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Naive CD4+ T lymphocytes differentiate into various Th cell subsets following TCR binding to microbial peptide:MHC class II (p:MHCII) complexes on dendritic cells (DCs). The affinity of the TCR interaction with p:MHCII plays a role in Th differentiation by mechanisms that are not completely understood. We found that low-affinity TCRs biased mouse naive T cells to become T follicular helper (Tfh) cells, whereas higher-affinity TCRs promoted the formation of Th1 or Th17 cells. We explored the basis for this phenomenon by focusing on IL-2R signaling, which is known to promote Th1 and suppress Tfh cell differentiation. SIRP⍺+ DCs produce abundant p:MHCII complexes and consume IL-2, whereas XCR1+ DCs weakly produce p:MHCII but do not consume IL-2. We found no evidence, however, of preferential interactions between Th1 cell–prone, high-affinity T cells and XCR1+ DCs or Tfh cell–prone, low-affinity T cells and SIRP⍺+ DCs postinfection with bacteria expressing the peptide of interest. Rather, high-affinity T cells sustained IL-2R expression longer and expressed two novel Th cell differentiation regulators, Eef1e1 and Gbp2, to a higher level than low-affinity T cells. These results suggest that TCR affinity does not influence Th cell differentiation by biasing T cell interactions with IL-2–consuming DCs, but instead, directly regulates genes in naive T cells that control the differentiation process.

Interleukin-1β (IL-1β) is a major cytokine that initiates and enhances inflammatory responses. Excessive IL-1β production is a characteristic of most chronic inflammatory diseases, including atherosclerosis, type 2 diabetes, and obesity, which affect a large proportion of the global population. The production of bioactive IL-1β is mediated by a caspase-1-activating complex known as an ‘inflammasome’. The NLRP3 inflammasome has been associated with several human inflammatory and autoimmune diseases and represents a potential therapeutic target for disrupting IL-1β production. We used molecular modeling guided by molecular dynamics simulations to design α-helical stapled peptides targeting the pyrin domain of the adaptor protein ASC to interrupt the development of its filament, which is crucial for NLRP3 inflammasome formation. The peptides were effectively internalized by human monocytic cells and efficiently suppressed the release of the inflammasome-regulated cytokines IL-1β and IL-18, following exogenous activation of the NLRP3 inflammasome. The peptides reduced ASC speck formation and caspase-1 processing thereby suppressing pro-IL-1β processing and release of active IL-1β. This is the first demonstration of the successful use of stapled peptides designed to target the adaptor protein ASC, and can be extended to other inflammatory pathways to disrupt excessive IL-1β production. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version

Phospholipids are an integral part of the cellular membrane structure and can be produced by a de novo biosynthetic pathway and, alternatively, by the Kennedy pathway. Studies in several yeast species have shown that the phospholipid phosphatidylserine (PS) is synthesized from CDP-diacylglycerol and serine, a route that is different from its synthesis in mammalian cells, involving a base-exchange reaction from preexisting phospholipids. Fungal-specific PS synthesis has been shown to play an important role in fungal virulence and has been proposed as an attractive drug target. However, PS synthase, which catalyzes this reaction, has not been studied in the human fungal pathogen Cryptococcus neoformans. Here, we identified and characterized the PS synthase homolog (Cn Cho1) in this fungus. Heterologous expression of Cn CHO1 in a Saccharomyces cerevisiae cho1Δ mutant rescued the mutant's growth defect in the absence of ethanolamine supplementation. Moreover, an Sc cho1Δ mutant expressing Cn CHO1 had PS synthase activity, confirming that the Cn CHO1 encodes PS synthase. We also found that PS synthase in C. neoformans is localized to the endoplasmic reticulum and that it is essential for mitochondrial function and cell viability. Of note, its deficiency could not be complemented by ethanolamine or choline supplementation for the synthesis of phosphatidylethanolamine (PE) or phosphatidylcholine (PC) via the Kennedy pathway. These findings improve our understanding of phospholipid synthesis in a pathogenic fungus and indicate that PS synthase may be a useful target for antifungal drugs. Published version

Upon Plasmodium falciparum infection of the red blood cells (RBCs), the parasite replicates and consumes haemoglobin resulting in the release of free heme which is rapidly converted to hemozoin crystallites. The bulk magnetic susceptibility of infected RBCs (iRBCs) is changed due to ferric (Fe3+) paramagnetic state in hemozoin crystallites which induce a measurable change in spin-spin relaxation (transverse relaxation) rate in proton nuclear magnetic resonance (NMR) of iRBCs. Earlier, our group reported that this transverse relaxation rate (R2) can be measured by an inexpensive, portable 0.5 Tesla bench top magnetic resonance relaxometry (MRR) system with minimum sample preparation and is able to detect very low levels of parasitemia in both blood cultures as well as animal models. However, it was challenging to diagnose malaria in human blood using MRR, mainly due to the inherent variation of R2 values of clinical blood samples, caused by many physiological and genotypic differences not related to the parasite infection. To resolve the problem of baseline R2 rates, we have developed an improved lysis protocol for removing confounding molecular and cellular background for MRR detection. With this new protocol and by processing larger volume of blood (>1 ml), we are able to reliably detect very low level of parasitemia (representing early stage of infection, ~0.0001%) with a stable baseline and improved sensitivity using the current MRR system.

Our understanding of the conformational and electrostatic determinants that underlie targeting of human leukocyte antigens (HLA) by anti-HLA alloantibodies is principally based upon in silico modelling. Here we provide a biochemical/biophysical and functional characterization of a human monoclonal alloantibody specific for a common HLA type, HLA-A*11:01. We present a 2.4 Å resolution map of the binding interface of this antibody on HLA-A*11:01 and compare the structural determinants with those utilized by T-cell receptor (TCR), killer-cell immunoglobulin-like receptor (KIR) and CD8 on the same molecule. These data provide a mechanistic insight into the paratope−epitope relationship between an alloantibody and its target HLA molecule in a biological context where other immune receptors are concomitantly engaged. This has important implications for our interpretation of serologic binding patterns of anti-HLA antibodies in sensitized individuals and thus, for the biology of human alloresponses., Anti-human leukocyte antigen (HLA) antibodies are important mediators of alloresponses, but structural insights on antibody:HLA interaction are still lacking. Here the authors provide a 2.4 Å structure of antibody:HLA complex, and also analyse HLA features important for other HLA-interacting molecules, to enhance our understanding of alloimmunity.

Our understanding of diseases has been transformed by the realisation that people are holobionts, comprised of a host and its associated microbiome(s). Disease can also have devastating effects on populations of marine organisms, including dominant habitat formers such as seaweed holobionts. However, we know very little about how interactions between microorganisms within microbiomes - of humans or marine organisms – affect host health and there is no underpinning theoretical framework for exploring this. We applied ecological models of succession to bacterial communities to understand how interactions within a seaweed microbiome affect the host. We observed succession of surface microbiomes on the red seaweed Delisea pulchra in situ, following a disturbance, with communities ‘recovering’ to resemble undisturbed states after only 12 days. Further, if this recovery was perturbed, a bleaching disease previously described for this seaweed developed. Early successional strains of bacteria protected the host from colonisation by a pathogenic, later successional strain. Host chemical defences also prevented disease, such that within-microbiome interactions were most important when the host’s chemical defences were inhibited. This is the first experimental evidence that interactions within microbiomes have important implications for host health and disease in a dominant marine habitat-forming organism.

Stapled-peptides have emerged as an exciting class of molecules which can modulate protein–protein interactions. We have used a structure-guided approach to rationally develop a set of hydrocarbon stapled-peptides with high binding affinities and residence times against the oncogenic eukaryotic translation initiation factor 4E (eIF4E) protein. Crystal structures of these peptides in complex with eIF4E show that they form specific interactions with a region on the protein-binding interface of eIF4E which is distinct from the other well-established canonical interactions. This recognition element is a major molecular determinant underlying the improved binding kinetics of these peptides with eIF4E. The interactions were further exploited by designing features in the peptides to attenuate disorder and increase helicity which collectively resulted in the generation of a distinct class of hydrocarbon stapled-peptides targeting eIF4E. This study details new insights into the molecular basis of stapled-peptide: eIF4E interactions and their exploitation to enhance promising lead molecules for the development of stapled-peptide compounds for oncology. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version

Pseudomonas aeruginosais one of the dominant species when it co-exists with many other bacterial species in diverse environments. To understand its physiology and interactions with co-existing bacterial species in different conditions, we established physiologically reproducible eighteen-species communities, and found thatP. aeruginosabecame the dominant species in mixed-species biofilm community but not in the planktonic community.P. aeruginosaH1 type VI secretion system was highly induced in the mixed-species biofilm community compare to its mono-species biofilm, which was further demonstrated to play a key role forP. aeruginosato gain fitness over other bacterial species. In addition, the type IV pili and Psl exopolysaccharide were shown to be required forP. aeruginosato compete with other bacterial species in the biofilm community. Our study showed that the physiology ofP. aeruginosais strongly affected by interspecies interactions, and both biofilm determinants and H1 type VI secretion system contribute toP. aeruginosafitness over other species in complex biofilm communities.ImportancePseudomonas aeruginosausually coexists with different bacterial species in natural environment. However, systematic comparative characterization ofP. aeruginosain complex microbial communities with its mono-species communities is lacking. We constructed mixed-species planktonic and biofilm communities consistingP. aeruginosaand seventeen other bacterial species to study the physiology and interaction ofP. aeruginosain complex multiple-species community. A single molecule detection platform, NanoString nCounter®16S rRNA array, was used to shown thatP. aeruginosacan become the dominant species in the biofilm communities while not in the planktonic communities. Comparative transcriptomic analysis and fluorescence-based quantification further revealed thatP. aeruginosaH1 type VI secretion system and biofilm determinants are both required for its fitness in mixed-species biofilm communities.

Antibiotic resistance threatens effective treatment of microbial infections globally. This situation has spurred the hunt for new antimicrobial compounds in both academia and the pharmaceutical industry. Here, we report how the widely used antitumor drug cisplatin may be repurposed as an effective antimicrobial against the nosocomial pathogen Pseudomonas aeruginosa. Cisplatin was found to effectively kill strains of P. aeruginosa. In such experiments, transcriptomic profiling showed upregulation of the recA gene, which is known to be important for DNA repair, implicating that cisplatin could interfere with DNA replication in P. aeruginosa. Cisplatin treatment significantly repressed the type III secretion system (T3SS), which is important for the secretion of exotoxins. Furthermore, cisplatin was also demonstrated to eradicate in vitro biofilms and in vivo biofilms in a murine keratitis model. This showed that cisplatin could be effectively used to eradicate biofilm infections which were otherwise difficult to be treated by conventional antibiotics. Although cisplatin is highly toxic for humans upon systemic exposure, a low toxicity was demonstrated with topical treatment. This indicated that higher-than-minimal inhibitory concentration (MIC) doses of cisplatin could be topically applied to treat persistent and recalcitrant P. aeruginosa infections.

The endosome-to-Golgi or endocytic retrograde trafficking pathway is an important post-Golgi recycling route. Here we show that amino acids (AAs) can stimulate the retrograde trafficking and regulate the cell surface localization of certain Golgi membrane proteins. By testing components of the AA-stimulated mTORC1 signaling pathway, we demonstrate that SLC38A9, v-ATPase and Ragulator, but not Rag GTPases and mTORC1, are essential for the AA-stimulated trafficking. Arl5, an ARF-like family small GTPase, interacts with Ragulator in an AA-regulated manner and both Arl5 and its effector, the Golgi-associated retrograde protein complex (GARP), are required for the AA-stimulated trafficking. We have therefore identified a mechanistic connection between the nutrient signaling and the retrograde trafficking pathway, whereby SLC38A9 and v-ATPase sense AA-sufficiency and Ragulator might function as a guanine nucleotide exchange factor to activate Arl5, which, together with GARP, a tethering factor, probably facilitates the endosome-to-Golgi trafficking., Amino acid levels are known to regulate anabolic and catabolic pathways. Here, the authors report that amino acids also affect membrane trafficking by stimulating endosome-to-Golgi retrograde trafficking and regulating cell surface localization of certain Golgi proteins through Ragulator and Arl5.

Signals arising from bacterial infections are detected by pathogen recognition receptors (PRRs) and are transduced by specialized adapter proteins in mammalian cells. The Receptor-interacting-serine/threonine-protein kinase 2 (RIPK2 or RIP2) is such an adapter protein that is critical for signal propagation of the Nucleotide-binding-oligomerization-domain-containing proteins 1/2 (NOD1 and NOD2). Dysregulation of this signaling pathway leads to defects in bacterial detection and in some cases autoimmune diseases. Here, we show that the Caspase-activation-and-recruitment-domain (CARD) of RIP2 (RIP2-CARD) forms oligomeric structures upon stimulation by either NOD1-CARD or NOD2-2CARD. We reconstitute this complex, termed the RIPosome in vitro and solve the cryo-EM filament structure of the active RIP2-CARD complex at 4.1 Å resolution. The structure suggests potential mechanisms by which CARD domains from NOD1 and NOD2 initiate the oligomerization process of RIP2-CARD. Together with structure guided mutagenesis experiments at the CARD-CARD interfaces, we demonstrate molecular mechanisms how RIP2 is activated and self-propagating such signal., The pathogen recognition receptors NOD1/2 recognize bacterial cell wall components and signal through their downstream adapter kinase RIP2 via a CARD (Caspase activation and recruitment domain) mediated oligomerization process. Here the authors present the cryo-EM structure of the active RIP2-CARD filament and discuss implications for NOD1/2-RIP2 signalling.

The slow and promiscuous properties of the CO2-fixing enzyme Rubisco constrain photosynthetic efficiency and have prompted the evolution of powerful CO2 concentrating mechanisms (CCMs). In eukaryotic microalgae a key strategy involves sequestration of the enzyme in the pyrenoid, a liquid non-membranous compartment of the chloroplast stroma. Here we show using pure components that two proteins, Rubisco and the linker protein Essential Pyrenoid Component 1 (EPYC1), are both necessary and sufficient to phase separate and form liquid droplets. The phase-separated Rubisco is functional. Droplet composition is dynamic and components rapidly exchange with the bulk solution. Heterologous and chimeric Rubiscos exhibit variability in their tendency to demix with EPYC1. The ability to dissect aspects of pyrenoid biochemistry in vitro will permit us to inform and guide synthetic biology ambitions aiming to engineer microalgal CCMs into crop plants., The microalgal pyrenoid has been reported to behave as a phase-separated liquid compartment. Here the authors demonstrate that the CO2-fixing enzyme Rubisco and the linker protein EPYC1 are necessary and sufficient to bring about a liquid-liquid phase separation that recapitulates the pyrenoid’s liquid-like behavior.

Although covalent protein binding is established as the pivotal event underpinning acetaminophen (APAP) toxicity, its mechanistic details remain unclear. In this study, we demonstrated that APAP induces widespread protein glutathionylation in a time-, dose- and bioactivation-dependent manner in HepaRG cells. Proteo-metabonomic mapping provided evidence that APAP-induced glutathionylation resulted in functional deficits in energy metabolism, elevations in oxidative stress and cytosolic calcium, as well as mitochondrial dysfunction that correlate strongly with the well-established toxicity features of APAP. We also provide novel evidence that APAP-induced glutathionylation of carnitine O-palmitoyltransferase 1 (CPT1) and voltage-dependent anion-selective channel protein 1 are respectively involved in inhibition of fatty acid β-oxidation and opening of the mitochondrial permeability transition pore. Importantly, we show that the inhibitory effect of CPT1 glutathionylation can be mitigated by PPARα induction, which provides a mechanistic explanation for the prophylactic effect of fibrates, which are PPARα ligands, against APAP toxicity. Finally, we propose that APAP-induced protein glutathionylation likely occurs secondary to covalent binding, which is a previously unknown mechanism of glutathionylation, suggesting that this post-translational modification could be functionally implicated in drug-induced toxicity. MOH (Min. of Health, S’pore) Published version

Overcoming multidrug resistance has always been a major challenge in cancer treatment. Recent evidence suggested epithelial-mesenchymal transition plays a role in MDR, but the mechanism behind this link remains unclear. We found that the expression of multiple ABC transporters was elevated in concordance with an increased drug efflux in cancer cells during EMT. The metastasis-related angiopoietin-like 4 (ANGPTL4) elevates cellular ATP to transcriptionally upregulate ABC transporters expression via the Myc and NF-κB signaling pathways. ANGPTL4 deficiency reduced IC50 of anti-tumor drugs and enhanced apoptosis of cancer cells. In vivo suppression of ANGPTL4 led to higher accumulation of cisplatin-DNA adducts in primary and metastasized tumors, and a reduced metastatic tumor load. ANGPTL4 empowered cancer cells metabolic flexibility during EMT, securing ample cellular energy that fuels multiple ABC transporters to confer EMT-mediated chemoresistance. It suggests that metabolic strategies aimed at suppressing ABC transporters along with energy deprivation of EMT cancer cells may overcome drug resistance. Electronic supplementary material The online version of this article (10.1186/s12943-018-0904-z) contains supplementary material, which is available to authorized users.

Actinomycetes are well-known prolific producers of bioactive secondary metabolites, including some most effective antibiotics in use today. The secondary metabolites isolated from actinomycetes accounts for 45% of natural product derived antimicrobial drugs. After the golden era of antibiotic discovery from 1940s to 1960s, the “Waksman platform” faced diminished returns. The rapid emergence of antimicrobial resistance among many pathogenic bacteria creates an urgent need for more potent antimicrobial compounds to fill the drug pipeline. Many researchers have ventured into underexplored environments to search for novels strains that has potential of producing novel compounds with interesting bioactivities. Little information is currently available for actinomycetes biodiversity in ecologically interesting sites in Singapore. The Sungei Buloh Wetland Reserve (SBWR) and Pulau Ubin Quarry Lake are considered to be unique in biotas because of different environmental conditions such as pressures, nutrient compositions and temperatures. In my Ph.D. research, I aimed to isolate unique actinomycetes strains from the two locations for the discovery of novel biosynthetic pathways and antimicrobial secondary metabolites. To uncover novel actinomycetes, I employed a variety of media recipes and in situ cultivation methods in the isolation processes, which yielded a total of 152 actinomycetes strains. The highest number of actinobacteria were found to be affiliated with the genus Streptomyces, followed by Micromonospora, Verrucosispora, Jishengella, Pseudonocardia, Isoptericola, Mycobacterium and Microbacterium. More than two third of the strains were tested to be bioactive against one or more clinical isolates such as Methicillin-resistant Staphylococcus aureus (MRSA). Taking advantage of the falling cost of genome sequencing, we performed genome sequencing and metabolite profiling to further prioritize the strains to identify the strains that have the highest potential to produce novel compounds. Several “high-potential” strains were identified based on the bioactivity assay, genome sequence and metabolite profiling results. We isolated and characterized two bioactive compounds from two such high-potential strains. While the first bioactive compound produced by Streptomyces sp. P19 is the known antimicrobial compound echinomycin, the second compound 4’,5-dihydroxy-7-methoxy-3-methylflavanone produced by Streptomyces sp. SD50 is a new compound and is a potent agonist of estrogenic receptor. We believe that many more novel biosynthetic pathways and microbial secondary metabolites will be uncovered from the high-potential strains identified by my research work. Hence, the research work described in the dissertation sets the stage for the downstream discovery of novel microbial natural products. Doctor of Philosophy (SBS)

Phospholipid homeostasis in biological membranes is essential to maintain cellular functions of organelles such as the endoplasmic reticulum (ER). Obesity has been associated to the perturbation of the ratio of the two most abundant phospholipids in the ER, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). PC/PE disequilibrium has also been linked to metabolic diseases such as non-alcoholic fatty liver disease and type II diabetes, but its biological significance remains unclear. This study aims to develop a model of how lipid perturbation (LP) can develop into diseased states. Previously, we reported that Saccharomyces cerevisiae adapts to lipid disequilibrium by upregulating several protein quality control (PQC) pathways such as the endoplasmic reticulum-associated degradation (ERAD) pathway and the unfolded protein response (UPR). We investigated the PQC pathways at their proteomic levels to investigate their effectiveness in response to LP. Surprisingly, we observed certain ER-resident transmembrane proteins (TPs), part of the UPR programme, to be destabilised under LP. Localisation and integration of the TPs to the ER remain unaffected, suggesting that the TPs might be destabilised by changes in ER membrane composition. The ER membrane was found to have undergone fatty acid remodelling and membrane stiffening. Among these, Sbh1, a member of the translocation complex, was prematurely degraded by dissociating from the Sec61 complex. Sbh1 is targeted for degradation through its highly conserved lysine residue near the membrane in a Doa10-dependent ERAD manner. Further investigations revealed Sbh1 destabilisation could result in the translocational defect under LP. Sbh1 overexpression fails to fix the translocational defect, but ameliorate the protein processing defects found under LP. This suggests a novel role of Sbh1 in protein processing. In addition, as LP compromises the UPR, disrupting phospholipid homeostasis may be exploited to target pathogens that upregulate the UPR for survival. Two lipid genes, PfFMP and PfPSD were characterised in Plasmodium falciparum by genetic complementation in yeast. Premature removal of key ER-resident TPs under prolonged LP might be an underlying cause of chronic ER stress in metabolic disorders. ​Doctor of Philosophy (SBS)

Predation by protozoa can impact bacterial communities by controlling their biomass and alter the community species composition. Bacterial communities are essential for the functioning of most ecosystems including engineered systems such as activated floccular sludge, where bacteria are responsible for biological nutrient removal and flocculation. Activated floccular sludge is often utilized to cultivate aerobic granules over long periods of time. Although the formation of granules has been optimized by controlling physical factors, the instability of aerobic granules remains a challenge for its implementation in full-scale wastewater treatment systems. While it has been hypothesised that protozoa are important in the formation of granules, no studies have characterized the abundance and diversity of protozoa during aerobic granulation. In this study, the impact of protozoa on microbial communities was monitored. Sessile ciliates were reported to be the most abundant protozoa that colonized the surfaces of granules. These ciliates consume suspended bacteria and are also hypothesized to act as a form of substratum for bacteria; colonization. Here, four bioreactors seeded with activated floccular sludge were operated for aerobic granulation for 11 weeks to better understand the roles of protozoa during the formation of aerobic granules. The abundance and diversity of protozoa decreased initially due to reduction in settling time. Upon the formation of granules, sessile ciliates became the dominant group of protozoa with gradual increase in abundance. However, microbial community analysis and correlation studies demonstrated that protozoa did not have a significant role in granule formation. In contrast, bacteria, particularly Candidatus Accumulibacter, were suggested to have a greater role in the formation of granules. The role of predation by protozoa on the formation of granules was further explored through the inhibition of protozoa in floccular sludge performed to investigate if aerobic granulation would be affected without protozoa. The absence of protozoa did not significantly affect the formation of granules from floccular sludge. Similar to the experiments following the changes in the community composition, it was also observed that Candidatus Accumulibacter was highly dominant when granules were formed in the treated sludge. In addition, inhibition of protozoa, including sessile ciliates on the surface of pre-formed granules, also did not result in disintegration of granules. Granules with and without sessile ciliates continued to maintain their granular appearance and size. Overall, the data suggested that protozoa did not play a dominant role in granulation and it was the change in sludge morphology that selected for the dominance of sessile ciliates in granules. Aerobic granules and activated floccular sludge comprised of high species diversity. Therefore, to better define the mechanisms that drive the interaction between microbial communities and predators, a mixed species biofilm system composed of Pseudomonas aeruginosa, Klebsiella pneumoniae and Pseudomonas protegens was subjected to protozoan grazing by the model ciliate, Tetrahymena pyriformis. It was found that grazing sensitive strains, K. pneumoniae and P. protogens, gained associational resistance from the grazing resistant P. aeruginosa. This resistance was partly due to the production of rhamnolipids, however results showed that there were other unidentified factors that provide P. aeruginosa resistance to predation. Doctor of Philosophy (IGS)

Atherosclerotic coronary artery disease (CAD)-related deaths will continue to rise along with global population ageing. Although established conventional risk factors are in place for the prediction of cardiovascular (CV) events, however, the absence does not necessarily imply zero CAD risk. Effective screening and diagnostic methods that stratify patient at high risk for CAD, before onset of acute events, remain unsatisfactory, necessitating the search for novel measurable protein biomarkers. Thankfully, biomarker discovery has taken a leap forward with the development of mass spectrometry (MS)-based proteomics approaches, which afforded the interrogation of the plasma proteome. The incomplete understanding in the mechanisms in plaque destabilization and rupture, creates a knowledge gap in the pathophysiological underlying atherosclerosis and myocardial infraction (MI). In addition, currently there is no plasma-based screening assay that effectively differentiates atherosclerosis from MI. The development of innovative techniques that improves the detectable range of plasma pro teo me, is essential in advancing the field of plasma-based biomarker discovery research. My PhD research work has been divided into four parts (I - IV), the first three parts showcased the clinical utility of the plasma proteome for CAD proteomics-based biomarker discovery, and in the final part, and the successful development of a novel enrichment strategy for plasma glycoproteins and extracellular vesicles (EV) was demonstrated. Herein, the careful selection and systematic integration of several proteomics strategies have proven useful in the discovery of novel plasma biomarkers of CAD, derived from (i) undepleted plasma proteome, (ii) EV plasma proteome and (iii) carbamylated plasma proteome. Functional linkage analyses have prompted the generation of new hypotheses into the pathophysiological states that specifies either for atherosclerosis or myocardial injury. The novel plasma sub-proteome enrichment technique described here have afforded the detection of low-abundant proteins with concentration ranging from pg/mL- ng/mL in crude plasma. In essence, this thesis demonstrates the applicability and reliability of the different proteomics platforms in the discovery and development of useful biomarkers that are predictive of CAD. ​Doctor of Philosophy (SBS)

Mycobacterium tuberculosis (Mtb) has the ability to persist within the human host for a long time in a dormant stage and re-emerges when the immune system is compromised. During infection, Mtb is exposed to a number of redox stresses inside macrophages, such as reactive oxygen species and reactive nitrogen intermediates, both having the potential to damage a number of cellular components, including lipids, proteins, and DNA [1]. The ability of Mtb to persist inside macrophages suggests the presence of elaborated machineries capable of preserving redox homeostasis and countering oxidoreductive stress. Mtb is equipped with sophisticated proteins and thiols (mycothiol) responsible for antioxidant defense. Most of these proteins as well as mycothiol are unique to mycobacteria and therefore lacking in the human host, like the mycothiol-dependent system composed of the enzymes mycothione reductase (Mtr), myco-redoxin1 (Mrx-1), and the alkyl hydroperoxidase subunit E (MtAhpE), responsible for the reduction of hydrogen peroxide, peroxynitrous acid, organic peroxides or S-mycothiolated mixed disulfides, generated by oxidative stress. Clearly, the protein ensemble and mycothiol of the antioxidant defense of Mtb differ substantially from that in the mammalian hosts and thus the chances for a selective inhibition of the mycobacterial antioxidant defense system will be high. In the effort to understand the biological mechanisms of the mycobacterial antioxidant defense orchestra, this work aims to provide structural, enzymatic and mechanistic insights into the Mtb NADPH dependent Mtr (MtMtr), structural details of Mtb Mrx-1 (MtMrx-1) and AhpE (MtAhpE) as well as mechanistic insights into the mycothiol/Mrx-1 dependent recycling of peroxiredoxin MtAhpE. In the first part of this thesis, the production of a stable MtMtr using a GroEL/ES chaperone-chaperonin system is described. The recombinant MtMtr provided a platform for the first low resolution solution structure by small-angle X-ray scattering (SAXS), representing a dimeric enzyme. Substrate induced conformational changes of MtMtr in the presence of NADPH and mycothiol were studied in solution by SAXS, demonstrating significant overall changes after NADPH-binding, interpreted by a shift of a dimeric to a tetrameric form of the NADPH-bound MtMtr. Genetically engineered mutants of MtMtr shed light into the importance of the flexibility of the linker, connecting the catalytic FAD-domain I with the NADPH-domain, as well as the linker, connecting the NADPH- with the FAD-domain II. Furthermore, attempts were made to crystallize MtMtr, resulting in needle showers. Together, with the linker mutants generated, these may provide a platform for crystallization approaches in the near future. Together with the generation of recombinant MtMrx-1, which was revealed to be monomeric in solution regardless of the redox state and concentration, the ensemble formation of MtMtr and MtMrx-1 was studied by NMR titration. The MtMtr-MtMrx-1 interaction was characterized by a fast exchange regime and critical residues involved in the protein-protein interaction were identified. In order to shed light into the complete ensemble of the the mycothiol-dependent system, recombinant MtAhpE was produced and purified. Its ensemble formation with MtMrx-1 was analysed by NMR spectroscopy and docking studies, providing insights into the interaction interface and mechanism of action. The interaction model presented revealed, that two molecules of monomeric MtMrx-1 interact with one dimeric MtAhpE with each molecule of MtMrx-1 reducing one monomer of MtAhpE. The last chapter describes in detail the structural and mechanistic characterisation of MtAhpE. The oligomeric state of MtAhpE was shown to be a dimer regardless of the redox condition. Interestingly, the formation of a higher molecular weight oligomer was indicated in dynamic light scattering and SAXS studies in the presence of equimolar H2O2. Using cysteine-labelling NMR experiments, it was possible to investigate the redox dynamics of the catalytic and peroxidatic cysteine in solution in the presence of various reducing agents. These data enabled to propose a new monothiolic pathway for the reduction of MtAhpE by mycothiol in the absence of MtMrx-1. Finally, the crystallographic structures of MtAhpE in the presence of mycothiol (2.43 Å resolution) and the mycothiol-analogue N-acetylcysteine (2. 2.7 Å resolution) were determined, respectively, to validate the newly proposed pathway in depth. Doctor of Philosophy (SBS)

Elizabethkingia anophelis is an emerging pathogen that causes life-threatening infections in neonates, severely immunocompromised and postoperative patients with extremely high mortality. Multi-drug resistance, biofilm formation and oxidative stress resistance imply its pathogenesis. However, the lack of genomic information on E. anophelis hinders our understanding of its mechanisms of pathogenesis. In this study, we report the complete genome sequence of a clinically isolated E. anophelis NUHP1 strain, which is the first complete genome of the Elizabethkingia genus. E. anophelis NUHP1 has a circular genome of 4,369,828 base pairs and 4,141 predicted coding sequences. Sequence analysis and the genome comparison between E. anophelis NUHP1 and assemblies of E. anophelis isolated from mosquito gut indicates that E. anophelis has well-developed systems for scavenging iron and stress response. Many putative virulence factors and antibiotic resistance genes were identified, underscoring the potential host–pathogen interactions and antibiotic resistance. RNA-sequencing-based transcriptome profiling indicates that expressions of genes involved in synthesis of a yersiniabactin-like iron siderophore and heme utilization are highly induced as a protective mechanism toward oxidative stress caused by hydrogen peroxide treatment. Chromeazurol sulfonate assay verified that siderophore production of E. anophelis is increased in the presence of oxidative stress. Moreover, the transcriptome profiling comparison between E. anophelis NUHP1 treated with hydrogen peroxide and with mouse blood further showed that E. anophelis NUHP1 displayed the similar protective mechanism associated with iron siderophore and heme utilization when contact with mouse blood, indicating that oxidative stress is the major stress for E. anophelis NUHP1 when infect hosts and the strong resistance to oxidative stress enables E. anophelis to be dominant in the blood meal feeding mosquitoes. Furthermore, we also showed that hemoglobin but not ferric iron greatly facilitates the growth, hydrogen peroxide tolerance and biofilm formation of E. anophelis NUHP1. Our study suggests that siderophore production and heme uptake pathways might play essential roles in stress response and virulence of the emerging pathogen E. anophelis. ​Doctor of Philosophy (SBS)

Oxidative stress, an imbalance between the amount of Reactive Oxygen Species (ROS) and antioxidant defences, is detrimental to the survival of both benign and pathogenic bacteria [1]. To keep ROS in balance bacteria have evolved various antioxidant enzymes, among them the alkyl hydroperoxide reductase (AhpR), consisting of 21 kDa AhpC and 57 kDa AhpF, which serve as a primary hydroperoxide scavenger that reduces hydroperoxide to water and its corresponding alcohol, and preventing thereby damages like protein oxidation, lipid peroxidation and DNA damage [2-5]. AhpC and -F represent one of the most efficient enzyme systems responsible for redox homeostasis in bacteria [6] To do that, the Escherichia coli AhpC (EcAhpC) directly donates electrons to peroxides reducing them to less harmful products. The oxidized AhpC is reduced by AhpF via the disulphide center (-CxxC-) in its N-terminal domain (NTD), with a catalytic turnover rate measured to be 4.0 x 10^7 M^-1 s^-1 in Salmonella typhimurium [7]. By employing biophysical approaches, this thesis set out to understand the mechanistic basis for this efficiency. The C-terminal tail of AhpC has been implicated as a hydroperoxide sensor in eukaryotes but not in most bacteria [8]. By generating C-terminally truncated mutants, EcAhpC_1-177, EcAhpC_1-182, EcAhpC_1-185, together with site-specific mutations EcAhpC_KJ86A and EcAhpC_1187G, the critical role of the C-terminus in oligomerization under reducing conditions has been examined. Since oligomerization has been associated with catalytic efficiency [7], NADH-dependent assays have been used to associate the C-terminal mutations to catalytic efficiency. In an attempt to gain structural insight into the C-terminal tail, the EcAhpC_1-182 crystal structure has been solved in its oxidised state that revealed five molecules in an asymmetric unit. The ring shape decamer conformation was generated by applying a symmetry calculation with the asymmetric unit cell. The absence of the C-terminal tail in the crystal structure indicates a flexibility of this region. Furthermore, the first interaction studies between the EcAhpC and EcAhpF have been performed. By generating constructs of the N- and C-terminal domains of EcAhpF, it has been confirmed that EcAhpF interacts entropically via its NTD with EcAhpC. The affinity for the EcAhpF_NTD-EcAhpC interaction was calculated to be 3.2 μM. The effect of the redox-state of EcAhpC has been found to be important to this interaction as only the oxidized AhpC interacts with the NTD. In comparison, reduction abolished the interaction altogether. Importantly, by using NMR spectroscopy, it has been revealed that the interaction of the full-length EcAhpC and the NTD is long-lived, whereas the C-terminal free AhpC (EcAhpC_1-172) interacts very dynamically with the NTD. This dynamic nature of the EcAhpC_1-172-EcAhpF_1-212 interaction enabled the observation of chemical shift perturbations of residues of the isotopically labelled NTD. Information obtained have been used to drive a HADDOCK docking of the complex and subsequently a binding epitope of the Cterminal tail of EcAhpC which it is proposed to wrap around the backside of EcAhpF_NTD. Taken together, this thesis has revealed a novel binding mechanism, where the C-terminus of AhpC wraps around the NTD to slow the dissociation rate upon complex formation for an efficient electron transfer process to occur, and a release mechanism mediated by a well-described conformational change of the Cterminus of EcAhpC upon reduction. In addition, by employing a high ionic buffer system, solution structures of the full-length oxidized EcAhpC have been solved using small angle x-ray scattering (SAXS) throwing light on the flexibility of the C-terminus and its importance to the EcAhpC-EcAhpF complex formation. In addition, the orientation of the full-length proteins in the complex in solution agrees very well with models obtained by NMR. ​Doctor of Philosophy (SBS)

The Kinesin-2 family member KIF3 forms a heterotrimeric complex comprising the motor subunits KIF3A, KIF3B and the cargo carrying subunit KAP3. N-cadherin and b-catenin are reported to be cargoes of KIF3 motor. Serine-689 residue at the C-terminal of KIF3A has been identified as a major phosphorylation site significant for the transport of cargoes. In an attempt to find out the effect of phosphorylation on KIF3A’s function, serine-689 was mutated to alanine (S689A) to preclude phosphorylation, and to aspartic acid (S689D) to mimic continuous phosphorylation. In this study, we showed that overexpressing S689A mutant impaired the transportation of N-cadherin and b-catenin to the cell-cell junction, indicating that the phosphorylation status of serine-689 residue plays a crucial role in the intracellular KIF3-mediated cargo transport. We also identified that the interaction between the KIF3A motor and tail domain was relieved by Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation in vitro and in vivo by different assays. Our results also suggest that KIF3A’s ATPase activity was inhibited by the motor-tail interaction. Phosphorylation on serine-689 residue relieves the motor-tail interaction to allow recovery of the motor’s ATPase hydrolysis activity. Computational simulation of KIF3A motor-tail complex under different phosphorylation states also supports our observations. Based on these findings, we hypothesize that KIF3A adopts a folded conformation resulting in an autoinhibited state, and phosphorylation on the serine-689 residue at the C-terminal tail domain of KIF3A subunit will relieve this autoinhibition and induce a conformational change to expose the microtubule and ATP binding regions at the motor domain. The activated KIF3 motor can thus slide on microtubules to transport its cargoes. Detailed structural information is needed to explain KIF3A’s mechanisms. In this study, a low-resolution structure of KIF3A’s motor domain was reconstructed by Small-angle X-ray scattering (SAXS) data. The screening of the proper protein crystal growing conditions for X-ray crystallography is ongoing. Promising conditions for crystallization will be discussed. ​Doctor of Philosophy (SBS)

The growing problem of antibiotic resistance in bacteria, along with the dearth of new antibiotics, has led to the search for alternative antimicrobial therapeutic agents. Since the primary reason for the quest for novel antibiotics is to stay one step ahead of the emergence of resistance, knowledge on how bacteria become resistant to a new antimicrobial agent is necessary to facilitate the development of new antimicrobials. Oligophenylenevinylene conjugated oligoelectrolytes (COEs) have π-conjugated backbones and terminal ionic pendant groups. They have a high affinity for the lipid bilayer and spontaneously intercalate into the membrane. The ability of phenylenevinylene COEs to perturb bacterial membranes varies according to the number of aromatic rings and the type of ionic fragment. It was observed that the COEs with pyridinium cationic groups, i.e. COE1-3Py, were more inhibitory than when tetraalkylammonium groups were present (i.e. COE1-3C). In this study, the antibacterial mechanism of these molecules was investigated along with the mechanism of resistance. By exposing Enterococcus faecalis OG1RF to increasing concentrations of COE1-3C or COE1-3Py, we were able to experimentally evolve resistant strains EFC3C and EFC3Py, respectively. Whole genome sequencing of resistant isolates revealed mutations in the liaFSR three component regulatory system. Specifically, EFC3Py had a mutation in liaR, while EFC3C had a mutation in liaF. Fatty acid analysis of EFC3C and EFC3Py revealed an increase in the levels of cis-vaccenic acid and cyclopropane, both of which are associated with increase in membrane order. Transcriptomic analysis of wild type E. faecalis treated with COE1-3C and COE1-3Py indicated that the compounds induced a general stress response along with changes in the expression of membrane transporters. In the EFC3C and EFC3Py strains, gene expression analysis revealed that most of the genes induced or repressed in EFC3C were also altered in EFC3Py. In both mutants, EFC3C and EFC3Py, transporters of the ATP binding cassette superfamily were the most significantly induced or repressed genes compared to the wild type. Overall, the significantly upregulated and downregulated transporter genes were generally involved in transport of ferrichrome, lipoproteins export, amino acids, riboflavin, mannitol/fructose, oligopeptide and choline. In addition, cell envelope response regulon (LiaFSR), dlt operon and penicillin binding proteins (PBPs) were also differentially expressed. The mutations in the liaFSR regulon correlate to the resistance to COEs as confirmed by the deletion and complementation studies. Collectively, the data suggest that the COEs target bacterial membranes and disrupt membrane function by membrane perturbation. The transcriptomic response of E. faecalis to conjugated oligoelectrolytes revealed the mechanisms adopted to tolerate the membrane stress elicited by COEs. Additionally, COE1-3C and COE1-3Py appear to be promising antimicrobials against E. faecalis and that there is a high barrier to the emergence of severely resistant strains constrained by biological limits of membrane adaptation that can occur in E. faecalis. Doctor of Philosophy (IGS)

Chaperone-mediated autophagy (CMA) is a selective autophagic pathway that degrades soluble cytosolic proteins. It utilizes a cascade of chaperones and co-chaperones to recognize and deliver the substrates to the lysosome for degradation. Basal CMA operates in all mammalian cells in order to maintain protein homeostasis and quality control as well as performing specific functions in some cells like kidney cell growth, neuronal survival, antigen presentation and selective degradation of specific transcription factors or metabolic enzymes. CMA is induced upon stress and performs various cellular functions like recycling of raw materials under nutrient starvation, protection against oxidative stress, toxic stress and hypoxic stress. The rate limiting step in CMA is substrate binding to the lysosomal receptor lysosome-associated membrane protein-2A (LAMP-2A) followed by formation of the multimeric LAMP-2A translocation complex. It has been reported that the mTORC2/Akt1/PHLPP1 pathway regulates CMA activity by influencing the formation the LAMP-2A translocation complex. CMA is impaired during aging resulting in metabolic failure and various neurodegenerative disorders. Thus there is tremendous interest in understanding the regulation and deriving ways to regulate CMA function to harness pathological benefits. Certain evidences suggest the role of ubiquitination in CMA pathway. For instance, rat liver lysosomes depleted of CMA promoted accumulation of ubiquitin-positive aggregates, K63-ubiquitination is necessary for CMA degradation of HIF-1α and accumulation of K48 ubiquitinated protein in CMA-abolished cells, however, the exact role of ubiquitination in targeting substrates to CMA pathway remains unclear. The current study has shown the novel role of the ubiquitin system in CMA regulation by uncovering the presence of ubiquitin and a plethora of ubiquitin modifying enzymes associated with the subgroup of lysosomes specifically dedicated to perform CMA (henceforth known as CMA-active lysosomes). Based on these observations, this project aims to understand the potential modulatory role of selected lysosome associated ubiquitin modifying enzymes- E4 ligase carboxyl-terminus of Hsp70 interacting protein (CHIP) and the deubiquitinating enzyme ubiquitin C-terminal hydrolase - L1 (UCH-L1) in different aspects of CMA pathway. CHIP and UCH-L1 have been previously implicated in regulating the levels of Protein kinase B (Akt) and PH Domain and Leucine Rich Repeat Protein Phosphatase 1 (PHLPP1), but it is not known whether this function of CHIP and UCH-L1 can regulate CMA activity. Addressing this query, the present study has revealed that CHIP is an activator of CMA while UCH-L1 inhibits CMA by regulation of Akt1 and PHLPP1 levels. Knockdown studies revealed that depletion of CHIP leads to changes in lysosomal levels of ubiquitin, CMA associated chaperones as well as accumulation of pAkt1 accompanied by a decrease in levels of PHLPP1. CHIP helps in ubiquitination of pAkt1 leading to its proteasomal degradation. In the absence of CHIP, pAkt1 accumulates which leads to downstream destabilization of LAMP-2A translocation complex resulting in CMA inhibition. In contrast, knockdown studies on UCH-L1 indicate that depletion of UCH-L1 leads to accumulation of PHLPP1 accompanied by a reduction in pAkt1 levels in the lysosomes leading to stabilization of the LAMP-2A complex and promoting CMA activity. Effectively, CHIP and UCH-L1 regulate CMA activity by manipulation of the mTOR/Akt1/PHLPP1 pathway. Subsequently, it was observed that CHIP regulates the levels of UCH-L1 at the lysosomes by ubiquitination and lysosomal degradation of UCH-L1. Thus, the two enzymes operate in the same pathway; their activities being interconnected. The evidence that the ubiquitin modifying enzymes associate with CMA-active lysosomes and regulate CMA activity could indicate a possible crosstalk between the ubiquitin proteasome system (UPS) and CMA pathway. In the current study, it was observed that upon inhibition of the proteasomal pathway, CHIP localized at the lysosomes, thus reducing lysosomal levels of UCH-L1, pAkt1 and increasing levels of PHLPP1. This could indicate a rise in CMA activity under proteasomal stress. Thus in this study, a novel mechanism of CHIP-mediated crosstalk between the UPS and CMA pathways upon conditions of proteasomal stress has been identified. This study has been advantageous in understanding the intricate connection between ubiquitination and phosphorylation pathways regulating CMA activity and can be exploited in developing therapeutic benefits by cell type specific studies as UCH-L1 is highly abundant in brain. The current study also provides an insight in understanding the crosstalk between the UPS and CMA degradation pathways both of which are implicated in various neurodegenerative diseases. ​Doctor of Philosophy (SBS)

Bacterial keratitis is the most important leading cause of corneal opacification and blindness around the world. Mycobacterial keratitis is very hard to eradicate while Pseudomonas keratitis is very common and prone to drug resistance problems. In this study, we investigated the Pseudomonas keratitis as a biofilm mode of infection that induces cyclic-di-GMP signaling and forms phagocyte-tolerant microcolonies on mouse cornea. The increased c-di-GMP content in P. aeruginosa was responsible for decreased levels in cytokine production. C-di-GMP reducing agent (Sodium Nitroprusside) was shown to enhance the efficacy of Colisitin treatment in Pseudomonas keratitis. Mycobacteria readily formed biofilms on mouse cornea comprising of large amounts of extracellular DNA. A strong synergism between Amikacin and Fluoroquinolones against Mycobacterium fortuitum has been observed. DNase addition further enhanced the efficacy of anti-mycobacterial drugs by breaking the mycobacterial biofilm matrix. New treatment strategy (combination of Amikacin, Fluoroquinolones and DNase) showed the best efficacy for converting mycobacterial infections. ​Doctor of Philosophy (SBS)

Telomeric chromatin consists of tandem ‘TTAGGG’ DNA repeats that associate with a telomere-specific complex, known as shelterin. Shelterin has an indispensable role in telomere length homeostasis and protection against genomic instability arising from erroneous DNA damage responses. Shelterin collectively consists of six proteins - Telomeric repeat binding factors 1, 2(TRF1 and TRF2), Protector of telomeres 1 (POT1), TRF1 interacting nuclear factor 1(TIN2), TIN2 POT1 interacting protein (TPP1) and Repressor/activator protein 1(RAP1). Specific mutations in shelterin proteins leading to telomere dysfunction have been reported in human diseases such as Dyskeratosis, Hoyeraal-Hreidarsson syndrome and cancer. Although structures of the domains of the individual proteins are well-studied, the intact shelterin complex and its subcomplexes remain largely uncharacterized. Results presented in this thesis discuss the biochemical purification of the shelterin subcomplexes TRF2-RAP1-TIN2S and the telomerase recruitment complex POT1-TPP1-TIN2S. The subcomplexes are further characterized by analysis of the stoichiometry and interaction with DNA. Structures of TRF2-RAP1-TIN2S and POT1-TPP1-TIN2S obtained through negative stain electron microscopy (EM) are presented. Cryo-EM imaging of DNA and nucleosome core particle (NCP) samples utilizing Volta phase plate is shown. Subsequent Volta phase plate imaging of POT1-TPP1-TIN2S is presented, showing promise for forthcoming structure determination using cryo-EM. Finally, approaches to obtain high-resolution structures of shelterin subcomplexes in the future are discussed. ​Doctor of Philosophy (SBS)

The complement system serves as the frontline of host defense for the human body against foreign entities, and is triggered via one or more of several signaling cascades that consist of interactions between soluble and cell surface-bound complement proteins. In this thesis, the objective is to explore how lipid membranes influence the initiation, amplification, termination and regulation of complement activation, utilizing supported lipid bilayer platforms as a model experimental system. The overall hypothesis of this work is that the supported lipid bilayer platform provides a useful model system to recapitulate key protein-lipid and protein-protein interactions involved in complement activation at lipid membrane interfaces, thereby establishing an experimental framework to decipher key trigger factors (e.g., membrane surface charge) as well as evaluate candidate inhibitors acting against complement convertases and membrane attack complex assemblies. Within this scope, four different experimental studies were conducted, leading to new insights into the mechanisms by which lipid membranes influence complement activation. In the first study, the influence of membrane surface change on the adsorption properties of different pattern recognition proteins involved in initiation of complement activation was investigated. In the second study, the effects of covalent and noncovalent tethering strategies on the self-assembly of the alternative pathway C3 convertase was monitored. It was identified that the C3b immobilization scheme is a critical factor governing convertase assembly, further enabling successful evaluation of a clinically relevant complement inhibitor, compstatin. In the third study, the assembly of the complement-terminating membrane attack complex was monitored onto supported lipid bilayer platforms, with particular attention to the influence of membrane surface charge. It was determined that the membrane attack complex assembly preferentially forms on negatively charged lipid membranes, indicating the importance of membrane electrostatics. In the fourth study, the inhibitory action of vitronectin and clusterin against the membrane attack complex assembly was evaluated, offering insight into the different mechanisms by which these two regulatory proteins inhibit complement activation. In summary, the findings presented in this thesis significantly contribute to the fundamental understanding of complement activation occurring at lipid membrane interfaces, and offer a new platform to characterize the mechanisms of action for a wide range of complement inhibitors including small molecules, antibodies, and peptides. Doctor of Philosophy (MSE)

Natural killer (NK) cells are key components of the innate immune system, and play critical roles in the control of acute infection by malaria parasites. It is known that the secretion of IFN-γ by NK cells is a key determinant for disease resistance. However, much less is known about its cytotoxicity on infected red blood cells (iRBC). Also, how NK cells identify and respond to iRBC remains to be characterized. Here we show that control of parasitemia by NK cells was contact-dependent and involved cytolytic granules. Video microscopy demonstrated osmotic lysis of iRBC by NK cells. Furthermore, NK cells preferentially lysed trophozoites and schizont-stage iRBC, and such lysis required complex interactions of multiple activation signals provided by LFA-1, TLR2 and TLR4, as well as signaling through MYD88-dependent and TBK/IKKε-dependent pathways. Interestingly, NK cells from about 30% of malaria naïve donors do not eliminate iRBC, and upregulate the inhibitory NKG2A receptor instead of activation molecules, like CD69 and CD25. KIR genotyping further demonstrated increased frequencies of KIR2DS1 and KIR2DS2 in these donors. We further show that NK cell can control P. knowlesi parasitemia, demonstrating species-transcending immune control of this zoonotic parasite. In addition, over-expression of surface variant antigens by iRBC appeared to aid iRBC evasion from NK cell lysis. Together our study demonstrates the multifaceted interactions of NK cells and P. falciparum iRBC, and identifies the interplay of activation and inhibitory signals in the control of parasitemia by NK cells. ​Doctor of Philosophy (SBS)

The surface-associated growth of microbial populations / communities has been revealed as the most ubiquitous lifestyle of microorganisms, which is usually referred to as biofilm. In biofilms, cells are encased within a self-produced matrix of extracellular polymeric substances (EPS). Biofilm matrix is highly structurally and physicochemically heterogeneous. The EPS matrix enhances bacterial tolerance to environmental stresses and mechanical and metabolic stability, rendering biofilms a promising workhorse in industrial applications. However, the heterogeneity of biofilm structures and metabolic activities together with uncontrollable biofilm dispersal poses a great challenge to the industrial application of biofilm-based bioprocesses. To enable the control of biofilm-based processes, this project is designed and organized to develop a platform towards controllable and reproducible biofilm development and biofilm-based bioprocesses. In this work, we demonstrated the concept of biofilm mimics supported by hydrogel matrix and evaluated the effects on cells by the artificial matrix in protein level. And we explored the potential of matrix functionalization by introducing signaling or functional molecules in the hydrogel matrix. Further, we designed a new device which was able to generate controllable and reproducible chemical gradients for biofilm development. Taken together, we successfully established the platform towards the reproducible and controllable biofilms and biofilm-based bioprocesses. First, by using hydrogel as artificial matrix, we developed biofilm mimics and elucidated the responses of bacterial cultures to the artificial entrapment. Using calcium-alginate as a model hydrogel matrix and Shewanella oneidensis as a model organism, bacterial colonies were developed within the artificial matrix. The growth, surface properties and protein expression of the biofilm mimics were evaluated, in comparison with the flow cell biofilms developed by the same strain. Briefly, there were no significant differences regarding both growth and surface properties. The proteomic analysis provided basic information on bacterial activities, such as ion uptake, energy metabolism and eDNA synthesis, advancing the understanding on bacterial responses to the artificial matrix and to the microenvironments provided by the matrix. This study showed the great potential to utilize the bacterial populations / communities developed within the artificial matrix as an alternative to flow cell biofilms. To better regulate the performance of hydrogel matrix supported biofilm mimics, we explored two approaches: (1) regulation through cell communication systems and (2) mediation through environmental chemical cues. And to better understand biofilm mimics-based responses to the regulation approaches, we explored and optimized the method of high quality RNA extraction from biofilm mimics for downstream transcriptomic analysis. Further, we investigated the responses of biofilm mimics to synthesized quorum sensing molecules using Pseudomonas aeruginosa and its mutated strain as model organism through transcriptomic analysis. We compared the quorum sensing regulated genes in biofilm mimics to those reported in biofilms, facilitating the understanding of QS regulation in the artificial matrix. To study biofilm mimics responses to highly dynamic environmental cues, we developed a high throughput platform of fluidic chamber which can generate defined and reproducible chemical gradients. We explored the application of this device in the biofilm development study using S. oneidensis as model organism, showing the biofilm formation trend along chemical gradients in the chamber. Meanwhile, we showed the great potential to study biofilm communities by using this gradient-generator flow cell system with two strains of Commamonas testosteroni as model organisms. Furthermore, this chamber can be integrated with the artificial matrix to generate reproducible external signals for biofilm mimics regulation, which will be explored in future work. Doctor of Philosophy (IGS)

Porous framework materials have been widely studied, due to their numerous attracted intrinsic properties. With the extensive development of this field, incorporating exotic functional anchors have become a new theme to enrich and develop the usage of such materials, which inspires much interest of fabricating hierarchical materials. The implantation of multiple functional anchors to host framework materials not only brings attracting structural backbone and physical properties but also opens new routes for the extensive development of such materials in chemistry, material science and engineering. The primary focus of this thesis has been made to compare the recent advancements of porous framework materials in structure and post-functionalization, and analyse their advantages and disadvantages in structural design and application performance so that some critical challenges can be summarized and research motivation can be thus inspired. Next, our three-works related to the porous framework materials design based on the above analysis were introduced and discussed in details, ranging from the decoration of structure, to external polymer incorporation, and to improve its conductivity via conjugated bonding. Subsequently, summary and outlook in this rapidly developing area are finally discussed. Clearly, such unique porous framework materials will continue to attract the increasing attention, and the display stage will be offered to them in future, owing to their superior application potential. ​Doctor of Philosophy (SPMS)

For more than 40 years, metformin has been used before and during pregnancy. However, it is important to note that metformin can cross the placenta and circulate in the developing foetus. Recent studies reported that the concentration of metformin in foetal cord blood ranges from half to nearly the same concentration as in the maternal plasma. Since metformin has anti-cell growth and pro-apoptotic effects, there are persistent concerns over the use of metformin in early pregnancy. Current human studies are limited by sample size, lack of controls or, short follow-up durations. In this review, we examine the settings in which metformin can be passed on from mother to child during pregnancy and address the current controversies relating to the cellular and molecular mechanisms of metformin. Our efforts highlight the need for more data on the effects of metformin on general offspring health as well as further scrutiny into foetal development upon exposure to metformin. NRF (Natl Research Foundation, S’pore) ASTAR (Agency for Sci., Tech. and Research, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Published version

Oil palm (Elaeis guineensis, Jacq.) is a key tropical oil crop, which provides over one third of the global vegetable oil production, but few genes related to oil yield have been characterized. In this study, a GDSL esterase/lipase gene, which was significantly associated with oil content, was isolated from oil palm and designated as EgGDSL. Its functional characterization was carried out through ectopic expression in Arabidopsis ecotype Col-0. It was shown that expression of EgGDSL in Arabidopsis led to the increased total fatty acid content by 9.5% compared with the wild type. Further analysis of the fatty acid composition revealed that stearic acid (18:0) increased in the seeds of the transgenic lines, but the levels of linoleic acid (18:2) plus 11-eicosenoic acid drastically declined. Quantitative real-time PCR (qPCR) revealed that in oil palm, EgGDSL was highly expressed in mesocarp followed by leaf, and the expression level was very low in the root. The expression level of EgGDSL gene began to increase at two months after flowering (MAF) and reached its peak by four MAF, then declined rapidly, and reached its lowest level during the mature period (6 MAF). The EgGDSL gene was more highly expressed in oil palm trees with high oil content than that with low oil content, demonstrating that the transcription level of EgGDSL correlated with the amount of oil accumulation. The gene may be valuable for engineering fatty acid metabolism in crop improvement programmes and for marker-assisted breeding.

To understand the roles of various gut dendritic cell (DCs) subsets in controlling intestinal inflammation, two DTR mice strains: Clec9A DTR and Clec4a4 DTR, capable of conditional in vivo ablation of respective colonic CD103+ CD11b- and CD103+ CD11b+ DCs have been generated. In a dextran sodium sulphate (DSS) model of colonic inflammation, ablation of CD103+ CD11b- colonic DCs resulted in exacerbated colitis, while ablation of colonic CD103+ CD11b+ DCs resulted in resistance to colitis when subjected to the same DSS regiment. Hence our results highlight the opposing roles of these two distinct colonic DC subsets in controlling DSS-induced colonic inflammation. A cross-talk between DCs, other gut immune cells and the colonic epithelia, involved in maintenance of a functional mucosal epithelial barrier, has been proposed to directly impact outcome of colonic inflammation. The mechanistic details of how these distinct DC subsets contribute to regulate colonic inflammation will be discussed. DOCTOR OF PHILOSOPHY (SBS)

Staphylococcus aureus (S. aureus) is a common pathogen that can cause high mortality and morbidity. Every year, many people die from S. aureus related infections. Methicillin resistant S. aureus (MRSA) strain is typically responsible for severe and complicated infections in human beings. For this reason it is also considered as one of the “Superbug”. It is resistant to many drugs and the glycopeptide vancomycin is the only available antibiotic that is active against MRSA infections. However, recent reports indicate that vancomycin intermediate-resistant MRSA strains are emerging worldwide. Therefore, there is a concern that MRSA would gradually become fully-resistant to vancomycin in the near future and newer treatment strategies or compounds that can effectively cure S. aureus related infections should be well underway before our last antibiotic runs out. Plants have been naturally used since the ancient times as medicine and still many plant compounds can be found in the treatment of major ailments such as malaria and cancer. Due to multi-drug resistance problems in clinical settings and the need for newer compounds, there is great interest in phytochemical research because of their hypothesized and proven antimicrobial activities. Combinations of synergistic phytochemicals and antibiotics are assumed to be a new option to solve the drug resistance problem. Their combination can increase the efficacy and the killing rate and the chance of resistance development can be delayed. In this study, the effects of using conventional antibiotics in combination with phytochemicals were studied by using checker board methods and time-kill assays using 12 S. aureus strains. Studies suggested that tannic acid was synergistic with fusidic acid, minocycline, cefotaxime and rifampicin and additive with ofloxacin and vancomycin. Quercetin showed synergism with minocycline, fusidic acid and rifampicin. In a particular treatment option, the stability of the compounds (drugs or phytochemicals) is important as it selects resistance development in bacteria. The stability and the effectiveness of conventional antibiotics or phytochemicals were studied by inducing single-step adaptive resistant development in MRSA. The duration it took to get phenotypic mutant strains for a particular drug/phytochemical concentration was compared individually or in combination. It was found that conventional antibiotics under study were less stable than phytochemicals and combination of synergistic phytochemicals together with antibiotics could delay resistance formation in MRSA. The effectiveness of using fixed sub-Minimal Inhibitor Concentration (MIC) of phytochemicals in combination with synergistic step-wise increasing concentration of antibiotics were observed in this study. It was found that giving sub-MIC concentrations of tannic acid, quercetin and gallic acid ethyl ester together with fusidic or rifampicin could overcome or delay the occurrence of adaptive resistance in MRSA strains. Combination therapies using two or more synergistic antibiotics have been used before and their effectiveness in resistance prevention is still questionable because of the stability and the dosing pattern of the compounds used. In this study, with the consideration of the effectiveness of phytochemicals in combination with synergistic antibiotics in resistance prevention or delaying effects on partner synergistic antibiotic, a new noble dynamic drugs dosing combination hypothesis was introduced and tested with 4 MRSA strains for a duration of 10 days and 30 days to understand short-term and long-term effects. Giving more than 20% (0.2 MIC) of tannic acid or quercetin together with synergistic fusidic acid was found to prevent adaptive or genetic mutations in MRSA strains. Phytochemicals are assumed to have multi-targeted actions. Many authors proposed possible mechanisms of action of phytochemicals and much research is done on individual proposed mechanisms. Possible mechanisms of action of synergistic phytochemicals under study were investigated by using the Ethidium Bromide Accumulation Assay. Tannic acid, quercetin and gallic acid ethyl ester exhibited efflux pump inhibiting activities against four MRSA strains. The effects of phytochemicals on MRSA mutant strains revealed that phytochemicals under study have the ability to lower severe mutant strains into less severe mutant strains by bringing down the MICs. This work opens up the research into the novel strategies but using currently available drugs and synergistic phytochemicals instead of depending on new drugs. This is particularly important because developing new drugs for the prevention of resistance adaptation of multi-drug resistant bacteria like MRSA is extremely time consuming, expensive and comes with a high failure rate. DOCTOR OF PHILOSOPHY (MAE)

In this study, confocal microscope observation coupled with the fluorescence staining was employed to monitor biofouling of Reverse Osmosis (RO) membranes using Pseudomonas aeruginosa and Pseudomonas fluorescence as model fouling organisms. A range of parameters, including the presence of feed channel spacers, nutrient level, flux and cross-flow velocity were compared to determine their roles in biofouling. In addition, novel biofouling controls (UV pretreatment and NO addition) were tested to determine their efficacy as biofilm control agents. It was found that biofilm formation resulted in a slow rise in TMP and followed by a sharp increase, called the ‘TMP jump’. The initial slow increase in TMP was most likely due to the formation of a biofilm on the membrane surface, which then accelerated the biofouling rate through cake-enhanced polarization of nutrients. It was observed that nutrient limitation slowed biofilm accumulation and delayed the increase in TMP. It was evident that the presence of the spacer improved the performance of the RO system. In the presence of the spacer, biofilm development was slower and the TMP showed a much slower rise. When a spacer was introduced into the membrane module, the biofilm primarily occurred on the membrane, not on the spacer. While the inclusion of spacers delayed fouling, a faster TMP rise was observed under conditions of higher flux and lower cross-flow velocity. Two approaches were examined to control biofilm development and hence, to improve the performance of the RO system, UV pretreatment to kill the bacteria in the feed and induction of biofilm dispersal using Nitric Oxide (NO). UV pretreatment effectively inactivated bacteria in the feed. While UV treatment delayed biofilm formation and fouling, it did not prevent biofilm formation. Preliminary data showed that the NO donor, Sodium NitroPrusside (SNP) could disperse biofilms formed on RO membranes. The results presented here suggest that improving hydrodynamics (e.g. reducing the flux, increasing cross-flow) in membrane modules in combination with limiting nutrient concentrations in the feed as well as rationally-designed feed spacers could be used to control biofouling and improve system performance. DOCTOR OF PHILOSOPHY (SBS)

Parasitic manipulation of host behavior is often seen as exploitative in nature. An example is Toxoplasma gondii that has shown to alter the innate fear in rats toward cat odors, increasing its chances of transmission to complete its two-stage life cycle. In this thesis, I studied another behavioral manipulation in the reproductive domain. I show that T. gondii manipulates mate choice, causing females to prefer infected male rats and is also sexually transmissible. I demonstrated that this mate choice manipulation is due to Major Urinary Proteins (MUPs) and that infected rats had increased levels of MUPs. Moreover, MUPs is sufficient and necessary to attract females in a dose dependent manner. Finally, MUPs was shown to be dynamic and condition dependent on three factors- age, health and social environment. Thus, a parasite that can enhance reproductive opportunities of a host will benefit from blunted selection pressure from the host and gain advantage of transmission. DOCTOR OF PHILOSOPHY (SBS)

Besides the common right-handed B- or A- structures, the alternate Z-conformation, which is left-handed, can be formed in both DNA and RNA molecules. It is in a higher energy state and well-studied mostly in vitro. However, formation of Z-conformation in living organisms and its biological significance remains largely elusive. Here, a protein probe that could specifically bind to Z-conformation was used to map the Z-DNA distribution in the human genome. The probe is derived from the Z-DNA binding domain of the human double-stranded RNA adenosine deaminase 1 (ADAR1), named ZαADAR1. During our experiments, evidence suggested that the primary target of ZαADAR1 in vivo is RNA rather than DNA. Further studies revealed that ZαADAR1 could bind to ribosomes and inhibit translation in both E. coli and mammalian systems in a Z-conformation dependent manner. Potential ZαADAR1 binding sites on ribosomes were identified. Several binding sites were conserved between E. coli and human ribosomes, revealing that formation of Z-like RNA conformations might be a conserved property of the dynamic ribosome structure during translation. The implications for understanding biological functions of the full length ADAR1 were discussed. In vitro Chromatin Affinity Precipitation (ChAP) experiments using ZαADAR1 were also performed to map the Z-DNA distribution in the human genome, using human embryonic stem (huES) cells as the cell source. The data revealed that the ZαADAR1 binding sites were found to be enriched in centromere regions and G-bands, suggesting that formation of Z-DNA might contribute to specific chromatin structures. The enrichment of Z-DNA in transcription start sites then suggested that formation of Z-DNA is associated with transcription processes. Enrichment of SNPs in ZαADAR1 binding sites, especially in the centromere region, further suggested that formation of Z-DNA might be mechanistically linked to genetic instability and thus contributes to the evolution of centromere regions. Surprisingly, the ZαADAR1 binding sites did not coincide with Z-DNA forming sequences predicted based on in silico analyzes. This indicates that formation of Z-DNA in living cells is not purely depended on DNA sequence contexts, but is strongly affected by on the local physical environment. DOCTOR OF PHILOSOPHY (SBS)

Bacteria in the environment exist mostly as sessile communities known as biofilms. Biofilms are widely found on various biological surfaces such as on the leaves and roots of plants as well as in the lungs of humans, where they are associated with diseases. Biofilms can also grow on abiotic surfaces of rocks, water pipelines, contact lenses and surgical implants. Some biofilms can form independent of a surface, called suspended biofilms, and are represented by either floes or granules that are important for the proper functioning of wastewater treatment plants. Biofilms, which are difficult to eradicate, have many negative impacts. For example, they are reservoirs for many pathogens and their activities also lead to biocorrosion of different surfaces. Biofilms are also essential, contributing to the biogeochemical cycling in the environment and are key players in processes such as sewage treatment and bioremediation. As a result, intensive research has been carried out with the aim to manipulate biofilms to our advantage. However, a main limitation of such research to date is the focus on mono species biofilms. In reality, biofilms in the environment consist of multiple interacting species, where interspecies interactions play an important role in determining the development, structure and function of the biofilms. Hence, a mixed species biofilm model that can be explored experimentally for research and development projects, with relevance to specific environmental, industrial and medical settings, is much needed. Here, a reproducible mixed species biofilm consisting of Pseudomonas aeruginosa PAOl, Pseudomonas protegens Pf-5 and Klebsiella pneumoniae KP-1 was studied and compared to the respective mono species biofilms. The three bacterial species were stably labeled with three different fluorescent proteins using a Tn7 transposon expression system. The development of the mixed species biofilms was delayed relative to all mono species biofilms. While mushroom and tower like microcolonies were observed for mature PAOl and Pf-5 mono species biofilms respectively, these structures were not seen for the mixed species biofilms. In contrast, KP-1 in the mixed species biofilms formed mound-like microcolonies, which were not observed when it was grown alone. The proportions and structures of the three bacterial species within the mixed species biofilms grown at the inlet end and outlet end of the continuous-culture flow cell also differed, most likely as a result of decreasing glucose concentration and associated changes in the specific growth rate for KP-1 along the channel. Most importantly, the mixed species biofilms were more resilient to stresses such as tobramycin and sodium dodecyl sulfate than the mono species biofilms. Intriguingly, such community level resilience was found to be contributed by the resistant species to the whole community rather than selection for the resistant species. In addition, community level resilience was not observed for mixed species biofilms with loosely associated members and for mixed species planktonic cultures, suggesting that community level resistance was unique to a structured biofilm community with closely associated members. The formation of morphotypic variants, which is typical of almost all biofilm forming bacteria to date, was also observed and quantified in this study. One morphotypic variant each was identified for PAO 1 and KP-1 while four morphotypic variants were observed for Pf-5. The formation of these morphotypic variants was specific to biofilms as planktonic cultures produced either no variants or a significantly reduced number of variants. The morphotypic variants were also stable after daily passaging, while sequencing of their genomes showed that they possessed insertions/deletions and single nucleotide polymorphisms that might lead to changes in their phenotypes and morphotypes. In fact, each morphotypic variant was shown to differ from its parent strain in various phenotypes such as swimming and swarming motilities, attachment, biofilm formation as well as pyoverdine and pyocyanin productions. In addition, the morphotypic variants also formed biofilms with distinct structures and outcompeted their parent strains in co-cultured biofilms. The morphotypic variants also outperformed their respective parent strains when grown with the other two species in a mixed species biofilm. Despite their fitness, the frequency and diversity of these morphotypic variants decreased in the mixed species biofilms. Since each strain sacrificed the production of individual variants in the presence of other species, the results suggest that interspecies diversity may be more important than intraspecies diversity in microbial biofilms. Notably, the addition of PA01 and KP-1 cell-free biofilm effluents to Pf-5 biofilms also decreased the frequency and diversity ofmorphotypic variants formed by Pf-5. Hence, molecules present in the biofilm effluents ofPA01 and KP-1 may be responsible for an interspecies cell-cell signaling that regulates self-generated genetic diversity. Subsequently, metatranscriptomic analyses were performed to determine the genes that might be involved in biofilm response to sodium dodecyl sulfate. The results showed that extracellular polymeric substances production and export as well as stress response proteins were found to be involved in the resistance ofPA01 and KP-1 biofilms to SDS. Particularly, the up regulation of siaA has been associated with the autoaggregation of PA01 cells during SDS treatment, thereby protecting them from lysis by SDS. Similarly, the induction of Cpx envelope stress response proteins in SDS treated KP-1 biofilms may be responsible for protecting KP-1 cells from lysis by SDS through ensuring proper folding of the envelope proteins and maintaining the integrity of the cells. The induction of extracellular polymeric substances and relevant stress response proteins was not observed for SDS sensitive Pf-5 biofi lms. In conclusion, results from this study have shown that mixed species biofilms differ from their mono species counterparts in terms of development, structure, resilience and intraspecies diversity. As a result, studies on mono species biofilms cannot predict the behaviours of mixed species biofilm communities in nature. The mixed species biofilm model used in this study is reproducible and is one of the few emperical studies that provided evidence for the proposition that interspecies diversity could substitute intraspecies diversity. Furthermore, all three bacterial species are genetically tractable and amenable to molecular techniques such as mutagenesis and 'omics' based approaches, thus making this mixed species model an excellent system to investigate interspecies interactions using both the top down and bottom up approaches. DOCTOR OF PHILOSOPHY (SBS)

Enzymes are indispensable in maintaining the biological system. They metabolize complex molecules to supply nutrients, to produce energy, to regulate transcription of gene expression, and to control the concentration of effective signaling molecules in a cell, thus maintaining the homeostasis of biological system. This thesis summarizes the study of the structure and function of two enzymes in lipid signaling family using integrative application of X-ray crystallography, solution NMR spectroscopy, light scattering, ITC and thermal shift assay. Lipocalin prostaglandin D synthase (L-PGDS) is a tissue specific prostaglandin D2 producing enzyme with a lipocalin fold. Apart from its enzymatic role, it is known to act as a lipophilic ligand carrier. Crystal structure of human L-PGDS and substrate analog altogether with NMR spectroscopy experiments revealed binding sites for substrate catalysis and entry. NMR titration experiments with membrane mimetic showed that L-PGDS has intrinsic membrane binding affinity depending on the ligand bound. These results allowed a model of substrate catalysis and product egression to be proposed, hence, converging the enzymatic and transporter role that has been reported in literature previously. Since prostaglandin D2 is a pivotal inflammatory signaling molecule, molecular understanding of L-PGDS is important to facilitate future regulation of the prostaglandin isomerase. The dynamics of substrate-product exchange may guide future design of this lipophilic carrier as vehicle for drug delivery. The second enzyme, human acid sphingomyelinase like 3a (SMPDL3a), belongs to a metallophosphodiesterase family and shares close sequence identity with human acid sphingomyelinase (aSMase). SMPDL3a’s structure is reported for the first time revealing its binuclear catalytic core site bound with Zn metal. Even though it was presumed to be part of the lipid hydrolase family, enzymatic assays showed that it metabolizes nucleotides and modified nucleotides like CDP-choline, CDP-ethanolamine and ADP-ribose. Subsequently, CDP-choline soaked crystal revealed 5’ cytidine monophosphate (CMP) ligand bound in the catalytic site due to spontaneous catalysis. Its α-phosphate forms key interactions with histidine residues in the binuclear center. Based on this CMP-enzyme structure, general catalytic mechanism of aSMase family can be proposed. Besides, SMPDL3a also serves as a template for aSMase catalytic domain homology modeling. Further study on enzymes in the acid sphingomyelinase family can now be guided by the newly available structural information. DOCTOR OF PHILOSOPHY (SBS)

Tumor cell migration and invasion involves actin cytoskeleton reorganization, which is regulated by N-WASP (Neural-Wiskott Aldrich Syndrome Protein) and its interacting proteins such as WASP interacting protein (WIP). Expression of WIP was found to be higher in metastatic cancer cell line compared to its non-metastatic parental cell line. Overexpression of WIP was found to enhance the proliferative, migratory and invasive ability as well as confer anchorage independent growth properties in A549 lung carcinoma cells indicating a pro-metastatic function of WIP. Expression of N-WASP is enhanced in cells undergoing epithelial mesenchymal transition induced by hypoxia. In order to identify mechanism responsible for the differential expression of N-WASP, the N-WASP promoter was characterized, which led to the identification of HRE (Hypoxia Response element) a regulatory region in N-WASP promoter. Our results suggest that transcription factor HiF1α regulates N-WASP expression in promoting metastasis under hypoxic conditions. DOCTOR OF PHILOSOPHY (SBS)

Malaria is an important human infectious disease affecting millions of people every year. Despite years of research, transcriptional regulatory mechanisms in Plasmodium are still poorly understood. The presence of a large number of zinc finger proteins amongst the potential specific transcription factors suggests the importance of these proteins. In this thesis, we characterized PF10_0083, a putative transcription factor that belongs to the CCCH type zinc finger family. Using over-expression and knock-out cell lines, we carried out molecular characterization of this protein. PF10_0083 protein level was found to peak during the schizont stage, and the peak protein levels coincided with its nuclear localization. PF10_0083 was found to be expressed in only a sub-population of schizonts. We demonstrated that the over-expression of PF10_0083 resulted in enhanced gametocyte production, while the knock-out of PF10_0083 caused decreased gametocyte production. In addition, we showed that PF10_0083 is capable of associating with DNA. We demonstrated that PF10_0083 preferentially binds to the IGRs on the genome, and that this binding positively correlated with the transcriptional up-regulation of the gametocyte genes. As such, we conclude that PF10_0083 promotes the differentiation of asexual parasites to sexual forms under stress conditions. DOCTOR OF PHILOSOPHY (SBS)

The conidial germ tube in Magnaporthe oryzae, the causal agent of the devastating rice-blast disease, responds to inductive surface cues and switches from polarized to isotropic growth to ultimately form the specialized infection structure known as the appressorium. The critical role played by signaling pathways, especially the G-protein/ cyclic AMP (cAMP) signaling in appressorium initiation was identified almost two decades ago, yet several fundamental questions remain unanswered. The purpose of this study was to 1) identify additional regulatory proteins that facilitate the maintenance of steady-state levels of cAMP; 2) decipher in-vivo mechanisms that function to control the activity of such regulatory proteins; 3) investigate the spatial and temporal dynamics of the cAMP signaling cascade and finally 4) understand how the signals are transmitted through the crowded intracellular network to specific sites of growth and/or signaling. Utilizing a combination of reverse genetics, biochemical analyses and cell biology techniques, in Chapter I, I identified and functionally characterized two cAMP phosphodiesterases (a high affinity- PdeH and a low affinity PdeL) in M. oryzae. I also demonstrate that PdeH is the major enzyme hydrolyzing and regulating intracellular levels of cAMP during asexual, pathogenic and in planta development. More importantly, the study identifies that cAMP levels are modulated by PdeH in a biphasic manner, i.e. up regulation during early stages (appressorium initiation), and down regulation at the late stages (host invasion) of pathogenic development. The data further implicates the existence of two distinct pools of cAMP (cytosolic and nuclear) in M. oryzae, which are differentially modulated by PdeH and PdeL respectively. Given that Regulator of G-protein signaling (RGS) proteins function as key negative regulators that control the intensity and duration of active G-protein signaling, in Chapter II, I sought to address and understand the mechanisms possibly involved in the regulation of a proto-typical member, namely Rgs1(a GAP for MagA). I found that Rgs1 was subjected to an endo-proteolytic cleavage yielding an N-terminal DEP-DEP fragment (that facilitated membrane targeting) and a C-terminal catalytic core RGS domain that was sequestered within vacuole in the absence of a functional DEP motif. Rgs1 cleavage likely represents an important means of ultimately modulating the duration and intensity of G-protein signaling. Based on the physical interaction with Rgs1 specifically during early stages of appressorium initiation, I demonstrate Pth11 to be a bona fide GPCR (albeit non-canonical) for cAMP signaling in M. oryzae. Finally, in Chapter III, I demonstrate that in the likely absence of AKAP-like anchor proteins in M. oryzae, the late endosomal compartments (comprising of a PI3P-rich dynamic tubulo-vesicular network) are capable of serving as scaffolds or platforms to anchor active MagA/GαS, Rgs1, Adenylate cyclase and Pth11. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Exogenous cAMP remarkably rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae, suggesting that the late endosomal HOPS complex potentially serves as a scaffold to anchor, compartmentalize and mobilize G-protein signaling components to sites of active growth and signaling, during early pathogenesis. Taken together, the results presented in this thesis suggest that M. oryzae utilizes a variety of complex cellular mechanisms to tightly regulate the steady-state levels as well as modulate the spatio-temporal dynamics of cAMP signaling in response to inductive cues as well as in the context of changing cellular geometry DOCTOR OF PHILOSOPHY (SBS)

The contribution of epigenetics in the regulation of leukocyte development and functions has gained increasing attention over the past years. Polycomb group protein Enhancer of Zeste homolog 2 (Ezh2) is a histone methyltransferase which regulates gene expression through the modification of chromatin structure via the addition of H3K27me3 marks. In this study, we aim to characterize the role of Ezh2 using Langerhans cells (LCs) and thymic dendritic cells (DCs) as model systems, and further elucidate the molecular mechanism and in vivo relevance underlying such regulation. Surprisingly, the well-established epigenetic repressor Ezh2 was found to control LC integrin-dependent migration via an epigenetic-independent mechanism involving the direct methylation of an extra-nuclear substrate Talin1. Deficiency in Ezh2 impairs the disassembly of adhesion structures which enhances the adhesive properties of LCs, leading to their reduced transmigration across the skin basement membrane. Using a mouse model for allergic contact dermatitis, we demonstrated that reduced migration of Ezh2-deficient LCs to the skin-draining lymph nodes resulted in the development of an exaggerated contact hypersensitivity (CHS) response despite tolerization with low dosage of an innocuous hapten 2, 4- dinitrothiocyanobenzene. Such breakdown in skin tolerance arises from the compromised activation of regulatory T cells, which consequently promotes an enhanced CD8+ T cell response in the sensitization phase of CHS. In addition, DC-intrinsic expression of Ezh2 was found to regulate early T cell developmental program particularly in adult mice. Deficiency of Ezh2 in thymic DCs favours the T cell lineage and leads to a profound developmental block characterized by a decrease in DP thymocytes, indicating their roles in directing lineage choices and DN thymocyte development. These developmental defects are accompanied by a change in the cytokine milieu of the thymus, suggesting the importance of DC-intrinsic Ezh2 in sustaining a proper thymic cytokine environment for normal T cell development. Collectively, our data highlighted the importance of Ezh2 in LCs for the regulation of their integrin-dependent migration, and also in thymic DCs for the maintenance of adult-stage early T cell development. DOCTOR OF PHILOSOPHY (SBS)

Social behavior can be viewed as a varying output of an approach-avoidance conflict. For example a potential mate elicits a strong ‘approach’ response and a predator induces the very opposite. Social behavior must be plastic to best adapt to a continuously changing (biotic and abiotic) environment. Different external states are mirrored by changes in levels of circulating sex hormones; presence of a mating opportunity elevates testosterone levels while presence of a stressor depresses it. I hypothesize that the plasticity in social behavior is a function of changes in circulating sex hormones; i.e. testosterone in males. Arginine vasopressin (AVP) is a neuropeptide widely implicated in social behavior. In the extrahypothalamic regions of the brain it is highly sensitive to testosterone. Castration causes a drop in AVP projections in the extrahypothalmic zone, medial amygdala (MEA); a change that can be attributed to an androgen dependent hypermethylation of the AVP promotor. For these reasons extrahypothalmic AVP was selected as a candidate target. I focused my studies on the MEA, a part of the social behavior network, which has a central role in social behaviors such as reproductive and defensive behavior. The infection of rats by Toxoplasma gondii is a well suited system to study social behavior. Infected male rats display an attraction to their feline predators. This is a shift of the approach-avoidance conflict from avoidance to approach. I show that this change can be generalized to sexual aspects of the social behavior, coupled with a corresponding increase in testosterone levels. The changes in behavior were apparent in a long-time frame rather than being transient, implying a long lasting robust proximate mechanism. This suggested epigenetic regulation as an ideal candidate. AVP promoter contains two androgen sensitive methylation sites. Infection decreased methylation at both these sites in a sub-nucleus of the MEA. Global hypomethylation (methionine injection; s.c.) in infected males was sufficient to reverse Toxoplasma gondii infection induced behavior changes. Post methionine treatment infected males were no longer attracted to cat odor. Furthermore, hypomethylation by treatment with a DNA methyl transferase inhibitor (RG108) in MEA decreased fear response towards cat odor. I demonstrate that testosterone is necessary to drive the infection induced changes in behavior; localized supplementation of testosterone in the MEA of castrates reduces their fear response. Further an overexpression of AVP in the MEA is sufficient to cause a reduction in the fear response. Based on these findings I propose a model for plasticity of social behavior wherein changes in external environment are mirrored by changes in testosterone levels in males. This in turn changes levels of AVP in the MEA via androgen dependent methylation sites. Decreased methylation of the AVP promoter regions corresponds to increased AVP expression and an ‘approach’ bias in social behavior and vice versa. Taken together these posit a central role for epigenetic modulation of AVP in the MEA as a mediator of plasticity in social behavior. In culmination I propose that social behavior is collectively governed by a trinodal ‘extended social behavior network’. This consists of reciprocally connected nodes; the environment, gonadal milieu and neural circuits. DOCTOR OF PHILOSOPHY (SBS)

Bcl-2 family proteins play central roles in the apoptotic regulation at the mitochondrial membrane. Bcl-XL belongs to the anti-apoptotic subfamily of the Bcl-2 family. It interacts with pro-apoptotic subfamilies to inhibit them from forming pores on the mitochondrial membrane. Our current understanding on the function of Bcl-XL is primarily based upon its structural features and molecular characteristics in solution. Currently the membrane structure of Bcl-XL and its molecular mechanism in membrane environment remain poorly understood. Understanding the mode of binding is essential to unravel the biological function of proteins. In this thesis research we have characterized Bcl-XL in a detergent micelle which mimics membrane environment by employing biophysical methods including NMR spectroscopy. We first assigned NMR resonances of Bcl-XL and determined structure of Bcl-XL in DPC micelle. Furthermore, the binding mechanism with other Bcl-2 family members was also examined in detergent micelle by employing NMR spectroscopy. The molecular binding analysis demonstrated that the BH3 peptides fail to bind with Bcl-XL in micelle, indicating that Bcl-XL undergoes structural transition when Bcl-XL anchors to the micelle. Even though truncated Bid maintains the binding ability to Bcl-XL in micelle, NMR cross-saturation data indicates that the binding interface is significant different from that in solution phase. Based on the results, we proposed an interaction model of Bcl-XL in apoptosis regulation. DOCTOR OF PHILOSOPHY (SBS)

The skin is the largest organ in the body and is made up of mainly the epidermis and dermis. The skin functions as a natural barrier against external stimuli, dehydration and injury. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is one member of the PPAR family of nuclear receptors, and the predominant subtype found in the epidermis and dermis. Epidermal PPARβ/δ has well-established roles in maintaining skin homeostasis, such as during wound healing. However, in the mammalian system, skin homeostasis depends on a complex crosstalk between the epithelial and mesenchymal compartments, underscoring the importance for a tight regulation of the complex epiethelial-mesenchymal interaction. Although epidermal PPARβ/δ in normal wound healing is well-studied, there has been no progress in clinical therapies for diabetic chronic wounds. This is also ensued with problems such as drug bioavailability and patients’ compliance. In our study, we showed that pharmacological activation of fibroblasts PPARβ/δ activity using microencapsulated ligands can promote the healing of diabetic wounds via the modulation of ROS production. The role of PPARβ/δ in other diseases, such as cancer, has also been controversial due to differences in experimental setups and genetic modifications of the PPARβ/δ gene. While epidermal PPARβ/δ has been widely investigated on in these diseases, the role of fibroblasts PPARβ/δ in skin homeostasis and disease has been neglected. Hence, we generated a novel mouse model with the PPARβ/δ gene deleted specifically in the fibroblasts. These animal exhibited skin abnormalities during development, such as dermal thickening and increased collagen production, recapitulating a morphea-like phenotype. In addition, the dermis manifest as a hotbed for inflammatory events. In our subsequent preliminary studies, we showed that cancer-associated fibroblasts from squamous cell carcinoma exhibit low levels of PPARβ/δ. Our novel mouse model will therefore serve as an appropriate model to study the role of fibroblasts PPARβ/δ in diseases, such as acute inflammation, wound healing and tumorigenesis in the future. DOCTOR OF PHILOSOPHY (SBS)

Peptide and protein biologics are therapeutics and prophylactics known for their desirability of high specificity and low toxicity. They display a wide range of biological activities such as antimicrobial, immunomodulation, neurotransmission and hormonal functions. Most biologics are derived from animal sources with few representatives from plants. In herbal medicine, these biologics have not received much attention with the common perception that they are unstable and unavailable as a source of active principles in decoctions. This bias is explained by the intrinsic instability of peptides and proteins during the concoctions preparation, poor absorption through the gastrointestinal tract and their susceptibility to enzymatic and acidic hydrolysis under harsh environment inside our stomach. My hypothesis is that plant biologics may constitute a group of unexplored active principles in traditional herbal medicine. Research in this thesis focuses on plant cysteine-rich peptides (CRPs) such as cyclotides, uncyclotides, knottins, heveins and plant defensins. These peptides have recently gained great interest among the scientific community due to their remarkable stability and diverse biological functions. The major aims of this project are: (1) discovery of novel biologics from medicinal plants using tandem mass spectrometry, (2) characterization of their thermal and enzymatic stabilities, tissue specificity, genetic structures and disulfide connectivities, (3) pharmacological profilings of their biological activities for therapeutic applications. The proposed study may provide lead compounds for drug development and aid in quantitative profiling of active ingredients in commercial viable medicinal herbs. In this thesis, I have successfully identified more than 120 novel peptides comprising of cyclotides, uncyclotides, defensins and various CRPs from several medicinally important plants. By far only four uncyclotides have been reported, and thus the discovery of over 20 uncyclotides in this work is a surprising and provides insights about the biosynthetic processing of cyclic peptides. I also provide the first description of defensin-like peptides in cyclotide-producing plants. In addition, two novel classes of cysteine-rich peptides, ginsentide and morintide, have been identified from ginseng and Morinda citrifolia, respectively. Furthermore, I also expand the distribution of cyclotides to a new plant family, the Fabaceae. Biophysical characterization of novel CRPs showed that most of them display high stability against heat and enzymatic digestions together with their amphipathic structures making them the likely active principles in herbal medicine. Activity screenings showed that many CRPs display potent antimicrobial, cytotoxic and hemolytic properties. Biologics with interesting pharmacological profiles could be used as lead compounds for drug development. DOCTOR OF PHILOSOPHY (SBS)

UBE3A encodes the ubiquitin ligase E6-AP and the point mutation of this gene causes severe neurological disorder called Angelman Syndrome (AS) in a young child. To elucidate the molecular basis of AS pathogenesis and promote the design of rational therapies for AS patient, an Ube3a null mutation mouse, which recapitulated the development and behavioural defects of human patients, was generated. The mice with maternal deficiency (m-/p+) for Ube3a display several features of AS, including microcephaly, motor dysfunction, inducible seizures, abnormal hippocampal EEG and deficits in context-dependent learning. Based on the knowledge of altered UBE3A expression profile and uncoordinated movement observed in AS patients, I hypothesized that the normal function and electrophysiological properties of Purkinje neurons (PNs), which provide the sole output of cerebellar cortex might be affected by the deletion of E6-AP. The electrophysiological studies on PNs of AS mouse would be able to provide meaningful information on deciphering the molecular basis for the learning and memory deficit in AS and be helpful for developing effective drugs. Thus, in the first part of this thesis, the physiological properties of cerebellar cortex in Ube3a null mutation mice were thoroughly examined. The data collected from acute cerebellar slices showed that the intrinsic excitability, the inhibitory synaptic transmission and basal excitatory synaptic transmission were normal in the mutant mice. There was also no alteration in a type of long-term potentiation (LTP) that is presynaptically expressed at PF (parallel fiber)-PN synapses. In contrast, another type of LTP, which expressed postsynaptically at the same PF-PN synapses, was impaired, and another type of synaptic plasticity, long-term depression (LTD), III ATTENTION: The Singapore Copyright Act applies to the use of this document. Nanyang Technological University Library

Because almost all peptides and proteins have only one N- and one C-terminus, modification targeting terminal residues is obviously an attractive way to introduce a functional group into a peptide or a protein at a single site as compared to the reactions on the amino acid side chains. This forms the rationale of our study. First, I aim to provide reliable information about the transamination reaction to make future use of this NT-specific protein modification reaction in a much more predictable way. Second, on the peptide CT-modification aspect, this study aims to develop new chemical and enzymatic methods to synthesize peptidyl thioesters and thioacids which are important derivatives to introduce new functional groups onto the C-termini of proteins and peptides. DOCTOR OF PHILOSOPHY (SBS)

The regulatory functions of Bcl-2 and Bcl-2-binding protein FKBP38 in apoptosis and cell cycle have been sufficiently studied by using various molecular and cellular experimental methods. DOCTOR OF PHILOSOPHY (SBS)