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Ensuring Access to Safe and Nutritious Food for All Through the Transformation of Food Systems
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European Heart Rhythm Association (EHRA)/Heart Rhythm Society (HRS)/Asia Pacific Heart Rhythm Society (APHRS)/Latin American Heart Rhythm Society (LAHRS) Expert Consensus Statement on the state of genetic testing for cardiac diseases.
RNA pull-down-confocal nanoscanning (RP-CONA), a novel method for studying RNA/protein interactions in cell extracts that detected potential drugs for Parkinsonās disease targeting RNA/HuR complexes
MicroRNAs (miRNAs, miRs) are a class of small non-coding RNAs that regulate gene expression through specific base-pair targeting. The functional mature miRNAs usually undergo a two-step cleavage from primary miRNAs (pri-miRs), then precursor miRNAs (pre-miRs). The biogenesis of miRNAs is tightly controlled by different RNA-binding proteins (RBPs). The dysregulation of miRNAs is closely related to a plethora of diseases. Targeting miRNA biogenesis is becoming a promising therapeutic strategy.
HuR and MSI2 are both RBPs. MiR-7 is post-transcriptionally inhibited by the HuR/MSI2 complex, through a direct interaction between HuR and the conserved terminal loop (CTL) of pri-miR-7-1. Small molecules dissociating pri-miR-7/HuR interaction may induce miR-7 production. Importantly, the miR-7 levels are negatively correlated with Parkinsonās disease (PD).
PD is a common, incurable neurodegenerative disease causing serious motor deficits. A hallmark of PD is the presence of Lewy bodies in the human brain, which are inclusion bodies mainly composed of an aberrantly aggregated protein named Ī±-synuclein (Ī±-syn). Decreasing Ī±-syn levels or preventing Ī±-syn aggregation are under investigation as PD treatments. Notably, Ī±-syn is negatively regulated by several miRNAs, including miR-7, miR-153, miR-133b and others. One hypothesis is that elevating these miRNA levels can inhibit Ī±-syn expression and ameliorate PD pathologies.
In this project, we identified miR-7 as the most effective Ī±-syn inhibitor, among the miRNAs that are downregulated in PD, and with Ī±-syn targeting potentials. We also observed potential post-transcriptional inhibition on miR-153 biogenesis in neuroblastoma, which may help to uncover novel therapeutic targets towards PD.
To identify miR-7 inducers that benefit PD treatment by repressing Ī±-syn expression, we developed a novel technique RNA Pull-down Confocal Nanoscaning (RP-CONA) to monitor the binding events between pri-miR-7 and HuR. By attaching FITC-pri-miR-7-1-CTL-biotin to streptavidin-coated agarose beads and incubating them in human cultured cell lysates containing overexpressed mCherry-HuR, the bound RNA and protein can be visualised as quantifiable fluorescent rings in corresponding channels in a confocal high-content image system. A pri-miR-7/HuR inhibitor can decrease the relative mCherry/FITC intensity ratio in RP-CONA. With this technique, we performed several small-scale screenings and identified that a bioflavonoid, quercetin can largely dissociate the pri-miR-7/HuR interaction. Further studies proved that quercetin was an effective miR-7 inducer as well as Ī±-syn inhibitor in HeLa cells.
To understand the mechanism of quercetin mediated Ī±-syn inhibition, we tested the effects of quercetin treatment with miR-7-1 and HuR knockout HeLa cells. We found that HuR was essential in this pathway, while miR-7 hardly contributed to the Ī±-syn inhibition. HuR can directly bind an AU-rich element (ARE) at the 3ā untranslated region (3ā-UTR) of Ī±-syn mRNA and promote translation. We believe quercetin mainly disrupts the ARE/HuR interaction and disables the HuR-induced Ī±-syn expression.
In conclusion, we developed and optimised RP-CONA, an on-bead, lysate-based technique detecting RNA/protein interactions, as well as identifying RNA/protein modulators. With RP-CONA, we found quercetin inducing miR-7 biogenesis, and inhibiting Ī±-syn expression. With these beneficial effects, quercetin has great potential to be applied in the clinic of PD treatment. Finally, RP-CONA can be used in many other RNA/protein interactions studies
School-based interventions to increase physical activity and reduce cardiometabolic risk in children
Innate immunity and metabolism in the bovine ovarian follicle
Postpartum uterine disease in dairy cows is associated with reduced fertility. One of the first and most prevalent bacteria associated with uterine disease is Escherichia coli. The bacterial endotoxin, lipopolysaccharide (LPS), accumulates in the ovarian follicular fluid of animals with uterine disease. The granulosa cells of the ovarian follicle respond to LPS by secreting pro-inflammatory cytokines, such as interleukin (IL)-1a, IL-1b and IL-8, and oocyte health is perturbed. Dairy cows also experience metabolic energy stress in the postpartum period, which is associated with an increased risk of developing uterine disease and ovarian dysfunction. This thesis explored the crosstalk between innate immunity and metabolic energy stress in bovine granulosa cells and cumulus-oocyte complex. Firstly, we found that glycolysis, AMP-activated protein kinase and the mechanistic target of rapamycin, regulate the innate immune responses to LPS in granulosa cells isolated from bovine ovarian follicles. Activation of AMP-activated protein kinase decreased the LPS-induced secretion of IL-1a, IL-1b, and IL8, and was associated with shortened duration of ERK1/2 and JNK phosphorylation. Next, we found that decreasing the availability of cholesterol or inhibiting cholesterol biosynthesis using short-interfering RNA impaired the LPS-induced secretion of IL-1a and IL-1b by granulosa cells. Furthermore, metabolic energy stress or inhibiting cholesterol biosynthesis in the bovine cumulus-oocyte complex modulated the innate immune responses to LPS, and perturbed meiotic progression during in vitro maturation. Finally, we explored an in vivo model of uterine disease in heifers, using RNAseq to investigate alterations to the transcriptome of the reproductive tract. We found that uterine disease altered the transcriptome of the endometrium, oviduct, granulosa cells and oocyte, several months after bacterial infusion; these changes were most evident in the granulosa cells and oocyte of the ovarian follicle. The findings from this thesis imply that there is crosstalk between innate immunity and metabolism in the bovine ovarian follicle
Investigating the mechanism of human beta defensin-2-mediated protection of skin barrier in vitro
The human skin barrier is a biological imperative. Chronic inflammatory skin diseases, such as Atopic Dermatitis (AD), are characterised by a reduction in skin barrier function and an increased number of secondary infections. Staphyloccocus aureus (S. aureus) has an increased presence on AD lesional skin and contributes significantly to AD pathology. It was previously demonstrated that the damage induced by a virulence factor of S. aureus, V8 protease, which causes further breakdown in skin barrier function, can be reduced by induction of human Ī²- defensin (HBD)2 (by IL-1Ī²) or exogenous HBD2 application. Induction of this defensin is impaired in AD skin. This thesis examines the mechanism of HBD2-mediated barrier protection in vitro; demonstrating that in this system, HBD2 was not providing protection through direct protease inhibition, nor was it altering keratinocyte proliferation or migration, or exhibiting specific localisation within the monolayer. Proteomics data demonstrated that HBD2 did not induce expression of known antiproteases but suggested that HBD2 stimulation may function by modulating expression of extracellular matrix proteins, specifically collagen- IVĪ±2 and Laminin-Ī²-1. Alternative pathways of protection initiated by IL-1Ī² and TNFĪ± stimulation were also investigated, as well as their influence over generalised wound healing. Finally, novel 3D human skin epidermal models were used to better recapitulate the structure of human epidermis and examine alterations to skin barrier function in a more physiological system. These data validate the barrier-protective properties of HBD2 and extended our knowledge of the consequences of exposure to this peptide in this context
Socio-endocrinology revisited: New tools to tackle old questions
Animalsā social environments impact their health and survival, but the proximate links between sociality and fitness are still not fully understood. In this thesis, I develop and apply new approaches to address an outstanding question within this sociality-fitness link: does grooming (a widely studied, positive social interaction) directly affect glucocorticoid concentrations (GCs; a group of steroid hormones indicating physiological stress) in a wild primate? To date, negative, long-term correlations between grooming and GCs have been found, but the logistical difficulties of studying proximate mechanisms in the wild leave knowledge gaps regarding the short-term, causal mechanisms that underpin this relationship. New technologies, such as collar-mounted tri-axial accelerometers, can provide the continuous behavioural data required to match grooming to non-invasive GC measures (Chapter 1). Using Chacma baboons (Papio ursinus) living on the Cape Peninsula, South Africa as a model system, I identify giving and receiving grooming using tri-axial accelerometers and supervised machine learning methods, with high overall accuracy (~80%) (Chapter 2). I then test what socio-ecological variables predict variation in faecal and urinary GCs (fGCs and uGCs) (Chapter 3). Shorter and rainy days are associated with higher fGCs and uGCs, respectively, suggesting that environmental conditions may impose stressors in the form of temporal bottlenecks. Indeed, I find that short days and days with more rain-hours are associated with reduced giving grooming (Chapter 4), and that this reduction is characterised by fewer and shorter grooming bouts. Finally, I test whether grooming predicts GCs, and find that while there is a long-term negative correlation between grooming and GCs, grooming in the short-term, in particular giving grooming, is associated with higher fGCs and uGCs (Chapter 5). I end with a discussion on how the new tools I applied have enabled me to advance our understanding of sociality and stress in primate social systems (Chapter 6)
Statistical Learning for Gene Expression Biomarker Detection in Neurodegenerative Diseases
In this work, statistical learning approaches are used to detect biomarkers for neurodegenerative diseases (NDs). NDs are becoming increasingly prevalent as populations age, making understanding of disease and identification of biomarkers progressively important for facilitating early diagnosis and the screening of individuals for clinical trials. Advancements in gene expression profiling has enabled the exploration of disease biomarkers at an unprecedented scale. The work presented here demonstrates the value of gene expression data in understanding the underlying processes and detection of biomarkers of NDs. The value of novel approaches to previously collected -omics data is shown and it is demonstrated that new therapeutic targets can be identified. Additionally, the importance of meta-analysis to improve power of multiple small studies is demonstrated. The value of blood transcriptomics data is shown in applications to researching NDs to understand underlying processes using network analysis and a novel hub detection method. Finally, after demonstrating the value of blood gene expression data for investigating NDs, a combination of feature selection and classification algorithms were used to identify novel accurate biomarker signatures for the diagnosis and prognosis of Parkinsonās disease (PD) and Alzheimerās disease (AD). Additionally, the use of feature pools based on previous knowledge of disease and the viability of neural networks in dimensionality reduction and biomarker detection is demonstrated and discussed. In summary, gene expression data is shown to be valuable for the investigation of ND and novel gene biomarker signatures for the diagnosis and prognosis of PD and AD
Macrophage: A Key Player of Teleost Immune System
Fish, the free-living organisms, residing in aquatic environment, are earliest vertebrates with fully developed innate and adaptive immunity. Immune organs homologous to those of mammalian immune system are found in fish. Macrophages are best known for their role in immunity, basic function of which being cytokine production and phagocytosis. Due to environmental adaptation and whole genome duplication, macrophages in teleost are differently modulated (pro-inflammatory, M1-type, and anti-inflammatory/regulatory, M2-type) and perform a variety of different functions as compared with those of mammals. Phagocytosis is a major mechanism for removing pathogens and/or foreign particles in immune system and therefore is a critical component of the innate and adaptive immune system. One of the most competent phagocytes in teleost is found to be macrophages/monocytes. Increasing experimental evidence demonstrates that teleost phagocytic cells can recognize and destroy antigens to elicit adaptive immune responses that involve multiple cytokines. A detail understanding of teleost macrophages and phagocytosis would not only help in understanding the immune mechanism but will also help in disease prevention in teleost
The association between neurodegeneration and local complement activation in the thalamus to progressive multiple sclerosis outcome
The extent of grey matter demyelination and neurodegeneration in the progressive multiple sclerosis (PMS) brains at postāmortem associates with more severe disease. Regional tissue atrophy, especially affecting the cortical and deep grey matter, including the thalamus, is prognostic for poor outcomes. Microglial and complement activation are important in the pathogenesis and contribute to damaging processes that underlie tissue atrophy in PMS. We investigated the extent of pathology and innate immune activation in the thalamus in comparison to cortical grey and white matter in blocks from 21 cases of PMS and 10 matched controls. Using a digital pathology workflow, we show that the thalamus is invariably affected by demyelination and had a far higher proportion of active inflammatory lesions than forebrain cortical tissue blocks from the same cases. Lesions were larger and more frequent in the medial nuclei near the ventricular margin, whilst neuronal loss was greatest in the lateral thalamic nuclei. The extent of thalamic neuron loss was not associated with thalamic demyelination but correlated with the burden of white matter pathology in other forebrain areas (Spearman r = 0.79, p < 0.0001). Only thalamic neuronal loss, and not that seen in other forebrain cortical areas, correlated with disease duration (Spearman r = ā0.58, p = 0.009) and age of death (Spearman r = ā0.47, p = 0.045). Immunoreactivity for the complement pattern recognition molecule C1q, and products of complement activation (C4d, Bb and C3b) were elevated in thalamic lesions with an active inflammatory pathology. Complement regulatory protein, C1 inhibitor, was unchanged in expression. We conclude that active inflammatory demyelination, neuronal loss and local complement synthesis and activation in the thalamus, are important to the pathological and clinical disease outcomes of PMS
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