Single cell isolation from helminth-infected murine intestines has been notoriously difficult, due to the strong anti-parasite type 2 immune responses that drive mucus production, tissue remodeling and immune cell infiltration. Through the systematic optimization of a standard intestinal digestion protocol, we were able to successfully isolate millions of immune cells from the heavily infected duodenum. To validate that these cells gave an accurate representation of intestinal immune responses, we analyzed them using a high-dimensional spectral flow cytometry panel and confirmed our findings by confocal microscopy. Our cell isolation protocol and high-dimensional analysis allowed us to identify many known hallmarks of anti-parasite immune responses throughout the entire course of helminth infection and has the potential to accelerate single-cell discoveries of local helminth immune responses that have previously been unfeasible.

Single cell isolation from helminth infected intestines has been notoriously difficult, due to the strong anti-parasite type 2 immune responses that drive mucus production, tissue remodeling and immune cell infiltration. Through the systematic optimization of a standard intestinal digestion protocol, we were able to isolate millions of immune cells from heavily infected tissues. Using this protocol, we validated many hallmarks of anti-parasite immunity and analyzed immune cells from the lamina propria and granulomas during helminth development, as well as acute and chronic worm infection.

No abstract

<p>Cancer is a disease characterised by the uncontrolled growth of mutated cells, and is one of the leading causes of death worldwide, with over a third of people diagnosed with cancer in their lifetime. Despite extensive investment of both time and money in cancer research, poor patient outcomes and quality of life, and the evolution of treatment resistant cancers indicates that continued research, and more efficacious therapies are required. A recent investigation identified a mycothiol analogue which displayed significant toxicity in the promyelocytic leukemia cell line (HL60). Designed as a negative control, no biological activity was expected from this compound and its cellular target and mode of action are unknown.  This thesis describes the synthesis of a toxic trichlorovinyl cysteine-containing analogue of mycothiol, and the attempted synthesis of a propynylated and fluorescent derivative of this. The research also details immunomodulatory investigations, which were undertaken to probe the mode of action of the lead compound, and to determine whether its precursor, N-Boc-S-trichlorovinyl cysteine, induced toxicity through the same mechanism. The lead compound demonstrated mild immunomodulatory activity in splenocytes isolated from euthanised C57BL/6 mice, and enzyme linked immunosorbent assays revealed a likely Th2 mediated response, induced by the production of IL-4. The precursor however appears to promote a strong pro-inflammatory response, by inducing IL-17a production, which is widely considered a deleterious immune response in cancer. Whilst further work is required to determine the cellular target of the lead compound, the research described demonstrates the potential for this compound as an anti-cancer agent, while the precursor appears inappropriate for further development.</p>

<p>Cancer is a disease characterised by the uncontrolled growth of mutated cells, and is one of the leading causes of death worldwide, with over a third of people diagnosed with cancer in their lifetime. Despite extensive investment of both time and money in cancer research, poor patient outcomes and quality of life, and the evolution of treatment resistant cancers indicates that continued research, and more efficacious therapies are required. A recent investigation identified a mycothiol analogue which displayed significant toxicity in the promyelocytic leukemia cell line (HL60). Designed as a negative control, no biological activity was expected from this compound and its cellular target and mode of action are unknown.  This thesis describes the synthesis of a toxic trichlorovinyl cysteine-containing analogue of mycothiol, and the attempted synthesis of a propynylated and fluorescent derivative of this. The research also details immunomodulatory investigations, which were undertaken to probe the mode of action of the lead compound, and to determine whether its precursor, N-Boc-S-trichlorovinyl cysteine, induced toxicity through the same mechanism. The lead compound demonstrated mild immunomodulatory activity in splenocytes isolated from euthanised C57BL/6 mice, and enzyme linked immunosorbent assays revealed a likely Th2 mediated response, induced by the production of IL-4. The precursor however appears to promote a strong pro-inflammatory response, by inducing IL-17a production, which is widely considered a deleterious immune response in cancer. Whilst further work is required to determine the cellular target of the lead compound, the research described demonstrates the potential for this compound as an anti-cancer agent, while the precursor appears inappropriate for further development.</p>