Biochemistry and Pharmacology - Theses
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ItemNo Preview AvailableUsing Artificial Intelligence to Improve the Diagnosis and Treatment of Cancer( 2022-12)Cancer is a complex and heterogeneous disease driven by the accumulation of mutations at the genetic and epigenetic levels—making it particularly challenging to study and treat. Despite Whole-genome sequencing approaches identifying thousands of variations in cancer cells and their perturbations, fundamental gaps persist in understanding cancer causes and pathogenesis. Towards this, my PhD focused on developing computational approaches by leveraging genomic and experimental data to provide fundamental insights into cancer biology, improve patient diagnosis, and guide therapeutic development. The increased mutational burden in most cancers can make it challenging to identify mutations essential for tumorigenesis (drivers) and those that are just background accumulation (passenger), impacting the success of targeted treatments. To overcome this, I focused on using insights about the mutations at the protein sequence and 3D structure level to understand the genotype-phenotype relationship to tumorigenesis. I have looked at proteins that participate in two DNA repair processes: primarily non homologous end joining (NHEJ) along with eukaryotic homologous recombination (HR), where missense mutations have been linked to many diverse cancers. The molecular consequences of these mutations on protein dynamics, stability, and binding affinities to other interacting partners were evaluated using in silico biophysical tools. This highlighted that cancer-causing mutations were associated with structure destabilization and altered protein conformation and network topology, thus impacting cell signalling and function. Interestingly, my work on NHEJ DNA repair machinery highlighted diverse driving forces for carcinogenesis among core components like Ku70/80 and DNA-PKcs. Cancer-causing mutations in anchor proteins (Ku70/80) impacted crucial protein-protein interactions, while those in catalytic components (DNA-PKcs) were likely to occur in regions undergoing iii purifying selection. This insight led to a consensus predictor for identifying driving mutations in NHEJ. While when assessing the functional consequences of BRCA1 and BRCA2 genes of HR DNA repair at the protein sequence level, this methodology underlined that cancer-causing mutations typically clustered in well-established structural domains. Using this insight, I developed a more accurate predictor for classifying pathogenic mutations in HR repair compared to existing approaches. This broad heterogeneity of cancers complicates potential treatment opportunities. I, therefore, next explored the properties of compounds potentially active against one or various types of cancer, including screens against 74 distinct cancer cell lines originating from 9 tumour types. Overall, the identified active molecules were shown to be enriched in benzene rings, aligning with Lipinski's rule of five, although this might reflect screening library biases. These insights enabled the development of a predictive platform for anticancer activity, thereby optimizing screening libraries with potentially active anticancer molecules. Similarly, I used compounds' structural and molecular properties to accurately predict those compounds with increased teratogenicity early in the drug development process and prioritize drug combinations to augment combinatorial screening libraries, potentially alleviating acquired drug resistance. The outcomes of this doctoral work highlight the potential benefits of using computational approaches in unravelling the underlying mechanisms of carcinogenesis and guiding drug discovery for designing more effective therapies. Ultimately, the predictions generated by these tools would improve our understanding of the genotype-phenotype association, enabling promising patient diagnosis and treatment.
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ItemReaction hijacking tyrosyl-tRNA synthetase as a new anti-infectives strategy( 2022)Malaria is a deadly disease of humans, with Plasmodium falciparum responsible for the most cases. Disappointingly, drug resistance is observed against current front-line therapies; thus, new drugs with novel mechanisms are urgently needed. ML901, a nucleoside sulfamate derivative, has been shown to possess good antimalarial efficacy and to specifically target P. falciparum tyrosyl-tRNA synthetase (PfYRS). PfYRS is a pivotal enzyme that participates in the protein synthesis pathway, in which tyrosine-charged tRNA is formed. ML901 appears to target PfYRS via a novel reaction hijacking mechanism in which PfYRS catalyzes the synthesis of a Tyr-ML901 adduct, which in turn poisons the enzyme. Human YRS is not susceptible to the reaction hijacking mechanism. This project sought to understand the molecular basis for the potency and specificity of ML901 and to determine if reaction hijacking could be exploited more widely. All YRS sequences harbor a conserved motif, referred to as “KMSKS”, in a loop that is reported to change conformation to facilitate ATP binding and the aminoacylation reaction. Sequence alignment across species reveals that most pathogenic parasites, including P. falciparum, possess a KMSKS motif, whereas higher eukaryotes possess an equivalent KMSSS motif. Structural analysis revealed that the motif in human YRS is part of a flexible (unstructured) loop while the equivalent loop is structured in PfYRS. Here we examined the role of the second lysine (K250) in determining loop flexibility and activity of PfYRS as well as the susceptibility of the mutant enzyme to reaction hijacking. Surprisingly, the X-ray crystal structure of recombinant PfYRS harboring the K250S mutation (PfYRSK250S) showed that the KMSSS loop is even more stable than the wildtype KMSKS loop. PfYRSK250S was found to consume substantively less ATP in the initial activation step. However, the weakly active PfYRSK250S is still susceptible to reaction hijacking by ML901. This study shows that the flexibility of the loop is not determined simply by the K250. Moreover, it shows that K250 plays an important role in enzymatic mechanism. Further investigations are required to understand the important factors that contribute to the particular susceptibility of PfYRS to reaction hijacking by ML901. Broad specificity nucleoside sulfamates, such as adenosine sulfamate (AMS), have previously been shown to have inhibitory activity against Gram-positive and Gram-negative bacteria. The equivalent of the KMSKS motif in the Escherichia coli YRS sequence is KFGKT. Here we explored the possibility that bacterial YRS might also be susceptible to inhibition via a reaction hijacking mechanism. A screen of a range of bacterial species revealed that AMS inhibits growth of E. coli and Enterococcus faecium. Targeted mass spectrometry confirmed the production of a range of amino acids adducts upon treatment in E. coli with AMS. Recombinant EcYRS was purified and expressed and shown to be inhibited via the reaction hijacking mechanism by AMS. These data suggest that bacterial amino acyl tRNA synthetases may be exciting new targets for reaction-hijacking nucleoside sulfamates.
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ItemDevelopment of an extracellular vesicle-based therapeutic for wound healing.( 2022)Chronic wounds are defined as wounds that fail to progress through the normal stages of wound healing and stay unhealed for longer than 4 weeks. Such wounds affect 1-2 % of the population are characterized by the presence of phenotypically abnormal cells and disruptions in the inflammatory and proliferative phases of normal wound healing. Current treatment options have been ineffective as they focus on wound management and do not adequately address the underlying physiological causes. For example, platelet-derivatives have been tested due to their rich growth factor content but have yielded varying efficacies. To date, chronic wounds are responsible for a lower-limb amputation every 30 seconds globally and no new treatments have been approved for use by the FDA since 1994, highlighting the need for a new and effective treatment modality. Although platelets are well-known for their role in coagulation following an injury, they are also potent inducers of tissue regeneration. More recently, platelets have been shown to exert these regenerative properties through the release of extracellular vesicles (EVs). Platelet-derived EVs (pEVs) are spherical vesicles surrounded by a lipid bilayer enclosing a myriad of bioactive molecules. pEVs represent a new therapeutic approach as they are not only enriched in numerous growth factors and nucleic acids but also possess unique biophysical properties that are advantageous as biologic medicines. However, the clinical development of EVs as a whole, has been hampered due to the lack of a suitable isolation method. For example, high-speed differential centrifugation is currently the most-widely used method for isolation of EVs and results in their aggregation and loss of functional activity. An ideal isolation method for the clinical manufacture of EVs would have to be effective, scalable, and amenable to Good Manufacturing Practice (GMP) without compromising the structural and functional integrity of the EVs isolated. To address this, Exopharm Ltd has developed a novel isolation technology based on ion-exchange chromatography (IEX) called Ligand Exosome Affinity Purification (LEAP) that could overcome the scalability and purity challenges posed by other methods. However, the use of LEAP to isolate pEVs had not been previously tested. Hence, the first aim of this study was to induce the release of pEVs from platelets using cold-activation and to isolate the resulting pEVs using LEAP. A proliferation assay using dermal fibroblasts was developed to assess LEAP-isolated pEVs for functional activity. pEVs isolated using LEAP were found to significantly induce fibroblast proliferation. pEVs were also characterized for size and morphology and were found to adhere to guidelines set by the International Society of Extracellular Vesicles (ISEV). The second aim of this study was to conduct proteomic and transcriptomic analyses on pEVs from cold-activated platelets and isolated using LEAP to characterize their content and identify key proteins and nucleic acids that positively modulate wound healing process. Proteomic analyses through mass spectrometry revealed the presence of growth factors such as IGF and TGF-b that induce pro-regenerative and -angiogenic properties in recipient cells. Transcriptomic analyses through RNA sequencing reported that pEVs are enriched for microRNAs such as mir-21 and mir-126 that have been previously found to be downregulated within chronic wounds. Hence, content characterization provided first evidence that pEVs from cold-activated platelets and isolated using LEAP contain proteins and microRNAs important for cellular functions involved in healthy wound healing. The third aim of this study was to assess the functional activity of pEVs in in vitro assays. pEV-treatment was found to significantly induce proliferation of dermal fibroblasts and bone-marrow derived mesenchymal stem cells, both cell types important for generating new tissue following wounding. pEVs were also shown to increase the migratory capacity of fibroblasts and angiogenic capacity of dermal endothelial cells. Hence, pEVs from cold-activated platelets were found to positively influence multiple cell processes that are necessary for the proliferative phase of wound healing, that are often impaired in chronic wounds. Lastly, the use of pEVs as therapeutic for wound healing was assessed in a Phase I clinical trial to observe safety and biological activity following administration. One participant was successfully enrolled in the study and was administered autologous pEVs following a skin punch biopsy. No adverse events were reported and pEV-treatment was deemed to be safe and well-tolerated. Furthermore, histological studies of wound tissues procured at Day 7 following treatment showed increased presence of macrophages, keratinocytes and proliferating cells within pEV-treated tissue compared to the untreated control. This study was the first-in-human application of pEVs from cold-activated platelets and support the clinical development of pEVs as a next-generation therapeutic for chronic wounds.
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ItemLegionnaires’ Disease - the impact of cigarette smoke and functional lung recovery( 2022)Legionnaires’ Disease is a severe type of pneumonia most commonly caused by the bacterial species Legionella pneumophila and Legionella longbeachae. Epidemiological studies show that cigarette smoke is a major risk factor for susceptibility to Legionnaires’ Disease, but the underlying mechanisms for this connection are not known. During pneumonia, efficient oxygen uptake in the lung is compromised due to pathogen invasion and the resulting immune response. Some types of immune cells are known to resolve inflammation and promote regeneration of lung structure and function. However, the underlying mechanisms by which immune cells aid in lung recovery from bacterial pneumonia are not well understood. We used a mouse model of acute cigarette smoke exposure and Legionella infection to assess how cigarette smoke could impact the immune response towards Legionella and confer more severe disease. This work provides evidence that acute cigarette smoke exposure alone depleted alveolar macrophages (AM) in lungs of wild-type mice, which is contrary to the currently accepted view that smoking causes accumulation of AM within the airways. We investigated the cell death pathways that could be activated by cigarette smoke in AM and found that, in smoke-treated ASC-/- and NLRP3-/- mice, the smoke-induced AM depletion observed in wild-type mice was reversed. These results suggest an important role of NLRP3-dependent pyroptosis, a type of inflammatory cell death, in driving smoke-induced AM death in vivo. Legionella sp. subvert host immunity to establish a protected vacuole for bacterial replication within AM. Concurrent infection of smoke-treated mice with L. pneumophila caused more severe disease progression and significantly delayed bacterial clearance. In a model of clodronate-induced AM depletion, L. pneumophila clearance was similarly delayed in later stages of infection, despite limited bacterial replication early after infection due to the depletion of the bacteria’s replicative niche. In contrast, after concurrently infecting smoke-exposed mice with L. longbeachae, bacterial clearance was slightly accelerated. Therefore, smoke-induced AM death may be a risk factor for Legionnaires’ Disease caused by L. pneumophila. How AM mediate bacterial clearance under normal circumstances remains subject of future investigation. Using the mouse model of Legionnaires’ Disease, we assessed how immune cells impacted functional lung recovery. Surprisingly, we observed that neutrophils play an important role in the re-establishment of efficient oxygen uptake during the recovery phase from L. longbeachae infection. Neutrophils promoted the proliferation of type II alveolar epithelial cells, local progenitor cells that regenerate the alveolar epithelium. Mechanistically, neutrophils contributed to the production of several cytokines and growth factors associated with epithelial cell proliferation after pulmonary infection such as amphiregulin, TNF-alpha, or IL-1beta. These results provide novel insights into a regenerative role of neutrophils in the recovery phase of bacterial pneumonia.
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ItemMacrophage lipid metabolism in the intracellular survival of Leishmania mexicana( 2022)The parasitic protist Leishmania causes a spectrum of diseases with significant global impact. Current antileishmanial drugs are limited and undermined by drug resistance. Macrophages act as the Leishmania replicative niche or clear intracellular parasites, depending on their activation state. Interventions regulating macrophage activation and/or permissiveness for parasite growth are increasingly viewed as attractive therapeutic strategies that are less vulnerable to development of resistance. This study examines the role of macrophage lipid metabolism in the intracellular survival of L. mexicana with particular focus on omega-3 and -6 polyunsaturated fatty acids (PUFAs) and associated receptors. In Chapter 3, a fluorescent parasite strain (L. mexicanaTurboRFP) was generated to enable the detection, measurement, and characterisation of infected host cells. L. mexicanaTurboRFP was used to develop a new flow cytometry-based method for quantifying in vitro L. mexicana infection and its use in an in vivo model of cutaneous leishmaniasis revealed that distinct non-lymphocyte immune populations and the parasite replicative niche vary during infection. In Chapter 4, macrophage PUFA biosynthetic enzymes were chemically inhibited to assess their role in parasite virulence, as measured by the in vitro assay established in Chapter 3. Antagonists of fatty acid desaturase 2 (FADS2), arachidonate lipoxygenase 5 (ALOX5), ALOX15 and phospholipase A2 (PLA2) reduced intramacrophage survival of L. mexicanaTurboRFP in two host cell types, but also inhibited the growth/survival of cultured amastigotes. These data suggest that PUFA metabolism in either the host cell or the parasite is important for parasite survival. In Chapter 5 a complementary genetic approach was used to assess the role of ALOX15 and ALOX5 in the pathogenesis of L. mexicana. Leishmania growth was restricted in macrophages lacking ALOX15 using low multiplicity of infection (MOI), but not with high MOI, suggesting that ALOX15 may facilitate intracellular Leishmania growth. Importantly, the antileishmanial effect of the ALOX15 inhibitor was shown to be independent of ALOX15, indicating a direct effect on parasite growth. In contrast, CRISPR/Cas9 mediated deletion of ALOX5 in THP1 macrophages resulted in increased intracellular infection, which was at least partially mediated by leukotriene B4 as this lipid partly restored the capacity of ALOX5 knockout macrophages to control parasite growth. Loss of ALOX5 also influenced the macrophage lipidome, including the abundance of lipid droplet (LD) components and arachidonic acid (AA) containing phospholipids. ALOX5 may therefore suppress intracellular parasite survival/replication through regulation of macrophage LD biology and membrane AA sequestration. In Chapter 6 the contribution of peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors implicated in PUFA/lipid mediator signaling, in the host response to Leishmania infection was assessed using both pharmacological and genetic knockout approaches. Again, attempts to utilise known agonist/antagonists of these proteins to modulate Leishmania infection were confounded by off-target effects. Leishmania infection and growth in PPARalpha deficient macrophages were initially lower than in control macrophages, but subsequently increased to control levels, suggesting that PPARalpha usually supports the establishment of infection and later adopts a parasite-suppressive role. This early role may be related to altered cellular membrane properties and perturbed phagocytosis/phagosome maturation in PPARalpha knockout macrophages, as indicated by depleted cholesterol and phospholipids containing PUFAs within these cells. The coincidence of changes in PPARalpha expression and infection induced LD accumulation suggest the secondary role of this receptor may be associated with LD regulation. Overall, these studies show that host PUFAs have a significant role in regulating intracellular growth of Leishmania parasites. While these findings open the way for developing new host-directed antileishmanial therapies, they also highlight the off-target effects of existing inhibitors and the complexity of host PUFA signaling. Importantly, these data build upon considerable work demonstrating radical and dynamic restructuring of host cellular metabolism upon Leishmania infection, an important consideration for the suitability of host metabolism pathways as drug targets
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ItemUbiquitination in the malaria parasite Plasmodium falciparum( 2022)Ubiquitin is a post-translational modification that plays a role in many cellular processes, including protein degradation, trafficking, and signaling. The ubiquitination machinery includes E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, E3 ubiquitin ligases, ubiquitin-binding domain-containing proteins, and deubiquitinases. In the malaria parasite P. falciparum, only a few ubiquitination proteins have been characterised and <10 more have been implicated in drug resistance. Post-translational mechanisms are known to be important in sexual development in Plasmodium, and so we investigated the role of selected ubiquitination proteins in differentiation into sexual forms called gametocytes. Using a CRISPR/Cas9 knockout strategy, we initiated the characterisation of selected ubiquitination genes that are upregulated in gametocytes compared to asexual parasites. We found two ubiquitination genes, encoding for a polyubiquitin binding protein and an E2 ubiquitin-conjugating enzyme, that play an important role on the regulation of sex-specific differentiation and stage development. Loss of the polyubiquitin binding protein produced gametocytes that reached late stages but lack a defined sex. Loss of the E2 ubiquitin-conjugating enzyme produced gametocytes with a morphological defect in the late stages and lack a defined sex. We also investigated the role of Kelch 13 (K13), a protein mutated in artemisinin-resistant parasites and hypothesised to be a ubiquitination protein and demonstrate that it is required for normal parasite uptake of haemoglobin. This work furthers our knowledge on the role of ubiquitination and of K13 in P. falciparum.
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ItemNo Preview AvailableUnderstanding virulence protein trafficking in the P. falciparum infected red blood cell( 2022)After invading the red blood cell (RBC), the malaria-causative parasite P. falciparum traffics an adhesin, erythrocyte membrane protein 1 (here referred to as EMP1), to the host cell surface. EMP1 is essential for parasite survival in vivo as it prevents splenic clearance. Moreover, variants of EMP1 that confer cytoadherence to cerebral or placental tissue can lead to fatal complications of the disease. In addition to EMP1, ~500 other parasite proteins are exported into the host cell compartment, localizing to parasite- induced membrane-bound and membraneless structures and knob-like protrusions at the RBC surface. These exported proteins do not resemble canonical trafficking machinery (e.g., ESCRT, SNARE, and Rab machineries), and there is no remnant secretory machinery for the parasite to co-opt, so how EMP1 is transported through the host cell cytoplasm remains unclear. Here we present functional characterization of two exported proteins with potential roles in EMP1 transport. First focusing on the gametocyte exported protein 7 (GEXP07), we found that GEXP07 localizes to the Maurer’s clefts, an intermediate compartment for EMP1 trafficking. In the absence of GEXP07, the clefts segment into smaller membrane- bound structures, the knobs are larger and clustered, and EMP1 transport to the host cell surface is reduced. The work confirms the critical role of Maurer’s clefts in EMP1 trafficking and reveals a previously unappreciated link between EMP1 transport and host cell remodeling. We also characterized the PfEMP1 trafficking protein 7 (PTP7). We found that PTP7 localizes to the Maurer’s cleft and associated structures, including vesicles and membraneless structures called J-dots. In the absence of PTP7, the clefts become decorated with budding vesicles - seemingly stalled in the process of fission. The knobs morphology is altered and forward trafficking of EMP1 from the cleft to the infected RBC surface is ablated. We show that the poly-asparagine repeat-containing C-terminal domain of PTP7 is essential for its function. The work leads to the intriguing suggestion that low complexity domains might be important for the function of these non-canonical trafficking proteins. We sought to further understand the physical processes that might drive trafficking of exported proteins and explored the role of intrinsically unstructured protein domains, which have been shown to drive some trafficking events in other organisms. As mobile, membrane-less structures, the J-dots resemble the phase separated biomolecular condensates observed in other eukaryotes. Here, we used an optogenetic technique in a heterologous mammalian cell system to explore the condensate-forming potential of full-length J-dot proteins and protein domains. We identified the central region of a protein called 0801 as having a propensity to phase separate. We generated sequence variants of this protein region and determined the molecular grammar responsible for condensate formation. This work points to the possibility that intrinsically unstructured protein domains could play a previously unrecognized role in protein trafficking and/or protein sequestration in P. falciparum. Overall, the work presented in this thesis adds new insights to our understanding of EMP1 trafficking. Also, in the context of the RBC cytoplasm which lacks canonical trafficking machinery, our preliminary findings regarding the phase separation capacity of some exported proteins may inform the wider field of protein transport. If validated in P. falciparum, our findings prompt a re-evaluation of the molecular requirements for coordinated protein transport in eukaryotes more broadly.
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ItemMacropinocytosis and the FcRn-albumin recycling system in immune cells( 2022)One of the defining features of eukaryotic cells is their ability to carry out endocytosis, the process of internalising extracellular material by invaginating plasma membrane and enclosing the material in a membrane-bound vesicle. Macropinocytosis is a pathway of endocytosis that allows cells to non-selectively internalise very large quantities of extracellular fluid, material and plasma membrane, and is implicated in numerous cellular processes including nutrient acquisition, antigen presentation and signalling. Macropinocytosis can also be exploited by pathogens such as bacteria and viruses to invade cells and can be exploited by cancer cells to acquire nutrients that sustain their proliferation. However, despite the importance of macropinocytosis, the levels and molecular mechanisms of macropinocytosis in different cell types remain poorly defined. Furthermore, specific pharmacological inhibition of macropinocytosis and its associated processes remains difficult due to the absence of specific inhibitors. The neonatal Fc receptor (FcRn) is a transmembrane receptor that regulates the trafficking of its ligands following uptake from the extracellular environment by macropinocytosis. FcRn ligands are immunoglobulin G (IgG), which plays a critical role in the immune response to pathogens, and albumin, which has a number of functions including maintenance of oncotic pressure within blood vessels and acting as a carrier for various molecules. FcRn binds to these ligands in acidic endosomal compartments and recycles them to the cell surface, thereby diverting these two ligands from lysosomal degradation. As a result, these proteins have unusually long half-lives of about 3 weeks in humans. The ability of FcRn to recycle IgG and albumin has been exploited to design novel IgG or albumin-conjugated therapeutics with prolonged half-lives. However, many questions pertaining to the FcRn recycling system remain, particularly for albumin, such as the contribution of different cell types to FcRn-mediated albumin recycling in vivo and the mechanisms of albumin uptake, trafficking and recycling in different cell types. In chapter 3, I have characterised immortalised RAW 264.7 mouse macrophages for macropinocytic activity and validated these cells as a robust cell line model of macropinocytosis. Using this model, I attempted to identify novel molecular regulators of macropinocytosis by pooled CRISPR screening. Although this attempt was unsuccessful, cells with diminished macropinocytosis following application of the pooled CRISPR library were observed. In addition, the ability of the novel macropinocytosis inhibitor imipramine to inhibit macropinocytosis in RAW 264.7 macrophages and primary bone marrow-derived macrophages (BMDMs) was validated. Imipramine was also shown to influence macropinocytosis-dependent processes in primary BMDMs, namely antigen degradation and mTORC1 signalling. In this chapter, I also assessed the levels of macropinocytosis in a number of cancer cell lines and demonstrated a broad range of macropinocytic activity across these lines. These cancer cell lines also showed differential sensitivity to imipramine with respect to both macropinocytosis and proliferation. In chapter 4, I developed and validated an ELISA to analyse the levels of labelled-albumin in mouse serum. Subsequently, WT and FcRn KO mice were used to generate bone marrow chimeras with FcRn absent in haematopoietic-derived immune cells and present in non-immune cells or present in immune cells and absent in non-immune cells. By analysing the levels of injected labelled-albumin in the serum over time in these chimeric mice, I was able to determine that immune cells play an important role in FcRn-mediated albumin homeostasis in vivo. In chapter 5, I analysed the uptake, trafficking and recycling of albumin in DCs, using the mouse CD8+ dendritic cell (DC) line, DC1940, as a model. I confirmed that DCs express FcRn and demonstrated that they internalise albumin by macropinocytosis. The effect of DC activation status on albumin uptake was evaluated, and it was shown that activated DCs internalise a greater quantity of albumin compared with immature DCs. Pulse-chase analyses revealed that internalised albumin exhibits slow disappearance kinetics in both immature and activated DCs, which contrasts with the rapid disappearance of internalised albumin in macrophages. The slow disappearance kinetics in DCs was attributed to slow proteolytic degradation, as the presence of protease inhibitors offset the disappearance over time and microscopy analysis revealed the presence of albumin in degradative compartments. In addition, recycling of albumin by immature DCs was also detected, which appeared to occur in both an FcRn-dependent and FcRn-independent manner. In summary, the work in this thesis has established systems to study macropinocytosis in macrophages, DCs and a number of cancer cell lines, and demonstrated the potential of imipramine in modulating macropinocytosis and its associated physiological processes. The findings presented have also shown that immune cells make a major contribution to FcRn-mediated albumin homeostasis in vivo and have provided insights into the FcRn-albumin recycling system in DCs.
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ItemInvestigating features and interactions of the childhood respiratory microbiome( 2022)The human microbiome is closely linked with the health of an individual and is implicated in numerous complex diseases including diabetes, inflammatory bowel disease, cancer, cystic fibrosis (CF), and asthma. There is growing evidence to suggest the respiratory microbiome influences risk and trajectory of respiratory disease from an early age. Hence, unravelling the biology of the childhood respiratory microbiome is critical to gain a comprehensive understanding of respiratory disease, and requires characterisation of both the aggregate community and individual community members. This thesis strengthens our understanding of the childhood respiratory microbiome through i) investigation of specific community members, Haemophilus influenzae and Haemophilus parainfluenzae, in the context of CF, and ii) exploration of upper respiratory tract (URT) microbiome development during the first year of life with a particular focus on microbe-microbe interactions. While morbidity and mortality of CF principally result from repeated respiratory infections by Pseudomonas aeruginosa, there is emerging evidence that respiratory tract colonisation by Haemophilus species during childhood induces early disease progression. I describe the detection, antimicrobial resistance (AMR), and genome sequencing of H. influenzae and H. parainfluenzae isolated from airway samples of children enrolled in the AREST CF program. This work revealed H. influenzae and H. parainfluenzae carriage rates and strain persistence among participants. Haemophilus isolates were genetically diverse and commonly resistant to antimicrobials with several putative novel resistance determinants identified. Finally, genomic data identified transmission of Haemophilus strains between participants. The association between the respiratory microbiome and respiratory disease has been established in several cohort studies. However, no work has been undertaken to compare preservation of respiratory microbiome dynamics or to reconcile differences between cohort studies. This thesis explores 16S rRNA gene survey data from four longitudinal childhood cohorts, with a focus on microbe-microbe interactions. The URT microbiome composition dynamics during the first year of life are shown to be well preserved across cohorts, and the aggregate data set is leveraged to reveal associations between specific community members and symptoms of acute respiratory illness. A foundation for microbe-microbe interactions during the first year of life is established, which facilitated discovery of two communities that dominate the URT microbiome. For both areas of focus presented in this thesis I additionally developed two novel software tools to support and enhance analysis: hicap, a tool for robust inference of H. influenzae serotype and cap locus structure from WGS data, and FastSpar, a tool for rapid and scalable correlation estimation from compositional data. Collectively, this thesis contributes to our understanding of the childhood URT microbiome in the context of CF and normal development. The results in this thesis provide the first insights into the population dynamics and genomic AMR determinants of H. influenzae and H. parainfluenzae strains in a paediatric CF cohort. The presented findings further recapitulate the most complete overview of URT microbiome development during the first year of life and provide the first foundation for microbe-microbe interaction dynamics. The tools developed and the analyses performed in this thesis provide an important framework for future studies to investigate features and interactions of the respiratory microbiome.
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ItemProbing the mechanisms of translation arrest caused by arginine-rich C9ORF72 ALS and FTD proteins( 2022)Hexanucleotide repeat expansion in C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Five types of dipeptide repeat proteins (DPRs) can be translated from this mutant RNA expansion. Recent studies have found that two of these DPRs, poly-glycine-arginine (poly-GR) and poly-proline-arginine (poly-PR), cause severe ribosome stalling during translation. It has been proposed that stalling on arginine-rich (R-rich) DPRs happens via electrostatic jamming of the ribosome exit tunnel by a positively charged dipeptide repeat sequence, however, the details of this mechanism are not well-studied. In this work, I have studied the relationship between stalling efficiency and the length of the R-rich DPR protein. Instead of having a length threshold after which elongation becomes severely aborted, both poly-GR and poly-PR have shown a length-dependent increase in their ability to stall ribosomes, and more than 40-50 repeats (80-100 amino acids) are required to induce moderate translation arrest. Based on these findings, I have a proposed a model of DPR-induced stalling that suggests that the positively charged nascent chain must partially exit the polypeptide exit tunnel in order to effectively inhibit elongation. To test the importance of the arginine residue for stalling, I have generated a library of dipeptide repeat proteins (40xGly-Xxx), where Arg was replaced with every other canonical amino acid and examined the ability of each DPR from this library to stall ribosomes. The substitution of Arg for Lys, another positively charged residue, but not uncharged amino acids residues, preserved the stalling phenotype, supporting the hypothesis that electrostatic interactions are the driving force of DPR-induced stalling. Ribosome stalling often occurs on defective mRNAs and serves as a trigger event for quality control machineries that clear arrested ribosome complexes and target aberrant mRNAs and stalled polypeptides for degradation. To explore how cells respond to stalling on R-rich DPRs, I have conducted a genome-wide CRISPR knockout screen for genes that can modulate the elongation efficiency of 102xPR-coding mRNA. In comparison, I have also screened for the regulators of translation arrest on polyadenosine mRNA, a canonical substrate for ribosome-associated quality control (RQC) pathway. Collectively, my findings indicate that the known cellular mechanisms that abolish translation arrest on defective mRNAs cannot efficiently respond to ribosomes stalled by poly-PR protein.
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