1,036 research outputs found

    Model-based Comparative Prediction of Transcription-Factor Binding Motifs in Anabolic Responses in Bone.

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    Understanding the regulatory mechanism that controls the alteration of global gene expression patterns continues to be a challenging task in computational biology. We previously developed an ant algorithm, a biologically-inspired computational technique for microarray data, and predicted putative transcription-factor binding motifs (TFBMs) through mimicking interactive behaviors of natural ants. Here we extended the algorithm into a set of web-based software, Ant Modeler, and applied it to investigate the transcriptional mechanism underlying bone formation. Mechanical loading and administration of bone morphogenic proteins (BMPs) are two known treatments to strengthen bone. We addressed a question: Is there any TFBM that stimulates both “anabolic responses of mechanical loading” and “BMP-mediated osteogenic signaling”? Although there is no significant overlap among genes in the two responses, a comparative model-based analysis suggests that the two independent osteogenic processes employ common TFBMs, such as a stress responsive element and a motif for peroxisome proliferator-activated receptor (PPAR). The post-modeling in vitro analysis using mouse osteoblast cells supported involvements of the predicted TFBMs such as PPAR, Ikaros 3, and LMO2 in response to mechanical loading. Taken together, the results would be useful to derive a set of testable hypotheses and examine the role of specific regulators in complex transcriptional control of bone formation

    PTH1R Signaling in Osteoblasts Stimulated with Functionally Selective Ligands: Phosphoproteomics Reveals Unique Signaling Networks

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    The past 20 years have seen G-protein coupled receptor (GPCR) theory advance significantly. Receptors are now thought of as adopting multiple conformations in a state of dynamic equilibrium. The study of GPCR biased agonism has emerged from this changing concept of receptors and introduced the field to “pluridimensional efficacy.” It is thought that a single readout of efficacy is no longer sufficient and multiple parameters of efficacy must be measured in drug screens to improve the ability to predict in vivo effects. While several GPCRs have multiple cognate ligands that elicit functionally-selective responses, the present study focused on biased signaling of the parathyroid hormone receptor (PTH1R). Parathyroid hormone (PTH) maintains serum calcium and is a key regulator of bone remodeling. Human PTH1-34 (Forteo) is the only FDA approved drug used for treatment of osteoporosis that acts via its anabolic actions on osteoblasts. However, the therapeutic utilization of PTH1-34 is limited by its catabolic effects, mediated in part by protein kinase A, which after two years culminate in net bone resporption through the activation of osteoclasts by RANKL. The experimental, biased ligand of the PTH1R, bovine parathyroid hormone residues 7-34 with D-Trp12 and Tyr34 (bPTH(7-34)), does not exhibit the catabolic effects of PTH1-34. In vivo administration of the conventional ligand, PTH 1-34, and the biased ligand, bPTH(7-34), for eight weeks increased bone mineral density (BMD) in mice. The anabolic effect of bPTH(7-34) in vivo was lost in β-arrestin deficient mice, revealing a dependence on β-arrestin mediated signaling. Further analysis of osteoblast and osteoclast number, transcript expression, and the generation of second messengers revealed the anabolic effect of each ligand was achieved by different mechanisms. To elucidate the unique, proximal signaling events activated by acute stimulation of the PTH1R with the biased agonist, this study focused on characterization and comparison of the phosphorylation-mediated signaling profiles of osteoblasts stimulated with each osteogenic agonist. Relative changes in phosphorylation were measured using a SILAC-based phosphoproteomic screen following acute stimulation of MC3T3- E1 preosteoblast cells with hPTH(1-34) or bPTH(7-34) for 5 minutes. The experiments were performed in proliferating preosteoblasts (Day 0) and differentiating osteoblasts (Day 10). Over ten thousand sites of phosphorylation were observed. Regulated phosphosites and phosphoproteins were examined for putative kinase activity, targeted signaling pathways, and biological processes. Differences were observed in the kinases stimulated by each agonist. For example, bPTH(7-34) treatment activated MAPK1 and increased phosphorylation of downstream substrates, while phosphorylation of predicted MAPK1 substrates were decreased with hPTH(1-34) activation. While both drugs regulated phosphorylation of proteins in signaling pathways involving GPCR signaling (PLC, MTOR, Rho GTPases); Ingenuity Pathway Analysis (IPA) also revealed discrete signaling networks engaged by each drug. PTH (1-34) treatment yielded regulated proteins involved in cytoskeletal dynamics and the Wnt/β- catenin pathway, whereas bPTH(7-34) treatment modulated pathways related to survival (ATM, CDKs, and p70S6K) and transcription (Jak/Stat, and PPARα). Cell-based assays confirmed hPTH(1-34) and bPTH(7-34) both confer resistance to etoposide-induced apoptosis and bPTH(7-34) increases proliferation in MC3T3-E1 cells. At the biological process level, both ligands modulated proteins involved in cell survival, migration, growth, and bone metabolism. Comparison of regulated phosphoproteins at two time points during osteogenic differentiation unexpectedly revealed that the bPTH(7-34) gave a more robust effect in proliferating preosteoblasts, whereas hPTH(1-34) stimulated more sites of phosphorylation in differentiating osteoblasts. This observation indicates the differential effects of each agonist may result from changes in signaling mediators that are expressed at these two time points. While the PTH receptor was present at both time points, β-arrestin was more highly expressed in proliferating preosteoblasts

    Semantic systems biology of prokaryotes : heterogeneous data integration to understand bacterial metabolism

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    The goal of this thesis is to improve the prediction of genotype to phenotypeassociations with a focus on metabolic phenotypes of prokaryotes. This goal isachieved through data integration, which in turn required the development ofsupporting solutions based on semantic web technologies. Chapter 1 providesan introduction to the challenges associated to data integration. Semantic webtechnologies provide solutions to some of these challenges and the basics ofthese technologies are explained in the Introduction. Furthermore, the ba-sics of constraint based metabolic modeling and construction of genome scalemodels (GEM) are also provided. The chapters in the thesis are separated inthree related topics: chapters 2, 3 and 4 focus on data integration based onheterogeneous networks and their application to the human pathogen M. tu-berculosis; chapters 5, 6, 7, 8 and 9 focus on the semantic web based solutionsto genome annotation and applications thereof; and chapter 10 focus on thefinal goal to associate genotypes to phenotypes using GEMs. Chapter 2 provides the prototype of a workflow to efficiently analyze in-formation generated by different inference and prediction methods. This me-thod relies on providing the user the means to simultaneously visualize andanalyze the coexisting networks generated by different algorithms, heteroge-neous data sets, and a suite of analysis tools. As a show case, we have ana-lyzed the gene co-expression networks of M. tuberculosis generated using over600 expression experiments. Hereby we gained new knowledge about theregulation of the DNA repair, dormancy, iron uptake and zinc uptake sys-tems. Furthermore, it enabled us to develop a pipeline to integrate ChIP-seqdat and a tool to uncover multiple regulatory layers. In chapter 3 the prototype presented in chapter 2 is further developedinto the Synchronous Network Data Integration (SyNDI) framework, whichis based on Cytoscape and Galaxy. The functionality and usability of theframework is highlighted with three biological examples. We analyzed thedistinct connectivity of plasma metabolites in networks associated with highor low latent cardiovascular disease risk. We obtained deeper insights froma few similar inflammatory response pathways in Staphylococcus aureus infec-tion common to human and mouse. We identified not yet reported regulatorymotifs associated with transcriptional adaptations of M. tuberculosis.In chapter 4 we present a review providing a systems level overview ofthe molecular and cellular components involved in divalent metal homeosta-sis and their role in regulating the three main virulence strategies of M. tu-berculosis: immune modulation, dormancy and phagosome escape. With theuse of the tools presented in chapter 2 and 3 we identified a single regulatorycascade for these three virulence strategies that respond to limited availabilityof divalent metals in the phagosome. The tools presented in chapter 2 and 3 achieve data integration throughthe use of multiple similarity, coexistence, coexpression and interaction geneand protein networks. However, the presented tools cannot store additional(genome) annotations. Therefore, we applied semantic web technologies tostore and integrate heterogeneous annotation data sets. An increasing num-ber of widely used biological resources are already available in the RDF datamodel. There are however, no tools available that provide structural overviewsof these resources. Such structural overviews are essential to efficiently querythese resources and to assess their structural integrity and design. There-fore, in chapter 5, I present RDF2Graph, a tool that automatically recoversthe structure of an RDF resource. The generated overview enables users tocreate complex queries on these resources and to structurally validate newlycreated resources. Direct functional comparison support genotype to phenotype predictions.A prerequisite for a direct functional comparison is consistent annotation ofthe genetic elements with evidence statements. However, the standard struc-tured formats used by the public sequence databases to present genome an-notations provide limited support for data mining, hampering comparativeanalyses at large scale. To enable interoperability of genome annotations fordata mining application, we have developed the Genome Biology OntologyLanguage (GBOL) and associated infrastructure (GBOL stack), which is pre-sented in chapter 6. GBOL is provenance aware and thus provides a consistentrepresentation of functional genome annotations linked to the provenance.The provenance of a genome annotation describes the contextual details andderivation history of the process that resulted in the annotation. GBOL is mod-ular in design, extensible and linked to existing ontologies. The GBOL stackof supporting tools enforces consistency within and between the GBOL defi-nitions in the ontology. Based on GBOL, we developed the genome annotation pipeline SAPP (Se-mantic Annotation Platform with Provenance) presented in chapter 7. SAPPautomatically predicts, tracks and stores structural and functional annotationsand associated dataset- and element-wise provenance in a Linked Data for-mat, thereby enabling information mining and retrieval with Semantic Webtechnologies. This greatly reduces the administrative burden of handling mul-tiple analysis tools and versions thereof and facilitates multi-level large scalecomparative analysis. In turn this can be used to make genotype to phenotypepredictions. The development of GBOL and SAPP was done simultaneously. Duringthe development we realized that we had to constantly validated the data ex-ported to RDF to ensure coherence with the ontology. This was an extremelytime consuming process and prone to error, therefore we developed the Em-pusa code generator. Empusa is presented in chapter 8. SAPP has been successfully used to annotate 432 sequenced Pseudomonas strains and integrate the resulting annotation in a large scale functional com-parison using protein domains. This comparison is presented in chapter 9.Additionally, data from six metabolic models, nearly a thousand transcrip-tome measurements and four large scale transposon mutagenesis experimentswere integrated with the genome annotations. In this way, we linked gene es-sentiality, persistence and expression variability. This gave us insight into thediversity, versatility and evolutionary history of the Pseudomonas genus, whichcontains some important pathogens as well some useful species for bioengi-neering and bioremediation purposes. Genome annotation can be used to create GEM, which can be used to betterlink genotypes to phenotypes. Bio-Growmatch, presented in chapter 10, istool that can automatically suggest modification to improve a GEM based onphenotype data. Thereby integrating growth data into the complete processof modelling the metabolism of an organism. Chapter 11 presents a general discussion on how the chapters contributedthe central goal. After which I discuss provenance requirements for data reuseand integration. I further discuss how this can be used to further improveknowledge generation. The acquired knowledge could, in turn, be used to de-sign new experiments. The principles of the dry-lab cycle and how semantictechnologies can contribute to establish these cycles are discussed in chapter11. Finally a discussion is presented on how to apply these principles to im-prove the creation and usability of GEM’s.</p

    Neural EGFL Like 1 as a Potential Pro-Chondrogenic, Anti-Inflammatory Dual-Functional Disease-Modifying Osteoarthritis Drug

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    Arthritis, an inflammatory condition that causes pain and cartilage destruction in joints, affects over 54.4 million people in the US alone. Here, for the first time, we demonstrated the emerging role of neural EGFL like 1 (NELL-1) in arthritis pathogenesis by showing that Nell-1-haploinsufficient (Nell-1+/6R) mice had accelerated and aggravated osteoarthritis (OA) progression with elevated inflammatory markers in both spontaneous primary OA and chemical-induced secondary OA models. In the chemical-induced OA model, intra-articular injection of interleukin (IL)1β induced more severe inflammation and cartilage degradation in the knee joints of Nell-1+/6R mice than in wildtype animals. Mechanistically, in addition to its pro-chondrogenic potency, NELL-1 also effectively suppressed the expression of inflammatory cytokines and their downstream cartilage catabolic enzymes by upregulating runt-related transcription factor (RUNX)1 in mouse and human articular cartilage chondrocytes. Notably, NELL-1 significantly reduced IL1β-stimulated inflammation and damage to articular cartilage in vivo. In particular, NELL-1 administration markedly reduced the symptoms of antalgic gait observed in IL1β-challenged Nell-1+/6R mice. Therefore, NELL-1 is a promising pro-chondrogenic, anti-inflammatory dual-functional disease-modifying osteoarthritis drug (DMOAD) candidate for preventing and suppressing arthritis-related cartilage damage. © 2019 Elsevier Lt

    The role of histone deacetylase 3 in chondrogenesis and osteoarthritis

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    PhD ThesisHistone deacetylases (HDACs) regulate the acetylation pattern of chromatin to control gene expression. Previous studies reported class I HDACs and specifically HDAC3 as a key regulator of MMP1/13 expression, the major collagenases in cartilage. Moreover, HDAC inhibitors were shown to have a chondroprotective role in in vivo models of osteoarthritis. The aim of this study was to examine the role of HDAC3 in catabolic gene expression in osteoarthritis and in chondrocyte differentiation in vivo and in vitro. Chemical inhibitors against all HDACs (e.g. TSA) and specifically against HDAC3 (Apicidin) were used in conjunction with HDAC3 RNAi in human SW1353 chondrocytes, stimulated with IL-1 and in a mesenchymal stem cell (MSC) chondrogenic differentiation model. Gene expression was quantified using qRT- PCR and protein by western blotting, immunohistochemistry and biochemical methods. RNA microarray analysis was performed following RNAi or Apicidin treatment, across a time-course of IL-1 stimulation in chondrocytes to identify critical regulators of catabolic gene expression. Subsequently the role of E2F-1 transcription factor was studied with gain or loss of function experiments. E2F-1 activity on MMP1/13 was assessed by luciferase reporter gene activation and interaction with HDAC3 determined by immunoprecipitation. The role of HDAC3 in murine development was determined by generating a cartilage specific conditional knockout mouse model. HDAC3 is essential for IL-1- induced MMP1/13 and FRA1 expression in chondrocytes. Microarray analysis suggests the E2F-1 transcription factor may mediate the effect of HDAC3 on gene expression regulation. Accordingly, modulation of E2F1 levels alters IL-1 induced MMP1/13 expression and HDAC3 appears to interact with E2F1. Additionally, HDAC3 regulates anabolic gene expression, including COL2A1 and ACAN, during MSC chondrogenesis and is required for normal endochondral ossification, since conditional deletion resulted in embryonic lethality. We propose a novel role for HDAC3 in the regulation of catabolic and anabolic gene expression.Arthritis Research U

    Coronectomy of deeply impacted lower third molar : incidence of outcomes and complications after one year follow-up

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    Objectives: The purpose of present study was to assess the surgical management of impacted third molar with proximity to the inferior alveolar nerve and complications associated with coronectomy in a series of patients undergoing third molar surgery. Material and Methods: The position of the mandibular canal in relation to the mandibular third molar region and mandibular foramen in the front part of the mandible (i.e., third molar in close proximity to the inferior alveolar nerve [IAN] or not) was identified on panoramic radiographs of patients scheduled for third molar extraction. Results: Close proximity to the IAN was observed in 64 patients (35 females, 29 males) with an impacted mandibular third molar. Coronectomy was performed in these patients. The most common complication was tooth migration away from the mandibular canal (n = 14), followed by root exposure (n = 5). Re-operation to remove the root was performed in cases with periapical infection and root exposure. Conclusions: The results indicate that coronectomy can be considered a reasonable and safe treatment alternative for patients who demonstrate elevated risk for injury to the inferior alveolar nerve with removal of the third molars. Coronectomy did not increase the incidence of damage to the inferior alveolar nerve and would be safer than complete extraction in situations in which the root of the mandibular third molar overlaps or is in close proximity to the mandibular canal

    Molecular and functional analysis of mutations in the transcription factor ZNF687 associated to Paget disease of bone

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    The zinc finger protein ZNF687 is a transcription factor containing various Cys2-His2 zinc finger domains that has been related to Paget’s disease of bone (PDB) associated with giant cell tumor of bone. Recently, four mutations have been independently identified in a Southern Italian population, and were determined as predisposal (p.Ser242Ile, p.Pro665Leu, and p.Gln784Glu,) or causal (p.Pro937Arg) for development of PDB. Moreover, it has been suggested that ZNF687 plays an important role in bone metabolism in both human and zebrafish, and that the function of this protein might be conserved throughout evolution. Nonetheless, the mechanism by which ZNF687 affects bone metabolism, and hence, PDB, remains to be elucidated. To contribute to respond to these questions we have proposed to perform in silico analysis in order to investigate how these four mutations could affect the protein conformation and function, and in vitro analyses to evaluate how mutant ZNF687 (p.Pro937Arg, mediated by site-directed mutagenesis), ZNF687 overexpression (mediated by transient transfection) and ZNF687 knock-down (mediated by CRISPR-Cas9), could affect the expression of genes involved in bone metabolism. We also assessed the mineralization process of knock-down clones and evaluated specific gene expression. Lastly, comparative in silico and in vitro analyses were performed in order to define the usefulness of zebrafish as a biological model for ZNF687 study. Therefore, the mutation analysis suggested alterations in protein-protein and protein-acid nucleic interactions that might distort ZNF687 target genes expression and lead to PDB. Moreover, preliminary results suggested that ZNF687 might regulate genes involved in osteoblastogenesis and osteoclastogenesis, such as RUNX2, OSX, RANK, SQSTM1 and OPTN, while its role in mineralization remained uncleared. Finally, by identifying significant similarities in genomic structure, protein domain, and molecular players affecting znf687a, znf687b and ZNF687 transcription, together with the identification and characterization of promoter’s regions that regulate the transcription of znf687a and human ZNF687 genes, we have confirmed that zebrafish is a useful biological model system to study ZNF687.A proteína dedo de zinco 687 (ZNF687) é um fator de transcrição que contem vários motivos Cis2-His2, sendo um dos motivos mais comuns de ligação ao DNA encontrados nos fatores de transcrições dos eucariotas. O gene ZNF687 humano tem sido associado à várias doenças, nomeadamente à leucemia mielóide aguda, à doença óssea de Paget (DOP) severa associada ao tumor de células gigantes do osso, e ao carcinoma hepatocelular, advogando um putativo papel oncogénico. Além disso, foi demostrado que a expressão de RNA mensageiro do ZNF687 era significativamente aumentada durante a osteoclastogénese e a osteoblastogénese, tanto no humano como no peixe zebra, sugerindo um papel importante no metabolismo do osso, e uma conservação quanto à função desta proteína ao longo da evolução, apesar da especiação. A doença óssea de Paget é um distúrbio metabólico crónico e raro, caracterizado por áreas focais com exagerada remodelação óssea. Com efeito, uma atividade anormal dos osteoclastos leva à reabsorção óssea excessiva, que é por sua vez, rapidamente compensada por uma hiperatividade dos osteoblastos, células responsáveis pela formação do osso. A doença pode ser monostótica ou poliostótica, mas em ambos os casos, a estrutura óssea resultante é desorganizada, desformada e frágil. A etiologia da DOP não é bem conhecida, mas um fator genético está fortemente associado à doença, existindo também um fator ambiental. Foram identificadas várias mutações em diferentes genes, nomeadamente no sequestossoma 1 (SQSTM 1), fator estimulante de colónia 1 (CSF-1), membro da superfamília do receptor do fator de necrose tumoral 11a (TNFRSF11A), superfamília 7 transmembrana 4 (TM7SF4), optineurina (OPTN), ras e intercatores rab 3 (RIN3), proteína contendo valosina (VCP), nucleoporina 205 (NUP205) e mais recentemente, ZNF687 e ribonucleoproteína nuclear heterogênea A2 / B1 (hnRNPA2B1). Recentemente, foram descritas, em estudos independentes, numa população do sul da Itália, quatro mutações associadas a DOP: três delas foram identificadas como não causais (p.Ser242Ile, p.Pro665Leu, e p.Gln784Glu) e uma delas (p.Pro937Arg) foi determinada como necessária e suficiente para desenvolver a DOP. O mecanismo pelo qual a ZNF687 afeta o metabolismo ósseo e deste modo pode causar a DOP, ainda não foi esclarecido. Para responder a estas questões, propusemos fornecer dados, realizando vários estudos in silico e in vitro. Deste modo, para estudar o efeito das mutações descritas na proteína ZNF687, efetuaram-se análises de comparação da estrutura da proteína normal versus a estrutura da proteína com as diferentes mutações. As mutações associadas a DOP não mostraram nenhuma alteração na estrutura secundária da proteína, no entanto, as diferenças nas propriedades entre os resíduos “wild-type” (WT) e os resíduos mutados podem levar às alterações, tanto na conformação local, como na interação de proteína-proteína ou de proteína-ácido nucleico. Contrariamente às mutações p.Ser242Ile e p.Pro665Leu, que não se encontram num domínio funcional, a mutação p.Glu784Gln ocorre na extremidade C-terminal de um domínio dedo de zinco (ZF), podendo afetar a interação aminoácido-DNA especifica deste local. A mutação p.Pro937Arg gera uma carga positiva adicional antes do local de sinalização nuclear (NLS). O resíduo mutado acaba por fazer parte deste NLS, tornando-o mais forte, o que pode levar a um aumento da translocação da proteína para o núcleo. Após estes resultados preliminares, fomos avaliar a expressão de genes putativos alvos da ZNF687, que estão descritos como estando envolvidos no metabolismo do osso. Para isto, a região codificante do gene, inserida num vetor de expressão, foi submetida ao processo de mutagénese dirigida para se reproduzir a mutação p.Pro937Arg. Esta construção foi posteriormente utilizada na transfeção de células SaOS-2, em paralelo com o vetor contendo a forma normal, para induzir a sobre-expressão da ZNF687 mutada e normal. Outra estratégia do nosso trabalho consistiu na obtenção de clones de células SaOS-2, em que efetuámos a repressão do gene ZNF687 através da ferramenta de edição génica, CRIPSR-Cas9. Tanto nas células submetidas a sobre-expressão como nos clones “knock-down”, o RNA foi extraído e a expressão dos níveis de mRNA de vários genes de interesse foi analisada por PCR quantitativo. Em seguida, estudámos também o processo de mineralização nos clones “knock-down” e avaliámos a expressão dos genes alvos da ZNF687. Os nossos dados preliminares sugeriram que a ZNF687 poderia desempenhar um papel regulador de RUNX2 e OSX, dois genes envolvidos na osteoblastogénese, e de RANK, SQSTM1 e OPTN, genes envolvidos na osteoclastogénese através da via de sinalização do NF-κB induzida por RANKL. No entanto, não foi possível determinar se a regulação do SQSTM1 e de OPTN pela ZNF687 dá-se de uma forma direta ou se é feita através da regulação de RANK. O mesmo pode acontecer no caso da regulação do OSX, podendo este ser diretamente regulado pelo RUNX2 e indiretamente pela ZNF687. O papel da ZNF687 no processo de mineralização também permanece incerto sendo que a diminuição dos níveis de expressão da osteocalcina (OCN) e da atividade da fosfatase alcalina (ALP), observados nos clones “knock-down”, poderia ser uma consequência da diminuição dos níveis de RUNX2, diminuição possivelmente causada pelo “knock-down” da ZNF687. Por outro lado, a expressão em células que sobre-expressam a forma mutada de ZNF687 demonstraram um padrão de expressão similar ao das células que sobre-expressam a forma WT. Anteriormente, foi descrito que a mutação p.Pro937Arg, mutação causal encontrada em pacientes com DOP, parece atuar como um ganho de função levando a um aumento da translocação de ZNF687 para o núcleo, onde a sua acumulação aumentará a expressão de genes alvos. Portanto, a alteração da expressão dos genes RUNX2, OSX, RANK, OPTN e SQSTM1, envolvidos na diferenciação dos osteoblastos e dos osteoclastos, poderá ser a causa do desenvolvimento da DOP em pacientes que apresentam essa mutação. Porém, após análises comparativas entre os genes znf687a e znf687b do peixe zebra com o gene ZNF687, foi possível sugerir que o gene znf687b era ortólogo do gene ZNF687 humano sendo que é aquele que apresentava maiores semelhanças na estrutura genómica, domínio proteico e fatores moleculares afetando a transcrição znf687a, znf687b e ZNF687. Finalmente, através da análise funcional da região promotora dos genes do peixe zebra e do humano identificou-se uma região no znf687a do zebrafish e duas regiões no ZNF687 humano como sendo responsáveis pela sua regulação. Além disso, o ensaio de co-transfecção demonstrou um efeito de repressão da transcrição da construção contendo o promotor do gene zbf687a devido ao fator de transcrição YY-1. Nos ensaios efetuados com a construção contendo o promotor do gene ZNF687 observou-se uma repressão da sua transcrição devido ao fator de transcrição AP-1. Estes resultados parecem sugerir que os promotores do gene ZNF687, tanto em humano como em peixe zebra, são regulados negativamente por factores de transcrição, que regulam genes envolvidos no desenvolvimento do osso. Estes resultados juntamente com a nossa análise in silico comparativa do locus genómico, do gene e da proteína entre as duas espécies permite-nos sugerir a potencialidade de utilizar o peixe-zebra como modelo biológico para o estudo da função da ZNF687

    Markers of Bone Turnover In Preclinical Development of Drugs for Skeletal Diseases

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    Skeletal tissue is constantly remodeled in a process where osteoclasts resorb old bone and osteoblasts form new bone. Balance in bone remodeling is related to age, gender and genetic factors, but also many skeletal diseases, such as osteoporosis and cancer-induced bone metastasis, cause imbalance in bone turnover and lead to decreased bone mass and increased fracture risk. Biochemical markers of bone turnover are surrogates for bone metabolism and may be used as indicators of the balance between bone resorption and formation. They are released during the remodeling process and can be conveniently and reliably measured from blood or urine by immunoassays. Most commonly used bone formation markers include N-terminal propeptides of type I collagen (PINP) and osteocalcin, whereas tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) and C-terminal cross-linked telopeptide of type I collagen (CTX) are common resorption markers. Of these, PINP has been, until recently, the only marker not commercially available for preclinical use. To date, widespread use of bone markers is still limited due to their unclear biological significance, variability, and insufficient evidence of their prognostic value to reflect long term changes. In this study, the feasibility of bone markers as predictors of drug efficacy in preclinical osteoporosis models was elucidated. A non-radioactive PINP immunoassay for preclinical use was characterized and validated. The levels of PINP, N-terminal mid-fragment of osteocalcin, TRACP 5b and CTX were studied in preclinical osteoporosis models and the results were compared with the results obtained by traditional analysis methods such as histology, densitometry and microscopy. Changes in all bone markers at early timepoints correlated strongly with the changes observed in bone mass and bone quality parameters at the end of the study. TRACP 5b correlated strongly with the osteoclast number and CTX correlated with the osteoclast activity in both in vitro and in vivo studies. The concept “resorption index” was applied to the relation of CTX/TRACP 5b to describe the mean osteoclast activity. The index showed more substantial changes than either of the markers alone in the preclinical osteoporosis models used in this study. PINP was strongly associated with bone formation whereas osteocalcin was associated with both bone formation and resorption. These results provide novel insight into the feasibility of PINP, osteocalcin, TRACP 5b and CTX as predictors of drug efficacy in preclinical osteoporosis models. The results support clinical findings which indicate that short-term changes of these markers reflect long-term responses in bone mass and quality. Furthermore, this information may be useful when considering cost-efficient and clinically predictive drug screening and development assays for mining new drug candidates for skeletal diseases.Luun biokemialliset merkkiaineet luustosairauksien prekliinisessä lääkekehityksessä Luun uudismuodostusta tapahtuu koko elämän ajan. Tässä prosessissa osteoklastit hajottavat vanhaa luuta ja osteoblastit muodostavat uutta luuta. Tasapainoon vaikuttaa ikä, sukupuoli ja perinnöllisyys, mutta myös monissa luustosairauksissa, kuten osteoporoosissa ja syövän luustometastaaseissa, tämä tasapaino on järkkynyt johtaen vähentyneeseen luun määrään ja lisääntyneeseen murtumaherkkyyteen. Luun biokemialliset merkkiaineet kertovat luun aineenvaihdunnasta eli hajotuksen ja muodostuksen välisestä tasapainosta. Merkkiaineita vapautuu luun uudismuodostuksessa ja niitä voidaan helposti ja luotettavasti mitata seerumista tai virtsasta immunomääritysmenetelmillä. Yleisesti käytettyjä luun muodostuksen merkkiaineita ovat tyypin I kollageenin aminoterminaalinen propeptidi (PINP) ja osteokalsiini, sekä luun hajotuksen merkkiaineita tartraatti-resistentti hapan fosfataasi alatyyppi 5b (TRACP 5b) ja tyypin I kollageenin karboksiterminaalinen telopeptidi (CTX). Näistä PINP on ainoa merkkiaine, jolle ei ole aiemmin ollut saatavilla prekliiniseen käyttöön soveltuvaa kaupallista immunomääritysmenetelmää. Tällä hetkellä biokemiallisten merkkiaineiden laajamittainen käyttö on vielä rajoittunutta, koska niihin liittyy paljon biologista ja analyyttistä variaatiota eikä niiden merkitsevyydestä ja käyttökelpoisuudesta pitkän aikavälin muutosta ennustavana tekijänä ole riittävästi näyttöä. Tämän tutkimuksen tavoitteena oli selvittää luuston biokemiallisten merkkiaineiden soveltuvuutta lääkemolekyylien tehokkuuden ennustajina prekliinisissä osteoporoositutkimusmalleissa. Tutkimuksessa karakterisoitiin ja validoitiin PINP:lle prekliiniseen käyttöön kehitetty immunomääritysmenetelmä. PINP:n, osteokalsiinin N-terminaalisen keskifragmentin, TRACP 5b:n ja CTX:n tasoja tutkittiin prekliinisissä osteoporoosimalleissa, ja saatuja tuloksia verrattiin perinteisillä menetelmillä kuten histologialla, tiheysmittauksilla ja mikroskopialla saatuihin tuloksiin. Kaikkien tutkittujen luuston merkkiaineiden alkuvaiheen muutosten havaittiin korreloivan kokeen lopussa nähtyihin luuston rakenteellisiin muutoksiin. TRACP 5b korreloi osteoklastien lukumäärään ja CTX osteoklastien aktiivisuuteen sekä in vitro että in vivo kokeissa. CTX/TRACP 5b suhteelle luotiin termi resorptio-indeksi, joka kuvaa osteoklastien keskimääräistä aktiivisuutta. Indeksi antoi tarkempaa tietoa kuin kumpikaan merkkiaine erikseen käytetyissä prekliinisissä osteoporoosimalleissa. PINP:n havaittiin korreloivan vahvasti luun muodostukseen, kun taas osteokalsiini kuvasi sekä luun muodostusta että hajotusta. Tämän tutkimuksen tulokset antavat uutta tietoa luun biokemiallisten merkkiaineiden soveltuvuudesta lääkemolekyylien tehokkuuden ennustajina prekliinisissä osteoporoosimalleissa. Havainnot tukevat kliinisiä tutkimuksia, joissa merkkiaineiden on havaittu korreloivan myöhemmin nähtävien luuston rakenteellisten muutosten kanssa. Tutkimuksessa saatu tieto auttaa suunniteltaessa kliinisen ennustettavuuden kannalta parempia määritysmenetelmiä, joiden avulla voidaan nopeammin ja tehokkaammin löytää uusia toimivia lääkemolekyylejä luustosairauksien hoitoon.Siirretty Doriast

    Mechanically-regulated microRNAs in articular cartilage

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    Introduction: The role of microRNAs (miRs) in articular cartilage is still not well established, however many studies have reported the differential expression of a number of miRs between healthy and osteoarthritic (OA) articular cartilage. These studies have focused on the OA pathology itself without considering the impact of mechanical load, which is one of the major risk factors implicated in the loss of cartilage integrity and the onset of OA development. Previous studies have already identified a number of mechanically-regulated miRs, therefore I hypothesised that (i) physiological and non-physiological magnitudes of compressive load differentially regulate the expression of mechano-sensitive miRs, and (ii) mechanically-regulated miRs differentially expressed in response to a non-physiological magnitude of load are implicated in the regulation of mechano-sensitive matrix molecule turnover and are involved in OA development. Results: Transcriptional assessment of selected mechanically-regulated matrix molecules demonstrated that loading regimes of 2.5MPa and 7MPa (1Hz, 15 minutes) induced homeostatic and catabolic responses at the gene level respectively, therefore they were selected to represent ‘physiological’ and ‘non-physiological’ magnitudes of loads which have the potential to induce biosynthetic and degradative protein responses if applied for prolonged periods of time. Next generation sequencing (NGS) of articular cartilage miRs libraries demonstrated that the alteration in expression of specific miRs occurs in a magnitude- and time-dependent manner. However, 24h post-load, according to the NGS data, seems to be the most appropriate to observe significant changes in miRs levels. Validation of a few miRs, important for cartilage integrity, at 24h post-load indicated up-regulation of miR-21-5p, miR-27a-5p, miR-221 and miR-222 and down-regulation of miR-483 in response to the ‘non-physiological’ 7MPa magnitude (1Hz, 15 minutes) whereas in explants subjected to a ‘physiological’ 2.5MPa magnitude (1Hz, 15 minutes) the level of these miRs remained unchanged. Identification of target genes of miR-21-5p, miR-221 and miR-222 performed by NGS of RNA extracted from primary articular chondrocytes transfected with specific miR inhibitors demonstrated a number of differentially expressed genes. qPCR validation of these potential miR target genes on RNA collected from cells transfected with functional miR-21-5p, miR-221 and miR-222 inhibitors or mimics identified TIMP-3 as a direct target of miR-21-5p, miR-221 and miR-222, whereas CPEB was targeted by miR-21-5p. Conclusion: This current study confirms the reported mechano-regulation of miR-221 and miR-222, and furthermore demonstrates the novel mechano-regulation of miR-21-5p, miR-27a-5p and miR-483 in cartilage explants. This work is the first to identify TIMP-3 as a target of miR-21-5p and miR-221/-222, and CPEB3 as a direct target of miR-21-5p in primary chondrocytes. An association between the identified differentially-regulated miRs in response to a non-physiological magnitude of load, with those that are expressed in OA cartilage and their regulatory effect on molecules important for cartilage integrity, described in this thesis may pioneer future studies aimed at identifying cartilage biomarkers of load-induced OA and provide therapeutic potential for OA treatment
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