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1
Morra, Erica, and Lisa Zenker. "Chapter 1: In Search of Innate Leadership : Discovering, Evaluating and Understanding Innateness." Thesis, Linnéuniversitetet, Institutionen för organisation och entreprenörskap (OE), 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-34622.
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Every individual is born with different natural competencies that can be honed by both voluntary and involuntary environmental stimuli. The response our genotype decides to make, if any, towards those stimuli, determines how well our competencies develop. Each person’s coding and variations of genes will result in unique qualities in their phenotype, or physical structure. As a result, a person has various traits that are displayed through their behavior. DNA is genetically shown to express itself through traits by up to 75%. This leaves a sort of buffer of around 25%. This region is available for us to adapt to our environmental stimuli. Your innate qualities will not reach their full potential without stimulation from the environment, in a leadership case, with education and training and therefore it can be argued that environmental exposure is necessary to fully expose the potentials and capabilities of an individual, rather than instill a new skill or develop a talent that was not existent before. Innate leadership is not a permanent state, on the contrary, it is a continuously adaptive situation demanding contextual evolutionary changes or resignation from the subject occupying the role. When the needs and demands of a society or era outweigh the relevance of the innate leaders' traits and competencies, an evolution of leadership is needed to maintain a positive relationship between all parties involved. As a result, the innate leader will begin to lose their innateness in their role and unless they evolve and adapt (because the two actions are not the same) to new contextual needs, their tenure as leader will begin to be detrimental and counter-functional. What we want to put forward is a real, universal and constructive understanding of what makes a human happy, motivated and productive and how an innate person in context is a much better solution in the short and long run, for those around them when put to a task.
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Pelissier, Kiara. "INNATE." VCU Scholars Compass, 2006. http://scholarscompass.vcu.edu/etd/1016.
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I often think of life as a tight rope stretching across an expanse. Our inner strength enables us to walk forward across it. When this fails us, we fall. But in those moments when we prevail, we soar and float as though weightless and timeless. As a gymnast I learned that control of one's insecurities results in a powerful and balanced presence of body. Give into them and the body becomes uncertain and clumsy. Rarely is life this transparent. Many forms of tension manifest themselves in physical, spiritual, and emotional unrest. How does the physical contour of the skin reflect the soul of a material body? Through the use of tension and balance, and with the aid of transparency, translucency, and opacity I alter the perception of surface, form, internal and external space. My work is a comment on the flux of my emotions and attitude towards daily life.
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Usui, Naoki. "Innateness and the mind." Thesis, University of Sheffield, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.617026.
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Ariew, Andre 1968. "Innateness: A developmental account." Diss., The University of Arizona, 1997. http://hdl.handle.net/10150/289478.
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Ascriptions of innateness are ubiquitous in the cognitive, behavioral and biological sciences. For example, some linguists think that humans possess an "innate" language aquisition device. Some ethologists think that a great number of animal behaviors are "innate". Implicit in these ascriptions is the belief that innateness is a well-understood biological phenomenon. The question I would like to address in this dissertation is, what makes a morphological, physiological or behavioral feature "innate"? According to some nay-sayers, innateness is not well-defined in biology and the practice of ascribing innateness to various biological traits should be dropped from respectable science. Proponents of this view think that the notion of innateness rests on a dichotomous conception of development that has been, through decades of powerful criticism, proven to be mistaken. Accordingly the burden of proof rests on those who employ the innateness concept to demonstrate that despite the criticisms there really is a biological phenomenon underlying the concept. In this dissertation I will attempt to shoulder this burden.
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Bachtel, April. "Innate Materiality." Kent State University Honors College / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1304282952.
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Merritt, Michele. "Minimally innate ideas." [Tampa, Fla.] : University of South Florida, 2007. http://purl.fcla.edu/usf/dc/et/SFE0001993.
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Hazeldine, Jon. "Mechanisms underlying innate immunesenescence." Thesis, University of Birmingham, 2013. http://etheses.bham.ac.uk//id/eprint/3965/.
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Although it is evident that physiological ageing is accompanied by marked alterations in the function of innate immune cells, little is known regarding the underlying mechanism(s). Furthermore, the effect of age on many novel aspects of innate immunity is unknown. This thesis has identified the mechanism(s) behind the well-documented age-related decline in natural killer (NK) cytotoxicity (NKCC) and demonstrated for the first time that human ageing is accompanied by a significant reduction in the generation of neutrophil extracellular traps (NETs). Following target cell recognition, it was found that NK cells from older adults secreted into the immunological synapse (IS) significantly lower levels of perforin, a pore-forming protein that plays a non-redundant role in NKCC. This impairment led to reduced perforin binding to the target cell surface, an event that correlated strongly with NKCC. Underlying the reduction in perforin secretion was defective polarisation of lytic granules to the IS, which was associated with delayed activation of extracellular signal-regulated kinase 1/2. Whilst no age-related difference was observed in NET production triggered by phorbol 12-myristate 13-acetate (PMA), neutrophils from older adults generated significantly fewer NETs when challenged with interleukin-8 or lipopolysaccharide, which was accompanied by a reduction in reactive oxygen species generation. As PMA activates cells independent of membrane receptors, aberrant intracellular signalling proximal to the neutrophil membrane may underlie the age-related impairment in NET production.
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Daley, Kenneth. "Concepts as constructions: Structure, content, and innateness." Connect to online resource, 2007. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3288722.
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Mukhopadhyay, Subhankar. "Innate immune activation of macrophages." Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414236.
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Vakakis, Emmanouil. "Innate immune responses to Picornaviridae." Thesis, University of Sussex, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.516152.
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Viral infections affect millions of people worldwide and pose a major threat to human health. Therefore efforts to understand the host defences against viruses are timely and useful. There are specific receptors on the host cells such as Pattern Recognition Receptors (PRR), which are capable of sensing infectious viruses and initiate reactions collectively known as innate immune responses by detecting motifs or molecular signatures. These responses include activation of antiviral cytokines and initiation of the adaptive immune response, thus inhibiting virus replication. The main two families of PRR involved in virus recognition are the Toll like receptors and the RIG-1 like receptors (RLRs; also known as RIG-1 like proteins or RNA helicases). This study was aimed to clarify the innate immune responses and recognition pathways of Picornaviridae by the host. Picornaviridae are single-stranded RNA viruses that can infect many tissues and organs and produce a variety of symptoms and illnesses to the host. The results from this study have shown that TLRs and RLRs and more specifically TLR7, TLR8 and MDA5 are involved in the detection of Picornaviridae such as Coxsackievirus A9 (CAV-9) and Human Rhinovirus 6 (HRV6) leading to the activation of antiviral cytokines by the host cells.
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Crowley, Thomas. "Innate immune memory in fibroblasts." Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8060/.
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The innate immune system is a generic response to infection or injury. Evidence shows the innate response has immunological memory capable of altering subsequent responses to stimuli. Fibroblasts are ubiquitous stromal cells capable of responding to inflammatory triggers, and of orchestrating endothelial cell and leukocyte behaviour during inflammation. Repeated challenge with cytokines (such as tumour necrosis factor (TNF) a) induced an augmented second response to stimulation. Fibroblasts from multiple anatomical locales significantly increased cytokine secretion upon second challenge with TNFa. The precise mediators augmented depended on fibroblast site of origin. Depending on site, memory was inherent, or only present in fibroblasts from chronically-inflamed tissue. This suggests a phenomenon intrinsic to some sites but pathological in others. The secreted mediators from the fibroblast initial or memory responses exerted differing effects on leukocytes, dependent upon fibroblast site of origin. Finally, examination of intracellular signalling showed the augmented response was at least partly due to prolonged activity of nuclear factor (NF) KB during the memory response. Innate immune memory exists in fibroblasts from multiple tissues, but may be pathologically acquired in some. The altered response to second challenge may represent a fibroblast mechanism for altering the recruitment and behaviour of the inflammatory infiltrate.
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Ahrens, S. "Extracellular actin in innate immunity." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1433762/.
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The innate immune system is capable of responding to tissue injury by detecting the abnormal exposure of intracellular, often ubiquitously expressed, molecules referred to as damage-associated molecular patterns (DAMPs). DAMPs are normally sequestered inside healthy cells but become exposed to the extracellular environment upon loss of membrane integrity during cell death. Exposed DAMPs are then recognised by receptors of the innate immune system. One such DAMP receptor is DNGR-1 (CLEC9A), which is expressed on CD8+ DCs, a rare but specialised subset of DCs involved in regulating T cell responses. Loss of DNGR-1 on CD8+ DCs impairs cross-presentation of dead-cell associated antigens to CD8+ T-cells indicating that DNGR-1 couples DAMP recognition to the generation of cytotoxic T cell immune responses. Prior to the work presented in this thesis, the DAMP ligand for DNGR-1 had not been identified. Using a variety of experimental approaches, I demonstrated that this ligand corresponds to filamentous actin (F-actin), a component of the cytoskeleton of all cells. Given its extreme evolutionary conservation, abundance and ubiquitous expression, as well as its association with tissue damage in a range of inflammatory conditions, actin possesses ideal DAMP characteristics. Thus, I further hypothesised that actin may engage receptors other than DNGR-1 and act as a universal and evolutionarily ancient sign of cell damage that is more generally detected by metazoans as a means of inducing sterile inflammation and/or tissue repair. In order to test this hypothesis, I made use of the Drosophila melanogaster model system. I found that actin injection into flies stimulates strong activation of the stress-induced JAK/STAT pathway without triggering immune defence pathways. Given the conservation of innate defence mechanisms in invertebrates and vertebrates, it is tempting to speculate that understanding the recognition of actin in Drosophila melanogaster will provide useful insights into the induction of inflammation in mammals.
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Kästele, Verena. "Intestinal migratory innate lymphoid cells." Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/30723/.
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Innate lymphoid cells (ILCs) mainly reside at mucosal sites, where they produce effector cytokines upon activation. ILCs are also found, at lower frequencies, in secondary lymphoid tissues. ILCs in the lymph nodes (LN) are located in the interfollicular area, which is rich in migratory dendritic cells (DCs) and recently activated T cells and B cells. We recently discovered that some intestinal ILCs traffic from the intestinal lamina propria to mesenteric lymph nodes (MLN). However, the functions of ILCs in the LNs are hardly understood. Here we provide for the first time a detailed description of the migratory ILCs in intestinal lymph of mice in homeostasis and immunity. To isolate migratory ILCs, we obtained lymph by thoracic duct cannulations of mice with or without a previous mesenteric lymphadenectomy. The ILC populations were then analysed by flow cytometry, and their transcriptomic profile was assessed by performing RNA sequencing. Our results show that a small number of migratory ILCs continuously traffic from the intestine to the MLNs. Whole genome analyses in the steady state revealed a shared global ILC signature between migratory ILCs in lymph and intestinal resident ILCs. We then demonstrated that all subsets of ILCs migrate in intestinal lymph, with ILC1 and ILC2 being most frequent. We compared the transcriptomic profile of migratory DCs, T cells and ILCs in afferent lymph and clearly identified a core migratory signature shared by all cell types. In order to investigate the impact of inflammation on this migratory ILC population, we used several infection and inflammation models, and assessed changes in the migratory ILC populations. We detected phenotypic changes of migratory ILCs following Salmonella infections of mice. Furthermore, although Salmonella infection did not increase total numbers of migratory ILCs in lymph, we observed an increase in T-bet+Roryt+ co-expressing ILCs in lymph of infected mice. After infection, changes in ILCs transcriptomic profile indicate an activated phenotype. Corresponding to the lymph ILC data, we also observed an accumulation of T-bet+Roryt+ co-expressing ILCs in the colonic draining LN (cMLN). Our data clearly demonstrate, for the first time, that inflammation can alter migratory ILC populations. This might be important for the initiation and regulation of adaptive immune responses in the draining LN. This first characterisation of migratory ILCs is important as it helps to understand how they contribute to shaping adaptive immune responses in the interfollicular area of the LNs.
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Scoville, Steven. "Human Innate Lymphoid Cell Development." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1459952541.
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Ramanathan, Balaji. "Innate immunity : receptors and effectors /." Search for this dissertation online, 2004. http://wwwlib.umi.com/cr/ksu/main.
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TRIGGIANESE, PAOLA. "Innate lymphoid cells: a potential innate functional equivalent of adaptive cells in infertility and inflammatory arthropathies." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2015. http://hdl.handle.net/2108/203137.
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Background. Studies over the past few years have indicated that adaptive immunity is most commonly associated with several autoimmune conditions such as unexplained infertility and inflammatory arthropathies. However, the identification of innate subsets that mimic T response has greatly advanced our understanding of how innate immune responses may orchestrate and shape the pathomechanisms involved in autoimmune diseases. Aim. Aim of the research was to evaluate the role of innate lymphoid cells (ILCs) in unexplained infertility and inflammatory arthropathies in order to explore whether ILCs control or induce autoimmune processes, i.e., whether an innate functional equivalent of T cells is in place. Methods. In an observational setting, women with unexplained infertility were evaluated for their peripheral blood natural killer (NK) cell levels: the associations between NK and clinical features as well as the effects of the prolactin (PRL) and the thyroid function (in terms of both autoimmune and non-autoimmune diseases) on NK cells were analyzed. In a prospective cohort study, patients with enteropathic spondyloarthritis (EspA) were enrolled in order to characterize peripheral blood T and non-T cells, and ILCs subsets. EspA patients were compared with healthy subjects and patients with inflammatory bowel disease (IBD) as controls. Interferon (IFN)-ɣ and interleukin IL)-17 expressing cells and their associations with the SpA disease activity were analyzed. Results. A higher percentage of peripheral blood NK cells were registered in infertile women compared with fertile controls. In multiple regression analyses, PRL was confirmed to be the only factor to have a significant effect on NK cell levels in infertile women. The most commonly associated thyroid dysfunction in infertile women was the non-autoimmune thyroid disorder while women with higher mean NK cells levels were mainly euthyroid. A higher proportion of women with abnormal NK cells (≥15%) was detected in women with non-autoimmune thyroid disorder. ESpA patients showed higher levels of IL-17 producing non T-cells with the respect to the controls. In this context, IL-17 producing nonT cells correlated with SpA disease activity. Levels of IL-17 expressing ILCs resulted significantly higher in ESpA patients compared with both healty controls and patients with IBD and positively correlated with IFN-ɣ expressing ILCs. Discussion. The higher levels of NK cells in patients with infertility and their correlation with PRL suggest a role of both nervous and endocrine systems in the regulation of the immune response. However, thyroid dysfunctions in infertility seem to be related with non-autoimmune thyroid disorders as well as with elevated peripheral blood NK cells as possible alloimmune mechanisms in reproductive failure. Peripheral blood of ESpA patients is enriched for IL-17 expressing ILCs with the respect to the controls. That evidence might support the role of IL-17 producing ILCs in SpA pathogenesis as well as their potential role as a biomarker for a SpA condition in IBD. Conclusion. Infertile women and ESpA patients represent two human models of well accepted conditions due to an inappropriate T cell response and where the inappropriate inflammatory response may not occur primarily or exclusively due to T cell dysfunction, but is probably promoted by a disturbed placental barrier or synovial tissues. Elucidating whether innate immune cells detectable in peripheral blood can be related with immunological outcomes and clinical features can be crucial. In the long term, a better understanding of the very first events that take place early on during the initiation of an immune response may be key in designing better strategies of diagnostic and therapeutic intervention.
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Kirby, Simon. "Function, selection and innateness : the emergence of language universals." Thesis, University of Edinburgh, 1996. http://hdl.handle.net/1842/21340.
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A central topic for linguistic theory is the degree to which the communicative function of language influences its form. In particular many so-called functional explanations argue that cross-linguistic constraints can be explained with reference to pressures imposed by processing. In apparent opposition to this is the innatist stance which claims that universals are properties imposed by an autonomous language module. This thesis approaches the issues raised by this conflict by examining the nature of the link between processing and universals. The starting point for the work, then, is not the discovery of new universals nor new explanations, but the question "exactly how do processing theories that have been proposed give rise to the universals that they claim to explain?" Careful investigation of this problem proves to be fruitful in highlighting the roles of innateness and function in explaining universals. The methodology chosen involves computational simulations of language as a complex adaptive system, in which language universals appear as emergent properties of the dynamics of the system and the influence of processing on use. This influence is characterised as a differential selection of competing variant forms. The simulation approach is first used to demonstrate the plausibility of a recent parsing explanation for word order universals. An extension of the model to deal with hierarchical universals relating to relative clauses leads to the conclusion that current explanations of hierarchies in general are incomplete. Instead, it is argued that implicational hierarchies are the result of competing processing pressures, in particular between morphological and parsing complexity. Further examination of relative clause processing and universals leads to an apparent flaw in the approach put forward. It is noted that not all processing pressures appear to show up as universals, challenging the explanatory adequacy of the functional explanations.
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McAuley, Julie Louise. "MUC1 in innate and adaptive immunity /." [St. Lucia, Qld.], 2005. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe19061.pdf.
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Goritschnig, Sandra. "Protein modification in plant innate immunity." Thesis, University of British Columbia, 2006. http://hdl.handle.net/2429/30887.
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Plant diseases cause major crop losses worldwide. Crop protection
strategies enhancing the plants' own defence mechanisms could be a sustainable
solution to ensure future food supply. This thesis describes my research effort to
better understand the innate defence mechanisms in plants.
Specific resistance responses towards invading pathogens are mediated by
Resistance (R) proteins. They recognize pathogen-derived molecules and activate
signalling cascades, initiating physiological responses to limit pathogen spread in
infected cells while minimizing harmful effects on the rest of the plant.
We use the unique gain-of-function R gene snd as a tool to identify
components of resistance signalling in Arabidopsis thaliana. In a screen for
suppressors of snc7-mediated constitutive resistance, we identified a number of
modifier of snd (mos) mutants. My thesis focuses on the identification and
characterization of mos5 and mos8. Both mutations partially suppress sndassociated
morphological phenotypes and revert susceptibility to virulent
pathogens to wild type levels.
mos5 contains a deletion in one of two ubiquitin activating enzyme genes in
Arabidopsis. The mutation in mos5 lies in a putative binding domain, potentially
disrupting interaction with downstream ubiquitin acceptors. The mos5 single
mutant displays enhanced susceptibility to virulent bacteria, as well as to bacteria
carrying the effector protease AvrRpt2, indicating a role of ubiquitination in both
specific and basal resistance. A mutation in the mos5 homolog UBA2 does not
affect resistance, however, a double mutant mos5 uba2 is lethal, indicating that the
two genes are partially redundant.
mos8 is allelic to enhanced response to abscisic acid 1 (eral), which
encodes the beta subunit of protein farnesyltransferase. Mutations in the gene are
known to affect development and abscisic acid signalling. mos8 displays enhanced
susceptibility to virulent and avirulent pathogens and acts additively with NPR1.
Defects in geranylgeranylation, a protein modification similar to farnesylation, do
not affect resistance responses against virulent or avirulent pathogens. Taken together, my data reveals the importance of post-translational
modification of yet to be identified regulatory proteins in plant innate immunity.
Further research will aim at unravelling the mechanisms by which mos5 and mos8
affect resistance signalling.Science, Faculty of
Botany, Department of
Graduate
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Linde, Annika. "Comparative studies on cardiac innate immunity." Diss., Manhattan, Kan. : Kansas State University, 2008. http://hdl.handle.net/2097/946.
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Haghayeghi, Amirhossein. "Pellino function in «Drosophila» innate immunity." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86930.
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The Toll pathway mediates innate immunity, the first line of defense against pathogens in vertebrates and invertebrates. In Drosophila the Toll pathway protects against Gram-positive bacteria and fungi by inducing antimicrobial peptides such as Drosomycin. Pellino is a highly conserved protein which biochemically interacts with Pelle/IRAK, a crucial kinase downstream of Toll. Here we report that the chemically induced Pellino 7T2 mutant does not affect Toll pathway function in early embryonic dorsoventral patterning, but it severely compromises induction of Drosomycin upon infection with Gram-positive Micrococcus luteus. In this study we identify Pellino as a novel component of Toll pathway-mediated innate immunity.La transduction de signal initiée par Toll active l'immunité innée. Cet événement constitue la première ligne de défense contre des agents pathogènes chez les vertébrés et les invertébrés. Dans la Drosophile, la transduction de signal initiée par Toll protège contre les bactéries Gram-positives et les champignons en induisant la production de peptides antimicrobiens tels que Drosomycin. Pellino est une protéine hautement conservée qui interagit biochimiquement avec Pelle/IRAK, une kinase essentielle activée par Toll. Ici, nous reportons que Pellino 7T2, un mutant chimiquement induit, n'affecte pas la fonction de Toll durant l'établissement de la polarité dorsoventral dans les embryons précoces. Cependant, Pellino affecte l'induction de Drosomycin suite à une infection avec les bacteries Gram-positive Micrococcus luteus. Dans cette étude, nous reportons un nouveau rôle à Pellino, notamment, celui d'un élément de la voie de signal initiée par Toll qui impacte l'immunité innée.
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Truman, William Matthew Donald. "Signalling pathways underylying plant innate immunity." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429264.
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Zilbauer, Matthias. "Innate immune defence to Campylobacter jejuni." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1445170/.
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Campylobacter jejuni is the most prevalent cause of bacterial diarrhoea worldwide and is frequently associated with severe post-infectious complications such as the Guillain-Barre syndrome. Despite the serious health burden caused by the bacterium disease pathogenesis remains ill defined. Human (3-defensins (hBDs)), a family of epithelial antimicrobial peptides, are a major component of host innate defence at mucosal surfaces. In the present study we investigated the effect of C. jejuni on intestinal epithelial innate responses. Up-regulation of IL-8, hBD-2 and hBD-3 gene and peptide expression was observed in Caco-2 and HT-29 cell-lines in response to C. jejuni strains 11168H and 81-176. Furthermore, recombinant hBDs were found to exhibit potent bactericidal activity against C. jejuni suggesting a major role for these peptides in disease pathogenesis. Secondly, we aimed to identify host receptor(s) involved in sensing of C. jejuni and initiating innate defence. Given the invasive nature of infection, we investigated the potential role of cytoplasmic nucleotide-binding oligomerisation domain (NOD) proteins. Using small interfering (si) RNA targeting NODI and transfection of NOD2 overexpression plasmids, we identified NODI as a major pattern recognition receptor involved in mediating innate host defence to C. jejuni while NOD2 was found to play a minor role. Additionally, reduced NODI expression resulted in an increased number of intracellular C. jejuni thus highlighting a critical role for NODI mediated antimicrobial defence in limiting infection. In the final part of the study an ex-vivo model of C. jejuni infection using human intestinal biopsies was developed. Additionally, a vertical diffusion chamber system was utilised to improve culture conditions in C. jejuni infection models. In conclusion, this study highlights the important role of intestinal innate host defence to C. jejuni. The development of new and improved models of infections has the potential to provide previously unavailable opportunities to study C. jejuni disease pathogenesis.
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Yim, Chi-ho Howard, and 嚴志濠. "Mechanisms of mycobacteria-induced innate responses." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45200981.
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Lunnon, Katie Sarah. "Innate inflammation's contribtion to chronic neurodegeneration." Thesis, University of Southampton, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.443033.
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Matsumiya, Magali Maya Laurence. "Innate immune responses to tuberculosis vaccines." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:bcec5004-fd34-4d36-b088-c0b7e31a87f8.
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Tuberculosis, caused by infection with Mycobacterium tuberculosis (M.tb), remains a global health problem. Drug resistance and high rates of HIV infection have fuelled the pandemic and, although a vaccine exists, its ability to protect from pulmonary tuberculosis varies between 0 and 80%. Bacille Calmette Guerin (BCG) has been administered to billions worldwide yet its protective mechanisms remain unknown, as do the reasons for its failure to protect in many parts of the world. Modified Vaccinia virus Ankara expressing antigen 85A (MVA85A) is a novel candidate vaccine designed to boost immune responses to BCG and improve protection. An aim of this thesis has been to characterise the innate immune response to an MVA85A boosting vaccination in both UK adults and South African infants. In the former, volunteers develop a strong innate response following vaccination however this does not always translate into a robust adaptive response to antigen 85A (Ag85A), which is determined in part by Treg expansion and the nuclear protein HMGB1 signaling through the TLR1-2-6 axis. By contrast, not all South Africa infants mount a strong innate immune response to MVA85A yet this response is correlated with the magnitude of the adaptive response. The immune response to BCG in both populations is also characterised and an association found between increased production of IL-17, IL-22 and IFN-γ in response to BCG stimulation and control of mycobacterial growth. The results presented here further the knowledge on the links between innate and adaptive responses to vaccination with BCG and MVA85A and the variation in mechanisms involved in different populations.
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Boughan, P. K. "Innate immune defence to Helicobacter pylori." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1444551/.
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Helicobacter pylori exhibits tropism for the human stomach causing a spectrum of complications ranging from gastritis to gastric cancer in susceptible individuals. The mechanism(s) that allow the bacteria to persist and cause disease are unfolding. p-defensins are a family of endogenous, epithelial anti-microbial peptides that engage in host defense most prominently at mucosal surfaces. We and others have previously shown that human p-defensin (hBD)-2 and -3 are potent bactericidal agents against H. pylori. At present the identity of signalling pathways involved in host-bacterial cross talk leading to modulation of host antimicrobial immunity are unknown. The present study firstly investigated the potential role of bacterial virulence factors in mediating human p-defensin gene expression during H. pylori infection. AGS gastric epithelial cells were infected with cytotoxic H. pylori strains (60190, 84-183) and isogenic mutant strains (cagA-, cagE-, vacA- and CagPAl-). Human p-defensin (hBD2 & -3) gene expression quantified by RT-PCR and p-defensin transcriptional regulation was followed by transient transfection studies utilising hBD2 and -3 promoter luciferase constructs. We found hBD2 induction was dependent upon an intact cagPAI and minimal involvement was observed for the bacterial virulence factors CagA and VacA in modulating P-defensin expression. We sought to investigate the bacterial component responsible for instigating epithelial innate immune responses. Through the use of siRNA for NODI we determined a role for NODI-dependent NF-kB activation in mediating hBD2 but not hBD3 expression. Experiments utilising specific inhibitors of the MAP Kinase pathways directed us to delineate the role of each pathway in modulating p-defensin expression by the activation of stably transfected conditional MAP Kinase mutants. These studies revealed critical involvement of ERK pathway in the regulation of hBD3 but not hBD2 gene expression. Signalling upstream of ERK was explored and revealed EGFR as the host receptor responsible for detection and initiation of hBD3 gene and peptide production. Our studies demonstrated a crucial role for NODI in H. /Ty/ort-mediated hBD2 but not hBD3 expression and implicate EGFR transactivation in mediating hBD3 but not hBD2 expression, thus indicating two distinct regulatory mechanisms at play during innate immune host response to H. pylori infection.
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Kar, Satwik. "Innate immune response to respiratory viruses." Thesis, Cardiff University, 2014. http://orca.cf.ac.uk/73374/.
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The innate immune system has a variety of ways of recognizing infection from pathogens such as viruses and bacteria. One of its first lines of defence is to detect Pattern Associated Molecular Patterns (PAMPs) using Pattern Recognition Receptors (PRRs) such as the Toll-Like Receptors (TLRs), the RIG-Like Helicases (RLHs) and the Nod-Like Receptors (NLRs). These receptors recognize certain molecular structures from the pathogens and lead to first line of defence which includes increased cytokines and IFNs. This study delineate the human body’s innate immune responses to human respiratory viruses such as HRV (Human Rhinovirus), RSV (Respiratory Syncytial Virus) and IAV (Influenza A virus). In Vitro experiments carried out on various kinds of lung tissues suggest that respiratory disease pathogenesis is related to inflammatory mediators including interleukins and cytokines produced by the host’s innate immune system. Virus induced respiratory tract infection are known to trigger bronchiolitis, wheezing and acute asthma exacerbations, as a result of inflammation of lung tissues and excessive release of pro- inflammatory cytokines such as IL-1β. This study identifies that intracellular macromolecular complexes called inflammasomes assemble as a result of viral trigger. Inflammasomes convert the inactive forms of the pro-inflammatory cytokines to their active forms. Although the exact mechanism of activations of these complexes was unknown. This study identified that Virus encoded proteins such as the 2B protein of HRV, the SH protein from RSV and the Influenza M2 which are also termed viroporins can activate the inflammasome by causing ion imbalance (across cells membranes and organelles). Thus providing a trigger for inflammasome assemblage. v Drugs which act as Ion channel blockers have been shown to block viroporin activity and hence reduce IL-1β production. Therefore in the future the use of ion inhibitors could be a possible therapeutic intervention in order to reduce lung inflammation and prevent associated respiratory disease such as COPD and Asthma.
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Elcombe, Suzanne E. "The innate response to fungal infection." Thesis, University of Dundee, 2013. https://discovery.dundee.ac.uk/en/studentTheses/bd524bb3-6273-4ea3-9faa-7ef7092755d3.
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In the healthy individual fungal infections are relatively benign, however in the rapidly increasing population of immunosuppressed patients fungal infections have become an increasing cause of morbidity and mortality. In the response to fungal pathogens, the innate immune system recognises a series of specific PAMPs via the Dectin-1 and TLR2 receptors, ultimately resulting in pro and anti-inflammatory cytokine production. Following Dectin-1 activation, I show that blocking SYK activity with SYK inhibitor II prevents MAPK and NF?B signalling, causing a reduction in both pro and antiinflammatory cytokine production. However, the clinically used inhibitor R406 (fostamatinib), which has been described as a SYK inhibitor, does not block these signalling pathways downstream of Dectin-1 activation, but is able to abolish cytokine production. As R406 has these effects in response to not only Dectin-1 ligand stimulation, but also TLR2 and TLR4 stimulation, it is likely that these events are the result of an off target effect of R406, and not a result of SYK inhibition. In line with this, R406 was found to inhibit multiple kinases in an in vitro kinase screening panel. To investigate signalling further downstream of Dectin-1, I attempted to elicit the kinase responsible for ERK1/2 activation. For most stimuli, Raf-1 activates MEK1/2 which activates ERK1/2, however in response to TLR signalling the kinase Tpl2 is required to activate MEK1/2. I show that the kinase responsible for ERK1/2 activation downstream of Dectin-1 is not Tpl2, but is an unidentified off target effect of the Tpl2 small molecule inhibitor SHN681. MSK1 and 2 have previously been shown to be important in limiting inflammatory cytokine production by macrophages in response to the TLR4 agonist LPS. This is in large part due to the ability of MSKs to regulate the production of the anti-inflammatory cytokine IL-10. In this thesis I show that MSKs are activated in macrophages by fungal ligands, including the Dectin-1 specific agonists curdlan and depleted zymosan, via the ERK1/2 and p38a MAPK pathways. Further, I show that although MSKs regulate Dectin-1 induced IL-10 transcription, this does not significantly affect pro-inflammatory cytokine production. This is in direct contrast to the inhibition of these pro-inflammatory cytokine that we see post LPS stimulation. Investigating further, I show that although IL-10 secreted in response to zymosan is unable to suppress pro-inflammatory cytokine production, it is still able to promote STAT3 phosphorylation. I suggest that Gfi1 is involved as I show that LPS can induce high levels of Gfi1 expression, whereas zymosan does not induce Gfi1. One possible explanation would be that without Gfi1 to inhibit PIAS3, PIAS3 is binding the activated STAT3 and not allowing it to bind to DNA. This would result in STAT3 being unable to function, and hence no repression of proinflammatory cytokine expression would occur, regardless of the level of IL-10 present. Finally, I show that activation of Dectin-1 in a SYK dependent fashion resulted in macrophage switching to a regulatory macrophage phenotype. As regulatory macrophages are thought of as essentially anti-inflammatory, this may help explain why many people suffer commensal fungal infections that can persist for long periods of time without developing the classical inflammatory signs of infection.
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McGlasson, Sarah Louise. "Regulation of the innate immune system." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/17911.
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The innate immune system is the first line of defence against pathogen invasion. The range of diseases that are caused by deficiencies in or deregulation of the innate immune system illustrates the importance of maintaining an effective balance between clearance of infectious agents and minimisation of inflammatory mediated tissue damage. This thesis explores the role of two proteins in the regulation of the innate immune system. Primarily, this work investigates the effect of human β-defensin 3 (hBD3) on the response to self-DNA and pathogenic DNA. HBD3 is an antimicrobial peptide (AMP), which has been shown to have a role in regulating the immune response; increased copy number of the region containing the gene for hBD3, DEFB103, is linked to an increased risk of psoriasis. Additionally, a similar cationic AMP, LL37, has been shown to exacerbate the pathogenesis of psoriasis by forming an immunogenic complex with self-DNA. This lead to the hypothesis that hBD3 may also affect the innate immune response to DNA. Therefore this project investigates what effect hBD3 has on the response of the innate immune system to self and pathogenic DNA. Flt-3 dendritic cells were used to show that whilst hBD3 increased cellular uptake of self-DNA, it did not convert self-DNA into an immune stimulus. However, hBD3 significantly exacerbated the response to bacterial DNA in a TLR9-dependent manner, also by increasing cellular uptake into FLDCs. The finding that hBD3 increased cellular uptake of both self- and pathogenic DNA suggests that at sites of infection or increased cell death, where DNA would be found in the extracellular environment, hBD3 may increase uptake into immune cells and could induce an increased immune response. Since increased hBD3 expression is induced by inflammatory stimuli, this process would cause a positive feedback loop of inflammation during bacterial infections. In conclusion, hBD3’s role in regulating the innate immune response to DNA is at the ligand-receptor level rather than affecting signalling pathways. Furthermore, hBD3 promotes the innate immune response to bacterial DNA by increasing the efficiency of cellular uptake possibly by inducing DNA aggregation. These results implicate a possible role for hBD3 in the earliest stages of psoriatic plaque development, which is often initiated or exacerbated by an infection, and this could be investigated further. Secondly, I investigated the innate immune function of an E3 ubiquitin ligase (E3L) not previously associated with human disease. Mutations in E3L have been identified in three microcephalic primordial dwarfism families; these patients also presented with recurrent respiratory illnesses. E3L has been implicated in the regulation of the innate immune system via interactions with signalling pathways downstream of the receptor, though its role is not clear. We hypothesised that E3L had a dual role both in regulating growth and cell division and in regulating the immune system. Primary patient fibroblasts did not demonstrate an altered cytokine response to bacterial or viral ligands, implying that E3L may have a specific function in immune cells. To investigate this further, and to provide a system to study E3L in vivo, two transgenic mouse lines were designed and engineered, firstly a conditional ‘knock-out’ designed to replicate some of the alternative isoforms of E3L seen in RT-PCRs, and secondly a ‘knock-in’ line to recapitulate the human mutation in exon 7 of E3L, R185X. These mouse lines should offer an insight into the developmental role for E3L, and contribute to establishing a potential role for E3L in the innate immune system. This thesis exemplifies the complexity of the innate immune system and the regulatory pathways that interact to maintain a delicate homeostasis preventing pathogenic inflammation. Understanding these regulatory mechanisms may shed light on the pathogenicity of diseases and identification of potential targets for therapeutics.
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Kiritsy, Michael C. "Functional Genomics of Mammalian Innate Immunity." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1102.
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The breadth of genetic diversity in the mammalian immune response stands out amongst the ubiquity of variation seen in the genome, evidence that microbial infections have been a major driver of evolution. As technology has facilitated an understanding of the etiology of immunological diversity, so too has it enabled the assessment of its varied functions. Functional genomics, with its ability to assess both cause and effect, has revolutionized our understanding of fundamental biological phenomena and recalibrated our hypotheses. We build upon the model of host immunity established by rare genetic variants that are causative of immunodeficiencies, but that incompletely consider the complexities of the genome. To expand our understanding, we performed a series of forward genetic screens to identify regulators of distinct functions of the innate immune system. Our studies discovered genes with novel functions in antigen presentation and immunoregulation, including several involved in central metabolism. Studies in macrophages and dendritic cells identified mitochondrial respiration as a positive regulator of the interferon-gamma response, and cells incapable of respiration failed to activate T cells. Notably, human mutations in several of these genes are responsible for immune dysfunction. In summary, this work uses new methods in genetic engineering to systematically assess the regulation of innate immunity. Our results suggest that variation in these regulatory pathways is likely to alter immunity in states of health and disease. Thus, our work validates a new approach to identify candidate genes relevant to immune dysfunction.
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Bryner, Charles Heather Marie. "Factors Regulating Insect Innate Immune Responses." Miami University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=miami149088600671424.
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Dayel, Iman Bin. "Probiotics, innate immunity & ageing (PRIMAGE)." Thesis, University of Reading, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.658873.
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Introduction: Ageing has often been associated with a marked reduction in numbers of bifidobacteria and an increase in numbers of clostridia. In addition, ageing is associated with alterations in immunity and poor response to vaccination. Pre- and probiotics are suggested to restore the gut micro flora ecosystem and modulate immune function. The current study evaluated the influence of a novel probiotic, Bifidobacterium longum by. infantis CCUG 52486, with a potential prebiotic (glucooligosaccharide; GIOS) on the gut microbiota composition and on innate immunity in young (18-35y) and older (60-85y) subjects undergoing influenza vaccination.
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Vajhi, Jafari N. "Defining innate immunity to Clostridium difficile." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1347959/.
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Clostridium difficile is a spore-forming anaerobic bacillus and a leading cause of nosocomial diarrhoea. C. difficile infection occurs often following antibiotic treatment leading to alteration of the gut microbiota enabling C. difficile to thrive and cause a range of symptoms from asymptomatic carriage to severe diarrhoea, PMC and death. C. difficile virulence is associated with production of toxins (A, B and CDT) nonetheless this bacteria harbours an array of other virulence factors. In the present study, C. difficile-mediated innate immunity response was investigated utilising four different strains including: R20291 a hypervirulent strain (A+B+, CDT+), 630 a fully sequenced strain (A+B+) and variant strains M68 and CF5 (A-B+). Initial studies showed that all strains shared comparable survival and sporulation capacity whereas strains R20291, M68, and CF5 achieved similar growth kinetics. R20291 showed significant adherence to IEC and was the most potent strain. 630 and M68 were found to be as cytotoxic leading to significant cell apoptosis, IEC TJ disruption and increased paracellular permeability. In contrast, CF5 had the least effect on cell death and IEC barrier integrity. Although all four strains induced antimicrobial immunity and pro-inflammatory cytokines, CF5 mediated the least pro-inflammatory responses. Similar findings were also found in an ex vivo model of infection utilising human colonic explants. C. difficile strains up-regulated murine DC maturation markers leading to a predominant anti-inflammatory IL-10 secretion, which is known to suppress IL-12 and IL-23 induction although C. difficile may generate a cytokine milieu that favours dual Th1/Th17 immunity. C. difficile toxin mutant strains showed DC activation and cytokine production in a toxin-dependent and independent manner and triggered an ASC-containing inflammasome causing the activation of caspase-1 and release of mature IL-1β. These findings indicated that multiple bacterial factors may play a role in initiating host innate immune responses to C. difficile infection.
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Camargo, Ramírez Rosany del Carmen. "Function of microRNAs in plant innate immunity." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/405716.
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Esta tesis aborda el estudio de miARNs en la inmunidad innata en plantas. El trabajo se ha desarrollado en arroz (Capítulo I y Capítulo II) y en Arabidopsis (Capítulo III), En el capítulo I se describe la identificación y caracterización funcional de nuevos miARNs de arroz en su interacción con el hongo Magnaporthe oryzae. Este hongo es responsable de la piriculariosis, una de las enfermedades más devastadoras para el cultivo del arroz a nivel mundial. A partir de la información generada mediante secuenciación masiva de bibliotecas de pequeños ARNs de arroz, se seleccionaron secuencias candidatas a representar nuevos miARNs de arroz, habiéndose estudiado 5 de estos candidatos (miR-64, miR-75, miR-96, miR-98 y miR-203). La obtención de líneas transgénicas de arroz ha permitido demostrar que la sobreexpresión de MIR-64 y MIR-75 confiere resistencia a M. oryzae, tratándose por lo tanto de miARNs que funcionan como reguladores positivos en la respuesta inmune de arroz. Por otra parte, la sobreexpresión de MIR-96, MIR-98 o MIR-203 aumenta la susceptibilidad a la infección por M. oryzae en plantas de arroz (reguladores negativos de la respuesta inmune). El análisis de mutantes de arroz afectados en la biogénesis de miARNs (mutantes dcl1, dcl3 y dcl4) indican que la producción del miARN maduro miR-64, miR-75 o miR-96 es dependiente de DCL3 y/o DCL4, lo cual apoya la idea de que se trata de nuevos miARNs de arroz. Además, mediante edición génica por CRISPR/Cas9, se ha comprobado que una delección de 22 nucleótidos en el precursor miR-75 resulta en un fenotipo de susceptibilidad a M. oryzae (Capítulo II), lo que concuerda con el fenotipo de resistencia que se observa en las plantas que sobreexpresan este miARN.
En el capítulo III se ha estudiado la función de miR858 en la inmunidad innata de Arabidopsis thaliana frente a la infección por hongos patógenos. Este miARN reprime la expresión de factores de transcripción de tipo MYB que actúan como activadores de la expresión de genes que participan en la biosíntesis de flavonoides. Cuando la actividad del miR858 se encuentra bloqueada por la expresión de un gen de imitación de díana (plantas MIM858), las plantas son resistentes a la infección por hongos patógenos (Plectosphaerella cucumerina, Fusarium oxysporum f. sp. Conglutinans and Colletotrichum higginsianum), mientras que la sobreexpresión de este miARN confiere mayor susceptibilidad a la infección. Además, la interferencia con la actividad de miR858, y consiguiente aumento de la expresión de genes MYB, en las plantas MIM858 afecta de manera importante el metabolismo de fenilpropanoides, priorizándose la síntesis y acumulación de flavonoides, a expensas de la síntesis de precursores de lignina. La actividad antifúngica que se observa para kaempferol, naringenina (flavonoides) y ácido p-cumárico, explicaría el fenotipo de resistencia a la infección por hongos que se observa en las plantas MIM858.
En su conjunto, los resultados obtenidos en este trabajo demuestran que los miARNs son componentes importantes en la resistencia/susceptibilidad a la infección por patógenos fúngicos en plantas de arroz y Arabidopsis. Un mayor conocimiento de función de miARNs en la inmunidad innata de las plantas, y de los procesos que son regulados por estos riboreguladores, puede ser de utilidad en el diseño de nuevas estrategias para el control de enfermedades en plantas.This thesis comprises the study of miRNAs in innate immunity in plants. The work has been developed in rice (Chapter I and Chapter II) and in Arabidopsis (Chapter III), model systems used in studies of functional genomics in monocotyledonous and dicotyledonous species, respectively. Chapter I describes the functional identification and characterization of new rice miRNAs in their interaction with the fungus Magnaporthe oryzae. This fungus is responsible for blast disease, one of the most devastating diseases for rice cultivation worldwide. From the information generated by high-throughput sequencing of small rice RNA libraries, candidate sequences to represent novel rice miRNAs were selected. In this work 5 of these candidates have been studied (miR-64, miR-75, miR-96, miR-98 and miR-203). Obtaining transgenic rice lines has demonstrated that the overexpression of MIR-64 and MIR-75 confers resistance to M. oryzae, therefore these miRNAs function as positive regulators in the rice immune response. Moreover, overexpression of MIR-96, MIR-98 or MIR-203 increase susceptibility to M. oryzae in rice plants (negative regulators of immune response). Analysis of rice mutants affected in the miRNA biogenesis (dcl1, dcl3 and dcl4 mutants) indicate that the mature miRNA production of miR-64, miR-75 or miR-96 depends on DCL3 and/or DCL4, which supports the idea that they are novel rice miRNAs. Furthermore, by gene editing using CRISPR/Cas9, it has been found that a 22 nucleotides deletion in miR-75 precursor results in a susceptibility phenotype under M. oryzae infection (Chapter II), in agreement with a resistance phenotype that was observed in overexpressor plants for this miRNA. In chapter III, the miR858 function in Arabidopsis thaliana innate immunity to infection by pathogenic fungi was studied. This miRNA represses the expression of MYB transcription factors, which act as activators of the expression of genes involved in flavonoids biosynthesis. Plants are resistant to infection by pathogenic fungi (Plectosphaerella cucumerina, Fusarium oxysporum f. sp. Conglutinans and Colletotrichum higginsianum) when the activity of miR858 is blocked by the expression of target mimicry (MIM858 plants), while the overexpression of this miRNA confers greater susceptibility to infection. Additionally, interference with miR858 activity and consequent increase of MYB gene expression in MIM858 plants significantly affects phenylpropanoids metabolism, favoring the synthesis and accumulation of flavonoids, and disfavoring the synthesis of lignin precursors. The antifungal activity that was observed for Kaempferol, naringenin (flavonoids) and p-Coumaric acid, would explain the resistant phenotype by fungi infection which is observed in the MIM858 plants. Altogether, the results obtained in this work demonstrate that miRNAs are an important component in the resistance/susceptibility to infection by pathogenic fungi in Arabidopsis and rice plants. Greater knowledge of miRNA function in plant innate immunity and processes that are regulate by these riboregulators, can be useful in the design of new strategies for the control of diseases in plants.
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Baldrich, Patricia. "Role of microRNAs in plant innate immunity." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/315463.
Full textAbstract:
Los pequeños ARNs son ARNs cortos no codificantes que regulan la expresión génica en la mayoría de eucariotas. Las plantas tienen dos clases de pequeños ARNs, los microARNs (miRNAs) y los pequeños ARNs de interferencia (siRNAs), que se diferencian por su biogénesis y su modo de acción.
Actualmente se estima que las pérdidas debidas a patógenos y plagas alcanzan el 50-80%, siendo uno de los factores limitantes de la producción y causando graves pérdidas económicas. Tenemos pues la necesidad de mejorar el conocimiento de los mecanismos de defensa y desarrollar nuevas estrategias para la protección de los cultivos. Con el fin de ampliar los conocimiento en este sector, se han llevado a cabo estudios con Arabidopsis y arroz, los dos sistemas modelo usados en genómica funcional en plantas dicotiledóneas y monocotiledóneas.
En el primer capítulo, se analizaron las alteraciones en la acumulación de pequeños ARNs, incluyendo miRNAs, en respuesta al tratamiento con elicitores en plantas de Arabidopsis. Entre los miRNAs regulados por elicitores se encuentra miR168, que regula ARGONAUTE1, el componente principal del complejo RISC (RNA-induced silencing complex) de la maquinaria de funcionamiento de miRNAs. Los microarrays permitieron, además de analizar miRNAs conocidos, la identificación de un pequeño ARN incorrectamente anotado como miRNA en el registro miRBase. Se demostró que este pequeño ARN es un pequeño ARN heterocromático (hc-siRNA) denominado siRNA415.
En el segundo capítulo, se usaron librerías de secuenciación masiva de pequeños ARNs para la identificación global de miRNAs de arroz regulados por elicitores fúngicos. Esto permitió también describir 9 miRNAs no caracterizados previamente. Combinando dichas librerías con el análisis de nuestros degradomas, se identificaron sus respectivos targets y se observó la existencia de redes reguladoras enriquecidas en miRNAs regulados por elicitores. Específicamente, se identificó un número importante de miRNAs y sus genes diana, implicados en las rutas de biosíntesis de pequeños ARNs, incluyendo miRNAs, hc-siRNAs y “trans-acting siRNAs” (ta-siRNAs). Así mismo se presentan evidencias de miRNAs y sus genes diana implicados en la señalización mediada por hormonas así como en la interacción entre rutas hormonales, demostrando así un gran potencial en la regulación de la inmunidad del arroz. Asimismo se describe la regulación de genes de arroz que contienen “Conserved-Peptide upstream Open Reading Frame” (CPuORF) mediada por miRNAs. Esto sugiere la existencia de una red reguladora nueva que integra la función de miRNAs y CPuORF en plantas. El conocimiento adquirido en este estudio ayudará al la comprensión de los mecanismos de defensa mediados por miRNAs, así como a desarrollar estrategias apropiadas a la protección del arroz.
En el tercer capítulo, se uso la combinación de herramientas bioinformáticas y experimentales para el análisis de miRNAs policistrónicos en arroz, observando la existencia de 23 loci con la habilidad de formar la típica estructura de horquilla de los precursores de miRNAs. Se presentan evidencias experimentales de 7 miRNAs policistrónicos que contienen tanto miRNAs homólogos como miRNAs no homólogos. Se demostró también un patrón de conservación en diferentes especies de arroz (Oryza sativa) cuyo genoma es AA, no presente en especies primitivas de arroz.
Conjuntamente, los resultados obtenidos en este trabajo apoyan la idea que los miRNAs pueden ser considerados como componentes de la respuesta de las plantas a la infección por patógenos, posiblemente actuando como nodos de regulación de diferentes procesos fisiológicos durante la adaptación de las plantas a la infección.Small RNAs (sRNAs) are short non-coding RNAs that guide gene silencing in most eukaryotes. Plants have two main classes of sRNAs, microRNAs (miRNAs) and small interfering RNAs (siRNAs), which are distinguished by their mode of biogenesis and mechanisms of action. In this day and age, crop losses due to pathogens and pests are estimated from 50% to 80%, factors limiting crop production and causing important economical losses. There is then an imperative need to improve our knowledge in defense mechanisms and to develop novel strategies for crop protection. To improve the understanding in this field, we carried out studies in Arabidopsis and rice plants, the two model systems used for functional genomic studies in dicot and monocot plant species. In the first chapter, we analyzed alterations on the accumulation of smRNAs in response to elicitor treatment, including miRNAs, in Arabidopsis plants. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA in the miRBase registry. We demonstrated that this smRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. In the second chapter, we used deep sequencing of small RNA libraries for global identification of rice miRNAs that are regulated by fungal elicitors. We also describe 9 previously uncharacterized miRNAs. Combined small RNA and degradome analyses revealed regulatory networks enriched in elicitor-regulated miRNAs supported by the identification of their corresponding target genes. Specifically, we identified an important number of miRNA/target gene pairs involved in small RNA pathways, including miRNA, heterochromatic and trans-acting siRNA pathways. We present evidence for miRNA/target gene pairs implicated in hormone signaling and cross-talk among hormone pathways having great potential in regulating rice immunity. Furthermore, we describe miRNA-mediated regulation of Conserved-Peptide upstream Open Reading Frame (CPuORF)-containing genes in rice, which suggests the existence of a novel regulatory network that integrates miRNA and CPuORF functions in plants. The knowledge gained in this study will help in understanding the underlying regulatory mechanisms of miRNAs in rice immunity and develop appropriate strategies for rice protection. In the third chapter, we used a combination of bioinformatic tools and experimental analyses for the discovery of new polycistronic miRNAs in rice, revealing 23 loci with the ability to form the typical hairpin structure of miRNA precursors in which two or more mature miRNAs mapped along the same structure. Evidence is presented on the polycistronic nature of 7 miRNA precursors containing homologous or non-homologous miRNA species. We also demonstrated a pattern of conservation in the genome of rice (Oryza sativa) species that have an AA genome, but not in primitive rice species. Collectivelly, results obtained in this work support the notion that miRNAs might be considered as components of the plant response to pathogen infection, possible acting as regulatory nodes of different physiological processes during plant adaptation to infection conditions.
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Taylor, Kristen Rea. "Activation of cutaneous innate defense by glycosaminoglycans." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3211370.
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Thesis (Ph. D.)--University of California, San Diego, 2006.Title from first page of PDF file (viewed June 13, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 142-162).
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Hyun, Jinhee. "RIP1 and FADD's Role in Innate Immunity." Scholarly Repository, 2011. http://scholarlyrepository.miami.edu/oa_dissertations/519.
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Rapid production of type I Interferon is pivotal to initiate cellular antiviral host defense and adaptive immunity. In order to facilitate innate immune processes, a cell harbors pattern recognition receptors (PRRs) which sense distinctive forms of pathogen associated molecular patterns (PAMPs). For example, Toll like receptors (TLRs) and RIG-I like receptors (RLRs) were discovered as PRRs for pathogen derived molecules and the production of type I Interferon (IFN). To induce type I IFN, several transcription factors such as nuclear factor-kappaB (NF-ĸB), interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), and activating protein-1 (AP-1) need to be stimulated through the specific signaling adaptors. Among them, our lab is interesting in the death domain (DD) containing proteins Receptor interacting kinase1 (RIP1) and Fas-associated death domain protein (FADD), which we showed were important for innate signaling processes. RIP1 and FADD were initially identified as Fas and TNFR interacting proteins which were involved in death receptor mediated apoptosis. Aside from apopotic function, recent publications indicate that RIP1 and FADD mediate cell survival, proliferation, and cytokine production through NF-ĸB activation. Here, we show that RIP1 and FADD are essential for efficient TLR-independent signaling. We report that RIP1 and FADD lacking MEF cells are sensitive to viral cytolysis and also exhibit impaired IFN production against dsRNA virus infection. RIP1 acts as a scaffolding protein for death receptor mediated apoptosis and NF-ĸB activation, necrosis, and innate immunity. As mentioned, we demonstrate that cells lacking RIP1 are sensitive to RNA virus infection. To understand the detailed mechanisms of RIP1 function in innate signaling, we first tested whether RIP1 is involved in RIG-I signaling. We found that RIP1 forms a complex with RIG-I in the presence of dsRNA. Additionally, we showed that RIP1 is required for optimal RIG-I and melanoma differentiation-associated protein 5 (MDA-5) activity. We also find that FADD, a RIP1 interaction protein, is implicated in innate immunity. To study the precise mechanisms of FADD in type I IFN signaling, we generated FADD variants and used luciferase reporter assays to indicate that the FADD death effector domain (DED) is crucial for IFN-β signaling. In order to identify interacting partners of FADD, yeast two hybrid assays were performed and indicated that FADD binds to protein inhibitor of activated STAT (PIAS1), part of the SUMO machinery. SUMOylation is a reversible post-translational modification of a protein by SUMO, a 100 amino acid protein. The consequence of SUMOylation alters specific proteins’ function by affecting activity, localization, stability or influencing molecular interactions by interfering with or linking to a target protein. To confirm FADD-PIAS interactions, we conducted in-vitro SUMOylation assays by using Ubc9 conjugated FADD and found possible FADD SUMOylation sites. We also discovered that FADD and SUMO are co-localized in the nucleus. This result reveals that FADD undergoes SUMOylations and its modification might regulate FADD’s function, including role in innate signaling. Furthermore, we report here that HTLV-1 Tax protein interacts with RIP1 and inhibits IFN-β inducing signaling by abrogating RIP1 and IRF7 interaction. This implies that RIP1 is involved in the regulation of IRF7 and is essential for IFN-β production. Collectively, our data demonstrate the significance of RIP1 and FADD in dsRNA recognition pathways in mammalian cells that are essential for the optimal induction of type I IFNs and other innate genes important for host defense.
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Parvatiyar, Kislay. "Ubiquitin Dependent Regulation of Innate Antiviral Signaling." Scholarly Repository, 2010. http://scholarlyrepository.miami.edu/oa_dissertations/651.
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Induction of type I interferons by the transcription factors IRF3 and IRF7 is essential in the initiation of antiviral innate immunity. Activation of IRF3/7 requires C-terminal phosphorylation by the upstream kinases TBK1/IKKi, where IRF3/7 phosphorylation promotes dimerization, and subsequent nuclear translocation to the IFN-beta promoter. Recent studies have described the ubiquitin-editing enzyme A20 as a negative regulator of IRF3 signaling by associating with TBK1/IKKi, however the regulatory mechanism of A20 inhibition remains unclear. Here we describe the adaptor protein, TAX1BP1, as a key regulator of A20 function in terminating signaling to IRF3. Murine embryonic fibroblasts (MEFs) deficient in TAX1BP1 displayed increased amounts of IFN-beta production upon viral challenge compared to WT MEFs. TAX1BP1 inhibited virus-mediated activation of IRF3 at the level of TBK1/IKKi. TAX1BP1 and A20 blocked antiviral signaling by disrupting K63-linked polyubiquitination of TBK1/IKKi independently of the A20 deubiquitination (DUB) domain. Furthermore, TAX1BP1 was required for A20 effector function as A20 was defective for the targeting and inactivation of TBK1 and IKKi in Tax1bp1/ MEFs. Additionally, we found the E3 ubiquitin ligase TRAF3 to play a critical role in promoting TBK1/IKKi ubiquitination. Collectively, our results demonstrate TBK1/IKKi to be novel substrates for A20 and further identifies a novel mechanism whereby A20 and TAX1BP1 restrict antiviral signaling by disrupting a TRAF3/TBK1/IKKi signaling complex. Several viruses utilize a number of strategies to evade the host innate immune response by inhibiting the production of type I interferons. The Human T-cell leukemia virus type 1 (HTLV-1) has been shown to block interferon signaling, however the mechanism of inhibition is poorly understood. We show here that the HTLV-1 encoded protein, Tax plays a critical role in blunting the activation of type I interferons. Tax expression rendered MEFs hyper-permissive in supporting virus replication. Correspondingly, Tax blocked the production of IFN-beta. Interestingly, Tax did not require NEMO interaction to inhibit antiviral signaling to IRF3/7. Instead, Tax targeted RIP1 and further blocked IRF7 K63-linked polyubiquitination. Altogether, we show that Tax inhibits IFN activation by disrupting the ubiquitin dependent activation of IRF7 mediated by RIP1.
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Cordeiro, Cristianne. "Eggshell Membrane Proteins provide Innate Immune Protection." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/33389.
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The microbiological safety of avian eggs is a major concern for the poultry industry and for consumers due to the potential for severe impacts on public health. Innate immune defense is formed by proteins with antimicrobial and immune-modulatory activities and ensures the protection of the chick embryo against pathogens. The objective of this project was to identify the chicken eggshell membrane (ESM) proteins that play a role in these innate immune defense mechanisms.
We hypothesized that ESM Ovocalyxin-36 (OCX-36) is a pattern recognition protein, and characterized purified ESM OCX-36. OCX-36 has antimicrobial activity against S. aureus and binds E. coli lipopolysaccharide (LPS) and S. aureus lipoteichoic acid (LTA). We additionally investigated the OCX-36 nonsynonymous single nucleotide polymorphisms (SNPs) at cDNA position 211. The corresponding isoforms (proline-71 or serine-71) were purified from eggs collected from genotyped homozygous hens. A significant difference between Pro-71 and Ser-71 OCX-36s for S. aureus LTA binding activity was observed. From these experiments, we confirmed the hypothesis that OCX-36 is a pattern recognition molecule. We also found that OCX-36 has anti-endotoxin properties and is a macrophage immunostimulator to produce NO and TNF-α. Digested OCX-36 down-regulated the expression of genes involved in LPS signaling and inflammatory responses. Moreover, OCX-36-derived peptides inhibited the production of LPS-induced pro-inflammatory mediators associated with endotoxemia in vivo.
Quantitative proteomics analysis of ESMs was performed to evaluate changes in ESM protein abundance during chick embryonic development. Bioinformatics analysis revealed enrichment of proteins associated with antimicrobial and immune protection, vascularization, calcium mobilization and lipid transport, which are vital for chick embryonic development. In unfertilized eggs, protease inhibitors and antimicrobial proteins were enriched.
In summary, the ESMs are enriched in proteins with antimicrobial, antioxidant and immune-modulatory properties, which aid in the development of the chick embryo and protect the embryo and unfertilized egg against pathogen invasion.
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Thakur, Nishant. "Integrated signaling networks in C.elegans innate immunity." Thesis, Aix-Marseille, 2016. http://www.theses.fr/2016AIXM4034/document.
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C. elegans est infecté par divers agents pathogènes ; bactéries, champignons et virus. Lors d'une infection fongique, C. elegans surexprime de nombreux gènes codant pour des peptides antimicrobiens (AMP). Le principal objectif de ma thèse était de construire un réseau de régulation génique intégré représentant l'induction de ces gènes AMP pendant l'infection. Pour trouver les principales composantes du réseau de régulation, via un criblage ARNi du génome entier (Zugasti et al 2016), nous avons identifié 278 clones Nipi (pour "Absence d'induction de peptides antimicrobiens après infection") qui abrogent l’induction d’AMP. En utilisant "CloneMapper" (Thakur et al. 2014), nous avons identifié 338 gènes cibles pour ces clones. Nous avons montré que les voies de MAPK sont au cœur de l'induction des AMP. Parmi les 50 gènes arbitrairement sélectionnés et surexprimant les AMP, nous en avons validé 48 en utilisant Fluidigm. Pour attribuer des fonctions aux gènes identifiés dans ces études à haut débit, nous avons développé un outil d'enrichissement fonctionnel pour la communauté C.elegans (MS en préparation). Nous avons utilisé cet outil pour analyser les cibles du criblage ARNi sur le génome entier et sur d'autres bases de données concernant divers agents pathogènes. Nous avons fait une analyse de l'enrichissement fonctionnel des cibles ChIPseq de CEBP-1, un facteur de transcription lié à la régulation de la réponse immunitaire innée (Kim et al, Soumis). Enfin, pour mieux comprendre l'interaction entre l'hôte et le pathogène, nous avons séquencé, assemblé, annoté et analysé le génome de D. coniospora. Nous avons identifié plusieurs facteurs de virulence potentiels dans ce génomeC. elegans is infected by diverse pathogens, including bacteria, fungi and viruses. Upon fungal infection, C. elegans up-regulates the expression of many antimicrobial peptide (AMP) genes. The main aim of my thesis was to build an integrated gene regulatory network representing the induction of these AMP genes upon infection. To find the main/backbone components of the regulatory network, through a genome-wide RNAi screen (Zugasti et al. 2016), we identified 278 Nipi (for “no induction of antimicrobial peptides after infection”) clones that abrogate AMP induction. Using “CloneMapper” (Thakur et al. 2014), we identified 338 target genes for these clones. We showed that MAPK pathways are central to the induction of AMPs. We also characterized the transcriptional changes provoked by infection using RNA-sequencing and identified more than 300 genes that are dynamically up-regulated after infection, including 13 AMPs. We validated 48 (96%) of 50 arbitrary selected up-regulated genes using Fluidigm. To assign functions to genes identified in these high-throughput studies, we developed a functional enrichment tool for C.elegans community (MS in preparation). We used this tool to analyse the genome-wide RNAi screen targets and other pathogen-related datasets. We did functional enrichment analysis of ChIPseq targets of CEBP-1, TF linked to the regulation of the innate immune response (Kim et al., submitted). Finally, to understand better the interaction between host and pathogen, we sequenced, assembled, annotated and analysed the D. coniospora genome (Lebrigand et al. 2016). We identified various potential virulence factors in the fungal genome
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Noursadeghi, Mahdad. "Studies of innate immunity to bacterial infection." Thesis, Imperial College London, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406424.
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Paveley, Ross A. "Imaging innate immune responses to schistosoma mansoni." Thesis, University of York, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.516640.
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Lee, Mark N. "Genomic Approaches to Dissect Innate Immune Pathways." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10692.
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The innate immune system is of central importance to the early containment of infection. When receptors of innate immunity recognize molecular patterns on pathogens, they initiate an immediate immune response by inducing the expression of cytokines and other host defense genes. Altered expression or function of the receptors, the molecules that mediate the signal transduction cascade, or the cytokines themselves can predispose individuals to infectious or autoimmune diseases. Here we used genomic approaches to uncover novel components underlying the innate immune response to cytosolic DNA and to characterize variation in the innate immune responses of human dendritic cells to bacterial and viral ligands. In order to identify novel genes involved in the cytosolic DNA sensing pathway, we first identified candidate proteins that interact with known signaling molecules or with dsDNA in the cytoplasm. We then knocked down 809 proteomic, genomic, or domain-based candidates in a high-throughput siRNA screen and measured cytokine production after DNA stimulation. We identified ABCF1 as a critical protein that associates with DNA and the known DNA-sensing components, HMGB2 and IFI16. We also found that CDC37 regulates stability of the signaling molecule, TBK1, and that chemical inhibition of CDC37 as well as of several other pathway regulators (HSP90, PPP6C, PTPN1, and TBK1) potently modulates the innate immune response to DNA and to retroviral infection. These proteins represent potential therapeutics targets for infectious and autoimmune diseases that are associated with the cytosolic DNA response. We also developed a high-throughput functional assay to assess variation in responses of human monocyte-derived dendritic cellsto LPS (receptor: TLR4) or influenza (receptors: RIG-I and TLR3), with the goal to ultimately map genetic variants that influence expression levels of pathogen-responsive genes. We compared the variation in expression between the dendritic cells of 30 different individuals, and within paired samples from 9 of these individuals collected several months later. We found genes that have significant inter- vs. intra-individual ariation in response to the stimuli, suggesting that there is a substantial genetic component underlying variation in these responses. Such variants may help to explain differences between individuals’ risk for infectious, autoimmune, or other inflammatory diseases.
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Smith, Christopher. "Avian innate immune responses to 'Campylobacter' infection." Thesis, University of Bristol, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432743.
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Barakat, Farah Mukhlis. "Protective innate immune responses against Cryptosporidium parvum." Thesis, Queen Mary, University of London, 2013. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8733.
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Cryptosporidiosis is a common infectious diarrhoeal disease of mammalian livestock and humans worldwide. The etiological organisms responsible are intestinal apicomplexans of the genus Cryptosporidium, including C. parvum, that infect intestinal epithelial cells. Immunocompromised or malnourished hosts develop severe life-threatening disease. Immunological elimination of Cryptosporidium requires CD4+ T cells and IFN-γ. Nevertheless, studies have shown innate immune responses have a significant protective role. Importantly, in T cell-deficient mice, IFN-γ is important for control of C. parvum infection. In innate immunity natural killer (NK) cells are major producers of IFN-γ and are activated by cytokines including type I IFNs but the roles of these components in immunity to Cryptosporidium infection have not been investigated. Therefore, the purpose of this project was to study the involvement of type I IFNs and NK cells in immunity to C. parvum employing in vitro and in vivo (murine) infection models. Enterocytes were shown capable of the production of type I IFNs in response to C. parvum infection. These cytokines directly inhibited parasite development in epithelial cells. Also, in neonatal SCID mice the level of infection increased after treatment with anti-type I IFN neutralising serum. A higher level of infection was observed in Rag2-/-γc-/- mice deficient in T, B and NK cells in comparison to Rag2-/- mice with a normal NK cell population and early mortality during chronic infection of adult animals was associated with the absence of NK cells. Using cultures of SCID mouse splenocytes, NK cells were the main source of IFN-γ in response to C. parvum antigen stimulation. However, IFN-γ was also found to have a protective role in Rag2-/-γc-/- mice, implying cells other than lymphocytes produce this cytokine. In conclusion, this is the first study to indicate important protective roles for type I IFNs and NK cells in innate immunity against C. parvum.
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Milioris, A. S. "Modulation of innate immunity by enteropathogen motifs." Thesis, University College London (University of London), 2016. http://discovery.ucl.ac.uk/1482091/.
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Bacterial gastroenteritis affects millions, especially in the developing world. While the mechanisms by which enteric pathogens trigger inflammation have been studied extensively, how they evade immune recognition is less understood. We hypothesised that the structural components of common enteric pathogens, such as Clostridium difficile and Campylobacter jejuni, facilitate immune evasion by engaging inhibitory receptors on innate immune cells. In the present study we investigated how C. difficile peptidoglycan (PGN) engages with monocytic cells and identified NOD2 as a critical innate sensor in this interaction. PGN affected cell maturation and in addition it modulated monocytic immune responses to other bacterial motifs, as PGN-exposed cells became unresponsive to subsequent Lipopolysaccharide (LPS) stimulation. This data suggests a role for C. difficile PGNNOD2 axis in trained immunity. We have previously reported on a novel interaction between C. jejuni flagellin pseudaminic acid moieties and the Siglec-10 receptor, an interaction that specifically targets the dendritic cell IL-10 axis. Herein, we studied the downstream signalling events involved in this crosstalk in human macrophages. Utilizing wild-type and isogenic mutant C. jejuni, we identified a potential role for the epidermal growth factor receptor (EGFR), the scaffolding SHP-2 and PI3K-AKT/MAPK kinase pathways in modulating IL-10 expression. We report for the first time novel engagement between C. jejuni 11168H capsule, lipooligosachharide (LOS) and host Siglec-5 and Siglec-9 receptors, pathways involved in bacterial adherence and phagocytosis. Collectively, this thesis provides evidence as to how common bacterial structures exert immunosuppressive effects, effects that may hold the balance between asymptomatic colonization and infection.
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Mazzon, M. "Pathogenesis of dengue : subversion of innate immunity." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/134036/.
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Dengue viruses (DENV) are mosquito-borne flaviviruses that cause a severe febrile illness, and sometimes a potentially lethal syndrome called dengue haemorrhagic fever (DHF). Despite decades of effort, the global resurgence of dengue is testament to the inadequacy of current control measures. Dengue has become an immense international public health concern: WHO estimates that there are 50-100 million dengue infections and 500,000 cases of DHF hospitalised each year. Thus dengue has a major economic impact in the developing world through loss of healthy life and utilisation of constrained health resources. Global dengue control is likely to require a combined approach based on the development of successful strategies for immunization and antiviral drugs, as well as vector control. Better understanding of DENV pathogenesis presents new opportunities for design of rationally attenuated vaccine candidates and antiviral therapies. This thesis focuses on understanding a critical step in DENV pathogenesis: evasion of human innate immune responses mediated by interferon. A lentiviral vector system was developed to express dengue non-structural (NS) proteins in human cells, and we used this to show that expression of dengue NS5 alone inhibited IFN-α, but not IFN-γ, signalling. The IFN-α signalling cascade is blocked downstream of Tyk2 phosphorylation: NS5 binds to the transcription factor STAT2 and inhibits its phosphorylation. The polymerase domain of NS5 is sufficient to block IFN-α induced signal transduction, and inhibition does not require NS5 nuclear translocation. We finally tested several hypotheses to explain why STAT2 degradation occurs in both DENV-infected and replicon-containing cells, but not when NS5 is expressed alone. The most important conclusion from this work is that DENV NS5 is a potent and specific type I IFN antagonist. The results of this study are an important step in defining the molecular pathogenesis of dengue, and provide clues to potential new approaches to combat this disease.
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Rigby, Rachel Elizabeth. "Ribonuclease H2, RNA:DNA hybrids and innate immunity." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/6509.
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The activation of the innate immune system is the first line of host defence against infection. Nucleic acids can potently stimulate this response and trigger a series of signalling cascades leading to cytokine production and the establishment of an inflammatory state. Mutations in genes encoding nucleases have been identified in patients with autoimmune diseases, including Aicardi-Goutières syndrome (AGS). This rare childhood inflammatory disorder is characterised by the presence of high levels of the antiviral cytokine interferon-α in the cerebrospinal fluid and blood, which is thought to be produced as a consequence of the activation of the innate immunity by unprocessed self-nucleic acids. This thesis therefore aimed to define the role of one of the AGS nucleases, the Ribonuclease H2 (RNase H2) complex, in innate immunity, and to establish if nucleic acid substrates of this enzyme were able to induce type I interferon production in vitro. The AGS nucleases may function as components of the innate immune response to nucleic acids. Consistent with this hypothesis, RNase H2 was constitutively expressed in immune cells, however, its expression was not upregulated in response to type I interferons. RNase H2-deficient cells responded normally to a range of nucleic acid PAMPs, which implied that a role for RNase H2 as a negative regulator of the immune response was unlikely, in contrast to the reported cellular functions of two other AGS proteins, TREX1 and SAMHD1. Therefore, no clear evidence was found for the direct involvement of RNase H2 in the innate immune response to nucleic acids. An alternative model for the pathogenesis of disease hypothesises that decreased RNase H2 activity within the cell results in an accumulation of RNA:DNA hybrids. To investigate the immunostimulatory potential of such substrates, RNA:DNA hybrids with different physiochemical properties were designed and synthesised. Methods to purify the hybrids from other contaminating nucleic acid species were established and their capacity as activators of the innate immune response tested using a range of in vitro cellular systems. A GU-rich 60 bp RNA:DNA hybrid was shown to be an effective activator of a pro-inflammatory cytokine response exclusively in Flt3-L bone marrow cultures. This response was completely dependent on signalling involving MyD88 and/or Trif, however the specific receptor involved remains to be determined. Reduced cellular RNase H2 activity did not affect the ability of Flt3-L cultures to mount a cytokine response against the RNA:DNA hybrid. These in vitro studies suggested that RNA:DNA hybrids may be a novel nucleic acid PAMP. Taken together, the data in this thesis suggest that the cellular function of RNase H2 is in the suppression of substrate formation rather than as a component of the immune response pathways. Future studies to identify endogenous immunostimulatory RNA:DNA hybrids and the signalling pathways activated by them should provide a detailed understanding of the molecular mechanisms involved in the pathogenesis of AGS and related autoimmune diseases.
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Dias, Antonio Gregorio. "Characterisation of the innate impune sensor MDA5." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:8593db28-b4d3-4880-8831-4ea7837937f6.
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Melanoma Differentiation-Associated gene 5 (MDA5) is a cytosolic RNA receptor. Its activation triggers the production of the antiviral cytokines type I and III Interferons (IFN). It is believed that MDA5 detects viral and/or cellular RNAs. The latter case may be relevant in the context of cancer treatment with DNA demethylating agents, and in autoinflammatory and autoimmune diseases. In all these settings, physiological MDA5 agonists have not been identified. To identify MDA5-stimulatory RNAs, we generated mouse monoclonal antibodies against human MDA5. We showed that some of these antibodies are specific and detect MDA5 protein by Western Blot, in immunofluorescence, and work in immunoprecipitation (IP). To identify MDA5's RNA ligands in living cells, we employed the UV-crosslinking and IP (iCLIP) technique. Interestingly, we found that MDA5 associated with RNAs regardless of virus infection. Sequencing and characterisation of these RNA species will be performed in the future. MDA5 overexpression led to IFN-I induction, which could be explained by MDA5 binding to cellular RNAs. Indeed, two bioassays developed here suggest that MDA5 can sense RNAs from uninfected human cells and tissues. In a second project, we demonstrated that Zika Virus (ZIKV) replication generated MDA5 and, RIG-I immunostimulatory RNAs. RIG-I-dependent IFN-I induction was sensitive to alkaline phosphatase treatment, in line with the requirement for 5'-ppp groups for RIG-I activation. Furthermore, by using a mini-screen of ZIKV proteins, we identified the non-structural protein 5 (NS5) as a potent inhibitor of the type I IFN signalling pathway. We show that this inhibitory mechanism is unrelated to: i) NS5's nuclear localisation, ii) mutations found in strains of the current outbreak, and iii) a conserved residue in the methyltransferase domain. In sum, we provide novel insights into the biology of MDA5, new reagents for the studies of MDA5, as well as data on the role of RNA sensors in detecting ZIKV infection and evasion of innate immunity by ZIKV.