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Wang, Mengxi. "Role of Map4k4 in Skeletal Muscle Differentiation: A Dissertation." eScholarship@UMMS, 2013. http://escholarship.umassmed.edu/gsbs_diss/675.
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Skeletal muscle is a complicated and heterogeneous striated muscle tissue that serves critical mechanical and metabolic functions in the organism. The process of generating skeletal muscle, myogenesis, is elaborately coordinated by members of the protein kinase family, which transmit diverse signals initiated by extracellular stimuli to myogenic transcriptional hierarchy in muscle cells. Mitogen-activated protein kinases (MAPKs) including p38 MAPK, c-Jun N terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) are components of serine/threonine protein kinase cascades that play important roles in skeletal muscle differentiation. The exploration of MAPK upstream kinases identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4), a serine/threonine protein kinase that modulates p38 MAPK, JNK and ERK activities in multiple cell lines. Our lab further discovered that Map4k4 regulates peroxisome proliferator-activated receptor γ (PPARγ) translation in cultured adipocytes through inactivating mammalian target of rapamycin (mTOR), which controls skeletal muscle differentiation and hypotrophy in kinase-dependent and -independent manners. These findings suggest potential involvement of Map4k4 in skeletal myogenesis.
Therefore, for the first part of my thesis, I characterize the role of Map4k4 in skeletal muscle differentiation in cultured muscle cells. Here I show that Map4k4 functions as a myogenic suppressor mainly at the early stage of skeletal myogenesis with a moderate effect on myoblast fusion during late-stage muscle differentiation. In agreement, Map4k4 expression and protein kinase activity are declined with myogenic differentiation. The inhibitory effect of Map4k4 on skeletal myogenesis requires its kinase activity. Surprisingly, none of the identified Map4k4 downstream effectors including p38 MAPK, JNK and ERK is involved in the Map4k4-mediated myogenic differentiation. Instead, expression of myogenic regulatory factor Myf5, a positive mediator of skeletal muscle differentiation is transiently regulated by Map4k4 to partially control skeletal myogenesis. Mechanisms by which Map4k4 modulates Myf5 amount have yet to be determined.
In the second part of my thesis, I assess the relationship between Map4k4 and IGF-mediated signaling pathways. Although siRNA-mediated silencing of Map4k4 results in markedly enhanced myotube formation that is identical to the IGF-induced muscle hypertrophic phenotype, and Map4k4 regulates IGF/Akt signaling downstream effector mTOR in cultured adipocytes, Map4k4 appears not to be involved in the IGF-mediated ERK1/2 signaling axis and the IGF-mediated Akt signaling axis in C2C12 myoblasts. Furthermore, Map4k4 does not affect endogenous Akt signaling or mTOR activity during C2C12 myogenic differentiation.
The results presented here not only identify Map4k4 as a novel suppressor of skeletal muscle differentiation, but also add to our knowledge of Map4k4 action on multiple signaling pathways in muscle cells during skeletal myogenesis. The effects that Map4k4 exerts on myoblast differentiation, fusion and Myf5 expression implicate Map4k4 as a potential drug target for muscle mass growth, skeletal muscle regeneration and muscular dystrophy.
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Clark, Daniel N. "Promoter Polymorphisms in Interferon Regulatory Factor 5." BYU ScholarsArchive, 2013. https://scholarsarchive.byu.edu/etd/4058.
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The promoter region of interferon regulatory factor 5 (IRF5) contains the rs2004640 T or G single nucleotide polymorphism (SNP) and a CGGGG indel. Both of these polymorphisms have been implicated as genetic risk factors for several autoimmune diseases, including systemic lupus erythematosus, whose pathology involves altered apoptosis and cytokine signaling. The polymorphisms' overall effect is to increase IRF5 levels. IRF5 is a transcription factor of several cytokines, including interferon, and is pro-apoptotic. Thus an alteration of cytokine levels and apoptosis signaling due to high IRF5 levels is the proposed source of autoimmune risk. Each of IRF5's four first exons (1A, 1B, 1C, 1D) has its own promoter and responds to specific stimuli. rs2004640 is a T or G polymorphism; T is the risk allele. The SNP creates a sequence-specific recognition site for the spliceosome, making exon 1B spliceable. Analysis of the 1B promoter showed putative p53 binding site. IRF5 and p53 are pro-apoptotic transcription factors, and the p53 site may provide a positive feedback loop. Apoptosis levels were altered in cells with the rs2004640 risk T/T allele when treated with DNA damaging agents (extrinsic apoptosis), but not when activating death receptors (intrinsic apoptosis). The 1B promoter was the only one to activate expression after inducing DNA damage in a luciferase reporter assay, and this activation was abolished after mutating the p53 site. The exon 1A promoter contains either three or four copies (4X) of CGGGG; the 4X variant is the risk allele. The 1A promoter is constitutively active and is responsive to the Toll-like receptor 7 agonist imiquimod. RNA folding analysis revealed a hairpin encompassing exon 1B. Mutational analysis showed that the hairpin shape decreased translation five-fold in a luciferase reporter assay. Cells with the CGGGG or rs2004640 risk allele exhibited higher levels of IRF5 mRNA and protein, but demonstrated no change in mRNA stability. Quantitative PCR in cell lines with either risk polymorphism demonstrated decreased usage of exons 1C or 1D, although no other correlated splicing events were observed. Also, several mRNA splice variants of IRF5 were sequenced. The risk polymorphisms altered cytokine signaling as well. Expression of interferon, Toll-like receptor, and B cell receptor pathways were affected by a risk haplotype which includes the rs2004640 SNP. The CGGGG polymorphism decreased the levels of CC-chemokine receptor 7. Specific transcription factor binding sites define promoter activity and thus first exon usage and transcription levels. Translation levels are affected by mRNA folding. Overall, the rs2004640 SNP and the CGGGG indel cause high levels of IRF5. High IRF5 expression causes altered cytokine and apoptosis signaling, and may bias the immune system toward autoimmunity.
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Kiefer, Julie Christine. "Analysis of myogenic regulatory factors and insulin-like growth factors in early somite myogenesis /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/9227.
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Khoyratty, Tariq. "Interferon regulatory factor 5 : a systematic study of macrophage gene regulation." Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:b820f612-ceb7-4e2c-af26-52999e368c41.
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Macrophages are multifaceted innate immune cells, able to adapt their phenotype to respond to a myriad of conditions, engaging in tissue-specific functions and mediating either inflammatory or anti-inflammatory responses depending on the encountered stimuli. They conduct key roles in the orchestration of immune responses; from pathogen recognition through sterilising inflammation to resolution and repair. The Udalova laboratory has previously demonstrated that IRF5 promotes a pro- inflammatory macrophage phenotype, leading to the secretion of TNF, IL-12, and IL-23, enhancing Th1/Th17-mediated immune responses, and described the cooperation between IRF5 and the transcription factor RelA, which mediate the production of pro-inflammatory genes. The aim of this thesis is to further characterise the activity of IRF5 in macrophage inflammatory responses. I demonstrate that IRF5 not only regulates the transcription of cytokines and chemokines in response to bacterial stimuli, but also anti-microbial peptides, whilst simultaneously down-regulating homeostatic and resolving macrophage functions. My data also suggests that IRF5 plays a role in enforcing monocyte to macrophage differentiation by up-regulating the transcription of key macrophages markers and repressing dendritic cell identity genes. To further characterise the mechanisms of the inflammatory response mounted by macrophages I used an unbiased approach; combining twenty-three transcription factor ChIP-seq data sets with chromatin accessibility information from ATAC-seq, uncovering RUNX1 as a novel partner of IRF5 that binds co-operatively to clusters of enhancers, which control the transcription of pro-inflammatory genes in a signal-dependent manner. This is the first study demonstrating a critical role for RUNX1 in activity of inflammatory macrophages.
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Siu, Ho-on. "The role of interferon regulatory factor 5 gene polymorphisms in systemic lupus erythematosus." Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/HKUTO/record/B39558393.
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Siu, Ho-on, and 蕭可安. "The role of interferon regulatory factor 5 gene polymorphisms in systemic lupus erythematosus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39558393.
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Seneviratne, Anusha. "Shear stress and interferon regulatory factor 5 modulate myeloid cell behaviour in atherosclerosis." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/45408.
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Rupture of 'vulnerable' atherosclerotic plaques and subsequent thrombosis cause acute cardiovascular events, and can develop upon exposure of the arterial wall to low shear stress. Myeloid cells - the main inflammatory cells within atherosclerotic plaques - are heterogeneous; ranging from 'classical' pro-inflammatory M1 macrophages to 'alternative' M2 macrophages and various subsets of dendritic cells. The activation of Toll-like receptors and downstream Interferon Regulatory Factors (IRFs) is involved in atherosclerosis. IRF5 polarises macrophages towards the M1 phenotype and modulates cytokine production by dendritic cells. I utilised two murine models of atherosclerosis: the hypercholesterolaemic ApoE-/- (Apolipoprotein E knockout) mouse strain, and a perivascular cast modifying shear stress patterns in the carotid artery. Firstly, I found the majority of macrophages in early and intermediate lesions of the aortic root and advanced oscillatory shear stress-modulated lesions express heme oxygenase-1 (HO-1). The representation of the M1 macrophage marker iNOS (inducible nitric oxide synthase) and IRF5 is more prevalent in low shear stress-modulated plaques, which resemble a vulnerable plaque, while M2 macrophage markers are elevated in oscillatory shear stress-modulated plaques resembling stable plaques. Secondly, I studied the effect of IRF5 deletion on the development of atherosclerosis by comparing the severity of atherosclerosis in ApoE-/- mice with ApoE-/-IRF5-/- mice. Atherosclerotic lesions in the aortic root of ApoE-/-IRF5-/- mice are reduced in size, and in all vascular regions they have smaller necrotic cores (a marker of plaque vulnerability), due to a reduction in efferocytosis, and an increase in atheroprotective macrophages. Lesions in ApoE-/-IRF5-/- mice also have a depleted content of cells expressing CD11c; therefore IRF5 is detrimental in atherosclerosis by skewing myeloid cell differentiation towards dendritic cells possibly via GM-CSF. My study provides a novel link between inflammatory signalling, efferocytosis and necrotic core formation.
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Krausgruber, Thomas. "Interferon Regulatory Factor 5 (IRF5) : an important player in macrophage polarization and TNF regulation." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/9045.
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Macrophages are dynamic and heterogeneous cells that can be divided into specific, phenotypic subsets. Based on Th1/Th2 polarization concept they are referred to as proinflammatory classical M1 (IL-12high, IL-23high, IL-10low) macrophages and anti-inflammatory M2 (IL-12low, IL-23low, IL-10high) macrophages. In contrast to T lymphocyte subsets, the transcription factor(s) underlying macrophage polarization remain largely unknown. My research has highlighted the importance of Interferon regulatory factor 5 (IRF5) for establishing the pro-inflammatory M1 macrophage phenotype. I was able to show that high expression of IRF5 is characteristic of M1 macrophages, in which it transcriptionally regulates M1-specific cytokines, chemokines and co-stimulatory molecules. Consequently, the depletion of IRF5 in human M1 macrophages results in down-regulation of M1-specific cytokines and further evidence for a role of IRF5 in effective immunity stems from my work using an in vivo model of polarizing inflammation. IRF5 deficient mice showed a significant reduction in serum levels of M1-specific cytokines compared to wild-type littermate controls. Therefore, the suppression of macrophage function via inhibition of IRF5 provides a new approach to attenuate the inflammatory response. Tumor necrosis factor (TNF) plays an essential role in the host defence against infections but is a major factor in the pathogenesis of chronic inflammatory diseases. The expression of TNF is therefore tightly regulated. I was able to demonstrate that IRF5 is not only involved in the induction of human TNF gene expression but also crucial for the late phase secretion of TNF by human myeloid cells. IRF5 is using a complex molecular mechanism to control the TNF gene with two spatially separated regulatory regions (5‟ upstream and 3‟ downstream of the gene) and two independent modes of action (direct DNA binding and formation of IRF5/RelA complex) being involved. The manipulation of the IRF5/RelA interaction could be a putative target for cell-specific modulation of TNF gene expression.
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Yang, Long. "Functional study of the innate-immune-signaling components, interferon regulatory factor 5 and nuclear factor kappa B essential modulator." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=95562.
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The success of innate host defence to viral infection depends on the ability of innate immunecells to detect the invading pathogen culminating in the production of cytokines and chemokinesthat disrupt viral replication and shape the platform for innate and adaptive immune responses.Rapid induction of type I IFN is a central event in establishment of the antiviral response, whichis tightly regulated by extracellular and intracellular signals that activate transcription factors(NF-KB, AP-l and IRF). The synergistic action of these transactivators on the promoters of type IIFN genes triggers the immediate early IFN response. which is further amplified by JAK-ST ATsignaling proteins. In the IRF family, IRF-5 is the central mediator in modulating the productionof pro-inflammatory cytokines in TLR-dependent signalings, whereas IRF-3 and IRF-7 are themasters of type I IFN in both TLR-dependent and TLR-independent signalings. Upon activation,IRF-5 like IRF-3 undergoes phosphorylation, dimerization and translocation to the nucleus.However, the mechanism of IRF-5 cytoplasmic-to-nuclear translocation remains unknown. InTLR-independent signalings, RIG-I is critical for detecting intracellular RNA virus infection.RIG-I senses viral-derived RNA through its helicase domain and relays downstream signals viaits N-terminal caspase recruitment domain (CARD). The mitochondrial antiviral signaling(MAVS) adaptor links RIG-I to the downstream canonical IKKs and the IKK-related kinases,IKKE and TBKl, culminating in the activation ofNF-KB and IRF3, respectively. Previous studieshave demonstrated that cross-talk between the canonical IKKs and IKK-related kinasesinfluences NF-KB activation. TBKI was first described as a kinase interacting with the TRAFfamily member-associated NF-KB activator (TANK) adaptor protein that was able to activate NFKB.However, whether the canonical IKKs influence IRF-3 and IRF-7 activation represents acritical missing link in the understanding of TLR-independentLa réussite de la défense immunitaire face a l'infection virale dépend de la capacité des cellules immunitaires a détecter les pathogènes et a produire des cytokines et des chemokines qui interrompent la réplication virale et établissent la base des réponses immunitaires innée et acquise. La production d'interféron (IFN) de type I est un élément central dans la réponse antivirale et est régule étroitement par des signaux extracellulaires et intracellulaires qui activent les facteurs de transcription NF-KB, AP-l et IRF. L'action synergique de ces protéines sur les promoteurs des gènes IFN active la réponse rapide et immédiate des IFN, amplifiée par la signalisation JAKSTAT. Parmi les IRF, IRFS est le principal régulateurs de !'induction des cytokines pro inflammatoires par les TLRs alors qu'IRF3 et IRF7 sont les médiateurs de ractivation des IFN de manière dépendante et indépendante des TLRs. IRFS, comme IRF3, est active par phosphorylation, dimerisation et translocation dans le noyau OU il active la transcription de ses gènes cibles. Le mécanisme moléculaire de la translocation nucleaire de IRFS est inconnu. RIG-I joue un role crucial dans la détection de l' ARN viral indépendante des TLRs. RIG-I reconnait I' ARN d' origine virale par son domaine helicase et signale par son domaine CARD. L'adaptateur MAVS transmet l'activation de RIG-I aux kinases canoniques IKKs et aux kinases apparentées IKKE et TBKI qui activent NF-KB et IRF3, respectivement. Des études précédentes ont démontre que la communication croisée entre les kinases canoniques et apparentées module l'activation de NF-KB. TBKI a été décrit originellement comme une kinase qui interagit avec l'adaptateur TANK qui active NF-KB. Neanrnoins, le rôle des kinases canoniques dans l'activation d'IRF3 et IRF7 représente un lien crucial inexploré dans la compréhension de la signalisation dépendante des TLRs. Dans cette étude, nous avons caractérise un mutant d'IRFS
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Stone, R. C., P. Du, D. Feng, K. Dhawan, Lars Rönnblom, Maija-Leena Eloranta, R. Donnelly, and B. J. Barnes. "RNA-Seq for Enrichment and Analysis of IRF5 Transcript Expression in SLE." Uppsala universitet, Reumatologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-194621.
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Polymorphisms in the interferon regulatory factor 5 (IRF5) gene have been consistently replicated and shown to confer risk for or protection from the development of systemic lupus erythematosus (SLE). IRF5 expression is significantly upregulated in SLE patients and upregulation associates with IRF5-SLE risk haplotypes. IRF5 alternative splicing has also been shown to be elevated in SLE patients. Given that human IRF5 exists as multiple alternatively spliced transcripts with distinct function(s), it is important to determine whether the IRF5 transcript profile expressed in healthy donor immune cells is different from that expressed in SLE patients. Moreover, it is not currently known whether an IRF5-SLE risk haplotype defines the profile of IRF5 transcripts expressed. Using standard molecular cloning techniques, we identified and isolated 14 new differentially spliced IRF5 transcript variants from purified monocytes of healthy donors and SLE patients to generate an IRF5 variant transcriptome. Next-generation sequencing was then used to perform in-depth and quantitative analysis of full-length IRF5 transcript expression in primary immune cells of SLE patients and healthy donors by next-generation sequencing. Evidence for additional alternatively spliced transcripts was obtained from de novo junction discovery. Data from these studies support the overall complexity of IRF5 alternative splicing in SLE. Results from next-generation sequencing correlated with cloning and gave similar abundance rankings in SLE patients thus supporting the use of this new technology for in-depth single gene transcript profiling. Results from this study provide the first proof that 1) SLE patients express an IRF5 transcript signature that is distinct from healthy donors, 2) an IRF5-SLE risk haplotype defines the top four most abundant IRF5 transcripts expressed in SLE patients, and 3) an IRF5 transcript signature enables clustering of SLE patients with the H2 risk haplotype.
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Singthongthat, Wanwisa. "Analysis and validation of Interferon Regulatory Factor 5 (IRF5) on circulating microparticles in patients with SLE." Thesis, Uppsala universitet, Institutionen för kvinnors och barns hälsa, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-415148.
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Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease that cause various inflammatory conditions in the body. The pathogenesis of this disease is yet unknown, and the diversity within the patients bring on major obstacle to clinical research for specific diagnostic markers. As a biomarker of SLE, both Interferon Regulatory Factor-5 (IRF5) and Microparticles (MP) have been suggested. Recently a study demonstrated higher concentration of IRF5+ MP in a small number of SLE patients compared to controls. Aim: The purpose of this study was to validate and analyze IRF5+ MPs in a larger number of SLE patients and compare the results to known SLE subgroup based on IRF5 concentration. Materials and methods: Totally 50 plasma samples from a larger cohort of SLE-patients (n=35) was analyzed together with population-based controls(n=15). Three different antibodies (in-house and commercial) were used for detection of IRF5+ MP with flow cytometry. Students t-test was used to investigate significant differences between SLE subgroup, controls and compared to the previous values. Results and Conclusion: The concentration of IRF5+ MP in SLE subgroup was significantly higher compared to controls (p<0,05). However, there were no correlations between our results and the values from the previous study, suggesting that both methods measure various forms of IRF5. These results imply that IRF5+ MP could be a possible biomarker for pathogenesis in SLE, but further studies are needed for a better understanding of IRF5, as well as of MP.
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Eames, Hayley. "The Interferon Regulatory Factor 5 (IRF5) interactome : investigating the role of co-factors in regulation of inflammation." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/29848.
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Interferon Regulatory Factor 5 (IRF5) is a key transcription factor that regulates inflammatory responses by acting as a mediator of macrophage plasticity. IRF5 is expressed at high levels in pro-inflammatory (M1) macrophages where it drives expression of characteristic M1 markers, such as IL-12p40, IL-12p35 and IL-23p19, and inhibits expression of typical markers of anti-inflammatory (M2) macrophages, like IL-10. In this way, IRF5 polarises macrophages towards a pro-inflammatory phenotype. There are two modes of IRF5 activity: 1) direct binding to Interferon Stimulated Response Element (ISRE) sites in the DNA, and 2) indirect recruitment via protein- protein interactions. IRF5 can be indirectly recruited via interactions with the RelA subunit of Nuclear Factor kappa B (NFκB) at multiple inflammatory loci genome- wide, including Tnf. This suggests protein-protein interactions between the two factors are of great importance for driving inflammatory gene expression. My research shows the IRF Association Domain (IAD) of IRF5 and the Dimerisation Domain (DD) of RelA form an interaction interface between the two proteins. I have identified a short peptide, a region of IRF5 IAD sequence, that can bind to RelA, and hypothesise this peptide could block IRF5-RelA interactions, potentially dampening expression of inflammatory mediators co-regulated by these two factors. I have also identified a novel co-factor of IRF5: Krüppel Associated Protein 1 (KAP1) by a proteomic screen consisting of affinity purification coupled to mass spectrometry. The IRF5-KAP1 complex is present in M1 macrophages, and absence of KAP1 in this cell type results in prolonged TNF secretion, suggesting that KAP1 is important for 'switching off' Tnf gene expression. Further investigation showed KAP1 is important for regulation of a heterochromatin (H3K9me3) environment downstream of the Tnf locus. By understanding the IRF5 interactome, we hope to unravel and potentially manipulate the determinants of IRF5's key role in inflammation in a cell-type and activity-specific manner.
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Junior, Erivan Schnaider Ramos. "Expressão e localização de fatores regulatórios miogênicos (MyoD e Miogenina) em músculos somíticos de ratos reinervados pela técnica de tubulização." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/25/25142/tde-03072009-101927/.
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As lesões dos nervos periféricos, que inervam os músculos esqueléticos, evoluem para perdas da propriocepção e alterações na morfologia e função das fibras musculares, causando um impacto negativo na qualidade de vidas dos indivíduos. Tais lesões implicam em alteração na expressão de genes específicos do músculo, como por exemplo, na MyoD e Miogenina, atuantes na ativação de células satélites e reguladores da massa muscular A técnica cirúrgica de tubulização é um recurso empregado na prática clínica para tratamento de músculos que sofreram desnervação. O objetivo do presente estudo foi analisar se a técnica de tubulização com o preenchimento de gordura altera a expressão de Myod e Miogenina, a morfometria do músculo sóleo de ratos e localização da Myod e Miogenina. Para isso, 57 ratos Wistar foram separados em grupos: controle inicial (GCI); final 45 (GCF45), final 150 (GCF150), desnervado 45 dias (GD45), desnervado 150 dias (GCD150) e grupos experimentais com veia vazia 45 dias (GESP45) e 150 dias (GESP150) e com veia preenchida de gordura 45 dias (GEG45) e 150 dias (GEG150). Para os procedimentos cirúrgicos de desnervação e reinervação e coleta do músculo os animais foram profundamente anestesiados. Após os devidos tempos experimentais, os animais foram sacrificados, o músculo sóleo foi dissecado, envolvido em meio de criopreservação e estocado a -80°C. A quantificação de mRNA do MyoD e Miogenina foi realizada por amplificação por reação em cadeia de polimerase (PCR) em tempo real (RealTimePCR) e a localização da produção de Myod e Miogenina foi realizada por microscopia confocal a laser e imunofluorescência. A morfometria foi realizada em lâminas coradas com HE, observadas em microscópio ótico e calculadas pelo software Image Pro-Plus 6.2. Os resultados do presente estudo mostraram que houve aumento da expressão do Myod e Miogenina nos grupos experimentais 45 dias quando comparados ao grupo controle inicial e um decréscimo da expressão de Myod e Miogenina para os grupos experimentais com 150 dias. A área da secção transversa nos grupos experimentais com 45 dias (GESP45 e GEG45) não apresentaram diferença estatística, quando comparado com grupo desnervado 45 dias (GCD45), enquanto que o grupo experimental com preenchimento de gordura 150 dias (GEG150) obteve os melhores resultados na medida da área da secção transversal do músculo sóleo. As lâminas observadas no microscópio confocal mostram a MyoD e Miogen localizadas no mionúcleo. Concluiu-se que o uso da gordura na técnica de tubulização do nervo ciático de ratos, interfere na regeneração do músculo sóleo.Peripheral nerve injuries can result in the loss of propioception, morphological and functional alterations of muscle fibers which causes a negative impact on the quality of life. These injuries elicit an alteration on the expression of muscle specific genes, like MyoD and Myogenin, involved in the satellite cell activation and muscle mass regulation. The vein graft tubulization is a well known technique for treatment of denervated muscle. The aim of this work was to investigate if vein graf tubulization filled with fat tissue changes the expression and localization of MyoD and Myogenin and to study if it can modify the morphometry of soleus muscle. Fifty seven Wistar rats were divided in initial control group (ICG), final control group 45 days and 150 days (FCG45; FCG150), denervated 45 days and 150 days (D45; D150) and experimental groups with vein graft 45 days and 150 days (VG45; VG150). and vein graft filled with fat tissue 45 days and 150 days (VF45; VF150). For denervation and reinervation procedures and muscle biopsy the animals were submitted to anaesthesia and after the experimental time they were euthanized. Soleus muscle was dissected, involved in criopreservation medium and stored at -80oC. It was performed RealTime polymerase chain reaction (RealTimePCR) for MyoD and Myogenin mRNA quantification. The localization of its production was analysed by laser confocal microscopy and immunofluorescence staining. The morphometric analysis were done by Hematoxilin-Eosin staining and examined at optical microscopy using the Image ProPlus 6.2 software. There was an upregulation on the expression of MyoD and Myogenin for the experimental groups at 45 days when compared to the initial control group. On the other hand, we found a downregulation on the MyoD and Myogenin expression in the same groups with 150 days. The area of transversal section in the 45 days experimental groups (VF45, VG45) did not show statistical difference compared with denervated group with 45 days (D45). Moreover, the group filled with fat tissue at 150 days (VF150) presented the best results in the transversal section area of soleus muscle. In addition, the slides analysed under confocal microscopy showed the localization of MyoD and Myogenin in the mionuclei. In conclusion, the application of vein graft filled fat tissue improves the soleus muscle regeneration.
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Jiang, Zhaozhao. "Dissecting the Role of Innate Pattern Recognition Receptors and Interferon Regulatory Factor-5 in the Immune Response to Human Metapneumovirus and other Pathogens: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/510.
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The Innate immune system is the first line of defense against invading microbial pathogens. It is a fast-acting and non-antigen-specific defense system, which employs germline encoded surveillance systems capable of responding to a broad-spectrum of pathogens. The innate immune system involves a variety of immune cells, which express different profiles of surveillance or detection receptors. Upon sensing pathogens, these receptors trigger cell signalling to turn on transcription of inflammatory cytokines, chemokines, anti-microbial peptides and type I Interferons. These effectors have direct effects on the control of pathogen load and also activate the adaptive immune system, which is ultimately required to clear infections. The type I interferons (IFNs) are the principal cytokines strongly induced during infection with viruses and are required for direct control of viral replication and modulation of cells of the adaptive immune response. The signalling pathways induced in order to activate type I IFNs are dependent on the interferon regulatory factors (IRFs). Striving for survival, microbes have evolved various strategies to subvert/impair these critical defense molecules.
In this thesis work, I have used Human Metapneumoviruses (HMPVs), a relatively newly described family of paramyxoviruses as model viruses to explore the role of pattern recognition receptors (PRRs) and the IRF family of transcription factors in the innate immune response. These studies revealed that the recognition of HMPV viral pathogen-associated molecular patterns (PAMPs) by immune cells is different in different cell types. Retinoic acid-inducible gene-I (RIG-I), a cytosolic RNA helicases senses HMPV-A1 virus for triggering type I IFN activation by detecting its 5’- triphosphate viral RNA in most human cells, including cell lines and primary monocytes. An exception to these findings was plasmacytoid dendritic cells (PDCs), where Toll-like receptor (TLR)-7 is the primary sensor involved in detecting HMPV viruses. By comparing the innate immune response to two HMPV strains, we found that these two closely related strains had very different immune stimulatory capabilities. HMPV-1A strain triggered type I IFNs in monocytes, PDCs and cells of epithelial origin. In contrast, a related strain, HMPV-B1 failed to trigger IFN responses in most cell types. Our studies suggested that the phosphoprotein (P) of HMPV-B1 could prevent the viral RNA from being detected by RIG-I, thus inhibiting the induction of type I IFN production in most cell type examined. This finding adds to our understanding of the mechanisms by which viruses are sensed by surveillance receptors and also unveils new means of viral evasion of host immune responses.
Although IRFs are extensively studied for their role in regulating type I IFN activation, especially in TLR and RIG-I like receptor (RLR) signalling pathways upon viral infection, a clear understanding of how this family of transcription factors contributes to anti-viral immunity was lacking. Studies conducted as part of this thesis revealed that in addition to IRF3 and IRF7, which play a central role in anti-viral immunity downstream of most PRRs (e.g. TLRs, RLRs, DNA sensors), the related factor IRF5 was also an important component of innate anti-viral defenses. Using IRF5-deficient mice we studied in detail the role of IRF5 in coordinating antiviral defenses by examining its involvement in signalling downstream of TLRs. These studies led us to examine the role of IRF5 in the regulation of type I IFNs as well as inflammatory cytokines in different cell types. While most TLRs that induced IFNβ showed normal responses in IRF5-deficient mice, CpG-B-induced IFNβ production in CD11c+CDCs isolated from mouse spleen but not those generated in vitro from bone marrow required IRF5. This was in contrast to responses with lipopolysaccharide (LPS) or polyriboinosinic polyribocytidylic acid (polyIC), ligands for TLR4 and 3, respectively. Moreover, we found that in contrast to IRF3 and/or IRF7, IRF5 was important in coordinating the expression of inflammatory cytokines such as TNFα downstream of some TLRs. In addition to our studies to examine the requirement for IRF5 in TLR signaling, we also showed that muramyl peptide (MDP) from Mycobacterium tuberculosis (Mtb) could activate type I IFNs via IRF5. This was the first evidence linking IRF5 to a non-TLR-driven pathway. IRF5 activation in this case was downstream of a novel nucleotide-binding oligomerization domain containing (NOD)-2/receptor-interacting serine-threonine kinase (RIP)-2 signaling pathway.
Collectively, the studies outlined in this thesis have assisted in providing a framework to understand the role of TLRs, RLRs and IRFs in the immune response to paramyxoviruses and have unveiled new mechanisms of activation of the IRFs as well as new mechanisms by which pathogens subvert or evade these important innate defense mechanisms.
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Sarver, Amy G. "The ontogeny of myogenic regulatory factor expression during muscle differentiation in the biceps femoris and pectoralis major muscles of the chicken Appendix I. Isolation and characterization of microsatellite DNA in rainbow trout ; Appendix II. Analysis of myostatin expression during embryogenesis of the rainbow trout /." Morgantown, W. Va. : [West Virginia University Libraries], 2001. http://etd.wvu.edu/templates/showETD.cfm?recnum=2080.
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Thesis (M.S.)--West Virginia University, 2001.Title from document title page. Document formatted into pages; contains vii, 78 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 52-62).
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Linga, Reddy MV Prasad. "The Genetics of Systemic Lupus Erythematosus : The Specificity of IRF5 to SLE." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8332.
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Hsiao, Sheng-Pin, and 蕭勝斌. "The modulation of myogenic regulatory factor transactivation activity by Bhlhe40." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/81544043634296185425.
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碩士國立中央大學
生命科學研究所
100
The differentiation of functional skeletal muscle cells is characterized by the acquirement of contractile filaments, cell cycle exit, and fusion of myoblasts to form voluntarily contractible multinucleated myotubes. This process is critically regulated by a family of basic helix-loop-helix (bHLH) transcription factors, including MyoD, Myf-5, Myogenin and MRF4, called as myogenic regulatory factors (MRFs) that work together with myocyte enhancer factors (MEF2s) to activate the expression of muscle-specific genes required for myogenic differentiation. In this study, we found that the expression level of Bhlhe40, a ubiquitously expressed bHLH transcriptional repressor, was up-regulated during terminal myogenic differentiation and it could repress the MRF-activated transcription of PGC-1α, M-cadherin, and Myogenin by directly binding to their promoters in vitro and in vivo. Furthermore, we found that Bhlhe40-mediated repression of MRF transactivational activity could be relieved by over-expression of P/CAF, an essential coactivator of MRFs. We demonstrated that Bhlhe40 repressed MRF-activated M-cadherin transcription through a HDAC independent pathway and the DNA binding activity of Bhlhe40 was required but not sufficient to repress the M-cadherin expression. We also found that Bhlhe40-mediated repression of M-cadherin promoter activity may not only through its direct binding to the E3-box in the M-cadherin proximal promoter but also could be mediated by unknown bHLH proteins. We are currently identifying the unknown proteins and observing the interaction among MRFs, Bhlhe40, P/CAF, and other co-factors (co-activators or co-repressors) acting on muscle-specific gene promoters. Based on our observations on the regulation of PGC-1α and M-cadherin transcription, we speculate that the specificity of MyoD targeting and transactivation can be significantly determined by the E-boxes and their binding proteins adjacent to the MyoD binding sites. We are currently identifying genome-wide targeting sites of both MyoD and Bhlhe40 to prove this hypothesis.
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Hung-Chieh, Lee. "Transcriptional regulation and biological Function of Zebrafish Myogenic Regulatory Factor, MYF5." 2006. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-1407200609214300.
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Lee, Hung-Chieh, and 李鴻杰. "Transcriptional regulation and biological Function of Zebrafish Myogenic Regulatory Factor, MYF5." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/4869qg.
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博士國立臺灣大學
分子與細胞生物學研究所
94
Myf5, one of the basic helix-loop-helix transcription factors, controls muscle differentiation and is expressed in somites during early embryogenesis. However, the transcription factors bound to the cis-elements of myf5 are poorly understood. In this study, we used the yeast onehybrid assay and found that Forkhead box d3 (Foxd3) interacted specifically with the -82/-62 cassette, a key element directing somite-specific expression of myf5. The dual-luciferase assay revealed that the expression of Foxd3 potently transactivated the myf5 promoter. Knocking down foxd3 with morpholino oligonucleotide (MO) resulted in a dramatic down-regulation of myf5 in somites and adaxial cells but not in the presomitic mesoderm. On the other hand, myod expression remained unchanged in foxd3 morphants. Foxd3 mediation of myf5 expression is stagedependent, maintaining myf5 expression in the somites and adaxial cells during the 7- to 18-somite stage. Furthermore, in the pax3 morphant, the expression of foxd3 was down-regulated greatly and the expression of myf5 was similar to that of the foxd3 morphant. Co-injection of foxd3 mRNA and pax3-MO1 greatly restored the expression of myf5 in the somites and adaxial cells, suggesting that pax3 induces foxd3 expression, which then induces the expression of myf5. This report is the first study to show that Foxd3, a well-known regulator in neural crest development, is also involved in myf5 regulation. To understand whether myf5 plays other roles than myogenesis during embryogenesis. We first observed that myf5 was expressed in the non-axial mesoderm at the shield stage. Knockdown of Myf5 resulted in abnormal expansion and disorder of the dorsal organizer. We proved that the segmentation of the hindbrain were affected severely in the myf5 morphants due to either lost or defective expression of krox20 and pax6,. The expression of neural crests markers was dramatically reduced in the myf5 morphants; five-day-old myf5 morphants had serious chondrodysplasia in craniofacial cartilage. The TUNEL assay showed that apoptosis occurred significantly in the head of the myf5 morphants. These findings suggest that the reduction of head size and the absence of pharyngeal cartilage formation induced by myf5 inactivation were due primarily to apoptosis. Of interest, the pharyngeal arch defects found in the myf5 morphants were identical to those of the fgf3-MO-injected embryos, and the expression of fgf3 and its down-regulators erm and pea3 was greatly reduced in the myf5 morphants. The fgf3 transcripts also were reduced in the myf5 morphants, but co-injection of fgf3 mRNA and myf5-MO1 into the embryos rescued the hindbrain patterning and the ceratobranchial cartilage defects; the apoptotic signals were also reduced. This evidence suggests that, myf5 is involved in axial and non-axial mesoderm interaction during gastrulation. The disrupted hindbrain segmentation affects the fgf3 signaling, thus causing the CNC to undergo apoptosis. Myf5 is necessary for dorsal organizer patterning, hindbrain segmentation, CNC survival, and cranial cartilage formation.
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Chen, Yau-Hung, and 陳曜鴻. "Molecular Structure, Transcriptional Regulation and Biological Characterizations of Zebrafish Muscle Regulatory Factor, MYF-5." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/22723744338724410537.
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博士國立臺灣大學
漁業科學研究所
89
The myf-5 is the one of muscle regulatory genes involved in the proliferation of myoblasts and differentiation of myogenic cells. We isolated a 1438 bp cDNA fragment that encoded MYF-5 myogenic factor of zebrafish. The deduced amino acid sequence contained 237 residues, including the basic helix-loop-helix domain that is conserved in all known MYF-5. The zebrafish myf-5 gene contained at least 3 exons and two introns. A 6212-bp upstream regulatory region was also cloned and sequenced. The zebrafish myf-5 transcripts were first detectable at 7.5 hours post-fertilization (hpf), increased substantially until 16 hpf, and then declined gradually to an undetectable level by 26 hpf. During somitogenesis, zebrafish myf-5 transcripts were distributed mainly in the somites and segmental plates. Prominent signals occurred transiently in adaxial cells in two-parallel rows, but did not extend beyond the positive-signal somites. Various lengths of upstream region of zebrafish myf-5 fused with EGFP gene were used to carry out transgenic analysis. Results showed that a small, 82 bp (nucleotide positions from -82 to -1) regulatory cassette is sufficient to control the somite- and stage-specific expression of zebrafish myf-5 during early development, and -62~-82 motif is an essential transcriptional regulatory element both for activation and repression. In addition, it was also found that stage-dependent cellular factors (complexes I, II and III), can bind to -62~-82 motif, controlling myf-5 expression. In order to better understand the biological functions of zebrafish myf-5 during embryogenesis, we injected myf5-morpholino (myf5-MO) into one- or two-cell-stage of zebrafish embryos. Results showed no phenotypic abnormalities following injections with 0.2 ng of myf5-MO, but 4 out of 68 (5.9%) surviving embryos injected with 1 ng of myf5-MO displayed such mild defects as abnormal somites and bent tails. Morphologically severe defects became more pronounced with increased dosages: 32 out of 336 (9.5%) surviving embryos injected with 4.5 ng of myf5-MO showed such abnormalities as the absence of eyes and abnormal brain formation in addition to the mild defects found in the low-dosage injection group. These evidences revealed that myf-5 gene might play an important role in somites patterning and brain formation during zebrafish embryogenesis.
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Lee, Wen-Chih, and 李文志. "Molecular Structure, Dynamic Expression and Promoter Analysis of Zebrafish (Danio rerio) Muscle Regulatory Factor, Myf-5." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/76780103661908870736.
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碩士國立臺灣大學
漁業科學研究所
88
The myf-5 is the one of muscle regulatory genes which is involved in the proliferation of myoblasts and differentiation of myogenic cells. Although myf-5 cDNAs were cloned and studied in carp, Xenopus, chicken, mouse and human, the cis-acting elements of myf-5 gene are still not available. Using reverse transcriptase-polymerase chain reaction, we isolated a cDNA fragment with 1438 bp which encoded a Myf-5 myogenic factor from zebrafish (Danio rerio) myf-5 embryonic cDNAs. The deduced amino acid sequence contained 236 residues including a basic helix-loop-helix domain that is conserved in all known Myf-5 proteins. To study the temporal and spatial distribution of the zebrafish myf-5 (myf-5) transcripts, whole-mount in situ hybridization demonstrated that the myf-5 transcripts were first detectable at 7.5 hpf, sustaintially increased at 16 hpf, and then gradually reduced to an undetectable level after 26 hpf. During somitogenesis, myf-5 transcripts distributed in adaxial cells, segmental plates and somites. To define the cis-acting elements required for specific expression of myf-5, various length of regulatory sequences were fused with green fluorescent protein cDNA, which served as a reporter gene. By transgenic analysis, we defined a 82 bp (-82~-1) minimal promoter, which was sufficient to control the tissue- and stage-specific expression of myf-5 in the early development of embryos. Moreover, the proximal enhance element(s) located at nucleotide position from —290 to —154 was also identified.
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Watkins, Amanda Ann. "The role of interferon regulatory factor-5 in systemic lupus erythematosus (SLE) and SLE-associated atherosclerosis." Thesis, 2014. https://hdl.handle.net/2144/14319.
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Gain-of-function polymorphisms in the gene encoding human interferon regulatory factor-5 (IRF5) are associated with an increase in risk for the development of the autoimmune disease Systemic Lupus Erythematosus (SLE). IRF5 is a transcription factor that participates in the activation of the immune system through its role in both innate and adaptive immune cells. To determine the role of IRF5 in lupus pathogenesis in vivo, we evaluated the effect of Irf5-deficiency in the MRL/lpr mouse lupus model. We find that Irf5-deficient (Irf5-/-) MRL/lpr mice develop much less severe disease than their Irf5-sufficient (Irf5+/+) littermates, demonstrating an important role for IRF5 in disease pathogenesis in vivo.
Patients with SLE are at increased risk for the development of atherosclerosis due in large part to poorly-defined lupus-specific risk factors. One such lupus-specific risk factor is thought to be chronic inflammation associated with the autoimmune process. As IRF5 is involved in pro-inflammatory responses we hypothesized that Irf5-deficiency would ameliorate atherosclerosis development in the context of autoimmunity. We therefore examined the role of IRF5 in the gld.apoE-/- mouse model of lupus and lupus-associated atherosclerosis. Irf5-deficiency led to a decrease in splenomegaly, lymphadenopathy, anti-nuclear autoantibody production and the severity of kidney disease. Surprisingly, despite the reduction in systemic autoimmunity, Irf5-deficiency led to a marked increase in the severity of atherosclerosis and to metabolic dysregulation characterized by hyperlipidemia, increased adiposity and insulin-resistance. Bone marrow chimera studies revealed that the pathogenic role of IRF5 in lupus was solely due to its expression in hematopoietic cells. The atheroprotective effect of Irf5 and the suppression of adiposity were found to be due to Irf5 expression in both hematopoietic and non-hematopoietic cells, whereas protection from hyperlipidemia was solely due to the expression of Irf5 in non-hematopoietic cells. Together, our results reveal a role for IRF5 in metabolic homeostasis, as well as in protection against atherosclerosis even in the setting of reduced lupus severity.
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Su, Ying-Fang, and 蘇盈方. "Zebrafish Dickkopf-3-related gene (Dkk3r) regulates the promoter activity of myogenic regulatory factor myf5 gene through interaction with membrane receptor of Integrin alpha 6b." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/59233074383649141970.
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碩士國立臺灣大學
分子與細胞生物學研究所
99
Myf5, one of the myogenic regulatory factors, plays important roles in the specification and differentiation of muscular cells during myogenesis. In zebrafish, an intronic microRNA (miR), miR-3906, located within myf5 intron I, has been reported to silence the translation of its target gene, dickkopf-3-related (dkk3r) gene. Dkk3r, a secretory protein, regulates the phosporylation of p38a to maintain Smad4 stability, which, in turn, enabling the Smad2/Smad3a/Smad4 complex to form and to activate the myf5 promoter in nucleus. However, the membrane receptor(s) bound by Dkk3r to control signal transduction is still unknown. After recombinant zebrafish Dkk3r tagged with Flag was produced by insect cells, we applied protein immunoprecipitation and mass spectromotry to screen the putative receptors of Dkk3r. We found that Integrin alpha 6b (Itga6b) might be one of receptors to interact with Dkk3r. To further confirm this hypothesis, we used whole-mount in situ hybridization and found that the transcripts of both dkk3r and itga6b were presented in somites at 16 hpf during myogenesis. By in vitro cell surface binding assay, we also observed that Dkk3r and Itga6b were co-expressed at the cell membrane of HEK-293T, indicating that the temporal and spatial expressions of dkk3r and itga6b are co-localized. Furthermore, in vivo luciferase assay demonstrated that the luciferase activity driven by myf5 promoter was 223% and 217% greater than that of control when the excessive dkk3r and itga6b mRNAs were injected into embryos, respectively. Interestingly, when we co-injected dkk3r and itga6b mRNAs into embryos, the luciferase activity was up-regulated as high as 397% greater than that of control embryos. This up-regulation of myf5 promoter activity mediated by interaction between dkk3r and itga6b was dosage-dependent. In contrast, when dkk3r was knockdown and co-injected with itga6b mRNA, the luciferase activity was down-regulated to 69% of control embryos, suggesting that the regulatory effect of Itga6b on the downstream activity is dependent on Dkk3r signal pathway. In addition, knockdown of itga6b by injection of itga6b-morpholinos resulted in abnormal shape of somites and weak or even absent expression of myf5 in somites at 16 hpf. Furthermore, knockdown of itga6b reduced the protein level of the phosphorylated p38a. Taken together, we concluded that it is highly likely that Itga6b functions as a receptor of Dkk3r. Their interactions drive the downstream signal transduction to regulate myf5 promoter activity in somites during the development of zebrafish embryos.
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Lok, Ling Ling. "Macrophage infiltration in the aortic roots in mouse models of lupus and atherosclerosis: the role of interferon regulatory factor 5." Thesis, 2016. https://hdl.handle.net/2144/16991.
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The pathogenesis of systemic lupus erythematosus (SLE) and cardiovascular disease (CVD) are tightly linked, and CVD is one of the leading causes of death in lupus patients. There are many risk factors that increase the risk of CVD in SLE patients, including endothelial dysfunction, lipid dysregulation, and abnormal regulation of innate and adaptive immunity. We have previously investigated the role of interferon regulatory factor 5 (Irf5), on atherosclerosis in lupus mouse models. Irf5 has a pro-inflammatory function by activating macrophage and cytokine recruitment and is thus being considered as a potential therapeutic target for the treatment of SLE. We hypothesized that Irf5 deficiency would ameliorate lupus disease as well as improve cardiovascular disease in the Irf5-deficient mouse model. However, while lupus disease did improve in the mouse model, the atherosclerotic plaques were found to be significantly increased in size. This poses a challenge to our current understanding of Irf5, as well as adds complexity to an already difficult clinical problem. Therefore, our aim of this study is to characterize the cells within the atherosclerotic lesions to examine their inflammatory potential. The focus of this study is the infiltration of macrophages into the mouse aortic root as determined by immunohistochemistry staining.
In a time-course study using apoE.Irf5-/- mice, we found that macrophages started to accumulate into aortic leaflets as early as two weeks after starting a Western diet. Macrophage infiltration into the site of leaflet attachment seemed to possibly be a precursor to atherosclerotic lesion formation and appeared as early as 4 weeks after starting Western diet. No apparent differences were found between Irf5 sufficient and Irf5 deficient mice at either two or four weeks on Western diet.
In a bone marrow chimera study, we examined the effects of Irf5 from bone marrow- and non-bone marrow-derived cells on the accumulation of macrophages on aortic leaflets and in the tunica intima in the gld.apoE-/- mouse model of lupus and atherosclerosis. Macrophage accumulation did not correlate with differences in Irf5 production. However, the finding of macrophage accumulation on aortic leaflets suggests a role of macrophages in Libman-Sacks endocarditis, an inflammatory disease of the mitral and aortic valves seen in patients with lupus.
Together, our results do not support nor refute a role of Irf5 in macrophage infiltration into the aortic root. More samples are needed, as are more methods of identifying macrophages and quantifying them. However, it is still likely that macrophages play a role in the pathogenesis of atherosclerotic lesion formation in a lupus mouse model, and it is an area of study worth exploring.
In a time-course study using apoE.Irf5-/- mice, we found that macrophages started to accumulate into aortic leaflets as early as two weeks after starting a Western diet. Macrophage infiltration into the site of leaflet attachment seemed to possibly be a precursor to atherosclerotic lesion formation and appeared as early as 4 weeks after starting Western diet. No apparent differences were found between Irf5 sufficient and Irf5 deficient mice at either two or four weeks on Western diet.
In a bone marrow chimera study, we examined the effects of Irf5 from bone marrow- and non-bone marrow-derived cells on the accumulation of macrophages on aortic leaflets and in the tunica intima in the gld.apoE-/- mouse model of lupus and atherosclerosis. Macrophage accumulation did not correlate with differences in Irf5 production. However, the finding of macrophage accumulation on aortic leaflets suggests a role of macrophages in Libman-Sacks endocarditis, an inflammatory disease of the mitral and aortic valves seen in patients with lupus.
Together, our results do not support a role of Irf5 in macrophage infiltration into the aortic root. However, it is still likely that macrophages play a role in the pathogenesis of atherosclerotic lesion formation in a lupus mouse model, and it is an area of study worth exploring.
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Baker, Margaret. "Generation of conventional dendritic cells from induced pluripotent stem cells for the study of the role of interferon regulatory factor 5 in systemic lupus erythematosus." Thesis, 2019. https://hdl.handle.net/2144/38586.
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Systemic lupus erythematosus (SLE) develops when genetically susceptible individuals lose tolerance to autoantigens, likely as a result of an environmental insult. The list of identified genetic susceptibilities is expansive, however variants in the interferon regulatory factor 5 (IRF5) gene have consistently and convincingly been shown to be associated with an increased risk of developing SLE across all ethnic and racial groups examined. These genetic variants are hypothesized to produce a gain-of-function phenotype due to increased IRF5 mRNA and increased stability of the IRF5 protein; however, definitive functional studies examining these polymorphisms in primary human cells are not possible given the genetic variation from patient to patient. IRF5 is a transcription factor that is constitutively expressed in a number of immune cells including B cells and dendritic cells. IRF5 has cell type specific roles; in dendritic cells, it primarily controls a proinflammatory program which directs T cell polarization. Dysfunctional conventional dendritic cells (cDCs) have been implicated in the onset and development of SLE due to their high capacity to activate and interact with autoreactive lymphoid cells via a number of different pathways; the exact type of dysfunction and mechanisms underlying it are still debated. Study of primary cDCs either from SLE patients or healthy controls is complicated by the low frequency of cDCs in peripheral blood (<0.1%). To better evaluate the role IRF5 plays in cDC dysfunction in SLE, I developed a method for generating cDCs from induced pluripotent stem cells (iPSCs). The cDCs derived from this protocol are similar in many respects to primary human cDCs based on their gene expression profiles, cytokine production, and ability to act as antigen presenting cells to activate T cells. I also generated a library of iPSCs with and without the IRF5 risk haplotype to enable future studies to delineate the role of IRF5 polymorphisms in human cDCs. To facilitate these future studies, I also made an IRF5 deficient iPSC line which will be essential in discerning the role of IRF5 in cDC function. More broadly, we describe herein a platform to study gene function in an isogenic model of human conventional dendritic cells.
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Brzoň, Ondřej. "Analýza regulačních oblastí genů v genomu oxymonády Monocercomonoides." Master's thesis, 2016. http://www.nusl.cz/ntk/nusl-343145.
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iv Abstract Regulation of gene expression is a key ability of every single cell in its development, differentiation and homeostasis. On the other hand, rather sparse amount of information is available for protists and our understanding of regulation of gene expression in eukaryotes is limited to a few model organisms. Our research is aimed at oxymonads, poorly studied group of anaerobic protists, which inhabit digestive tract of some animals. In this study we focus on the genus Monocercomonoides. Gene expression is modulated at multiple levels by many mechanisms. This thesis is focused on structure of promoter regions, 5' untranslated regions and basal transcription and translation initiation factors. Our results are compared to the closest studied relatives of Monocercomonoides - Trichomonas vaginalis and Giardia intestinalis. We have identified several conserved motifs in promoter regions of Monocercomonoides, including TATA box and TATA-like motif. These motifs potentially play a role in the transcription regulation. 5' untranslated regions are relatively short (typically 20 - 30 nucleotides) and GC content in these regions is low compared to model organisms. In selected genes, the quality of the automatic prediction of UTR was verified by RACE. We have annotated sets of basic transcription (23 proteins)...