Kindlin-3 is a haematopoietic adaptor protein best known for regulating integrin activation. This thesis investigated its role in interleukin-2 receptor (IL-2R) signalling. Kindlin-3 preferentially associated with IL-2Rβ, with the interaction mapped to the kindlin-3 F0 subdomain and IL-2Rβ residues 530 to 551. Bio-Layer Interferometry confirmed direct binding between these regions with a dissociation constant of approximately 6 μM. Functionally, kindlin-3 promoted IL-2Rβ retention at the plasma membrane in an LFA-1-dependent manner, and this required the integrin-binding function of kindlin-3 and its native F0 subdomain. Kindlin-3 also supported sustained IL-2-induced STAT5, AKT and ERK1/2 signalling following cytokine withdrawal and promoted NK92 cell survival under limiting IL-2 conditions. Collectively, these findings identify kindlin-3 as a molecular link between LFA-1 and IL-2Rβ that coordinates cytokine receptor retention and sustained IL-2 signalling in haematopoietic cells.
Matrin-3 is an RNA- and DNA-binding protein associated with amyotrophic lateral sclerosis. The protein has no known yeast homologs and forms nuclear puncta in yeast, but loss of its RNA-recognition motif (RRMs) causes Matrin-3 to phase-separate into liquid, shell-like condensates. Taking advantage of the relatively simple chaperone system in yeast, I discovered that Hsp70 proteins and J-domain proteins (JDPs) are enriched in the cores of Matrin-3 multiphase condensates. RRM-deficient Matrin-3 transitions from uniform puncta to multiphase condensates over time. In yeast, I identified Sis1 and Sse1 as a critical JDP and nucleotide exchange factor, respectively, in Matrin-3 multiphase separation. Thus, the complete ATP/ADP cycle of the yeast Hsp70 system is required for Matrin-3 multiphase condensation. Additionally, I discovered that the zinc finger 2 domain of Matrin-3 is critical for its self-assembly and that disease-associated Matrin-3 mutations can perturb multiphase condensation in a yeast model. Other proteins, such as TDP-43 and REV-ERBα, have also been reported to form shell-like multiphase condensates enriched in chaperones. Thus, shell-like multiphase condensates may represent a general phenomenon driven by chaperone activity.
Pseudomonas aeruginosa is an opportunistic human pathogen capable of infecting a wide range of tissues and organs. Its virulence is largely attributed to biofilm formation during chronic infection, a process critically dependent on several extracellular polysaccharides, including alginate. The HtrA-like periplasmic serine protease MucD is one of the key regulators of bacterial virulence, stress response, and alginate production, yet its molecular mechanism has remained largely unclear. Here, we for the first time identified alginate biosynthesis proteins AlgK and AlgX as its protein substrates and characterized their degradation. Subsequently, we determined the cryo-EM structure of MucD in the apo and substrate bound forms, offering atomic insights into its substrate recognition and specificity. Together with structure-based mutation and biochemical assays, our results further revealed that the proteolytic activity of MucD is governed by an oligomeric equilibrium. Crucially, our in vivo data demonstrated that MucD represses alginate biosynthesis post-translationally. Our work elucidates the structural basis for the unique activation feature and a distinct regulatory role of MucD, providing novel insights into the regulation of alginate production and the functional role of HtrA-like serine proteases in bacteria.
Epithelial tissues rely on apical–basal polarity to maintain their structural integrity while undergoing dynamic changes during development. The Crumbs complex, comprising key components including CRB3, PALS1, PATJ and MPDZ, plays a central role in organizing epithelial polarity and coordinating cellular signalling. This thesis investigates how these scaffold proteins regulate tissue architecture and developmental signalling, using MDCKII epithelial cells and zebrafish embryos as experimental models. In MDCKII cells, the loss of PATJ or PALS1 disrupts apical–junctional organization but leads to distinct changes in YAP and WNT signalling, suggesting that these scaffold proteins regulate the cellular context of signalling rather than individual pathways. In zebrafish, PALS1a deficiency progressively disrupts cardiac architecture and the spatial organization of WNT and BMP signalling at the atrioventricular canal. Heartbeat inhibition abolishes the Klf2–WNT signalling programme regardless of PALS1a status, indicating that mechanical input is essential for this signalling programme, while PALS1a contributes to its proper spatial organization. Additionally, investigations into PATJ and MPDZ reveal distinct developmental requirements for these two scaffold proteins. Together, these findings highlight the importance of Crumbs-complex scaffolds in connecting epithelial architecture, tissue mechanics and spatial developmental signalling during morphogenesis.
Lipid bilayer asymmetry is maintained by a large variety of transbilayer lipid transporters. P4-ATPases are unidirectional, ATP-dependent lipid flippases that mediate the exoplasmic to cytosolic movement of lipids. Through recent advances in protein sample preparation and cryoEM methods, high resolution structural data has been able to reveal the functional characteristics of a large variety of yeast and mammalian flippases. However, the role of P4-ATPases in insect membrane environments remains poorly understood. The Drosophila melanogaster flippase ATP8B is an essential modulator of olfaction and has been known to maintain olfactory sensory neurons. In this study, a comprehensive investigation of dATP8B via biochemical and cryo-EM based methods is presented. Nineteen high resolution cryo-EM structures, lipid-dependent activity assays and the effect of modulatory lipids are reported. An unprecedented configuration of P4-ATPases is identified, and a rationale for possible role in activation is presented.
Seeds are important organs that are fundamental to agriculture by ensuring crop propagation while providing humans with nutrients and raw materials for food and industrial applications. Despite their importance, the molecular mechanisms underlying seed development and maturation remain elusive. In the first chapter, we demonstrate that BBX32 interacts with WRI1, a central transcriptional regulator of seed oil biosynthesis. BBX32 enhances the transcriptional activity of WRI1, leading to elevated expression of downstream lipid biosynthetic genes and increased seed oil accumulation. In the second chapter, we identified a previously uncharacterized role for DREB2G in regulating multiple seed traits, including oil accumulation, seed morphology and germination. Transcriptomic analysis revealed that DREB2G overexpression promotes germination by upregulating genes involved in cell-cycle and cell-wall loosening, such as the D-type cyclins and EXPANSINs. Collectively, these findings uncover two novel transcriptional regulatory mechanisms governing seed traits, identifying promising targets for genetic improvement of seed quality and productivity.
The Polycomb group protein Ezh2 is an important epigenetic regulator that has been extensively studied for its role as an H3K27 methyltransferase in various biological mechanisms. My preliminary data showed that Ezh2 deficiency in dendritic cells (DCs), the major professional antigen-presenting cells, leads to the accelerated thymic involution and an increase in CD8 T cells with a virtual memory phenotype (CD8 TVM). In addition, mice with Ezh2-deficient DCs (Ezh2-KO) showed significant morphological and functional changes in central nervous system (CNS) microglia and astrocytes, indicative of a brain aging phenotype. Single-cell transcriptomic of brain cells unravelled an expansion of pro-inflammatory microglia clusters in mutant mice as well. Ezh2-KO mice also demonstrated increased lymphocyte subsets, particularly CD8 TVM, in the brain, suggesting the role of CD8 TVM in the observed brain aging progress. Increased myeloid infiltration and Evans blue leakage was observed in the brain of Ezh2-KO mice indicating the blood-brain barrier disruption. Further studies will elucidate the precise mechanisms of CD8 TVM in the aging brain through their potential interaction with microglia. Collectively, these findings unravel the relationship between thymic involution and neuroinflammation, shed light on the potential impact of the CD8 TVM cells in the CNS, and identify potential mediators of thymus-brain crosstalk in the aging context.
Small molecules are essential in targeted cancer therapies and are abundantly present in medicinal plants, making phytotherapy a promising source for anticancer drug discovery. Clinical observations and patient medical records suggesting improved cancer outcomes following the consumption of Elephantopus-based integrative formulations provided the rationale for systematic investigation of their pharmacological value. Accordingly, this thesis aimed to validate the anticancer potential of Elephantopus tomentosus and to elucidate the mechanisms of action of its active molecular components. Two bioactive compounds, NYH001 and NYH101, were isolated from E. tomentosus and shown to exhibit anticancer activity. Structure–activity relationship-guided chemical modification generated the derivatives NYH002 and NYH102, which were subsequently subjected to extensive in vitro and in vivo characterization, including pharmacokinetic and biodistribution analysis. Mechanistic investigations revealed that NYH002 exerts potent anticancer effects by inducing replication stress and DNA double-strand breaks, with enhanced vulnerability observed in homologous recombination-deficient cancer cells. This mechanism enabled synergistic interactions with DNA-damaging chemotherapeutic agents and was supported by favorable systemic exposure, including blood–brain barrier penetration. In contrast, NYH102 exhibited pronounced anti-metastatic activity by perturbing RNA polymerase II-mediated transcription, thereby suppressing cancer cell migration and invasion. Overall, this thesis presents a systematic investigation linking clinical observations to molecular mechanisms and identifies distinct small-molecule leads for future combination therapies and medicinal chemistry optimization.
Short microbial generation times, large population sizes, and high mutation rates mean that microbes have the potential to accumulate genetic modifications so rapidly that these changes could impact simultaneous changes in species composition (presence and abundance of species and strains caused by environmental constraints) and in their interactions with each other and the environment. Microbial communities can rapidly respond to environmental change (e.g. threat of extinction), but the proportion of this response that can be attributed to evolutionary processes, rather than species composition or gene expression shifts, remains unresolved. Population metagenomics approaches are needed to address this question.
Metagenomes constitute pooled genomes of populations which in extreme cases may consist of thousands of species, are largely sparse, and prone to erroneous data. Conducting an accurate population genetic analysis with them requires adjustments to the existing statistics and development of novel methods. Therefore, I first focused on developing theory, then on extending the developed and existing methods to metagenomes, and then used these novel tools to analyse rhizobiome community data to understand eco-evolutionary processes and plant-soil interactions.
This 4th Annual Symposium ― Mycobacteria 2026: Translating Biology into Cures will bring together leading scientists, clinicians, pharma and innovators from around the globe to share the latest breakthroughs in Tuberculosis (TB) and non-tuberculous mycobacteria (NTM) research and drug discovery. The goal of this event is to discuss novel concepts and to provide a platform for vibrant and open discussions between industry, clinicians and senior scientists, postdoctoral fellows and students from academia.
Registration is free but compulsory and from the submitted abstracts short oral presentations will be selected.
Age-related macular degeneration (AMD) is linked to retinal pigment epithelium (RPE) dysfunction, where cellular senescence disrupts proteostasis and endoplasmic reticulum (ER) homeostasis. Here, a tert-butyl hydroperoxide (TBHP)-induced senescence model in human RPE1 cells was established to investigate how senescence alters IRE1-mediated unfolded protein response (UPR) signaling. Senescent cells exhibited a markedly weakened UPR under both proteotoxic and lipid bilayer stress. Although XBP1 mRNA splicing was induced, XBP1s protein was completely absent, indicating impaired translation. Senescence also intrinsically activated the regulated IRE1-dependent decay (RIDD) pathway, supported by basal degradation of the canonical RIDD substrate BLOC1S1 and RNA-seq-based identification of additional putative RIDD targets for future validation. Structurally, senescent cells showed severe ER fragmentation, which was aggravated by IRE1 loss but rescued by XBP1s overexpression. These findings demonstrate that senescence impairs IRE1 signaling, weakens ER stress resilience, and may contribute to AMD pathogenesis, highlighting IRE1-XBP1s restoration as a potential therapeutic strategy.
The function of many plant genes cannot be elucidated from their coding sequences, as many of them are lineage-specific, orphan genes, or possess multiple functions. In recent years, the genomes of plant species have been continually sequenced, and public plant gene expression data (in the form of public RNA-seq accessions) are rapidly accumulating owing to the advancement and increasing affordability of sequencing technologies. Gene Co-expression Networks (GCNs) can be used to take advantage of this unprecedented wealth of public transcriptomic data available for the Plant kingdom to supercharge the prediction of gene functions. However, there exist methodological hurdles that impede the utilisation of GCNs for this purpose. My research focuses on addressing these gaps.
Malaria and cancer continue to pose major global health challenges, and the emergence of drug resistance limits the long-term effectiveness of current therapies. The work of my thesis concerns the use of chemical synthesis and enzymatic protein modification approaches, respectively, for the design and engineering of anti-malaria and anti-cancer drug molecules that have the potential to overcome the resistance problem of current therapies.
The chemical approach involves systematic structure–activity relationship study on a previously discovered antimalarial lead compound. The lead compound possesses structural features indicative of DNA binding ability. Qualitative and quantitative mechanistic studies were performed to assess DNA recognition and binding as one potential mode of action (MOA) for its antimalarial activity. Results from this study suggest DNA as an underexplored target space for discovering antimalarial molecules with MOA beyond classical protein-targeted mechanisms and less likelihood to develop drug resistance.
The enzymatic protein modification approach involves the use of peptidyl asparaginyl ligases (PALs) for site-specific antibody functionalization, specifically for the preparation of antibody-drug conjugates (ADCs). Orthogonal protein modification strategies were established by combining chemoselective and enzymatic PAL-mediated conjugation strategies, allowing precise and independent installation of two different cytotoxic payloads at distinct sites of the antibody molecule while preserving antibody binding activity and payload function. In addition, PAL-mediated ligation was expanded to thioester substrates, allowing ligation at non-Asn/Asp junctions and enabling applications such as D-peptide cyclization and antibody dual labelling.
In conclusion, these studies integrate medicinal chemistry and enzymatic approaches to generate antimalarial and anticancer molecules with unique mechanisms of action and multifunctional architectures for addressing drug resistance.
Cancer is a complex disease characterized by the uncontrolled proliferation of abnormal cells and remains a leading cause of mortality worldwide. Immunotherapy has emerged as an effective strategy to combat cancer and is broadly classified into active and passive approaches. Active immunotherapy aims to stimulate or restore the patient’s immune system to recognize and eliminate tumour cells, whereas passive immunotherapy involves the administration of exogenously prepared immune effectors, such as antibodies or immune cells, independent of endogenous immune activation. Although monoclonal antibodies are widely used in passive immunotherapy, their large size and long serum half-life limit tissue penetration, restricts access to certain epitopes. To overcome these limitations, nanobodies have been developed. Their small size (~15 kDa) and extended, flexible CDR3 loop confer high affinity and enable access to previously inaccessible epitopes. Furthermore, conjugation of nanobodies with diverse payloads using the VyPAL2–hQC cascade system expands their applications as drug conjugates, imaging probes, and PET tracers.
Plants have evolved two receptor systems to detect microbial threats and initiate successive defenses. Cell-surface pattern-recognition receptors (PRRs) perceive conserved pathogen-associated molecular patterns (PAMPs) and trigger pattern-triggered immunity (PTI). Intracellular nucleotide-binding leucine-rich repeat (NLR) proteins monitor pathogen-delivered effectors and activate effector-triggered immunity (ETI). Phytopathogenic bacteria, fungi, oomycetes, and insects deploy diverse effectors to undermine host defenses. Many effctors have enzymatic domains that modify host targets for function perturbation. However, prior work has largely overlooked intrinsically disordered regions (IDRs) within effectors, which may confer unique activities without folded domains. Here, we focused on XopR, a highly disordered effector from Xanthomonas campestris pv. campestris. Using an effector-less pathogen, we developed a new delivery method for XopR. Leveraging this tool, in vitro reconstitution, live-cell imaging, and biochemical assays, we found that XopR’s multivalent binding induced clusters of PRR complex components at the plasma membrane. Such clustering impaired endocytic turnover and sustained PAMP signaling. We also observed that XopR interacted with NADPH oxidase and blocked the conformational activation required for reactive oxygen species production. To identify XopR partners systematically, we performed a TurboID proximity-labeling screen and uncovered RIN4, a key guardee for the NLRs RPM1 and RPS2. We demonstrated that XopR remodeled RIN4 biomolecular condensates, altering RIN4’s material properties and immune activation. Finally, we employed hydrogen–deuterium exchange/mass spectrometry to map molecular dynamics within XopR–RIN4 coacervates. We identified several critical regions that contained previously known RIN4 regulatory sites, enabling further validation. Overall, we showed that IDR-rich effectors subverted host immunity by driving multivalent clustering, perturbing protein conformations, and reshaping the material properties of host biomolecular assemblies.
Plants have evolved two receptor systems to detect microbial threats and initiate successive defenses. Cell-surface pattern-recognition receptors (PRRs) perceive conserved pathogen-associated molecular patterns (PAMPs) and trigger pattern-triggered immunity (PTI). Intracellular nucleotide-binding leucine-rich repeat (NLR) proteins monitor pathogen-delivered effectors and activate effector-triggered immunity (ETI). Phytopathogenic bacteria, fungi, oomycetes, and insects deploy diverse effectors to undermine host defenses. Many effctors have enzymatic domains that modify host targets for function perturbation. However, prior work has largely overlooked intrinsically disordered regions (IDRs) within effectors, which may confer unique activities without folded domains. Here, we focused on XopR, a highly disordered effector from Xanthomonas campestris pv. campestris. Using an effector-less pathogen, we developed a new delivery method for XopR. Leveraging this tool, in vitro reconstitution, live-cell imaging, and biochemical assays, we found that XopR’s multivalent binding induced clusters of PRR complex components at the plasma membrane. Such clustering impaired endocytic turnover and sustained PAMP signaling. We also observed that XopR interacted with NADPH oxidase and blocked the conformational activation required for reactive oxygen species production. To identify XopR partners systematically, we performed a TurboID proximity-labeling screen and uncovered RIN4, a key guardee for the NLRs RPM1 and RPS2. We demonstrated that XopR remodeled RIN4 biomolecular condensates, altering RIN4’s material properties and immune activation. Finally, we employed hydrogen–deuterium exchange/mass spectrometry to map molecular dynamics within XopR–RIN4 coacervates. We identified several critical regions that contained previously known RIN4 regulatory sites, enabling further validation. Overall, we showed that IDR-rich effectors subverted host immunity by driving multivalent clustering, perturbing protein conformations, and reshaping the material properties of host biomolecular assemblies.
The 6th meeting of the Singapore Malaria Network intends to foster new ideas and concepts by bringing together scientists working in multiple aspects of malaria.
Understanding genome regulation requires linking 3D chromatin organization with cellular responses to perturbations, yet current experimental approaches are limited by cost, sparsity, and noise. This work presents two data-efficient computational frameworks to address these challenges. Small-ChINNpredicts chromatin interactions from small-scale Hi-C libraries by combining DNA sequence features and genomic distance, enabling recovery of biologically meaningful interactions without deep sequencing. The method generalizes across cell types, assays, and clinical samples, and performs robustly even in sparse single-cell datasets. Complementarily, VirtuGraph-Cell models single-cell perturbation responses using graph transformers, capturing gene regulatory structure and predicting responses to unseen perturbations. Together, these approaches demonstrate that genome regulation can be learned from limited data when appropriate structural priors are incorporated, providing a foundation for scalable, predictive modeling of genome function in both research and clinical settings.
Acinetobacter baumannii (Ab) is a pathogen of concern for its high resistance against antibiotics resulting in a high death toll, against which a novel and specific treatment is required. Ab relies on the F1FO-ATP synthase for ATP formation, maintaining ATP/ADP as well as pmf homeostasis. This thesis demonstrates that the AbF-ATP synthase regulates ATP hydrolysis via its ε subunit, with the εI134–Q139 as the major inhibitory element. Cryo-EM studies reveal multiple conformational changes in the ATPase-active enzyme, as well as in respond to ATP synthesis conditions. Functional analysis into the proton translocating FO-domain of the enzyme highlights the front entry in subunit a as the main proton uptake pathway, key residues involved in proton translocation and the proton exit channel, as well as the ratchet function of the unique a-subunit helix. Together, these structural and functional insights shed light into novel AbF-ATP synthase targets for inhibitor design.
The 3rd Annual Symposium: Innovations in anti-mycobacterial Drug Discovery will include presentations from speakers from around the world who have made significant advances in TB- and NTM research and –drug discovery. The goal of this event is to present novel concepts and to provide a platform for vibrant and open discussions between industry, senior scientists, postdoctoral fellows and students from academia.
The symposium is organized by members of TELMabNet (Targeting energy of life for the development of drug combinations to eradicate antibiotic-tolerant Mycobacterium abscessus, a clinical nightmare, Network), an excellence cluster funded by the Singapore National Research Foundation.
Understanding the mechanical properties of plants and their response to environmental factors is essential for developing innovative and sustainable solutions to address the challenges facing agriculture and forestry. 'Plant Biomechanics for Sustainable Agriculture and Forestry’, co-organized by Nanyang Technological University-Singapore, and Zhejiang Agriculture & Forestry University-China, will delve into the critical intersection of plant biomechanics and its impact on sustainable agriculture and forestry. The conference scheduled to take place from May 19-22, 2024 in Singapore at NTU. The event will feature a distinguished lineup of Plenary Speakers and host presentations from invited speakers representing prestigious institutions, ensuring a comprehensive and diverse range of perspectives.
This conference aims to foster collaboration, knowledge exchange, and networking opportunities among researchers, scientists, and industry professionals working in the field of plant biomechanics. It will provide a platform for the dissemination of the latest research findings, innovative methodologies, and sustainable practices that contribute to the advancement of agriculture and forestry.
We invite all participants to engage in this enriching and rewarding experience, with the goal of fostering meaningful discussions and collaborations that will contribute to the sustainable future of agriculture and forestry. We look forward to welcoming you to this important gathering and to the valuable insights and contributions you will bring to the conference.
The workshop is funded by the Zhejiang Agriculture & Forestry University (ZAFU), China and Nanyang Technological University, School of Biological Sciences (SBS).
Join us in the celebration of our achievements through this research symposium where we invite our alumni, graduate students and postdocs to share their discoveries.
Speaker:
Professor Ernst H.K. Stelzer Bunchmann Institue for Molecular Life Sciences, Goethe Universität Frankfurt am Main
Hosted By:
Prof. Peter Török, A/P Li Hoi Yeung
