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  • (2022) Bello, Idris
    Thesis
    The inflammatory artery diseases atherosclerosis and abdominal aortic aneurysm (AAA) are major causes of morbidity and mortality and there is significant attention towards identifying and targeting prominent inflammatory mediators underpinning these cardiovascular diseases. In the first chapter, the role of the pro-inflammatory and pro-oxidant enzyme myeloperoxidase (MPO) in inflammatory artery disease was studied. A clinical study showed that while circulating plasma MPO levels were not different in AAA patients versus healthy controls, immunohistochemistry showed that the MPO protein was prevalent in human AAA tissue. In the angiotensin II (AngII)-infusion model of AAA and atherosclerosis in apolipoprotein-E gene-deficient (ApoE–/–) mice, administration of 2-thioxanthines (2-TX), a clinically-trialled MPO inhibitor, significantly inhibited AAA but not atherosclerosis. Paradoxically, MPO gene-deficiency did not affect AngII-induced AAA but attenuated atherosclerosis. Notably, 2-TX significantly inhibited AAA in ApoE–/–MPO–/– mice, indicating 2-TX protects against aortic disease in the absence of MPO. The role of MPO in the diabetes-accelerated atherosclerosis in ApoE–/– mice was also examined. While MPO gene-deficiency did not impact on the degree of diabetes it significantly reduced diabetes-accelerated atherosclerosis at the brachiocephalic artery and aortic sinus, but not aortic arch, indicating that MPO exhibits site-specific effect on atherosclerosis. A second chapter focused on semicarbizide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1), a pro-inflammatory enzyme that facilitates the vascular recruitment of activated leukocytes. Treatment of AngII-infused ApoE–/– mice with a clinically-viable SSAO/VAP-1 inhibitor significantly protected against AAA and atherosclerosis, independent of alterations to plasma lipid levels. A third chapter tested the therapeutic efficacy of apolipoprotein A-I (ApoA-I), the major cardioprotective protein in high-density lipoproteins, and a class of immunomodulatory nanoparticles (INPs), which selectively target and disable a pro-inflammatory monocyte subset. Although ApoA-I treatment did not impact on the development of arterial disease in AngII-infused, aged ApoE–/– mice, INPs provided significant protection against AAA and atherosclerosis in these mice. This novel research provides new insights on the roles of MPO and SSAO in AAA and atherosclerosis and identified clinically-viable inhibitors of MPO and SSAO and a class of biodegradable immunomodulatory nanoparticles as potential new therapeutics for treating inflammatory artery disease.

  • (2024) Rouaen, Jourdin
    Thesis
    Anti-GD2 immunotherapy has significantly enhanced survival of high-risk neuroblastoma patients however efficacy is strongly hampered by the immunosuppressive tumour microenvironment. Given the emerging link between copper and immune evasion, we assessed the impact of copper chelation therapy on neuroblastoma using the agents tetraethylenepentamine (TEPA) and the clinically approved analogue triethylenetetramine (TETA; marketed as Cuprior®). Using the preclinical TH-MYCN model, we performed single-cell RNA sequencing supported by OPAL multiplex immunohistochemistry and cytokine profiling to assess cellular and molecular changes occurring with TEPA treatment in the neuroblastoma tumour microenvironment. Copper chelation was observed to successfully deplete intratumoural copper to reinvigorate anti-tumour immunity as signalled by increased infiltration and activity of pro-inflammatory immune cells, specifically N1 neutrophils. Mechanistic in vitro assays reveal sequestration of copper by neuroblastoma cells causing dysregulated neutrophil function, with successful reversal upon TEPA treatment. Findings propose a novel mechanism of immune evasion, highlighting copper chelation as a therapeutic strategy to counteract immunosuppression. Copper chelation is further shown to increase GD2 expression, while also enhancing neutrophil antibody-dependent cellular cytotoxicity in vitro. Using the TH-MYCN model, TEPA was found to potentiate anti-GD2 therapy to achieve durable tumour control. This was associated with increased Fc-receptor-bearing natural killer and CD11B+ myeloid cells capable of performing antibody-dependent cellular cytotoxicity. Finally, we evaluated TETA for repurposing as a novel immunomodulatory agent. Comparative in vitro studies with TEPA confirm the ability of TETA to deplete intratumoural copper and downregulate immune checkpoint proteins Programmed Death-Ligand 1 (PD-L1) and indoleamine-pyrrole 2,3-dioxygenase (IDO). Using the preclinical NXS2 model, TETA exhibited an exceptional safety profile without altering copper levels or GD2 expression in healthy nerve tissue. Combination with anti-GD2 therapy achieved durable tumour control with no relapses occurring after treatment cessation and was similarly associated with infiltration of pro-inflammatory immune cells. Collectively, study findings credential copper chelation as a non-toxic strategy to overcome the immunosuppressive tumour microenvironment and improve neuroblastoma patient outcomes.

  • (2024) Santos Rodriguez, Gabriela
    Thesis
    An important question in Biology is how biological systems' complexity has expanded while the number of protein-coding genes has remained mostly stable. Previous research has shown that increased biological complexity has arisen partly from the dynamic generation of unique cell-specific transcriptomes. However, studies of tissues across distant animal lineages have shown that gene expression is highly conserved between the same tissues in different species. Hence, gene expression alone is unlikely to explain the expansion in complexity across vertebrate evolution. Instead, it is becoming evident that the plethora of post-transcriptional mechanisms expanding transcriptomic diversity underlies these advances. Among these, alternative splicing (AS), the process by which multiple distinct transcripts and protein variants express a single gene, appears to be the most widespread, as it regulates more than 95% of multi-exon genes. However, further layers of post-transcriptional regulation are known to impact tissue-specific evolution. These include the creation of non-coding RNAs, alternative promotor usage (APU), and alternative cleavage and polyadenylation (APA). In this thesis, I first focus on circular RNAs (circRNAs) as they are the product of back-splicing, affecting transcriptome complexity. Comparisons of circRNAs in human and mouse have shown that most human circRNAs are absent in mouse, suggesting a rapid expansion of circRNA molecules during evolution. Thus, I used transcriptomes of primate species to understand the evolutionary dynamics of circRNA in closely related species. I found that most circRNAs across primates are species-specific. Still, a small subset has a conserved neural expression. Such conserved circRNAs have longer downstream introns due to the insertion of novel retrotransposons than non-conserved circRNAs. Another limitation in the literature is the usage of is the use of short-read sequencing technology. Therefore, the conservation of full transcript expression and the coordinated regulation of AS, APA and APU events across evolution remains unknown. Thus, in the present thesis, I used comparative transcriptomics across vertebrate species to assess the conservation of mRNA isoforms and coordinated splicing events of AS, APA and APU using direct RNA Nanopore long-read sequencing. I found that most conserved coordinate splicing events are associative and tissue-specific. Still, a set of conserved coordinate splicing events is expressed across multiple tissues and regulates basic cellular functions. Altogether, in my thesis, I showcase the effect of distinct evolutionary processes affecting transcriptome complexity and its impact on phenotypic complexity.