsible for the most lethal form of malaria in humans, as its pre-erythrocytic stages are challenging to study. Plasmodium sporozoites are injected in the dermis by the bite of an infected mosquito. They make their way from the skin to the bloodstream and finally the liver, where they invade and replicate within a hepatocyte. The sporozoite’s journey from the skin to the host liver is enabled by a remarkable process called cell traversal that allows parasites to migrate and penetrate deeper into host tissues by entering and then rupturing host cells. Little is known about the key molecular interactions involved in this mechanism especially with respect to the host cell. There is a lack of knowledge about the importance of host factors and proteins involved in sporozoite infectivity. A deeper understanding of cell traversal and hepatocyte invasion could lead to novel interventions. This work aimed to identify key proteins involved in cell traversal and hepatocyte invasion by P. falciparum. A robust sporozoite production protocol was initially established to ensure the feasibility of the project. Host factors involved in cell traversal were systematically investigated using a whole genome CRISPR/Cas9 knock-out screen. The unbiased screen was enabled by the design of a new positive selection cell traversal assay that kills traversed hepatocytes, permitting the enrichment of traversal-resistant cells. Validation of more than one hundred curated hits identified several human genes involved in infection by other pathogens that are putative proteins involved in P. falciparum cell traversal. Finally, antibodies targeting different regions of the most abundant P. falciparum sporozoite surface protein — the circumsporozoite protein (CSP) — were characterised for their inhibition potential. To do so, an improved method allowing both cell traversal and hepatocyte invasion by P. falciparum sporozoite to be quantified by flow cytometry was established before inhibition assays were performed. Different inhibition profiles were identified, highlighting a role for the N-terminus of CSP in hepatocyte invasion. Identifying essential factors and parasite-host interactions during this first step of the malaria parasite lifecycle will provide more insight into support of a prophylactic treatment for malaria.