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Mucosa associated lymphoid tissue (MALT) lymphoma is characterized by t(11;18)(q21;q21)/API2-MALT1, t(1;14)(p22;q32)/BCL10-IGH andt(14;18)(q32;q21)/IGH-MALT1, which commonly activate the NF-κB pathway. Gastric MALT lymphomas harbouring such translocation do not respond to Helicobacter pylori eradication, while those without translocation can be cured by antibiotics. To understand the molecular mechanism of MALT lymphoma with and without chromosome translocation, 24 cases (15 translocation-positive and 9 translocation-negative) of MALT lymphomas together with 7 follicular lymphomas and 7 mantle cell lymphomas were analysed by Affymetrix gene expression microarray platform. Unsupervised clustering showed that cases of MALT lymphoma were clustered as a single branch. However, within the MALT lymphoma group, translocation-positive cases were intermingled with translocation-negative cases. Gene set enrichment analysis (GSEA) of the NF-κB target genes and 4394 additional gene sets covering various cellular pathways, biological processes and molecular functions showed that translocation-positive MALT lymphomas were characterized by an enhanced expression of NF-κB target genes, particularly TLR6, CCR2, CD69 and BCL2, while translocation-negative cases were featured by active inflammatory and immune responses, such as IL8, CD86, CD28 and ICOS. Separate analyses of the genes differentially expressed between translocation-positive and negative cases and measurement of gene ontology term in these differentially expressed genes by hypergeometric test reinforced the above findings by GSEA. The differential expression of these NF-κB target genes between MALT lymphoma with and without translocation was confirmed by quantitative RT-PCR and immunohistochemistry or Western blot. Expression of TLR6, in the presence of TLR2, enhanced both API2-MALT1 and BCL10 mediated NF-κB activation in vitro. In addition, there was cooperation between expression of BCL10, MALT1 or API2-MALT1, and stimulation of the antigen receptor or CD40 or TLR in NF-κB activation as shown by both reporter assay and IκBα degradation. Interestingly, expression of BCL10 but not API2-MALT1 and MALT1, in the presence of LPS stimulation, also triggered IκBβ degradation, suggesting activation of different NF-κB dimers between these oncogenic products. Study by co-immunoprecipitation showed that BCL10 directly interacts with MALT1. Sub-cellular localisation experiments in BJAB B-cells, showed that BCL10 localisation was affected by MALT1. When BCL10 was over-expressed, the protein was predominantly expressed in the nuclei, but when MALT1 was over-expressed, BCL10 was mainly localised in the cytoplasm. When both BCL10 and MALT1 were over-expressed, BCL10 was expressed in the cytoplasm in the early hours when the protein level was low, but in both the cytoplasm and nuclei after 9 hours when the protein level was high. Over-expression of API2-MALT1 did not shown any apparent effect on BCL10 sub-cellular localisation in vitro. Finally, comparison of MALT lymphoma expression microarray with other lymphomas showed lactoferrin to be highly expressed in MALT lymphoma. This was confirmed by qRT-PCR, showing lactoferrin to be significantly over-expressed in MALT lymphoma compared to FL and MCL. Thus lactoferrin may be a potential marker for MALT lymphoma.

Bacterial chondronecrosis with osteomyelitis (BCO), a leading cause of lameness in broiler chickens, is characterized by infection, inflammation, and bone attrition. There are currently no effective treatments and positive diagnosis is only possible through necropsy evaluations. Lameness is also a rising animal welfare and economic concern, making prevention and detection of BCO all the more critical. These challenges are exacerbated by a lack of mechanistic understanding of BCO’s etiology. The question I asked during my dissertation was how bacteria induce bone attrition in BCO pathology. My research has shown that mitochondrial dysfunction is characteristic of BCO conditions along with autophagy machinery dysregulation. This autophagy dysregulation is also seen to a result of in vitro infection with known BCO-isolates and affecting bone cell viability. The local bone and systemic blood profile of cytokines, chemokines, inflammasomes, and relevant FGFs were also evaluated. This revealed a unique signature of BCO detectable within circulation and in local bone. Additionally, this signature was made up of factors which negatively affect bone cell viability. It was also shown that primary avian chondrocytes exhibiting optimal phenotypes could be successfully isolated form chicks. These primary cells could provide an improved, highly relevant model for in vitro analysis of avian bone diseases and infections. Finally, the potential roles of two factors regulating energy and lipid metabolism were preliminarily explored as a future target for BCO research. These findings provide novel insight into mechanisms of etiology and means of non-invasive detection while also improving upon current methods of avian growth-plate research

As a first and second messenger, calcium (Ca2+) regulates numerous biological processes. Its homeostasis is modulated by intracellular Ca2+ stores, membrane channels, pumps, and/or receptors (etc. calcium sensing receptor (CaSR)). To integrate multi-level Ca2+ dynamics, which exhibit a wide range of temporal kinetics, there is a pressing need to integrate Ca2+ signaling intracellularly and extracellularly, using a novel class of genetically encoded Ca2+ indicators (GECIs) with rapid kinetics and fully understanding the molecular mechanism of CaSR, respectively. In the present study, we first report a green Ca2+ indicator called “G-CatchER+” that specifically reports rapid local ER Ca2+ dynamics. G-CatchER+ exhibits a superior Ca2+ on rate than G-CEPIA1er. G-CatchER+ also reports agonist/antagonist triggered Ca2+ dynamics in several cell types including primary neurons that are orchestrated by IP3Rs, RyRs, and SERCAs. Transgenic expression of G-CatchER+ in Drosophila muscle demonstrates its utility in stimulus-evoked SR local Ca2+ dynamics. Second, we developed a red Ca2+ indicator based on mApple. R-CatchER showed superior kinetics in vitro, and in multiple cell types. It captured spatiotemporal ER Ca2+ dynamics in neurons and hotspots at dendritic branchpoints. It also enabled the first report of ER Ca2+ oscillations mediated by CaSR and revealed ER Ca2+-based functional cooperativity of CaSR. Then we described a general strategy to rationally design a new class of genetically encoded Ca2+ indicators (GECIs), with a single Ca2+-binding site and fast kinetics, by tuning rapid protein dynamics to modulate the chromophore conformational ensemble and alter the electrostatic around the chromophore. G-CatchER2 (an improved version of G-CatchER) and R-CatchER represent a general proof-of-concept. Third, we use multiple in silico and in situ approaches including various spectroscopic and functional assays and computational modeling to provide important insights into the molecular mechanism of the receptor functions. We established a working model that several Ca2+-binding sites with endogenous ambient amino acids within the CaSR-ECD cooperatively activate the receptor. Specifically, we demonstrate the different allosteric mechanisms of TNCA and AMG-416 to modulate the receptor with the disease mutations. These studies represent a milestone for visualizing and integrating Ca2+ dynamics, and facilitate drug discovery related to ER, CaSR, and Ca2+ dysfunction.

The FDA-approved amyotrophic lateral sclerosis (ALS) drug Riluzole has great potential in treating Alzheimer’s disease (AD) based on promising animal data as well as its known action on modulating synaptic transmission. However, its detailed mechanism of action is not fully understood. Here, we proposed work aiming to address this aspect via focus- ing on the Heat Shock Factor 1 (HSF1)-dependent mechanisms. We found that Riluzole could increase HSF1 and BDNF (Brain-Derived Neurotrophic Factor) expression both at transcriptional and translational levels. CA1 (Cornu Ammonia, the first region in the hip- pocampal circuit), is its main target. We also demonstrated a protective role of Riluzole on rat primary neuronal culture which was abolished by a HSF1 inhibitor. Current data together suggest that Riluzole’s synaptic-protective mechanism is highly possible through a HSF1-BDNF axis. The positive outcome from this study will facilitate filling our knowl- edge gap and interpretation of the ongoing clinical trials of Riluzole in AD in which the final data will be released later this year.

The rapid accumulation of Genome Wide Association Studies (GWAS) and association studies of intermediate molecular traits provides new opportunities for comparative analysis of the genetic basis of complex human phenotypes. Using a newly developed statistical framework called Sherlock-II that integrates GWAS with eQTL (expression Quantitative Trait Loci) and metabolite-QTL data, we systematically analyzed 445 GWAS datasets, and identified 2114 significant gene-phenotype associations and 469 metabolites-phenotype associations (passing a Q-value cutoff of 1/3). This integrative analysis allows us to translate SNP-phenotype associations into functionally informative gene-phenotype association profiles. Genetic similarity analyses based on these profiles clustered phenotypes into sub-trees that reveal both expected and unexpected relationships. We employed a statistical approach to delineate sets of functionally related genes that contribute to the similarity between their association profiles. This approach suggested common molecular mechanisms that connect the phenotypes in a subtree. For example, we found that fasting insulin, fasting glucose, breast cancer, prostate cancer, and lung cancer clustered into a subtree, and identified cyclic AMP/GMP signaling that connects breast cancer and insulin, NAPDH oxidase/ROS generation that connects the three cancers, and apoptosis that connects all five phenotypes. Our approach can be used to assess genetic similarity and suggest mechanistic connections between phenotypes. It has the potential to improve the diagnosis and treatment of a disease by mapping mechanistic insights from one phenotype onto others based on common molecular underpinnings.

Environmental endocrine disrupting chemicals (EDCs) are a group of exogenous compounds that may interfere with the functioning of endogenous systems and affect human health. Bisphenol A (BPA) is one of most ubiquitous EDCs in the manufacturing industry as a plasticizing agent used in polycarbonate plastics and epoxy resins. It is well-documented that human exposure to BPA is extremely wide spread. It was demonstrated that BPA, at human-exposure relevant doses, rapidly promoted cardiac arrhythmias in female rat hearts. However, the molecular mechanisms underlying BPA’s pro-arrhythmic effects remain unclear.As a result of banning BPA’s use in various consumer products, bisphenol S (BPS) is increasingly used as a substitute agent for BPA. Human populations are reported to be widely exposed to BPS, but the biological activities and potential toxic effects of BPS are not well understood.The objective of this dissertation is to investigate the cardiac impact of EDCs including BPA and BPS, with a focus on their cardiac arrhythmogenesis and underlying cellular and molecular mechanisms. Of particular interest, was to elucidate the signaling cascades and protein targets underlying BPA’s rapid alteration of myocyte Ca2+ handling and promotion of arrhythmogenic-triggered activities in female rodent hearts; and to evaluate how BPS affects cardiac arrhythmogenesis in comparison to BPA.It was demonstrated that protein kinase A (PKA) and Ca2+/Calmodulin-dependent protein kinase II (CAMKII) signaling pathways are the two major signaling pathways activated by BPA. In isolated female rat ventricular myocytes, BPA exposure rapidly increased phosphorylation of the ryanodine receptors by PKA but not by CAMKII. BPA exposure also rapidly increased the phosphorylation of phospholamban by CAMKII but not PKA. These two pathways are mediated by estrogen receptor ß but not estrogen receptor α and are shown to be localized. Functional analysis also showed that both PKA and CAMKII were necessary contributors to the arrhythmogenesis of BPA on cardiomyocytes. This study identified the unique signaling cascades of BPA in the heart, and elucidated its novel effects on key Ca2+ handling proteins.Also of interest is the cardiac impact of BPS, especially on the electrical aspect of the heart. It was shown that in female rat hearts, BPS rapidly increased heart rate and promoted ventricular arrhythmias under stress conditions. BPS increased arrhythmogenic-triggered activities in isolated female myocytes via alteration of Ca2+ handling, in particular by increasing spontaneous sarco/endoplasmic reticulum Ca2+ release. BPS exposure increased phosphorylation of two key Ca2+ handling proteins, the ryanodine receptor and phospholamban. Additionally, the pro-arrhythmic effects of BPS were demonstrated to be female-specific, characterized by an inverted-U dose response curve. These results provide important mechanistic insights into the rapid cardiac arrhythmogenesis of BPS in female hearts, and contribute to the evaluation of the potential cardiac toxicity of BPS.Furthermore, the cardiac effects of probenecid were investigated. Collaboratively, it was shown that probenecid increased myocardial contractility using in vivo echocardiography, ex vivo Langendorff perfused heart and isolated myocyte system. The inotropic effect is likely mediated by transient receptor potential vanilloid 2 channels via enhanced sarco/endoplasmic reticulum Ca2+ release.