457 research outputs found

    Investigating Molecular Mechanisms behind Bacterial Chondronecrosis with Osteomyelitis (BCO) Pathogenesis in Modern Broilers

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    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 researc

    Stimulation of Piezo1 by mechanical signals promotes bone anabolism

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    Mechanical loading, such as caused by exercise, stimulates bone formation by osteoblasts and increases bone strength, but the mechanisms are poorly understood. Osteocytes reside in bone matrix, sense changes in mechanical load, and produce signals that alter bone formation by osteoblasts. We report that the ion channel Piezo1 is required for changes in gene expression induced by fluid shear stress in cultured osteocytes and stimulation of Piezo1 by a small molecule agonist is sufficient to replicate the effects of fluid flow on osteocytes. Conditional deletion o

    Teil 1: "Studies on the extracellular matrix enzyme lysyl oxidase (LOX) in epithelial cells" Teil 2: "Gene expression signatures of circulating peripheral blood cells associated with early-onset coronary artery disease"

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    Teil 1: The aim of the first part of my PhD project was to investigate the role of the extracellular matrix enzyme lysyl oxidase (LOX) in epithelial cells and its potential implications for cancers of epithelial origin. Using two well-characterized epithelial cell lines, i.e. MDCK II and MCF-10A, an in vitro model system was established to study the function of LOX in epithelia. For the first time the presence of mature LOX protein was demonstrated within cytoplasmic fractions. In addition, the enzyme was found to be secreted from the cells and BAPN-inhibitable LOX enzymatic activity was detected in concentrated fractions of conditioned cell medium. This result suggests that epithelial cells can express catalytically active LOX. Notably, extracellular expression and enzymatic activity of LOX were significantly elevated in post-confluent compared to pre-confluent cell cultures. This finding may indicate that LOX expression is elevated in differentiated epithelia. Although large amounts of mature LOX protein were observed in cytoplasmic fractions by western blotting, it was not possible to recover intracellular BAPN-inhibitable LOX enzymatic activity. LOX protein expression was shown to increase during scattering of MDCK II cells after hepatocyte growth factor (HGF) treatment. This process recapitulates important cellular characteristics of tumor metastasis in vivo. The higher LOX protein level correlated with a two-fold increase of LOX mRNA in HGF-treated compared to control cells as revealed by quantitative PCR. Therefore, the scatter assay may represent a suitable model system to study the recently reported role of LOX during cancer progression in vitro. Two strategies were followed to generate stable MDCK II lines that express recombinant LOX under a CMV promoter in order to determine whether constitutive LOX over-expression can induce changes of the epithelial phenotype. The first strategy comprised transfection of LOX-EGFP constructs. However, this approach did not yield detectable amounts of recombinant protein. Steric hindrance of the relatively large EGFP-tag (30 kD) may be a reason for the failure to express LOX- EGFP fusion proteins. Nevertheless, one clone transfected with full-length LOX-EGFP displayed an elongated morphology that resembled mesenchymal cells. The second approach used LOX-V5 expression constructs. Neither intra- nor extracellular expression of LOX induced significant phenotypical/morphological changes in MDCK II cells. In addition, no increase of LOX enzyme activity was detected in either, cytoplasmic fractions or conditioned cell medium of stably transfected cell lines. The difficulties in expressing recombinant LOX in this study represents a general challenge of the field and may result from its biochemical properties as well as the lack of more specific and sensitive assays to reliably determine its catalytic activity. Even though preliminary, the results of this study can serve as a promising basis for future investigations with the goal to decipher the precise function of LOX in epithelial cells. Teil 2: The aim of the second part of my PhD project was to identify differential gene expression signatures in circulating blood cells that are associated with coronary artery disease. Using a microarray-based whole genome expression profiling approach the transcriptome of circulating peripheral blood cells from individuals diagnosed with atherosclerotic coronary artery disease (CAD) was analyzed. The study cohort was assembled from a large clinical database hosted by the Veterans Administration Pacific Islands Healthcare System (VAPHICS). Samples from patients with early myocardial infarction (MI) prior to age 50 and clinically diagnosed with CAD were compared to a healthy control group treated with the same cardiac-related medication. Gene expression profiles from whole blood mRNA samples that were depleted for β-globin transcripts was assessed by microarray analysis. A novel algorithm called LOTEST that was specifically designed to detect heterogenous and sparse signals embedded within gaussian noise, identified 1203 differentially expressed genes between patients and controls. Out of these, 195 genes were excluded from downstream analysis as they were represented by only two individuals within the patient group. The gene ontology databases „Protein Analysis Through Evolutionary Relationships“ (PANTHER) and „Ingenuity Pathway Analysis“ (IPA) were then applied to analyze functional grouping within the identified differential gene expression pattern. The analysis revealed over-representation of genes that are associated with inflammation and immune system function. A list of potential candidate genes with the most consistent expression pattern across the sample groups was confirmed by quantitative real-time PCR analysis and included up-regulated pro- inflammatory genes (PTX3, LGALS3, CAMP), down-regulated anti-inflammatory genes (IL12RB1, JAG1), decreased expression of genes that protect from auto-immunity (IL2RA, CCR7) and genes not previously implicated in immune system function (GSTT1, NGFR). The results of this study suggest persistent ongoing inflammation in CAD patients of the study cohort despite treatment with anti-inflammatory medication. The outcome of this small-scale study can serve as a basis for future investigations in order to discover new biomarkers and/or potential targets for drug development against so far unknown disease mechanism(s). Certainly, the realization of these tasks would require substantial epidemiological and basic research efforts

    【研究分野別】シーズ集 [英語版]

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    [英語版

    Single Cell Analysis

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    Cells are the most fundamental building block of all living organisms. The investigation of any type of disease mechanism and its progression still remains challenging due to cellular heterogeneity characteristics and physiological state of cells in a given population. The bulk measurement of millions of cells together can provide some general information on cells, but it cannot evolve the cellular heterogeneity and molecular dynamics in a certain cell population. Compared to this bulk or the average measurement of a large number of cells together, single-cell analysis can provide detailed information on each cell, which could assist in developing an understanding of the specific biological context of cells, such as tumor progression or issues around stem cells. Single-cell omics can provide valuable information about functional mutation and a copy number of variations of cells. Information from single-cell investigations can help to produce a better understanding of intracellular interactions and environmental responses of cellular organelles, which can be beneficial for therapeutics development and diagnostics purposes. This Special Issue is inviting articles related to single-cell analysis and its advantages, limitations, and future prospects regarding health benefits

    The Fifth National Technology Transfer Conference and Exposition

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    Carbon nanotubes micro-arrays: characterization and application in biosensing of free proteins and label-free capture of breast cancer cells

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    Circulating tumor cells (CTCs) are cells released into the bloodstream from primary tumors and are suspected to be one of the main causes behind metastatic spreading of cancer. The ability to capture and analyze circulating tumor cells in clinical samples is of great interest in prevailing patient prognosis and clinical management of cancer. Carbon nanotubes, individual rolled-up graphene sheets, have emerged as exciting materials for probing the biomolecular interactions. With diameter of about 1 nm, they can attach themselves to cell surface receptors through specific antibodies and hold a great potential for diagnostic cellular profiling. Carbon nanotubes can be either semiconducting or metallic, and the electronic properties of either type rivals the best known materials. Small size of nanotubes and the ability to functionalize their surface using 1-Pyrenebutanoic Acid, Succinimidyl Ester (PASE), enables a versatile probe for developing a platform for capture and analysis of cancer biomarkers and circulating tumor cells. Although nanotubes have previously been used to electrically detect a variety of molecules and proteins, here for the first time we demonstrate the label free capture of spiked breast cancer cells using ultra-thin carbon nanotube film micro-array devices in a drop of buffy coat and blood. A new statistical approach of using Dynamic Time Warping (DTW) was used to classify the electrical signatures with 90% sensitivity and 90% specificity in blood. These results suggest such label free devices could potentially be useful for clinical capture and further analysis of circulating tumor cells. This thesis will go in-depth the properties of carbon nanotubes, device fabrication and characterization methodologies, functionalization protocols, and experiments in buffy coats and in blood. Combination of nano and biological materials, functionalization protocols and advanced statistical classifiers can potentially enable clinical translation of such devices in the future
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