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Microglia are a tissue-resident immune cell of the central nervous system known to possess functions involved in immune surveillance and tissue homeostasis. Transcriptomics has characterised microglia and enabled discovery of microglial heterogeneity in addition to core microglial transcriptional programmes. However, investigation of microglia during severe inflammatory contexts has been challenging because no markers reliably discriminate them from the monocyte populations that ingress during inflammation. Nonetheless, candidate markers have been identified; these show promise in specific microglial identification yet remain to be widely validated. Within the literature, there are conflicting reports on how microglia regulate or promote inflammation depending on the tissue insult. However, it is well-recognised that the homeostatic state of microglia is altered yet it remains unknown if this state is restored post-resolution. Furthermore, understanding the plasticity of microglial responses to both acute and persistent inflammation within the eye will help to determine the extent to which different pathways are perturbed. The purpose of this thesis was to investigate the transcriptional changes that occur in retinal microglia in response to inflammation and whether the homeostatic threshold remains perturbed after acute and/or chronic inflammation. The data presented herein demonstrates how an ultra-low input mRNA-Seq approach was optimised and validated to permit transcriptomic assessment of low numbers of cells isolated from individual retinas. The Cx3cr1CreER:R26-tdTomato mouse line was then validated as microglial-specific during inflammation. mRNA-Seq was utilised to profile the temporal kinetics of the microglial transcriptome in the acute endotoxin-induced uveitis (EIU) model. Restoration of the microglial homeostatic state was confirmed, and key marker changes were orthogonally validated. Furthermore, C5AR1 was validated as a marker for differentiating microglial subsets during inflammation. The next steps have begun to examine microglial behaviour in experimental autoimmune uveitis (EAU), a model of chronic inflammation, and new approaches are being optimised to better understand tissue heterogeneity.

Female adults of many mosquito species possess distinct physiological features adapting to blood feeding for successful reproduction. The disease pathogens that are transmitted by mosquitoes have evolved to take advantages of the indispensable blood feedings to complete their transmission cycles and to survive attacks from the mosquito's innate immune system. Normal egg development and mosquito immunity are tightly controlled by tissue- and stage-specific gene expression and coordinated by many signal molecules in the mosquito. Understanding gene regulation affecting mosquito reproduction and malaria parasites infection is of paramount importance for developing novel malaria control strategies. A growing body of evidence indicates that microRNAs (miRNAs) are involved in egg maturation and immune reactions against invading pathogens in mosquitoes. However, the molecular mechanisms by which specific miRNAs selectively modulate reproduction and the survival of pathogens are largely unknown. The miRNA-induced gene-silencing pathway in mosquitoes was mostly extrapolated from the studies of flies. To explore the dynamics of miRNAs in reproduction, I used small RNAs sequencing to monitor miRNAs expression and their association with Argonaute 1 (Ago1) and Argonaute 2 (Ago2) in the malaria mosquito Anopheles gambiae (An. gambiae) during the 72-h period immediately after blood feeding. I found the abundance and Ago loading of most of the mature miRNAs were relatively stable after blood ingestion. However, miRNAs of the miR-309/286/2944 cluster were considerably upregulated after blood feeding. I confirmed that miR-309 is essential for normal egg development by depletion of endogenous miR-309 with a specific antagomir. In addition, my results showed that the Ago association of some miRNAs was not proportional to their cellular abundance implying additional regulation at miRNA integration. To investigate the functional roles of miRNAs and define context-dependent miRNA-mRNA interactions during the reproductive process, I have applied an innovative experimental approach to study miRNA-mRNA interactome. CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP can generate miRNA-mRNA chimeras from UV-irradiation stabilized Ago-miRNA-mRNA complex. My results have defined tens of thousands of miRNA-mRNA interactions in mosquitoes, including novel targets for mosquito-specific miRNAs. Verification of the predicted interactions using mRNA-seq, ribosome-profiling, and luciferase reporter assay revealed a reliable miRNA-mRNA interaction network. Based on the detected interactions, I refined the paring rules for mosquito miRNAs and illustrated the dynamic pairing between different regions of miRNAs with their targets in vivo. The miRNA-mRNA interactions were compared using this approach at multiple time points before and after blood feeding. Importantly, this study showed that the interactions were dynamic and enriched in genes that are involved in metabolisms, supporting the proposed functions of miRNAs in coordinating the gene regulation in mosquito reproduction. Plasmodium falciparum (P. falciparum) is a major human malaria parasite. To understand the functions of miRNAs in the mosquito resistance to Plasmodium infection, we analyzed the miRNA-mRNA interactions after female mosquitoes taking a P. falciparum-infected blood meal or an uninfected blood meal. Comparison of the interactions revealed enhanced miRNA-mRNA interactions after P. falciparum infection involving a group of immunity-related genes. In summary, this study has provided a systematic view and significantly advanced our understanding of the miRNA functions in mosquito reproduction and P. falciparum infection.