A systems approach identifies co-signaling molecules of early growth response 1 transcription factor in immobilization stress

Adaptation to stress is critical for survival. The adrenal medulla, the major source of epinephrine, plays an important role in the development of the hyperadenergic state and increased risk for stress associated disorders, such as hypertension and myocardial infarction. The transcription factor Egr1 plays a central role in acute and repeated stress, however the complexity of the response suggests that other transcription factor pathways might be playing equally important roles during acute and repeated stress. Therefore, we sought to discover such factors by applying a systems approach. Using microarrays and network analysis we show here for the first time that the transcription factor signal transducer and activator of transcription 3 (Stat3) gene is activated in acute stress whereas the prolactin releasing hormone (Prlh11) and chromogranin B (Chgb) genes are induced in repeated immobilization stress and that along with Egr1 may be critical mediators of the stress response. Our results suggest possible involvement of Stat3 and Prlh1/Chgb up-regulation in the transition from short to repeated stress activation

Traumatic Brain Injury Induces Genome-Wide Transcriptomic, Methylomic, and Network Perturbations in Brain and Blood Predicting Neurological Disorders.

The complexity of the traumatic brain injury (TBI) pathology, particularly concussive injury, is a serious obstacle for diagnosis, treatment, and long-term prognosis. Here we utilize modern systems biology in a rodent model of concussive injury to gain a thorough view of the impact of TBI on fundamental aspects of gene regulation, which have the potential to drive or alter the course of the TBI pathology. TBI perturbed epigenomic programming, transcriptional activities (expression level and alternative splicing), and the organization of genes in networks centered around genes such as Anax2, Ogn, and Fmod. Transcriptomic signatures in the hippocampus are involved in neuronal signaling, metabolism, inflammation, and blood function, and they overlap with those in leukocytes from peripheral blood. The homology between genomic signatures from blood and brain elicited by TBI provides proof of concept information for development of biomarkers of TBI based on composite genomic patterns. By intersecting with human genome-wide association studies, many TBI signature genes and network regulators identified in our rodent model were causally associated with brain disorders with relevant link to TBI. The overall results show that concussive brain injury reprograms genes which could lead to predisposition to neurological and psychiatric disorders, and that genomic information from peripheral leukocytes has the potential to predict TBI pathogenesis in the brain

Epidermis and Re-epithelialization in <i>Schmidtea mediterranea</i>

Epidermal layer is crucial for organism’s survival as its ability to close the wound is essential for tissue recovery. Planarian epidermis enables animal recovery and survival after virtually any body part amputation. Nevertheless, neither the epidermis nor the mechanisms endowing such a remarkable wound healing capacity is described in detail in planarians. Our work introduces live imaging methodology, which allows following epidermal cells and their response to tissue damage or tissue loss for extended time (hours) and in high resolution. Using our methods, we followed planarian cells live for the first time and in conjunction with electron microscopy analysis we described epidermal cell behaviors during tissue maintenance, response to tissue damage and tissue loss. Our data provides comprehensive description of cellular wound response, wound closure as well as preexisting tissue contribution to tissue restoration. In addition, we performed epidermal expression profile analysis to identify the candidate list of epidermally expressed genes to depict the machinery endowing these epidermal cell behaviors. In the pilot functional (RNAi) screen an array of transcription factors with a tissue maintenance phenotypes were identified. Our work established tools for subsequent functional studies of other epidermal expressed genes and paved the way to dissect the mechanisms of the epidermis’ maintenance and efficient wound healing in planarians

Genomic Approaches to Identify Important Traits in Avian Species

This dissertation focusses on identifying different molecular markers that have impact on overall poultry production. Chapter one reviews microRNA (miRNA), copy number variation (CNV) and single nucleotide polymorphism (SNP) as markers suggested in different avian species by various studies. It reviews modern genomic approaches that are employed for next generation sequencing data analysis and verification. Chapter two seeks to identify and validate the muscle specific miRNAs in the breast muscle of modern broilers and its foundational chicken line. Small RNA sequencing was performed to identify differentially expressed mature miRNAs in the breast muscles of these two chicken lines. Results showed that nine different mature miRNAs were differentially expressed (DE) in the breast muscle of modern broilers compared to foundational chicken lines. Target genes of DE miRNAs were involved in MAP/ERK1/2, calcium signaling, axonal guidance signaling and NRF2-mediated oxidative response pathways suggesting their roles in muscle growth and development. Chapter three is focused on identifying and validating copy number variation in the whole genome of two divergently selected high and low stress quail lines. Whole genome sequencing was performed, and data were analyzed for copy number variation detection in genome of the quail lines. Results showed the unique sets of copy number variable regions and genes in the genomes of high stress and low stress birds. Importantly, these genes were involved in development of nervous/endocrine systems, and humoral/cell-mediated immune responses suggesting that they could be potential biomarkers for understanding effects of stress in the well-being and growth performance of avian species and other animals. Chapter four focuses on identifying SNPs in whole genome of Arkansas Progressor (AP) and Regressor (AR) chicken lines selected for tumor progression and tumor regression upon v-src oncogene induction. Whole genome sequencing was performed, SNPs were analyzed and validated using allele-specific PCR. Results showed the unique sets of SNPs in AP and AR lines. Based on the functional studies, the candidate SNPs were associated with ubiquitylation, and PI3K and NF-kB signaling pathways, suggesting their role in tumor regression in AR chickens

Identification and characterization of small molecules inducing cellular reactive oxygen species accumulation

The human cell is constantly confronted with reactive oxygen species (ROS), which are oxygen-containing chemical intermediates readily reacting with different biomolecules. ROS can originate from exogenous sources, but are also produced within cells, e.g. as part of their metabolism. Depending on the concentration, ROS can either act as important signaling molecules or induce oxidative stress, thereby harming cellular components. Thus, cells utilize a multitude of antioxidant mechanisms to maintain redox homeostasis. As ROS play an important role in the development and propagation of cancer, modulation of cellular ROS levels, e.g. via small molecules, may have the potential to induce cancer-selective cytotoxicity. In the course of this thesis, novel fast-acting small-molecule inducers of cellular ROS accumulation were identified by means of phenotypic screening. A counter-screen for glutathione-reactive compounds led to the discovery of 4,5-dihalo-2-methylpyridazin-3-ones and 2,3,4,5(6)-tetrachloro-6(5)-methylpyridines as potent depleters of cellular glutathione. Furthermore, an in-depth characterization of a 2,4-diaminopyrimidine-based compound class was conducted to identify the molecular target and elucidate the mode of action. Structure-activity relationship studies improved both potency and water solubility of the original hit compound and eliminated bioactivities unrelated to its ROS-inducing properties. The optimized compound DP68 was found to strongly elevate cellular superoxide levels without affecting hydrogen peroxide concentrations, indicating an interference with superoxide dismutation. However, an inhibition of the enzymatic activity of superoxide dismutases by DP68 was not observed. Target identification studies of DP68 were conducted, using affinity-based chemical proteomics and different profiling techniques to investigate the compound’s influence on cellular morphology, thermal stability of proteins and the (phospho)proteome. Thereby, DP68 was identified as novel antagonist of the σ1 receptor, which however does not mediate ROS induction. Furthermore, DP68 was found to interact with HEAT repeat-containing proteins, including phosphatidylinositol 3-kinase-related kinases. In addition, DP68 induced a phosphorylation of superoxide dismutase 1, which may influence its cellular localization. DP68 induced cytotoxicity in cancer cell lines and non-malignant primary cells, which may depend on its superoxide-inducing properties.Die menschliche Zelle gerät dauerhaft mit reaktiven Sauerstoffspezies (ROS) in Kontakt. Dabei handelt es sich um sauerstoffhaltige chemische Intermediate, die spontan mit verschiedensten Biomolekülen reagieren können. ROS können sowohl aus exogenen Quellen stammen, aber auch in Zellen selbst, z.B. als Nebenprodukt ihres Metabolismus, entstehen. Abhängig von ihrer Konzentration können ROS sowohl als Signalmoleküle fungieren, oder oxidativen Stress auslösen und zelluläre Komponenten schädigen. Aus diesem Grund verfügen Zellen zur Erhaltung ihrer Redoxhomöostase über eine Vielzahl antioxidativer Mechanismen. Da ROS auch bei der Entstehung und Ausbreitung von Tumoren wichtige Rollen spielen, könnte ihre Modulation, zum Beispiel durch niedermolekulare Substanzen, das Potential zur Induktion krebsselektiver Zytotoxizität innehaben. Im Rahmen dieser Doktorarbeit wurden mithilfe eines phänotypischen Screens neuartige schnellwirksame niedermolekulare Substanzen identifiziert, die die zelluläre ROS-Konzentration erhöhen. Ein weiterer Screen zur Detektion glutathionreaktiver Substanzen führte zur Identifizierung von 4,5-Dihalogen-2-methylpyridazin-3-onen und 2,3,4,5(6)-Tetrachlor-6(5)-methylpyridinen, die eine zelluläre Glutathiondepletion induzieren. Des Weiteren wurden ausführliche Studien einer 2,4-Diaminpyrimidin-basierten Substanzklasse durchgeführt, um das Zielmolekül zu identifizieren und den Wirkmechanismus aufzuklären. Die Untersuchung der Struktur-Aktivitätsbeziehung führte zur Verbesserung der Potenz sowie der Wasserlöslichkeit und zur Eliminierung von Bioaktivitäten, die nicht mit der ROS-induzierenden Eigenschaft in Verbindung stehen. Untersuchungen der optimierten Substanz DP68, zeigte eine starke Erhöhung zellulärer Superoxidlevel ohne Änderung der Wasserstoffperoxidkonzentration, welches darauf hinweist, dass die Substanz mit der Superoxiddismutation interferieren könnte. Eine Inhibition der enzymatischen Aktivität von Superoxiddismutasen durch DP68 wurde jedoch nicht festgestellt. Zur Identifizierung der molekularen Zielstruktur von DP68 wurden Studien mittels affinitätsbasierter chemischer Proteomik und verschiedener Profilingtechniken, die den Einfluss der Substanz auf die zelluläre Morphologie, die thermische Stabilität von Proteinen und das (Phospho)proteom untersuchen, durchgeführt. Dabei wurde DP68 als neuartiger Antagonist des σ1-Rezeptors identifiziert, jedoch steht diese Interaktion nicht mit der ROS-Induktion in Verbindung. Desweiteren interagiert DP68 mit verschiedenen Proteinen, die „HEAT repeat“-Strukturmotive enthalten, z.B. mit Kinasen der „Phosphatidylinositol 3-kinase-related kinase“-Familie. Zusätzlich wurde die eine Phosphorylierung der Superoxiddismutase 1 nach Behandlung mit DP68 festgestellt, welches einen Einfluss auf dessen zelluläre Lokalisation haben könnte. DP68 induzierte Zytotoxizität sowohl in Krebszelllinien, als auch in nicht-malignen Primärzellen, welche auf seiner superoxidinduzierenden Eigenschaft beruhen könnte

Bioreator de perfusão para a análise high-throughput de combinações de biomateriais/células estaminais

Mestrado em Biotecnologia - Biotecnologia Industrial e AmbientalA engenharia de tecidos combina células humanos, materiais e engenharia de modo a induzir respostas biológicas com o objetivo de proporcionar uma regeneração rápida e correta do tecido danificado. O uso de matrizes tridimensionais (3D) para suportar o crescimento celular, ao contrário dos convencionais materiais 2D, é de grande importância para a simulação da organização estrutural de tecidos biológicos. Outros aspetos da matriz extracelular (ECM), para além da sua arquitetura são conhecidos por afetar a resposta celular. Fatores biomecânicos apresentados às células através das proteínas da ECM influenciam a adesão celular e fenómenos tais como manutenção do fenótipo, diferenciação celular e proliferação. Estudos in vitro muitas vezes falham na apresentação de fatores fisiológicos que incluem dinâmica de fluidos, o qual pode levar a uma correta oxigenação do biomaterial com células incorporadas, bem como a fenómenos de mecanotransdução. Neste trabalho, propomos um sistema que revela o efeito de 32 combinações de proteínas da ECM na adesão e expressão da alcalina fosfatasse (ALP) em células estaminais derivadas da coluna óssea (MSCs), tanto em ambiente estático como dinâmico. Um bioreator foi desenhado de modo a permitir um estudo high-throughput, para que fossem analisadas 32 combinações biomaterial-célula simultaneamente. Este bioreactor foi construído a partir de material de laboratório comum e de baixo custo (incluindo tubos e seringas descartáveis). As MSCs foram semeadas em scaffolds de quitosano poroso, modificado covalentemente com proteínas da ECM do osso, assim como proteínas responsáveis por contacto célula-célula e componentes do esmalte. Uma análise fatorial permitiu correlacionar a presença das várias combinações proteicas com melhor adesão celular ao biomaterial, assim como uma expressão de ALP após 24 horas e 5 dias de cultura. Os dados foram analisados tanto para ambiente estático, como dinâmico na presença de um pequeno fluxo, previamente comprovado como potenciador da diferenciação osteogénica de MSCs. O sistema desenvolvido foi útil na interpretação da grande complexidade das interações célula-ECM, e poderá ter possível aplicação no desenvolvimento de biomateriais para regeneração óssea, bem como em futuras aplicações como modelos de doença.Tissue engineering combines human cells, materials and engineering to induce biological responses seeking the rapid and accurate healing of damaged tissues. The use of three-dimensional (3D) matrices to support cellular growth, in opposition to traditionally used two dimensional (2D) materials, are of utmost importance to emulate the structural organization of biological tissues. Other aspects of the extracellular matrix (ECM) beyond its architecture are known to affect cell response. The biochemical cues presented to cells by ECM proteins influence cell adhesion and phenomena as cell phenotype maintenance, cell differentiation and proliferation. In vitro studies often lack physiological-like cues that include slow fluid dynamics, which may impair the correct oxygenation of the biomaterial-cells construct. Here, we engineered a system to disclose the effect of 32 different ECM protein combinations on the adhesion and alkaline phosphatase (ALP) expression of bone marrow-derived mesenchymal stem cells (MSCs), both under static and flow perfusion conditions. A novel bioreactor was designed to enable a high-throughput study, that allowed to withdraw data from 32 biomaterial-cell combinations in one single test. The bioreactor was assembled from widely available affordable labware (including plastic tubes and disposable syringes). MSCs were seeded on chitosan porous scaffolds covalently modified with bone ECM proteins, as well as cell-cell contact proteins and enamel components. A factorial analysis study allowed correlating the presence of single and combinations of proteins with improved cell adhesion to biomaterials, as well as improved ALP quantification after 24 hours and 5 days of culture. The data was analyzed both for static culture conditions, as well as in the presence of a slow perfusion rate, previously shown to potentiate MSCs osteogenic differentiation. The developed system has proven to be useful in the interpretation of the wide complexity of cells-ECM interactions, and may find application in the development of biomaterials for tissue regeneration or as disease model platforms

Germin and germin-like proteins: evolution, structure, and function

Germin and germin-like proteins (GLPs) are encoded by a family of genes found in all plants. They are part of the cupin superfamily of biochemically diverse proteins, a superfamily that has a conserved tertiary structure, though with limited similarity in primary sequence. The subgroups of GLPs have different enzyme functions that include the two hydrogen peroxide-generating enzymes, oxalate oxidase (OxO) and superoxide dismutase. This review summarizes the sequence and structural details of GLPs and also discusses their evolutionary progression, particularly their amplification in gene number during the evolution of the land plants. In terms of function, the GLPs are known to be differentially expressed during specific periods of plant growth and development, a pattern of evolutionary subfunctionalization. They are also implicated in the response of plants to biotic (viruses, bacteria, mycorrhizae, fungi, insects, nematodes, and parasitic plants) and abiotic (salt, heat/cold, drought, nutrient, and metal) stress. Most detailed data come from studies of fungal pathogenesis in cereals. This involvement with the protection of plants from environmental stress of various types has led to numerous plant breeding studies that have found links between GLPs and QTLs for disease and stress resistance. In addition the OxO enzyme has considerable commercial significance, based principally on its use in the medical diagnosis of oxalate concentration in plasma and urine. Finally, this review provides information on the nutritional importance of these proteins in the human diet, as several members are known to be allergenic, a feature related to their thermal stability and evolutionary connection to the seed storage proteins, also members of the cupin superfamily

Living Cell Microarrays: An Overview of Concepts

Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays.Niedersächsische Krebsgesellschaft e.V.BIOFABRICATION FOR NIFE InitiativeLower Saxony ministry of Science and CultureVolkswagen Stiftun