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1
Shields, Robert Colquhoun. "Extracellular DNA in head and neck biofilms." Thesis, University of Newcastle upon Tyne, 2014. http://hdl.handle.net/10443/2498.
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Extracellular DNA (eDNA) is a ubiquitous component of the extracellular matrix of microbial biofilms. It has a number of functions that include a role as an adhesin during biofilm attachment, and facilitating matrix stability in mature biofilms. Increasingly, deoxyribonuclease (DNase) enzymes have been shown to reduce the colonisation of many microbial biofilms, both bacterial and fungal. Biofilms are estimated to be responsible for around 60% of bacterial infections, including many chronic diseases. The aim of this work was to determine the role of eDNA in chronic mixed-species biofilm infections, and to explore the potential of DNase enzymes for biofilm control. This included three major areas of research, focusing on chronic rhinosinusitis, tracheoesophageal speech valves (TESVs), and dental plaque. An important aspect was to test the efficacy of a novel bacterial nuclease, NucB, isolated from a seaweed-associated strain of Bacillus licheniformis, against microbial biofilms. The colonisation of speech valves by micro-organisms was studied using scanning electron microscopy (SEM). In keeping with previous observations, these biofilms were co-aggregations of fungal and bacterial species. Using confocal laser scanning microscopy, eDNA was observed in the biofilm matrix. Extracellular DNA was extracted and quantified from TESV biofilms. All six biofilms studied had detectable nucleic acids, as measured by NanoDrop spectrophotometry. The eDNA was apparently heavily degraded, and produced smears by agarose gel electrophoresis. Nevertheless, eDNA appeared to be providing biofilm stability since micro-organisms were liberated from the surface of the valves following treatment with NucB in over 60% of the TESVs tested. To assess the role of eDNA in biofilms associated with chronic rhinosinusitis, ‘obstructive mucin’ and sinus mucosa biopsy samples collected during functional endoscopic sinus surgery at the Freeman Hospital, Newcastle, were analysed for the presence of biofilms and biofilm-forming micro-organisms. An average of 3.75 bacterial species per patient were cultured from obstructive mucin. The most commonly isolated micro-organisms were Staphylococcus aureus, coagulase-negative staphylococci and α-haemolytic streptococci. Micro-organisms were not detected by transmission electron microscopy of the obstructive mucin and this material appeared to originate through a host inflammatory response. However, bacteria were visualised on the surface of sinus mucosa using a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) universal bacteria probe. Twenty-four bacterial isolates were iv assessed for their ability to form biofilms in a microtitre plate model. All micro-organisms tested formed biofilms, and 14 of 22 were susceptible to NucB. In total, 15 of 24 microbial species produced eDNA that was detectable by agarose gel electrophoresis. By SEM, cellular colonisation was lower in treated samples and, in the case of Streptococcus constellatus FH20 stringy, matrix-like material was not present after DNase treatment. The role of eDNA in matrix stability and initial biofilm attachment was also studied in oral bacteria. Streptococcus gordonii DL1, Streptococcus mutans GS-5, Fusobacterium nucleatum 25586 and Actinomyces oris MG1 were examined using DNase treatment, CLSM, and eDNA extraction. Of these species, all except S. gordonii appeared to rely on high molecular weight (HMW) eDNA for biofilm attachment and biofilm stability. Although S. gordonii did not produce detectable HMW eDNA, nucleic acids were detectable by NanoDrop spectrophotometry. Furthermore, this species produces an extracellular nuclease which may degrade the HMW eDNA in the conditions used to culture biofilms. Interestingly, four S. mutans strains differed in their sensitivity to DNase treatment. Oral biofilms were also modelled in a BioFlux microfluidics device using flowing human saliva. Mixed-species biofilms and single species biofilms of S. mutans UA159 and S. gordonii DL1 were cultured using this technique, to determine whether this model would allow more realistic experiments for DNase testing. Finally, the extracellular nuclease, SsnA, of S. gordonii DL1 was characterised. A nuclease-deficient mutant did not produce extracellular nuclease activity on DNase Test agar or during a Forster Resonance Energy Transfer (FRET) assay. Nuclease activity was cell-wall-associated as predicted from the predicted amino acid sequence of SsnA. Allelic exchange mutagenesis determined that ssnA expression was regulated by CcpA in response to repressing sugars. However, in planktonic culture non-repressing carbon sources also inhibited enzyme activity during a FRET assay. Further experiments using acidic buffers replicated the inhibition of SsnA without the presence of sugars. SsnA was purified as a GST-tagged fusion protein in an Escherichia coli protein expression system, and had anti-biofilm activity against S. mutans GS-5. However, this species is strongly acidogenic and therefore it is hypothesised that although SsnA may be a competition biofilm factor, acid production by S. mutans may reduce its efficacy in vivo. In conclusion, this thesis has provided strong evidence for the role of eDNA in facilitating biofilm formation and mature biofilm stability of clinically relevant v biofilms. Nucleic acids were present in biofilms associated with a chronic infection, medical implant biofouling and dental plaque. A variety of DNase enzymes (NucB, DNase I, and SsnA) were capable of reducing biofilm colonisation. Given the adhesive function of eDNA in biofilm matrices it is proposed that DNase enzymes may be beneficial for controlling healthcare-related biofilms.
2
Fisher, Mark. "Intra and extracellular responses to DNA damage." Thesis, Queensland University of Technology, 2021. https://eprints.qut.edu.au/214106/1/Mark_Fisher_Thesis.pdf.
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Dos, Santos Goncalves Marina. "Rôle des exopolysaccharides et de l'ADN extracellulaire dans le développement du biofilm par Klebsiella pneumoniae." Thesis, Clermont-Ferrand 1, 2014. http://www.theses.fr/2014CLF1PP02.
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Le biofilm est défini comme une communauté microbienne adhérant à une surface biotique ou abiotique, et engluée dans une matrice extracellulaire auto-produite. Les biofilms naturels sont composés de plusieurs espèces microbiennes et leurs interactions, qu'elles soient synergiques ou antagonistes, jouent un rôle important dans le développement, la composition et le fonctionnement des consortia impliqués. De plus, ces relations impliquent souvent la production de molécules antagonistes limitant la croissance ou l'adhésion bactérienne. Enfin la composition de la matrice extracellulaire joue un rôle important dans la robustesse du biofilm. Au cours de ce travail, l'étude des interactions au sein de biofilms mixtes constitués par K. pneumoniae et S. epidermidis a permis d'isoler un polysaccharide produit par K. pneumoniae, capable d'inhiber l'adhésion aux surfaces abiotiques de plusieurs autres espèces bactériennes à Gram-négatif et à Gram-positif. La caractérisation physico-chimique de cette molécules de haut poids moléculaire a permis de mettre en évidence qu'elle était composée de galactose, glucose, rhamnose et d'acide glucuronique. Par ailleurs, l'analyse d'extraits de mutants déficients pour la production de capsule a montré que ce polysaccharide correspondait à la capsule de K. pneumoniae. Son mode d'action consisterait à inhiber les interactions initiales entre bactéries et surface. Le suivi dans le temps de la formation de biofilm monospecies par K. pneumoniae avec la technique Biofilm Ring Test® a également permis de mettre en évidence un phénotype original. En effet, la détection initiale d'agrégats bactériens est suivie par une modification apparente de leur structure, qui serait liée à des changements de leur robustesse face aux forces d'aimantation magnétique. La présence d'ADN extracellulaire au sein de la matrice du biofilm pourrait jouer un rôle dans la survenue de ce phénotype comme l'indique les mesures effectuées en présence de l'enzyme DNAse I. En parallèle, l'observation de biofilm formés par K. pneumoniae dans des modèles expérimentaux cinétiques a mis en évidence des décrochements massifs de biomasse au cours de la maturation du biofilm, qui pourraient être corrélées aux modifications internes de robustesse de la matrice. L'ensemble de ces données permet de mieux caractériser les interactions intimes survenant à l'intérieur de biofilms constitués par K. pneumoniae et à terme de mieux caractériser et donc prévenir leur formation et disséminationBiofilms are defined as microbial communities adhering to biotic or abiotic surfaces and embedded in a self-produced extracellular matrix. Natural biofilms are composed of several microbial species and their interactions, synergistic or antagonistic, play important roles in development, composition and functioning of the consortia. Furthermore, the relationships often involve the production of antagonist molecules that impair competitors' growth or adhesion. The composition and evolution of the extracellular matrix plays also an important role in the biofilms' robustness. In this work, study of the interactions within biofilms formed by K. pneumoniae and S. epidermidis led to the isolation of a polysaccharide produced by K. pneumoniae able to inhibit the adherence to abiotic surfaces of several Gram-negative and Gram-positive bacterial species. The physico-chemical characterization of this high molecular weight molecule showed it was composed of galactose, glucose, rhamnose and glucuronic acid. This data, together with the analysis of extracts from capsule-deficient mutants, indicated that the capsule of K. pneumoniae was responsible for the biofilm inhibition phenotype, probably by inhibiting the initial interactions between bacteria and surface. By monitoring the formation of monospecies biofilm by K. pneumoniae with the Biofilm Ring Test® technique, we were able to detect an original phenotype. Indeed, detection of bacterial aggregates still occurred after a few hours of incubation but in a different way, probably related to changes of the biofilm robustness towards magnetic forces. The presence of extracellular DNA in the matrix of the biofilm is likely to play a role in the occurrence of this phenotype, as indicated by the assays performed in presence of the enzyme DNase I. At the same time, observations of biofilm formed by K. pneumoniae in kinetic experimental models showed massive detachment events during biofilm maturation, which could be correlated to changes in internal strength of the matrix. All these dtat contribute to a better characterization of the intimate interactions occuring within biofilms formed by K. pneumoniae and will ultimately lead to the development of efficient anti-biofilm strategies
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Carrera, Samantha. "Influence of extracellular factors on p53-mediated DNA damage responses." Thesis, University of Leicester, 2013. http://hdl.handle.net/2381/27796.
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Cells have evolved sophisticated mechanisms to maintain genomic stability after cellular stress. Activation of DNA damage response pathways, and most importantly p53, leads to adaptive responses that can be influenced by different extracellular factors. The aim of this project was to study how extracellular factors modulate p53 cell-fate decisions after DNA damage, with particular interest in oxygen tensions and vitamin A metabolites. First, we focused on how physiological oxygen tensions (5% 02) may influence cellular responses to genotoxic stress. We showed that normal and cancer cells cultured at 5% 02 had a reduction in p53-mediated apoptosis after exposure to different genotoxic stresses. This was not due to a decrease p53 protein levels or its transactivation activity, and the oxidative damage caused by DNA damaging agents was not affected by oxygen tensions. We also found a p53-independent activation of MAPK at 5% 02, which when inhibited restored levels of p53-induced apoptosis. HIF-1α, a transcription factor induced at lower O2 concentrations, was present and active at 5% 02. However, this did not affect MAPK activation and HIF-1α was not involved in the resistance to apoptosis under these conditions, although MAPK was necessary for HIF-1α expression and activation. We next explored the effect of the vitamin A (retinol) pathway on the cellular responses to DNA damage. We showed that Stra6, a retinol-inducible gene, is upregulated by p53 after DNA damage. While overexpression of Stra6 sensitized cells to p53-induced apoptosis independently of the retinoic acid signalling, its inhibition resulted in decreased apoptosis after DNA damage and less induction of oxidative stress. This shows that both oxygen tensions and vitamin A metabolites, through Stra6, are potent modulators of the p53 responses to DNA damage.
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Tursi, Sarah Anne. "Curli-Extracellular DNA Complexes: Pathogenicity and Role in Enteric Biofilms." Diss., Temple University Libraries, 2018. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/511656.
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Microbiology and ImmunologyPh.D.
The first recorded observation of bacterial biofilms dates back to the 17th century by Antoine Van Leeuwenhoek. Today, biofilms are known as bacteria encapsulated within a self-produced extracellular matrix adherent to biotic or abiotic surfaces. Since the initial discovery of biofilms, research has explored the structure and function of biofilms. Only until recently has the role of biofilms within the medical setting become apparent. Here, we investigate the role of curli-extracellular DNA (eDNA) complexes in disease pathogenicity and explore the ability to target bacterial amyloid curli as a novel anti-biofilm therapeutic target. Biofilms of enteric bacteria, such as Escherichia coli and Salmonella enterica serovar Typhimurium, are composed of various components that act in consortium to fortify the extracellular matrix. One of the main components of enteric biofilms is amyloid curli. Curli, one of the best characterized bacterial amyloids, is a protein with a conserved cross beta sheet structure that forms basket like structures encapsulating the bacteria. Within the biofilm, curli serves to fortify the extracellular matrix, aids in bacterial attachment and protects bacteria from harsh environmental conditions. Extracellular DNA (eDNA) is another integral component of enteric biofilms. Recent reports from our lab has suggested that curli forms irreversible complexes with eDNA. Even with exposure to DNases, co-localized curli and eDNA can be observed. Other components of enteric biofilms include cellulose and Biofilm Associated Protein A. Biofilms of S. Typhimurium have been associated with significant disease pathologies. In addition to identifying the existence of curli-eDNA complexes within S. Typhimurium biofilms, our lab has also reported that curli-eDNA complexes of S. Typhimurium potentiate the autoimmune disease Systemic Lupus Erythematosus (SLE). SLE is an autoimmune disease characterized by the production of type I interferons and autoantibodies, although the etiology remains unknown. Systemically, curli binds to and activates the Toll like Receptor (TLR)1/2 complex leading to a pro-inflammatory response. In these studies we aimed to identify the innate immune mechanisms leading to the autoimmune phenotype following stimulation with curli-eDNA complexes. As TLR9 is activated by unmethylated bacterial DNA CpG DNA sequences leading to the production of type I interferons we hypothesized a potential role for TLR9 in recognizing eDNA of the curli-eDNA complex leading to the generation of the hallmarks of SLE. To investigate this hypothesis, we stimulated wild-type, TLR2 knockout, TLR9 knockout and TLR2-9 double knockout immortalized macrophages with curli-eDNA complexes purified from S. Typhimurium biofilms. We observed a significant reduction in the transcript level of type I interferons (IFN), Ifnβ, Isg15 and Cxcl10, upon stimulation of TLR2 knockout, TLR9 knockout and TLR2-9 double knockout immortalized macrophages implicating a role in TLR9 recognition of the curli-eDNA complex. As there was a significant reduction of type I interferon levels upon stimulation of TLR2 knockout macrophages, we hypothesized that TLR2 may serve as a carrier to bring the curli-eDNA complex into the endosome containing TLR9. To inhibit phagocytosis, we pretreated cells with endocytosis inhibitors and stimulated wild-type macrophages with curli-eDNA complexes. We observed a reduction in the transcript level of Ifnβ suggesting that curli-eDNA complexes gain access to endosomal TLR9 via TLR2 engagement. Finally, to explore the role of TLR2 and TLR9 in the production of autoantibodies, curli-eDNA complexes were intraperitoneal injected twice weekly for six weeks into C57BL/6 wild-type, TLR2 knockout, TLR9 mutant and TLR2 knockout-TLR9 mutant mice. We observed a robust generation of anti-double stranded autoantibodies within the first three weeks, however the production of autoantibodies was significantly decreased and delayed in the TLR2 knockout, TLR9 mutant and TLR2 knockout-TLR9 mutant mice. Overall, these data suggest that curli acts as a carrier for DNA to elicit an autoimmune response via TLR2 and TLR9. Within biofilms of S. Typhimurium, curli is the main proteinaceous component. Biofilms lacking curli destabilize and fail to form mature biofilms. Recent research has shown that in response to the production of host amyloids, the body will generate anti-amyloid antibodies in the serum. Monoclonal antibodies (mAb) generated from serum antibodies have been shown to have pan anti-amyloid properties in vitro and in vivo due to the β-sheet conformational epitope. As amyloids from both human and bacterial origin share a β-sheet conformational structure, we hypothesized as to if the anti-amyloid mAbs can eradicate S. Typhimurium biofilms by targeting curli. We incubated S. Typhimurium biofilms in the presence of various mAbs (ALZ.4A6, ALZ.4GI, ALZ.2C10 and ALZ.3H3) and observed a significant reduction of biofilm thickness and curli content within the biofilm. We deduced that ALZ.3H3 conferred the greatest anti-biofilm response. When we visualized the three-dimensional architecture of biofilms incubated with ALZ.3H3, we observed that ALZ.3H3 induced the formation of a loose architecture compared to untreated biofilms that were dense and compact. The resulting loose biofilm architecture induced by incubation with ALZ.3H3 enhanced the susceptibility of the biofilms to antibiotic exposure and macrophage clearance. We also observed enhanced biofilm eradication in vivo when catheters precoated with S. Typhimurium biofilms were inserted into the back flanks of mice that were percutaneously injected with ALZ.3H3. Both in vitro and in vivo, combination therapy of ALZ.3H3 and antibiotic enhanced biofilm clearance. In summary, we propose a novel anti-biofilm strategy by targeting the amyloid component of the biofilm, thus satisfying an unmet need in the art of biofilm prevention. Overall, these data in summation significantly broadens our understanding of disease pathogenicity and the role of curli-eDNA complexes in S. Typhimurium biofilms. As amyloid-eDNA complexes may be found in other biofilms, these results may extend beyond enteric bacteria proving novel insight into host-microbe interactions and the generation of novel anti-biofilm therapeutics.
Temple University--Theses
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Bußkamp, Holger [Verfasser]. "From New DNA Conjugation Approaches to 3D DNA Networks for an Artificial Extracellular Matrix / Holger Bußkamp." Konstanz : Bibliothek der Universität Konstanz, 2015. http://d-nb.info/1112745408/34.
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Susevski, Vanessa. "Development of DNA Aptamers Targeting Breast Cancer Derived Extracellular Vesicles for Biomarker Discovery." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/41035.
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Detection of cancer at the early stages greatly increases the chance for successful treatment and favourable prognosis for patients. However, a liquid-based biopsy has yet to be developed for most cancers. Extracellular vesicles (EVs) are an attractive candidate for early cancer detection since their surface proteome mirrors the cell of origin. Thus, there is a need for the development of reliable probes that can detect cancer derived EVs. In this thesis, the VBS-1 aptamer was developed to selectively bind to triple-negative breast cancer cell line derived EVs. Initially, several EV isolation methods were compared and isolated EVs were validated and characterized. Aptamer clones were developed by Systematic Evolution of Ligands by Exponential Enrichment to EVs isolated by differential ultracentrifugation and their binding was validated by flow cytometry. The binding partner of the selected VBS-1 aptamer was identified by LC-MS/MS to be the transmembrane protein ATP1A1. The presence of an ATP1A1-positive EV population was validated by flow cytometry. The selected aptamer may find further application in biosensors for the detection of EVs as cancer biomarkers in biological fluids.
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Rapsinski, Glenn James. "Immune Recognition of S. Typhimurium Biofilms via Amyloids and Extracellular DNA." Diss., Temple University Libraries, 2016. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/273605.
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Microbiology and ImmunologyPh.D.
Salmonella enterica serovar Typhimurium is an important cause of gastroenteritis in the United States and the developing world. Biofilm growth is an significant mechanism, which S. Typhimurium utilizes to contaminate food products and survive in the environment. Biofilms are also an important part of the infectious process for many pathogenic bacteria. As part of the biofilm, S. Typhimurium produces an extracellular matrix consisting of cellulose, extracellular DNA, and most importantly, the amyloid protein curli. Similar to amyloids associated with human diseases, curli is recognized by the innate immune system through Toll-Like Receptors (TLRs). Here, we studied the immune receptors recognizing curli as well as interactions between eDNA and curli during biofilm development in order to glean a better understanding of these complex bacterial communities and the immune response to them. Recently, our lab demonstrated that curli fibers are recognized by the TLR2/TLR1 complex. CD14 has been shown to be a common adaptor protein for TLR2/TLR1 complex in response to one of its ligands, tri-acylated lipopeptide, Pam3CSK4. In order to study the role of CD14 in the immune receptor complex recognizing curli, we utilized HeLa 57A cells, a human cervical cancer cell line that has a stably transfected luciferase reporter for Nf-κB activation. When these cells were transiently transfected with TLR2 and TLR1 together or with the addition of membrane-bound CD14, NfκB activation was enhanced by the presence of CD14 in response to purified curli, GST-tagged curli subunit (GST-CsgA), and the control lipopeptide Pam3CSK4. Soluble CD14 also increased NfκB activation in response to purified curli. Bone marrow derived macrophages (BMDM) from wild type (C57BL/6) mice produced more IL-6 and nitric oxide in response to stimulation with purified curli, GST-CsgA, and Pam3CSK4, than BMDMs deficient in CD14. Binding assays demonstrated direct binding of curli to all members of this hypothesized trimolecular complex, TLR2, TLR1, and CD14. Utilizing synthetic peptides corresponding to the fourth and fifth repeat of the CsgA monomer, CsgA R4-5, and its modified version, CsgA R4-5N122A deficient in forming amyloid fibers, we also showed that binding to CD14, and CD14 enhancement of IL-6 production required the fibrillar amyloid structure of curli. To study interactions between curli and eDNA in biofilms and the resulting immune response generated to composites formed by these ECM components, we analyzed biofilms of GFP expressing S. Typhimurium using confocal laser scanning microscopy (CLSM). Staining for amyloids with Congo Red revealed the presence of curli in the biofilms and staining with propidium iodide demonstrated the presence of extracellular DNA in the biofilms. Co-staining with TOTO-1, a nucleic acid stain, and Congo Red showed co-localization of the fluorescent signal for these molecules within the biofilms. DNase I treatment of the biofilms produced no significant change in biofilm thickness by confocal microscopy signifying that the biofilm, possibly eDNA, was resistant to DNase treatment. This was further confirmed by the presence of DNA in purified curli fibers, which were treated twice with DNase and RNase. Polymerization assays showed acceleration of amyloid polymerization in the presence of DNA from both bacteria and salmon sperm. CLSM of bone marrow derived dendritic cells demonstrated that DCs are able to sample antigens from biofilms. BMDCs also produced robust quantities of proinflammatory cytokines in response to wild type, msbB, and ΔfliCfljB S. Typhimurium biofilms and purified amyloid/DNA composites as measured by ELISA. Using BMDCs deficient in TLR2 and TLR9, we found that this cytokine production was partially dependent on TLR2, but did not require TLR9. Together, these findings significantly broaden our understanding of S. Typhimurium biofilms and the immune response to ECM components present in its biofilms. We now understand that a trimolecular complex of TLR2/TLR1/CD14 is required for full response to curli by innate immune cells. We also discerned that interactions between biofilm components aid biofilm development and create composites that are highly immunogenic. This new information enhances the need to explore the interaction between composite ligands and the immune system rather than only studying ligands individually.
Temple University--Theses
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Apel, Falko. "Recognition of Neutrophil Extracellular Traps by the Cytosolic DNA Sensor cGAS." Doctoral thesis, Humboldt-Universität zu Berlin, 2019. http://dx.doi.org/10.18452/19727.
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Neutrophile Granulozyten produzieren „Neutrophil Extracellular Traps“ (NETs), ein mit antimikrobiellen Molekülen bestücktes Netzwerk aus Chromatinfasern, das während eines Zelltodprogramms namens „NETosis“ von den sterbenden Neutrophilen ausgestoßen wird. Ihre netzartige Struktur erlaubt es ihnen, eine weitere Verbreitung des Infektionserregers zu verhindern; zudem erzeugen sie eine hohe lokale Konzentration an toxischen Molekülen, die Mikroorganismen töten können. Unter normalen Bedingungen werden NETs von Nukleasen zerkleinert und anschließend von Makrophagen entfernt. Wenn dieser Aufräummechanismus gestört ist, aktivieren NETs das Immunsystem und führen zur Produktion von Autoantikörpern oder entzündungsfördernden Zytokinen. NETs werden mit einer wachsenden Liste von inflammatorischen und Autoimmunerkrankungen in Verbindung gebracht. Wie genau dabei NETs durch das Immunsystem erkannt werden, ist noch nicht bekannt.
In der vorliegenden Arbeit zeige ich, dass NETs durch den zytosolischen DNA Sensor „cyclic GMP-AMP synthase“ (cGAS) detektiert werden können und dass dadurch die Expression von Typ I Interferonen (TIIFN) induziert wird. Zu Beginn demonstriere ich, dass NETs durch rekombinantes cGAS erkannt werden und dass mit isolierten NETs stimulierte Immunzellen cGAS-abhängig TIIFN produzieren. Des Weiteren zeige ich, dass Neutrophile, die NETosis begehen, in Nachbarzellen ebenfalls cGAS-anhängig TIIFN induzieren können. Abschließend konnte ich diese Ergebnisse in einem in vivo Mausmodel für systemische NET-Produktion bestätigen. Die vorliegende Arbeit zeigt einen Mechanismus, wie NETs durch das Immunsystem erkannt werden und dadurch sowohl zur Entstehung als auch zur Progression von Krankheiten beitragen kann. Sie ermöglicht dementsprechend die Entwicklung neuer Interventionsstrategien, welche zur Heilung oder Linderung einer Vielzahl von Erkrankungen beitragen können.The first line of cellular defense of the immune system are neutrophils. They are the most abundant white blood cell, which exert an array of antimicrobial effector functions. Neutrophils release neutrophil extracellular traps (NETs), a composite of chromatin and antimicrobial molecules, into the extracellular space during a form of regulated cell death called NETosis. Their net-like structure prevent further dissemination of the invader and establishes a high local concentration of toxic molecules that mediate pathogen killing. NETs provide a platform for undesired immune activation and contribute to the production of autoantibodies and pro-inflammatory cytokines. NETs are implicated in a growing list of inflammatory and autoimmune diseases, but the exact mechanism how NETs are recognized by the immune system is not fully understood. In this study, I demonstrate that the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) senses NETs and induces the production of type I interferons (TIIFN). I first showed that NETs are recognized by recombinant cGAS and that cells treated with isolated NETs produce TIIFN in a cGAS dependent mechanism. Secondly, I demonstrate that neutrophils undergoing NETosis are taken up by neighboring immune cells and induce cGAS-dependent TIIFN expression. Lastly, I confirmed our in vitro results in a mouse model of systemic NET induction. Wildtype mice injected with Concanavalin A significantly upregulate the expression of interferon stimulated genes, while cGAS-/- mice and Cybb-/- mice, which are incapable of producing NETs, fail to induce this response.
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Lawler, Danielle Suzanne. "The role of respiratory viral infection and extracellular DNA in allergic sensitisation." Thesis, Lawler, Danielle Suzanne (2018) The role of respiratory viral infection and extracellular DNA in allergic sensitisation. Honours thesis, Murdoch University, 2018. https://researchrepository.murdoch.edu.au/id/eprint/43144/.
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The correlation between allergic disease and viral infections has been well established. While respiratory viral infections are strongly correlated with the development of allergic asthma, it is not known precisely how viral infection may produce, or alternatively protect, against allergic disease. Furthermore, little is known about the impact of viral infection on IgE sensitisation, due to conflicting and limited research. Recently, neutrophil extracellular traps (NETs) have been observed in viral infections and shown to induce a Th2 response. We aim to explore the link between virus infection and allergy and test if the link is mediated through NETs. We hypothesise that NETs are induced during viral respiratory infections, but that they do not adequately control the viral disease. Instead, they contribute to the development of allergic disease, and their removal will be beneficial to patients.
This hypothesis was addressed by sensitising rats to ovalbumin (OVA), during a respiratory viral infection and re-exposing the animals to OVA one week after sensitisation. To test the involvement of NETs, DNase-I was administered during viral infection to degrade NETs. OVA-specific IgE serum levels, along with cellular infiltrates into the airways, were compared between groups to assess allergic sensitisation before and after OVA re-exposure.
Although no effect on IgE sensitisation was observed with viral infection, DNase treatment reduced the risk of IgE sensitisation and increased T regulatory cell (Treg) proportions in the airways, which also displayed higher levels of FoxP3. We did not observe a difference between any of the groups in respect to allergic recall response suggesting that the achieved sensitisation was not sufficient to induce clinical disease. Our findings nevertheless suggest that DNase treatment induces a regulatory response, which may protect against allergic disease. Future studies should explore this immunoregulatory response, as a novel strategy for allergy protection.
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Chambard, Marie. "Analyse génomique de l'ADN extracellulaire du Root Extracellular Trap (RET) et caractérisations omiques des "root Associated Cap-Devrived Cells" (AC-DC) chez le soja Glycine max (L.) Merr.1917." Thesis, Normandie, 2020. http://www.theses.fr/2020NORMR020.
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Le soja, culture d’intérêt agronomique mondiale et bientôt Normande, doit faire face à l’attaque de nombreux phytopathogènes et notamment à l’oomycète Phytophthora sojae Kaufm. & Gerd., qui engendrent chaque année d’importantes pertes économiques. Le RET (Root Extracellular Trap) est situé à l’apex racinaire, est constitué de cellules détachées de la racine appelées cellules frontières ou AC-DC (root Associated Cap-Derived Cells) et de leur mucilage associé. On retrouve au sein de ce mucilage des glycomolécules, des protéines, ou encore de l’ADN extracellulaire (ADNex). Cet ensemble, formant un territoire racinaire particulier, va permettre la protection de la racine et notamment de son apex contre les stress biotiques et abiotiques. Afin de mieux comprendre les mécanismes de défense racinaires, et notamment de certains acteurs du RET, une analyse transcriptomique et protéomique des AC-DC et de la racine et une analyse génomique de l’ADNex ont été réalisées en conditions témoin et élicitée avec PEP-13, un éliciteur provenant des oomycètes du genre Phytophthora sp. Les analyses transcriptomique et protéomique ont montré une spécificité des AC-DC par rapport aux racines et une réponse à PEP-13 qui semble différente entre ces deux zones. Le séquençage génomique de l’ADNex du RET a été réalisé afin de déterminer l’origine de celui-ci et savoir s'il existe une spécificité de séquences entre les deux conditions. Il semblerait que l'ADNex ait une meilleure couverture lors de l'alignement sur l’ADN mitochondrial et l’ADN plastidial comparé à l’ADN chromosomique. Cette différence de couverture peut indiquer une différence de persistance de l’ADNex dans le RET en fonction de son origine (chromosomique ou mitochondrial et plastidial), ou une libération dans le RET par ces organites. Il semblerait également qu’il n’y ait pas de différences entre les séquences d’ADNex du RET en condition élicitée avec PEP-13 ou témoinSoybean, a crop of Normand and world agronomic interest, is threatened by numerous phytopathogens like the oomycete Phytophthora sojae Kaufm. & Gerd., wich generate high levels of economical losses. The RET (root extracellular trap) is located at the root apex and is composed of border cells or AC-DC (root associated cap-derived cells) and their mucilage. This mucilage is made up of glycomolecules, proteins or also extracellular DNA (exDNA). The RET play a role in root protection against biotic stresses. In order to better understand the role of the RET in root protection, a transcriptomic and a proteomic analysis where done on AC-DC and roots in controle condition and in elicited condition with PEP-13 (an elicitor from Phytophthora sp.). The results show a specificity of AC-DC compared to the root, and an answer to PEP-13 wich seems to be different between these two tissues. An other experiment was to sequence RET exDNA in controle and elicited conditions, in order to define the origin of this exDNA. We show that the coverage of mitochondrial and plastidial DNA where much better than the coverage of chromosomic DNA. It could mean that chromosomic DNA isn’t conserved as well as organelles DNA, or exDNA could originate from organelles. Furthermore, results seems to show no differences between the sequences of elicited or control exDNA
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Finke, Alexander [Verfasser]. "Development of DNA-Based Materials as Mimicry of the Extracellular Matrix / Alexander Finke." Konstanz : KOPS Universität Konstanz, 2019. http://d-nb.info/1183573391/34.
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Wen, Fushi. "Root Border Cell Development and Functions of Extracellular Proteins and DNA in Fungal Resistance at the Root Tip." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/195136.
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Soilborne plant pathogens are responsible for many of the major crop diseases worldwide. However, plant root tips are generally resistant to pathogen infections. The goal of this dissertation research is to understand the mechanism of this natural resistance by testing the hypothesis that root caps and root border cells control the rhizosphere community through the biological products which they deliver to the soil. Specific objectives of this dissertation project are 1) identifying, isolating, and characterizing the genes important for border cell development and for root exudates delivery, and 2) analyzing the function of extracellular macromolecules in root exudates in root tip-fungal pathogen interaction. The expression of a primary cell wall synthesis gene, PsFut1, encoding Pisum sativum fucosyltransferase, was characterized during border cell production, and the impact of silencing this gene on border cell development was examined. Another gene, BRDgal1, encoding β-galactosidase, was identified and characterized in Pisum sativum during this study. It was shown that this β-galactosidase is specifically produced in and secreted from root border cells. The microarray transcriptional profiling in M. truncatula and mRNA differential display analysis in pea plants were carried out following the induction of border cell production to gain a broader understanding of the genes which potentially influence border cell development. In order to study the commonality of border cell production across different plant species, the expression of rcpme1, the marker gene for border cell production, was compared between the garden pea and a gymnosperm species, the Norway spruce (Picea abies). To accomplish the second objective, the focus of this study was shifted from border cell development to mucilaginous root exudates excreted by border cells and root cap cells. This resulted in a breakthrough in the understanding of the mechanisms of root tip resistance. The presence of extracellular DNA in the root mucilage was discovered and its requirement for root tip resistance to fungal infection was demonstrated. Extracellular proteins in the root mucilage were identified and they were shown to be also required for the root tip resistance to fungal infection. This work provided new insights into understanding plant defense mechanisms.
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Peters, Dimetrie Leslie. "Evaluation of eukaryotic cultured cells as a model to study extracellular DNA / D.L. Peters." Thesis, North-West University, 2011. http://hdl.handle.net/10394/6929.
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The diagnostic value of extracellular occurring DNA (eoDNA) is limited by our lack of
understanding its biological function. eoDNA exists in a number of forms, namely vesicle
bound DNA, histone/DNA complexes or nucleosomes and virtosomes. These forms of DNA
can also be categorized under the terms circulating DNA, cell free DNA, free DNA and
extracellular DNA. The DNA can be released by means of form–specific mechanisms and
seem to be governed by cell cycle phases and apoptosis. Active release is supported by
evidence of energy dependant release mechanisms and various immunological– and
messenger functions. Sequencing has shown that eoDNA sequences present in the nucleome
reflects traits and distribution of genome sequences and are regulated by ways of release
and/or clearance. eoDNA enables the horizontal transfer of gene sequences from one cell to
another, over various distances. The ability of eoDNA to partake in horizontal gene transfer
makes it an important facet in the field of epigenetic variation. Clinical implementation of
eoDNA diagnostics requires that all of the subgroups of eoDNA be properly investigated. It
is suggested that eoDNA is the result of the metabolic fraction of DNA that is released by the
cell. Various observations indicate that eoDNA may also be incorporated into the genome of
a cell, from where it may affect cell function. Therefore horizontal gene transfer in higher
organisms is a real possibility. In this study, variations and increases in eoDNA levels over
time correlate with stressors that are subjected to 143B human osteosarcoma cells. It seems
viable to assume that a stressor is met by a change in the molecular machinery of a cell,
required to neutralise the onset of metabolic instability. This may be done by amplification of
necessary cistrons, producing metabolic DNA, that may then be observed after its release as
eoDNA. The presence of hydrolysing enzymes gives an updated real time picture of the state
of eoDNA. The eogenics hypothesis emanating from this study, suggests that amplification
and horizontal transfer of cistrons affect tissue and organ function over long periods of time,
in order for an organism to evolve one or more a specialized genomes.Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.
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Moulière, Florent. "Etude de la structure et de l'origine des ADN circulants : application à la mise au point d'un test de détection des mutations KRAS et BRAF dans le cancer colorectal." Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20245/document.
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Les ADN circulants extracellulaires (ADNcf) sont considérés comme des biomarqueurs potentiels non invasifs de la progression tumorale. Ils présentent l'avantage d'être porteurs des altérations génétiques des tumeurs dont ils sont issus. Les connaissances sur les formes, les mécanismes de libération et les actions biologiques des ADNcf sont cependant encore peu caractérisées.Nous avons émis l'hypothèse que se focaliser sur l'étude de la structure et des origines des ADNcf issus des tumeurs permettrait d'ouvrir de nouvelles perspectives d'applications en génomique personnalisée.Nos travaux ont démontré à l'aide d'un animal modèle que les ADNcf issus des tumeurs de cancers colorectaux sont hautement fragmentés à des tailles inférieures à 145 bp. Cette observation a été confirmée sur plasma humain en réalisant par AFM la première image directe d'ADNcf issu de tumeurs. Nous avons déterminé que les proportions d'ADNcf mutés varient fortement dans la circulation sanguine, mais que près d'un tiers des individus présentaient des proportions d'ADNcf mutés supérieures à 25 % de tous les ADNcf retrouvés dans le sang. Ces découvertes nous ont permis de participer au développement d'une méthode d'analyse spécifique des ADNcf du plasma permettant de déterminer par Q-PCR la concentration en ADNcf, sa fragmentation ainsi que la présence des mutations KRAS et BRAF. Cette méthode a été validée cliniquement sur 79 échantillons de patients atteints de cancer colorectal métastatique en la comparant avec une concordance de 96 % à la technique de référence clinique utilisant l'ADN de tissu tumoral. L'utilisation des ADNcf en tant que « biopsie liquide » devrait être un biomarqueur central dans l'approche de génomique personnalisée des années à venir et les résultats de ces travaux de thèse participer au développement de cette nouvelle approcheCell-free circulating DNA (cfDNA) are considered as potentials non invasive biomarkers of tumor progression. They present the advantage to exhibit the genetic alterations from their tumor of origin. Knowledge on the forms, mechanism of release, and biological effect of cfDNA are however still less characterized. We have hypothesized that focalizing on the study of cfDNA structure and origin will open new perspectives of application in personalized genomic. Our works demonstrated, with an animal model, that cfDNA from colorectal cancer tumor are highly fragmented at size lower than 145 bp. This observation was confirmed on human plasma with AFM by realizing the first direct picture of tumor-derived cfDNA. We have determined that cfDNA proportion highly varied in bloodstream, but more than a third of individual exhibit proportions larger than 25 % of blood total cfDNA.These discoveries let us participate to the development of a specific analysis method of plasma cfDNA owing to determinate by Q-PCR the cfDNA concentration, its fragmentation and the presence of KRAS and BRAF mutation. This method has been clinically validated on 79 samples of metastatic colorectal cancer patients by comparing it, with a concordance of 96 %, with the technique of reference using DNA from tumoral tissue.cfDNA could be used as « liquid biospy » and could be a central biomarker in the personalized genomic for the future years, and this thesis work participate to the development of this new approach
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Silva, Eliane Pereira da. "Influência da temperatura, de enzimas degradantes de DNA e do sobrenadante de cultura de estafilococos na formação de biofilme por Listeria monocytogenes em superfície abiótica." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-19122013-103644/.
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A listeriose é uma infecção rara e grave, transmitida principalmente por alimentos. É causada pela bactéria Listeria monocytogenes e acomete principalmente mulheres grávidas e pessoas comprometidas imunologicamente. Este patógeno é reconhecido como um problema para as indústrias de alimentos devido à sua capacidade em formar biofilmes. Os biofilmes são comunidades microbianas aderidas a superfícies bióticas ou abióticas embebidas em uma matriz de polímeros extracelulares produzidos pelas próprias células. Estas estruturas são resistentes a procedimentos de higienização e desinfecção, contribuindo para a persistência de L. monocytogenes em ambientes processadores de alimentos. Desta forma, há grande interesse em estratégias para prevenir a formação de biofilmes por L. monocytogenes. No presente trabalho foi investigada a formação de biofilmes por L. monocytogenes em superfície abiótica, sob diferentes temperaturas, na presença de enzimas degradantes de DNA (DNAses) e na presença de sobrenadante de cultura de estafilococos com e sem atividade de DNAse termoestável (termonuclease). Foram utilizadas técnicas de cultivo e de microscopia e os resultados demonstraram que L. monocytogenes aderiu à superfície de aço inoxidável, em média 105-106 UFC/cm2. A temperatura de incubação influenciou na adesão de L. monocytogenes à superfície de aço inoxidável, mas outros fatores em conjunto, como a cepa bacteriana e o tipo de sistema de cultivo utilizado, também podem ter contribuído para os resultados observados. Por microscopia de fluorescência foi constatada a formação de biofilmes maduros por L. monocytogenes, contendo canais provavelmente envolvidos em fluxo de nutrientes e também, cavidades características de dispersão celular. A mesma técnica permitiu constatar um predomínio de células metabolicamente ativas envoltas por matriz extracelular polimérica contendo DNA extracelular (DNAe), coradas por laranja de acridina. Por microscopia confocal a laser, foram observadas microcôlonias contendo DNAe e foram visualizadas também camadas homogêneas pouco espessas de células viáveis, coradas por SYTO9 e DDAO. O DNAe foi observado ainda em locais sem células, característicos de dispersão celular. O sobrenadante de cultura de S. aureus com atividade de termonuclease inibiu L. monocytogenes livre no meio de cultura e interferiu na formação de biofilme por este patógeno por até 24h a 37ºC. As DNAses comerciais não interferiram na formação de biofilme por L. monocytogenes, indicando ser improvável a ação da termonuclease de S. aureus na inibição da forma planctônica ou séssil de L. monocytogenes. Foi constatada a produção de uma proteína termoestável por S. aureus, capaz de inibir L. monocytogenes em teste de antagonismo em ágar, mas estudos posteriores são necessários para a completa caracterização de tal substância inibitória.Listeriosis is a rare and serious mainly food-borne infection. It is caused by the bacterium Listeria monocytogenes that primarily affects pregnant women and immunologically compromised individuals. This pathogen is recognized as a problem for the food industry due to its ability to form biofilms. Biofilms are microbial communities adhered to biotic or abiotic surfaces embebed by self produced extracellular polymers. These structures are resistant to cleaning and disinfection procedures, allowing the survival and persistence of L. monocytogenes in food processing environments. Thus, several strategies have been adopted to prevent and control the formation of biofilms on food contact surfaces. The present study investigated biofilm formation by L. monocytogenes on abiotic surface at different temperatures, in the presence of DNA degrading enzymes (DNAses) and in the presence of culture supernatant with and without staphylococcal thermonuclease activity. For this purpose, we used culture techniques and microscopy. The results showed that L. monocytogenes was able to adhere on stainless steel surface on average 105-106 CFU per cm2. The different incubation temperatures affected the adhesion of L. monocytogenes on stainless steel surface, although other factors in combination were also involved, such as the bacterial strain and the type of system used for assay. By fluorescence microscopy, we noticed the formation of mature biofilms by L. monocytogenes, with channels likely involved in flow of nutrients and holes typical of cell dispersion. Furthermore, we found a predominance of metabolically active cells surrounded by extracellular matrix polymer containing extracellular DNA (eDNA) stained with acridin orange. By laser confocal microscopy, we observed the formation of microcolonies containing eDNA and homogeneous thin layer of viable cells stained with SYTO 9 and DDAO. The eDNA was also observed in locations with absence of cells characteristics of cell dispersion. The culture supernatant of S. aureus with thermonuclease activity was able to inhibit L. monocytogenes free on culture medium and interfered with the formation of biofilm by the pathogen for up to 24h at 37°C. The commercial DNAses did not affect biofilm formation by L. monocytogenes, possibly excluding the action of thermonuclease of S. aureus on the inhibition of planktonic or sessile forms of L. monocytogenes. It has been found a production of a thermostable protein by S. aureus and it was able to inhibit L. monocytogenes in agar antagonism assays but further studies are needed to characterize such inhibitory substance.
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Cioccoloni, Andrea. "Pro-inflammatory effect of extracellular vesicles released by senescent-like C2C12." Doctoral thesis, Urbino, 2021. http://hdl.handle.net/11576/2688889.
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Brundin, Malin. "Stability of bacterial DNA in relation to microbial detection in teeth." Doctoral thesis, Umeå universitet, Institutionen för odontologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-82735.
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The fate of DNA from dead cells is an important issue when interpreting results from root canal infections analysed by the PCR technique. DNA from dead bacterial cells is known to be detectable long time after cell death and its stability is dependent on many different factors. This work investigated factors found in the root canal that could affect the recovery of microbial DNA. In an ex vivo experiment, DNA from non-viable gram-positive Enterococcus faecalis was inoculated in instrumented root canals and recovery of DNA was assessed by PCR over a two-year period. DNA was still recoverable two years after cell death in 21/25 teeth. The fate of DNA from the gram-negative bacteria Fusobacterium nucleatum and the gram-positive Peptostreptococcus anaerobius was assessed in vitro. DNA from dead F. nucleatum and P. anaerobius could be detected by PCR six months post cell death even though it was clear that the DNA was released from the cells due to lost of cell wall integrity during the experimental period. The decomposition rate of extracellular DNA was compared to cell-bound and it was evident that DNA still located inside the bacterium was much less prone to decay than extracellular DNA. Free (extracellular) DNA is very prone to decay in a naked form. Binding to minerals is known to protect DNA from degradation. The fate of extracellular DNA was assessed after binding to ceramic hydroxyapatite and dentine. The data showed that free DNA, bound to these materials, was protected from spontaneous decay and from enzymatic decomposition by nucleases. The main conclusions from this thesis were: i) DNA from dead bacteria can be detected by PCR years after cell death ex vivo and in vitro. ii) Cell-bound DNA is less prone to decomposition than extracellular DNA. iii) DNA is released from the bacterium some time after cell death. iv) Extracellular DNA bound to hydroxyapatite or dentine is protected from spontaneous decomposition and enzymatic degradation.
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Mavropalias, Georgios. "Muscle damage and adaptations induced by eccentric cycling in relation to extracellular matrix." Thesis, Edith Cowan University, Research Online, Perth, Western Australia, 2020. https://ro.ecu.edu.au/theses/2336.
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Both the acute damage and resulting adaptations induced by eccentric exercise have been well documented, but less is known about their underpinning mechanisms. It is possible that muscle extracellular matrix (ECM), including both collagenous and costameric (e.g. integrins) components, plays a major role in both damage and adaptation, but this has not been extensively investigated. Eccentric cycling (ECC) was chosen as a model to investigate these mechanisms, since it consists only of eccentric muscle actions, and can induce typical characteristics of muscle damage such as prolonged decreases in muscle function and delayed onset muscle soreness (DOMS); while ECC training has been shown to induce increases in muscle function and muscle mass, even at low intensities. The purpose of this PhD project was to investigate the muscle damage and adaptations induced by the ECC in relation to collagen breakdown and integrin signalling pathways, which may shed light on the connection between ECM-mediated muscle changes.
This research project consisted of three studies. In Study 1, high- (HI) and low-intensity (LI) ECC with the same mechanical work were compared for changes in muscle function and DOMS, while the repeated bout effect was examined after subsequent HI ECC. Eleven men performed HI-ECC (1 min × 5 at 20% of peak power output: PPO) for two bouts separated by 2 weeks (H-H), and nine men performed LI-ECC (4 min × 5 at 5% PPO) for the first bout and then HI-ECC for the second bout (L-H). At 24 h after the first bout, both groups showed similar decreases in maximal isometric (70° knee angle, -10.6±11.8%) and isokinetic (-11.0±8.2%) knee extensor (KE) torque, and squat (-7.7±10.4%) and counter-movement (-5.9±8.4%) jump heights (P
In Study 2, plasma CK activity, hydroxyproline (Hyp) and cell-free DNA (cfDNA) concentrations were measured before, immediately after and 24 – 72 h after HI-ECC and LI-ECC, and examined in relation to changes in maximum voluntary isometric contraction strength (MVIC) and DOMS. The participants were the same as those in Study 1. Plasma CK activity increased at 24 h (135%), and Hyp concentration increased (40–53%, P
In Study 3, changes in the expression of integrin – integrin-linked kinase (ILK) – rapamycin-insensitive companion of mTOR (RICTOR) protein complex in VL before and after 8 weeks of HI-ECC training were examined along with possible associations to changes in muscle function and VL cross-sectional area (CSA) from pre- to post-training. Eleven young men completed 24 sessions of ECC with progressive increases in intensity and duration, resulting in a 2-fold increase in work from the first 3 sessions (75.4±14.1 kJ) to the last 3 sessions (150.7±28.4 kJ). Lower-limb lean mass (dual-energy X-ray absorptiometry), VL CSA (extended field-of-view ultrasonography), lower-limb static strength measured on the ergometer (breaking strength against the force provided by the crank pushing the foot), peak and average cycling power output were measured, and VL micro-biopsy samples were obtained before and after training. Significant (P
The findings from the three studies provide new insights into the mechanisms underlying muscle damage and adaptations induced by eccentric exercise. The Study 1 results showed that even low-intensity eccentric muscle actions affected the ECM, as indicated by DOMS, which has been shown by previous studies to be more associated with connective tissue damage and inflammation. ECC did not induce extensive muscle fibre damage, as indicated by the small increases in plasma CK activity. It is interesting that the increases in plasma Hyp concentration were similar between HI-ECC and LI-ECC, despite a significant difference in DOMS magnitude. ECC training increased integrin-ILK-RICTOR expression, but it was not associated with the increased CSA or muscle function. It seems possible that the ECM is involved in muscle damage and adaptations, but this was not endorsed by the present studies. It is necessary to investigate further histological changes in ECM after the initial and repeated bouts of eccentric exercise, how eccentric exercise induces ECM remodelling, and how this affects changes in muscle architecture including muscle CSA or mass and muscle function.
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Liu, Yang. "Transcriptioal [sic] and post-transcriptional regulation of extracellular enzyme production in Erwinia carotovora subsp. Carotovora /." free to MU campus, to others for purchase, 2000. http://wwwlib.umi.com/cr/mo/fullcit?p9999305.
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Tanos, Rita. "Développement de tests diagnostiques par détection d'ADN extracellulaire." Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTT054.
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Après sa découverte en 1948, l'ADN circulant (ADNcir) a été étudié dans divers domaines. Il est devenu un biomarqueur émergent, en particulier en oncologie, un domaine dans lequel plusieurs travaux ont récemment cherché à étudier son intérêt dans le dépistage et la détection précoce du cancer. La première partie de ma thèse a été consacrée à l’étude des caractéristiques quantitatives et structurelles de l’ADNcir, en prenant en compte son origine (ADNcir nucléaire et mitochondrial) et sa structure (fragmentation et profil de taille), pour le dépistage et la détection précoce du cancer. Deux paramètres, le Ref A 67 (concentration totale d’ADNcir nucléaire) et le MNR (Rapport entre la concentration de l’ADNcir mitochondrial et nucléaire), ont été quantifiés par q-PCR dans un modèle murin puis validés dans les milieux des cellules en culture pour évaluer leur potentiel à discriminer un état sain d’un état cancéreux. Ces deux paramètres ont été quantifiés chez l’Homme, en prenant en compte d'autres paramètres quantitatifs et structurels de l'ADNcir, après réajustement en fonction de l’âge, dans le plasma de 289 individus sains, 99 individus à risque de cancer colorectal (CCR) et 983 patients atteints de CCR (n = 791), de cancer du sein (n = 169) et d'autres cancers (hépatocellulaire, pancréatique, ovarien et lymphome) (n = 23). Par une approche d’apprentissage automatique, nous avons combiné ces différents paramètres dans un modèle de prédiction en utilisant des arbres de décision pour la classification des patients sains et cancéreux. Nous avons obtenu des résultats très encourageants, en particulier pour les cancers de stades précoces. Cette méthode semble prometteuse pour une détection précoce et non invasive du cancer. L'ajout d'autres biomarqueurs, comme le profil de taille ou le profil de méthylation de l'ADNcir, pourrait encore en augmenter le potentiel. La deuxième partie de ma thèse a été consacrée à l’étude de la relation entre la quantité d’ADN extracellulaire d’origine nucléaire et mitochondriale dans le milieu de culture d’embryons, et la qualité de ces embryons lors d’une fécondation in vitro (FIV). En effet, il a été montré qu’un embryon libère de l’ADN extracellulaire dans le milieu de culture lors d’une FIV, et que cet ADN pourrait être un biomarqueur prédictif de la qualité de l’embryon et servir comme test génétique préimplantatoire (PGT) non invasif. Nous avons détecté le gène SRY dans le milieu de culture afin de déterminer le sexe de l’embryon, ce qui constitue une information importante dans les cas des maladies génétiques liées au sexe. Nous avons également entrepris de détecter la présence de la mutation Delta F508 du gène CFTR responsable de la mucoviscidose par analyse de l’ADN extracellulaire issu d’embryons à risque, afin d’évaluer son potentiel en tant que PGT non invasifAfter its discovery in 1948, circulating DNA (cirDNA) was studied in various fields. It has become an emerging biomarker, particularly in oncology, and several studies have recently sought to investigate its interest in cancer screening and early detection. The first part of my thesis was devoted to the study of the quantitative and structural characteristics of cirDNA, taking into account its origin (nuclear and mitochondrial cirDNA) and its structure (fragmentation and size profile), for the screening and early detection of cancer. Two cirDNA parameters, the Ref A 67 (total nuclear cirDNA concentration) and the MNR (Mitochondrial to Nuclear Ratio), were quantified by q-PCR in a mouse model and further validated in cell culture media to assess their potential to discriminate between a healthy and a cancerous state. These two variables were evaluated by taking into account other quantitative and structural parameters of cirDNA, after age adjustment, in the plasma of 289 healthy individuals, 99 individuals at risk of colorectal cancer (CRC) and 983 patients with CRC (n = 791), breast cancer (n = 169) and other cancers (hepatocellular, pancreatic, ovarian and lymphoma) (n = 23). Through a machine learning approach, we combined these different parameters into a prediction model using decision trees for the classification of healthy and cancer patients. We have obtained very encouraging results, especially for early-stage cancers. This method seems promising for early and non-invasive cancer detection. The addition of other biomarkers such as the size profile of the cirDNA or the detection of methylation markers could further increase its potential.The second part of my thesis was devoted to the study of the relationship between the quantity of extracellular DNA of nuclear and mitochondrial origin in the embryo culture medium, and the quality of these embryos during in vitro fertilization (IVF). It has been shown that an embryo releases extracellular DNA into the culture medium during IVF, and that this DNA could be a predictive biomarker of embryo quality and thus be used as a non-invasive preimplantation genetic test (PGT). We detected, as well, the SRY gene in the culture medium to determine the sex of the embryo, which is an important information in the case of gender-related genetic disorders. We also tried to detect the presence of the Delta F508 mutation of the CFTR gene responsible for cystic fibrosis, by analyzing extracellular DNA from high-risk embryos to assess its potential as a non-invasive PGT
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Strachan, Sarah [Verfasser], and Dirk [Akademischer Betreuer] Reinhardt. "Investigating the biomarker potential of extracellular vesicle nucleic acids in cancer, and the role of extracellular vesicle DNA in cell-to-cell communication / Sarah Strachan ; Betreuer: Dirk Reinhardt." Duisburg, 2021. http://d-nb.info/1228270414/34.
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GRISTINA, Valerio. "Circulating cell-free DNA (cfDNA) and extracellular vesicles (EVs) as prognostic and predictive biomarkers in patients with advanced Non-Small Cell Lung Cancer (NSCLC): the LEXOVE prospective study." Doctoral thesis, Università degli Studi di Palermo, 2022. https://hdl.handle.net/10447/571952.
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Muthukrishnan, Uma. "The release of histone proteins from cells via extracellular vesicles." Licentiate thesis, Umeå universitet, Umeå centrum för molekylär medicin (UCMM), 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-147409.
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Histones are chromatin-associated proteins localized to the nucleus. However, extracellular histones are present in biofluids from healthy individuals and become elevated under disease conditions, such as neurodegeneration and cancer. Hence, extracellular histones may have important biological functions in healthy and diseased states, which are not understood. Histones have been reported in the proteomes of extracellular vesicles (EVs), including microvesicles and exosomes. The main aim of this thesis was to determine whether or not extracellular histones are secreted via EVs/exosomes. In an initial study (Paper I), I optimized methods for human embryonic kidney (HEK293) cell culture, transfection and protein detection using western blotting. In the main study (Paper II), I used oligodendrocyte cell lines (rat OLN-93 and mouse Oli-neu) to investigate the localization of histones to EVs. Western blotting of EVs purified from OLN-93 cell-conditioned media confirmed the presence of linker and core histones in them. Immunolocalization and transmission electron microscopy confirmed that histones are localized to EVs, as well as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). This suggests that histones are secreted via the MVB/exosome pathway. Localization of histones in EVs was investigated by biochemical/proteolytic degradation and purification followed by western blotting. Surprisingly, histones were associated with the membrane but not the luminal fraction. Overexpression of tagged histones in HEK293 cells confirmed their conserved, membrane localization. OLN-93 cell EVs contained both double stranded and single stranded DNA but nuclease and protease digestion showed that the association of histones and DNA with EVs was not interdependent. The abundance of histones in EVs was not affected by differentiation in Oli-neu cells. However, histone release was upregulated as an early response to cellular stress in OLN-93 cells and occurred before the release of markers of stress including heat shock proteins. Interestingly, a notable upregulation in secretion of small diameter (50-100 nm) EVs was observed following heat stress, suggesting that a sub-population of vesicles may be involved specifically in histone secretion in response to stress. Proteomic analyses identified the downregulation of endosomal sorting complex required for transport (ESCRT) as a possible mechanism underlying increased histone secretion. In Paper III, I developed methods to quantify extracellular histone proteins in human ascites samples from ovarian cancer patients. In summary, we show for the first time that membrane-associated histones are secreted via the MVB/exosome pathway. We demonstrate a novel pathway for extracellular histone release that may have a role in both health and disease.
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Ehrens, Alexandra [Verfasser]. "Eosinophil Extracellular DNA Trap Cell Death (EETosis) occurs as a life-cycle stage-specific response to filariae / Alexandra Ehrens." Bonn : Universitäts- und Landesbibliothek Bonn, 2020. http://d-nb.info/1239729839/34.
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Dubash, Adi Dara Burridge Keith. "Regulation of RhoA GTPase signaling by guanine nucleotide exchange factors in response to extracellular matrix adhesion and DNA damage." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2009. http://dc.lib.unc.edu/u?/etd,2407.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.Title from electronic title page (viewed Sep. 3, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell and Developmental Biology within the School of Medicine." Discipline: Cell and Developmental Biology; Department/School: Medicine.
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Apel, Falko [Verfasser], Arturo [Gutachter] Zychlinsky, Klaus [Gutachter] Osterrieder, and Benedikt [Gutachter] Beckmann. "Recognition of Neutrophil Extracellular Traps by the Cytosolic DNA Sensor cGAS / Falko Apel ; Gutachter: Arturo Zychlinsky, Klaus Osterrieder, Benedikt Beckmann." Berlin : Humboldt-Universität zu Berlin, 2019. http://d-nb.info/1182541429/34.
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Van, Andre P. "Characterisation of chromatin extracellular traps in rainbow trout (Oncorhynchus mykiss)." Thesis, University of Stirling, 2018. http://hdl.handle.net/1893/27930.
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One of the greatest challenges in finfish aquaculture is combating losses caused by infectious bacterial diseases, and a better understanding of the interactions between the host immune system and pathogens is essential for developing new methods to manage infections and outbreaks. Extracellular traps (ETs) are decondensed nuclear chromatin released by neutrophils into the extracellular matrix that can ensnare and kill microbes. Since the discovery of ETs in humans, these innate immune effectors have been characterised across the animal kingdom, including in some fish species, though their existence the salmonids has yet to be confirmed. Therefore, the aim of this thesis was to confirm and characterise the release of ETs in the rainbow trout (Oncorhynchus mykiss) and investigate the interaction of these structures with fish pathogenic bacteria. To do this, a triple-layer Percoll gradient technique was employed to give highly enriched cell suspensions of polymorphonuclear cells (PMNs) derived from head-kidney tissue preparations. Treatment of PMN-enriched cell suspensions with the nucleic-acid-specific stain, SYTOX Green, revealed the presence of ET-like structures that had been released without stimulation. These ET-like structures were confirmed by immunostaining techniques to contain the diagnostic proteinaceous markers of ETs: neutrophil elastase, myeloperoxidase and the H2A histone. Previously characterised inhibitors and inducers of ET release from phagocytic immune cells in other animals confirmed that calcium ionophore (CaI), flagellin, and cytochalasin D shared similar activities for ET-release by rainbow trout PMNs. However, interestingly, as the common ET-inducer phorbol-myristate acetate (PMA) and ET-inhibitor diphenyleneiodonium (DPI) did not exert their expected potency in ET release assays with the PMNs, perhaps indicating that these fish cells are less dependent on NADPH oxidase signalling for ET release compared to mammals and most invertebrate species. The PMN-derived ETs were demonstrated to bind to and trap the extracellular nuclease-deficient bacterial fish pathogen, Vibrio anguillarum (Vib 87) when co-cultured. Finally, extracellular nuclease activity produced by a V. anguillarum isolate (Vib 6) during culture was able to degrade ETs released by rainbow trout PMNs in a dose-dependent manner. Moreover, viable colony counts, fluorescent and phase contrast microscopy demonstrated that V. anguillarum Vib 6 eluded trapping by ETs, while an extracellular nuclease-deficient isolate did not. These observations are consistent with the suggestion that nucleases are a microbial virulence factor during host infection. Confirming the existence and antimicrobial potential of extracellular traps released by rainbow trout PMNs may provide a platform towards the development of novel therapeutics to reduce mortalities in finfish aquaculture caused by infectious microbial pathogens.
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Susin, Michelle Fernanda. "Análise funcional das proteínas HrcA, GroES/GroEL e DnaK/DnaJ em Caulobacter crescentus." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-14062016-171416/.
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O operon groESL de C. crescentus apresenta dupla regulação. A indução deste operon por choque térmico é dependente do fator sigma de choque térmico σ32. A temperaturas fisiológicas, a expressão de groESL apresenta regulação temporal durante o ciclo celular da bactéria e o controle envolve a proteína repressora HrcA e o elemento CIRCE (controlling inverted repeat of chaperonin expression). Para estudar a atividade da proteína repressora in vitro, produzimos e purificamos de E. coli a HrcA de C. creseentus contendo uma cauda de histidinas e a ligação especifica ao elemento CIRCE foi analisada em ensaios de migração retardada em gel de poliacrilamida (EMRGP). A quantidade de DNA retardada pela ligação a HrcA aumentou significativamente na presença de GroES/GroEL, sugerindo que estas proteínas modulam a atividade de HrcA. Corroboração desta modulação foi obtida analisando fusões de transcrição da região regulatória de groESL com o gene lacZ, em células de C. crescentus produzindo diferentes quantidades de GroES/EL. HrcA contendo as substituições Pro81 AJa e Arg87Ala, aminoácidos que se localizam no domínio putativo de ligação ao DNA da proteína, mostraram ser deficientes na ligação a CIRCE, tanto in vitro como in vivo. Em adição, HrcA Ser56Ala expressa na mesma célula juntamente com a proteína selvagem produziu um fenótipo dominante-negativo, indicando que a HrcA de C. crescentus liga-se a CIRCE como um oligômero, provavelmente um dímero. As tentativas de obtenção de mutantes nulos para os genes groESL ou dnaKJ falharam, indicando que as proteínas GroES/GroEL e DnaK/DnaJ são essenciais em C. crescentus, mesmo a temperaturas normais. Foram então construídas no laboratório as linhagens mutantes condicionais SG300 e SG400 de C. crescentus, onde a expressão de groESL e de dnaKJ, respectivamente, está sob controle de um promotor induzido por xilose (PxyIX). Estas linhagens foram caracterizadas quanto á sua morfologia em condições permissivas ou restritivas, assim como quanto à capacidade de sobrevivência frente a vários tipos de estresse. As células da linhagem SG300, exauridas de GroES/GroEL, são resistentes ao choque térmico a 42°C e são capazes de adquirir alguma termotolerância. Entretanto, estas células são sensíveis aos estresses oxidativo, salino e osmótico. As células da linhagem SG400, exauridas de DnaKlJ, são sensíveis ao choque térmico, à exposição a etanol e ao congelamento, e são incapazes de adquirir termotolerância. Além disso, tanto as células exauridas de GroES/GroEL quanto as exauridas de DnaK/DnaJ apresentam problemas na sua morfologia. As células de SG300 exauridas de GroES/GroEL formam filamentos longos que possuem constrições fundas e irregulares. As células de SG400 exauridas de DnaK/DnaJ são apenas um pouco mais alongadas que as células pré-divisionais selvagens e a maioria das células não possuem septo. Estas observações indicam bloqueio da divisão celular, que deve ocorrer em diferentes estágios em cada linhagem.In Caulobacter crescentus, the groESL operon presents a dual type of control. Heat shock induction of the operon is dependent on the heat shock sigma factor σ-32. At physiological temperatures, groESL expression is cell cycle regulated and the control involves the repressor protein HrcA and the element CIRCE (controlling inverted repeat of chaperonin ~xpression). To study the activity of HrcA in vitro, we produced and purified from E. coli a histidine-tagged version of the protein, and specific binding to the CIRCE element was analyzed in electrophoretic mobility shift assays (EMSA). The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that these proteins modulate HrcA activity. Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C. crescentus cells producing different amounts of GroES/GroEL. The mutants proteins HrcA Pro81Ala and HrcA Arg87Ala, that contain amino acid substitutions in the putative DNA-bindíng domain of the protein, were found to be deficient in binding to CIRCE in vitro and in vivo. Furthermore, HrcA Ser56Ala expressed together with the wild type protein within the same cell, produced a dominant-negative phenotype, indicating that C. crescentus HrcA binds to CIRCE in an oligomeric form, most likely as a dimer. Attempts to obtain null mutants for groESL or dnaKJ were unsuccessful indicating the importance of GroES/GroEL and DnaK/lDnaJ to the survival of C. crescentus cells. Conditional mutants were then constructed in our laboratory in which groESL and dnaKJ expression is under the control ofaxylose inducible promoter (PxyIX) , giving rise to strains SG300 and SG400, respectively. These strains were characterized in regard to their morphology under permissive and restrictive conditions, as well as their viability under different types of environmental stresses. SG300 cells depleted of GroES/GroEL are resistant to heat shock at 42°C and can acquire some thermotolerance, but they are sensitive to oxidative, saline and osmotic stresses. SG400 cells depleted of DnaKlJ are quite sensitive to heat shock, ethanol and freezing, and are unable of acquiring thermotolerance. Cells depleted of either GroES/EL or DnaKlJ also present morphological problems. SG300 cells depleted of GroES/EL form long and pinched filaments. SG400 cells depleted of DnaKlJ are only somewhat more elongated than wild-type predivisional cells and most cells do not present septum. These observations indicate a cell division arrest, which should occur at different stages in each strain.
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Shi, Chongxu [Verfasser], and Hans-Joachim [Akademischer Betreuer] Anders. "Extracellular DNA contributes to cholesterol crystal embolism-induced clot formation, acute kidney injury, and tissue infarction / Chongxu Shi ; Betreuer: Hans-Joachim Anders." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2021. http://d-nb.info/1233600680/34.
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Sun, Wenchao. "DNA Nanoparticles for Non-viral Gene Therapy: Mechanistic Studies and Targeting." Case Western Reserve University School of Graduate Studies / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=case1327614453.
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Cui, Kui, Christopher Ardell, Nataly Podolnikova, and Valentin Yakubenko. "The prevention of αDβ2-mediated macrophage adhesion to inflamed extracellular matrix thwarts macrophage retention during chronic inflammation." Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/asrf/2019/schedule/210.
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Chronic inflammation is a triggering mechanism for many metabolic diseases including atherosclerosis and diabetes. A critical step in the development of chronic inflammation is the accumulation of classically activated pro-inflammatory macrophages in the extracellular matrix (ECM) of peripheral tissues.
Recently, we demonstrated that adhesion receptor integrin αDβ2 is upregulated on macrophages in atherosclerotic lesions and inflamed adipose tissue, and promotes the development of atherosclerosis and insulin resistance. This pathophysiological mechanism is mediated by αDβ2-dependent strong adhesion of macrophages to the inflamed ECM, which promotes macrophage retention at the site of inflammation. Typical healthy ECM has a limited ligand capacity for integrin αDβ2. However, we recently found that the end-product of DHA oxidation, 2-(ω-carboxyethyl)pyrrole (CEP) serves as ligand for αDβ2. CEP is preferentially generated during inflammation-mediated oxidation and forms adduct with ECM proteins. CEP-modified proteins are detected in inflamed tissue during atherosclerosis, insulin resistance and pathological angiogenesis.
In this project, we propose a new strategy for the treatment of chronic inflammation by targeting macrophage retention in the inflamed tissue by focusing on the development of the inhibitor, which is exclusively specific for αDβ2-CEP interaction. The advantage of CEP as a new therapeutic target resides in its unique formation in inflamed tissue.
Using specially designed peptide library, protein-protein interaction measured by Biacore and adhesion assay with integrin-transfected HEK293 cells, we identified a sequence (called P5-peptide), which significantly inhibited αD-CEP binding. In vitro three-dimensional migration assay demonstrated that P5 peptide regulates macrophage migration within ECM but not the transendothelial migration of monocytes. The injection of cyclic P5 peptide in the model of thioglycollate-induced peritoneal inflammation led to 3-fold reduction in the number of macrophages accumulated in the peritoneal cavity after 72 hours. Interestingly, P5 peptide injection had no effect on the accumulation of macrophages in αD-deficient mice, that confirmed the specificity of inhibition. This inhibition only affects the recruitment of macrophages, while it has no effect on the efflux of macrophage from the peritoneal cavity in our in vivo studies. The tracking of adoptively transferred fluorescently-labeled WT and αD-/- monocytes in mice on a high fat diet revealed that αD-deficiency reduced 3 folds the accumulation of macrophages in the adipose tissue. The injection of P5 peptide in this model demonstrated the marked reduction of adoptively transferred WT macrophages in adipose tissue.
Taken together, these results demonstrate the importance of αDβ2-CEP interaction for the accumulation of infiltrating macrophages during inflammation and propose P5 peptide as a potential inhibitor of atherogenesis and diabetes. Further studies are required to develop these results.
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Binnenkade, Lucas [Verfasser], and Kai [Akademischer Betreuer] Thormann. "Molecular Control of Extracellular DNA Release and Degradation in Shewanella oneidensis MR-1 Biofilms: The Role of Phages and Nucleases / Lucas Binnenkade. Betreuer: Kai Thormann." Marburg : Philipps-Universität Marburg, 2015. http://d-nb.info/1080299068/34.
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Schuster, Anna-Kathrin [Verfasser], Ulrich [Akademischer Betreuer] Szewzyk, Ulrich [Gutachter] Szewzyk, and Isolde [Gutachter] Röske. "Production of extracellular DNA (eDNA) of the γ-Proteobacterium Rheinheimera sp. F8 in biofilms / Anna-Kathrin Schuster ; Gutachter: Ulrich Szewzyk, Isolde Röske ; Betreuer: Ulrich Szewzyk." Berlin : Technische Universität Berlin, 2017. http://d-nb.info/1156184126/34.
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Koenig, Michael J. "LKB1 Loss in Lung Adenocarcinoma." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1555503848137523.
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Keup, Corinna [Verfasser], and Sabine [Akademischer Betreuer] Kasimir-Bauer. "Comprehensive molecular characterization of circulating tumor cells, extracellular vesicles and cell-free DNA as matched multi-parametric liquid biopsy for therapy management in metastatic breast cancer patients / Corinna Keup ; Betreuer: Sabine Kasimir-Bauer." Duisburg, 2020. http://d-nb.info/1221960350/34.
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Vaubourg, Camille. "Développement de nouvelles approches thérapeutiques pour la dystrophie musculaire de Duchenne, basées sur l'utilisation de vésicules extracellulaires Minimal Consequences of CMAH and DBA/2J Background on a FKRP Deficient Model." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL012.
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La dystrophie musculaire de Duchenne est une maladie génétique rare causée par des mutations au sein du gène DMD, codant pour la dystrophine qui affecte les muscles squelettiques, cardiaque et respiratoires. Les patients présentent un phénotype dystrophique réduisant leur mobilité et leur espérance de vie. A l'heure actuelle, aucun traitement curatif n'est disponible pour l'ensemble des patients. Ce travail de thèse a visé à développer de nouvelles approches thérapeutiques basées sur l'utilisation de vésicules extracellulaires (VE), des particules de taille variant entre 40 et 500 nm, sécrétées par la majorité des types de cellules, qui jouent un rôle dans la communication intercellulaire par transfert de biomolécules. Elles sont en parallèle de plus en plus étudiées comme nouveau système de vectorisation non-viral de molécules thérapeutiques car elles présentent de nombreux avantages : une biocompatibilité, une faible immunogénicité, une capacité à moduler des voies de signalisation, la possibilité de les charger en molécules thérapeutiques de différentes natures ou la possibilité de modifier leur tropisme tissulaire. Au cours de ce travail de thèse, nous nous sommes intéressés à la vectorisation, par les VE de cellules souches mésenchymateuses (MSCs), de deux molécules agissant sur deux voies majeures du processus dystrophique (Smad7 et des siARN contre NF-kB) mais aussi de la nucléase du système CRISPR/Cas9 pour agir sur l'expression de la dystrophine. Avant le développement de sa vectorisation par les VE, le potentiel thérapeutique de Smad7 a été évalué par transfert de gène par vecteur AAV. Une augmentation de la masse musculaire sans réduction du niveau de fibrose a été observée. En parallèle, les protocoles de production, isolement et caractérisation des VE ont été définis et validés. Les VE de MSCs sur le processus dystrophique a été évalué et a montré un effet sur les éléments régulateurs de type microARN. Egalement, le chargement de siARN dans les VE a été initié et celui de l'endonucléase spCas9 a été validé. Le transfert in vitro de cette dernière a été validé, avec toutefois une fonctionnalité qui reste à prouverDuchenne muscular dystrophy is a rare genetic disease caused by mutations in the DMD gene, coding for dystrophin, affecting skeletal, cardiac and respiratory muscles. Patients show a dystrophic phenotype reducing their mobility and life span. Today, no cure is available for all patients. This thesis work aimed to develop new therapeutic approaches based on the use of extracellular vesicles (EVs), particles of 40-500 nm of diameter secreted by the majority of cell types and playing a role in intercellular communication by transferring biomolecules. The EVs are more and more studied as new non-viral delivery system of therapeutic molecules as they present a number of advantages: biocompatibility, low immunogenicity, ability to modulate some signalling pathways, the possibility to load them with different kind of molecules and to target them to tissues of interest. In this work, we were interested in the vectorization, by mesenchymal stem cells (MSCs)-derived EVs, of two molecules acting on main pathways of the dystrophic process (Smad7 and siRNA against NF-kB) as well as the nuclease of the CRISPR/Cas9 system for acting on dystrophin expression. Before the development of Smad7 vectorization by EVs, its therapeutic potential have been evaluated by AAV gene transfer. An increase of the muscle mass without any reduction of the fibrosis level has been observed. In parallel, EVs production, isolation and characterization protocols have been set up and validated. The effect of MSCs-derived EVs on dystrophic process have been assessed and showed an impact on regulatory elements as microRNA. Also, the loading of siRNA in EVs have been initiated and the one of the spCas9 have been validated. The in vitro transfer of the last one have also been validated, but its functionality has to be confirmed
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Malmlöf, Maria. "Mdm2 phosphorylations : characterization and applications /." Stockholm : Karolinska institutet, 2007. http://diss.kib.ki.se/2007/978-91-7357-265-1/.
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Al-Maalouf, Samar Wadih. "Exploration of a mammary epithelial cell model for the study of epithelial inflammation and mechanisms of anti-inflammatory activity in medicinal plants." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1166806742.
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Bustos, Silvina Odete. "Resposta celular associada à expressão de galectina-3 em linhagens de melanoma expostas a irradiação." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-21052014-110608/.
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O câncer de pele é um dos mais frequentes entre humanos, sendo o melanoma o tipo menos comum, mas com grande importância devido à agressividade que ele apresenta. Um dos principais agentes etiológicos deste tipo de tumor é a radiação ultravioleta proveniente da luz solar. A fração de radiação ultravioleta B (UVB) gera dano no DNA e induz alterações nas células da pele após a exposição prolongada e sem proteção. A resposta à luz UVB em melanócitos e melanomas é diferente, mostrando a importância do perfil celular. O efeito genotóxico da luz UVB pode alterar a expressão de moléculas como galectina-3 e MAPKs, desencadeando respostas UVB-dependentes. Galectina-3 é uma lectina que reconhece beta-galactosídeos e está envolvida na regulação de diversos processos celulares que modificam a viabilidade celular e a proliferação. Esta molécula é ubiquamente expressa apresentando um comportamento específico dependendo da sua localização subcelular. No presente trabalho mostramos que a distribuição de galectina-3 em melanoma e melanócitos é ampla, encontrando-se tanto no núcleo como no citoplasma, podendo ser modificada após irradiação UVB ou ainda secretada para o meio extracelular. Além disso, observamos que a luz UVB ativa a via de MAPKs, proteínas quinases ativadas por mitógenos envolvidas no crescimento, sobrevivência, diferenciação e resposta a estresse, em melanócitos e em melanomas poucos minutos após a exposição à UVB. Uma maior atividade de p38 e de ERK é evidenciada em melanomas, enquanto que em melanócitos a via de p38 é a mais ativa, corroborando a noção de que a resposta celular à luz UVB difere entre melanócitos e melanoma. As moléculas p38 e JNK são proteínas quinases ativada pelo estresse (SAPK). A via de JNK não é tão responsiva em alguns melanomas, mas ativação desta molécula parece estar envolvida com a sobrevivência celular e a translocação mitocondrial após UVB. Em adição, a inibição de JNK leva ao aumento de morte celular em linhagens melanocíticas irradiadas e não irradiadas, e em melanoma induz morte e aumenta autofagia após irradiação. Esta molécula parece interagir com galectina-3 em modelos murinos, mas não em melanomas humanos, enquanto que ERK interage fisicamente com galectina-3 em melanócitos e melanomas humanos, independente de UVB. Através do silenciamento de galectina-3 pela técnica de RNA de interferência, mostramos o aumento da ativação da via de ERK após irradiação e de proteínas downstream de ERK, promovendo a proliferação celular em melanomas nessas condições. Em melanócitos parece existir uma regulação negativa da via de ERK por galectina-3 acompanhada de uma diminuição da viabilidade celular após o silenciamento dessa lectina, independente de UVB. Estes resultados mostram que galectina-3 é uma importante reguladora de eventos associados com sobrevivência e morte celular em melanoma. Por outro lado, em melanomas a ausência de galectina-3 induz aumento da proliferação associada à ativação de ERK, evidenciando a importância do tipo celular na ação de galectina-3Skin cancer is the most common cancer among humans, melanoma being the least common type but very important due to its aggressive behavior. A major etiologic agent of this type of tumor is ultraviolet radiation from the sunlight. The ultraviolet B rays (UVB) cause DNA damage and induce alterations over the skin cells after prolonged exposition without protection. The UVB response in melanocytes and melanoma cells is different. This shows the importance of the cellular profile. The genotoxic effect of UVB light can alter the expression of molecules such as galectine-3 and MAPKs and also triggers multiple responses UVB-dependent. Galectin-3 is a lectin that recognizes beta-galactosides. It is involved in the regulation of many cellular processes that modify cellular viability and proliferation and presents specific behavior depending on its subcellular localization. In the present study we showed that galectine-3 distribution in melanoma cells and melanocytes is large, lying both in the nucleus and in the cytoplasm. After UVB irradiation this distribution could be modified or even galactine-3 secreted itself into the extracellular space. Moreover, we observed that UVB light activates the mitogen-activated protein kinase pathway (MAPK) involved in growth, survival, differentiation and stress-response in melanocytes and in melanoma cells just a few minutes after exposure. An increased activity of p38 and ERK was observed in melanomas, while in melanocytes just p38 pathway was highly active, supporting the notion that the cellular response to UVB light differs between melanocytes and melanoma cells. The molecules p38 and JNK are stress-activated protein kinases (SAPK). The JNK pathway is not responsive in some melanoma cells, but the activation of this molecule appears to be involved in cell survival and mitochondrial translocation after being exposed to UVB. Inhibition of JNK leads to increased cell death in irradiated and non-irradiated melanocytic lineage, but in melanoma cells induces cell death and increased autophagy only after irradiation. This molecule seems to interact with galectin-3 in mouse models but not in human melanomas, whereas ERK physically interacts with galectin-3 in human melanocytes and melanoma cells, regardless of UVB exposure. Through the knockdown of galectin-3 by siRNA, we showed increased activation of the ERK and its downstream pathway after irradiation, thus inducing cell proliferation. In melanocytes seems to be a negative regulation of the ERK pathway by galectin-3 accompanied by a decrease in cell viability after its knockdown regardless of UVB exposure. These results show that galectin-3 is an important regulatory molecule of events associated with cell death and survival in melanoma, which has different behavior depending on the cell type
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PIETRAMELLARA, GIACOMO. "Fenomeni di scambio genetico nel suolo: Interazioni tra DNA batterico extracellulare e minerali argillosi." Doctoral thesis, 1996. http://hdl.handle.net/2158/676453.
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Studio sulle interazioni tra il DNA extracellulare ed i minerali argillosi e loro conseguenze sull'efficienza biologica del DNA nei fenomeni di trasformazione. Lo studio ha preso in considerazione anche problematiche metodologiche legate alla determinazione dei valori di adsorbimento del DNA sui minerali argillosi
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Mantilla, Calderon David. "Antibiotic resistance genes and antibiotic resistant bacteria as emerging contaminants in wastewater: fate and persistence in engineered and natural environments." Diss., 2018. http://hdl.handle.net/10754/631716.
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The emergence and rapid spread of antimicrobial resistance (AMR) is a phenomenon that extends beyond clinical settings. AMR has been detected in multiple environmental compartments, including agricultural soils and water bodies impacted by wastewater discharges. The purpose of this research project was to evaluate what factors could influence the environmental persistence of antibiotic resistance genes (ARGs), as well as to identify potential strategies employed by human pathogens to survive in secondary environment outside the host.
The first part of this dissertation describes the incidence of the New Delhi metallobeta lactamase gene (blaNDM-1) – an ARG conferring resistance to last resort antibiotics – in the influent of a wastewater treatment facility processing municipal wastewater from Jeddah, Saudi Arabia. Detection of blaNDM-1 was followed by the isolation of a multi-drug resistant strain of E. coli (denoted as strain PI7) at a frequency of ca. 3 x 104 CFU/m3 in the untreated municipal wastewater.
Subsequently, we described the decay kinetics of E. coli PI7 in microcosm experiments simulating biological treatment units of wastewater treatment plants. We identified that transition to dormancy is the main strategy prolonging the persistence of E. coli PI7 in the microcosm experiments. Additionally, we observed slower decay of E. coli PI7 and prolonged stability of extracellular DNA in anoxic/anaerobic conditions. In the last chapter of this thesis, the fate of extracellular DNA is further explored. Using as a model Acinetobacter baylyi ADP1, we describe the stimulation of natural transformation frequencies in the presence of chlorination disinfection byproducts (DBPs). Moreover, we demonstrate the ability of BAA to stimulate transformation is associated with its capacity to cause DNA damage via oxidative stress.
Overall, this dissertation addresses important knowledge gaps in our current understanding of ARB and extracellular ARG persistence in the environment. The results from this project highlight the importance of retrofitting the existing water treatment process with advance membrane filtration units, and the need to relook into the current disinfection strategies. Wastewater treatment technologies should be assessed for their efficacies in not only inactivating ARB and ARGs, but also whether unintended consequences such as stimulated horizontal gene transfer would occur.
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Beauchemin, Karine. "Dissection moléculaire de l’interaction de la DNA topoisomérase I avec la matrice extracellulaire et les fibroblastes." Thèse, 2009. http://hdl.handle.net/1866/3202.
Full textAbstract:
La sclérose systémique est une maladie autoimmune dont l’une des complications majeures est la fibrose. La DNA topoisomérase I (topo) est l’un des principaux autoantigènes associés à cette maladie. Toutefois, aucun lien n’a encore pu être établi entre la présence des anti-topo et le développement de la fibrose.
Les travaux antérieurs du laboratoire d’accueil ont montré une interaction directe de la topo avec la surface des fibroblastes et la matrice extracellulaire. Nous avons voulu caractériser ces interactions du point de vue moléculaire. La topo a donc été exprimée sous forme de 5 fragments, déterminés à partir de ses principaux domaines structuraux et de ses épitopes majeurs, chez E. coli. Les fragments purifiés ont été analysés pour leur interaction avec l’héparine, représentant les héparane sulfates de la surface des fibroblastes, et avec des protéines purifiées de la matrice extracellulaire.
Nous avons montré que le fragment topo-N est le principal responsable de l’interaction avec l’héparine, ce qui suggère donc l’implication potentielle de ce domaine dans l’interaction de la topo avec la surface des fibroblastes. Le fragment topo-DIDII est responsable de l’interaction avec la plupart des protéines de la matrice extracellulaire étudiées, alors que le fragment topo-H15 n’interagit qu’avec la vitronectine. Aucune interaction des fragments topo-DIII et topo-C n’a été décelée.
Ces résultats pourront maintenant servir à mieux comprendre le rôle potentiel de la topo et des autoanticorps circulants anti-topo dans la fibrose présente chez les personnes atteintes de sclérose systémique en contribuant à l’identification de la cible de la topo sur les fibroblastes.Systemic sclerosis is an autoimmune disease in which one of the major complications is fibrosis. DNA topoisomerase I (topo) is a major autoantigen associated with this disease. However, no link has yet been established between the presence of anti-topo and the development of fibrosis. Previous work of the host laboratory showed a direct interaction of the topo with the surface of fibroblasts and extracellular matrix. We wanted to characterize these interactions at the molecular level. Topo was expressed in 5 fragments, determined from its main structural domains and its major epitopes, in E. coli. The purified fragments were analyzed for their interaction with heparin, representing heparan sulfates on the surface of fibroblasts, and with purified proteins of the extracellular matrix. We have shown that the topo-N fragment is responsible for interaction with heparin, suggesting hence, potential involvement of this domain in the interaction of topo with the surface of fibroblasts. The topo-DIDII fragment is responsible for the interaction with most proteins of the extracellular matrix studied, whereas the topo-H15 fragment only binds to vitronectin. No interaction of fragments topo-DIII and topo-C was found. These results can now be used to better understand the potential role of topo and circulating anti-topo autoantibodies in the fibrosis present in patients with systemic sclerosis in helping to identify the target of topo on fibroblasts.
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LIOU, HENG-YU, and 劉姮妤. "The RNA Contents Of Extracellular Vesicles In HeLa Cells Treated With Polyethyleneimine-DNA Complexes." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/s4q68g.
Full textAbstract:
碩士嘉南藥理大學
藥學系
105
Poly(ethylenimine) (PEI) is one of the most intensively used cationic polymers in non-viral nucleic acid delivery. Despite of great potentials of the applications in gene delivery systems, PEI has been shown to influence the regulations of gene expression in vitro and in vivo. Previous studies on PEI-induced gene regulation have primarily been based on encoding mRNAs that are translated into proteins. However, recent studies have demonstrated that non-coding RNAs are closely related to complex cellular development systems and various human diseases. Among these non-coding RNAs, microRNAs (miRNAs) are approximately 22 nucleotides long and primarily play important roles in the post-transcriptional regulation of gene expression, making them potential targets for therapeutic applications. Therefore, we explored the regulated miRNAs and identified their target genes in PEI-treated mouse embryonic fibroblast cells. The pathway analysis of target genes was performed using DIANA miRPath v.3.0, which is based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Our study may provide a deeper insight into the molecular pathways in cells caused by PEI.
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Madeira, Catarina Alexandra Catanas. "Activity of the major autolysin of Staphylococcus aureus Atl in the presence of extracellular DNA." Master's thesis, 2018. http://hdl.handle.net/10362/53142.
Full text
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"Discovery of an extracellular stress sensory protein in Beauveria bassiana and identification of photolyase encoding phr-1 sequences in five entomopathogenic fungi." Thesis, 2013. http://hdl.handle.net/10388/ETD-2013-08-1210.
Full textAbstract:
Entomopathogenic fungi, including Beauveria bassiana are being developed as an alternative to chemical insecticides. Their effectiveness can be enhanced through understanding of the mechanisms of response to environmental stresses and conditions. An aspect of repair of ultraviolet radiation induced DNA damage and response to high temperature were studied here. A region of the photolyase gene (phr-1), encoding cyclobutane pyrimidine dimer photolyase (CPD-PHR), an enzyme pivotal to DNA repair, was cloned, sequenced and identified for species of the genera Beauveria, Isaria, Lecanicillium, Metarhizium and Tolypocladium. The DNA and deduced amino acid sequences were analysed using several in silico methods and annotated for functionality. The data suggested that the DNA encoded a protein with conserved residues known in CPD-PHR function, which had structural homology with other CPD-PHRs and molecular phylogeny that was generally consistent amongst this group of fungi. These results are the first for a phr-1 from the genera Isaria, Lecanicillium and Tolypocladium.
In bacteria and yeasts, tolerance to environmental stress was shown to be aided through an inducible phenomenon that involves extracellular sensory component (ESC) proteins in Escherichia coli, which have yet to be purified or sequenced. The presence of an ESC-like factor (ELF) was examined in cell-free filtrate (CFF) from B. bassiana cultures. It was revealed that the tolerance of conidiospores and blastospores (BS) to ultraviolet radiation or heat could be increased by preheated CFF, respectively, but not after pretreating the CFF with trypsin. Several novel polyacrylamide-based in situ and binding bioassays were developed to screen for and characterize ELF candidate (EC) proteins. Two were detected (EC1 and EC2) and EC1 was found to interact with BS, while bioassays with purified ECs showed that EC1 could increase heat tolerance in BS.
De novo peptide sequencing revealed that the ECs were the same protein, but differed by EC1 being glycosylated. An expressed sequence tag from B. bassiana, encoding six peptides that were also found in the ECs, was identified in the public data base. This sequence information was exploited to amplify the remaining coding regions of the suspected ELF gene (elf) using polymerase chain reactions. Through this a 741 nucleotide open reading frame was cloned and sequenced. Structure-function analyses of the amino acid sequence encoded by the open reading frame revealed features that were consistent with the ECs, such as eight shared peptides, its nascent derived size (26 kDa), potential glycosylation sites and secretion signal peptide. In addition, other features such as the high proportion of cysteine residues and internal amino acid repeats will be discussed.
The elf gene was inserted into an expression vector and introduced into the methylotrophic yeast Pichia pastoris for its controlled over-expression. Heterologously expressed ELF conferred elevated tolerance to heat in BS to similar levels produced by ELF synthesized by B. bassiana. Several functional and molecular features of the ELF system have certain commonalities with many agonist-receptor systems involved in signal transduction, but remain to be detailed. This is the first report of the cloning and functional analyses of elf and ELF, respectively, from any organism.
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Medapati, Manoj Reddy. "Extracellular S100A4 induces human thyroid cancer cell migration." 2013. http://hdl.handle.net/1993/22120.
Full textAbstract:
Human thyroid cancer is the most commonly occurring cancer of the endocrine gland having good survival rate, but some patients show recurrence with an invasive phenotype and treatment failures. The mechanisms behind this invasive phenotype are not well understood in TC. Previously our group has identified a pro-migratory role of relaxin-like peptides in thyroid cancer that is mediated by S100A4. We have observed in human TC cells that extracellular S100A4 induces migration and activates ERK1/2, JNK/SAPK and NFkB signaling pathways. Employing immunohistochemistry and immunofluorescence we have identified the expression of RAGE in human TC primary cells, cell lines, and in tumor tissues but not in normal thyroid tissues. We showed that S100A4 binds to RAGE in TC cells and that RAGE and its cytoplasmic partner Dia-1 mediate the S100A4-induced migration of TC cells. This study identified a crucial role of RAGE in TC cell migration induced by S100A4.
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Hsu, Chi Yu, and 徐啟育. "Vancomycin Promotes the Bacterial Autolysis, Release of Extracellular DNA, and Biofilm Formation in Vancomycin-non-Susceptible Staphylococcus aureus." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/16108511334120609795.
Full textAbstract:
碩士長庚大學
醫學生物技術暨檢驗學系
100
Staphylococcus aureus, an important human pathogen, is particularly adept at producing biofilms on implanted medical devices. Although antibiotic treatment of non-susceptible bacteria will not kill these strains, the consequences should be studied. The present study focuses on investigating the effect of vancomycin on biofilm formation by vancomycin-non-susceptible S. aureus. Biofilm adherence assays and scanning electron microscopy demonstrated that biofilm formation was significantly enhanced following vancomycin treatment. Bacterial autolysis of some subpopulations was observed and was confirmed by the live/dead staining and confocal laser scanning microscopy (CLSM). A significant increase in polysaccharide intercellular adhesin (PIA) production was observed by measuring icaA transcript levels and in a semi-quantitative PIA assay in one resistant strain. We show that the release of extracellular DNA (eDNA) via cidA-mediated autolysis is a major contributor to vancomycin-enhanced biofilm formation. The addition of xenogeneic DNA could also significantly enhance biofilm formation by a PIA-overproducing S. aureus strain. The magnitude of the development of the biofilm depends on the balance between the amounts of eDNA and PIA. In conclusion, sublethal doses of cell wall-active antibiotics like vancomycin induce biofilm formation through an autolysis-dependent mechanism in vancomycin-non-susceptible S. aureus.
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Sanchez, Torres Viviana. "Escherichia coli Enhanced Hydrogen Production, Genome-wide Screening for Extracellular DNA, and Influence of GGDEF Proteins on Early Biofilm Formation." Thesis, 2010. http://hdl.handle.net/1969.1/ETD-TAMU-2010-12-8889.
Full textAbstract:
Escherichia coli is the best characterized bacterium; it grows rapidly, and it is easy to
manipulate genetically. An increased knowledge about the physiology of this model organism
will facilitate the development of engineered E.coli strains for applications such as production of
biofuels and biofilm control. The aims of this work were the application of protein engineering
to increase E. coli hydrogen production, the identification of the proteins regulating extracellular
DNA production (eDNA), and the evaluation of the effect of the proteins synthesizing the signal
3'-5'-cyclic diguanylic acid (c-di-GMP) on biofilm formation.
The Escherichia coli hydrogen production rate was increased 9 fold through random
mutagenesis of fhlA. Variant FhlA133 (Q11H, L14V, Y177F, K245R, M288K, and I342F)
enhances hydrogen production by increasing transcription of the four transcriptional units
regulated by FhlA. The amino acid replacements E363G and L14G in FhlA increased hydrogen
production 6 fold and 4 fold, respectively.
The complete E. coli genome was screened to identify proteins that affect eDNA
production. The nlpI, yfeC, and rna mutants increased eDNA production and the hns and rfaD
mutants decreased eDNA production. Deletion of nlpI increases eDNA 3 fold while
overexpression of nlpI decreases eDNA 16 fold. Global regulator H-NS is required for eDNA with E. coli since deletion of hns abolished eDNA production while overexpression of hns
restored eDNA to 70 percent of the wild-type levels. Our results suggest that eDNA production in E.
coli is related to direct secretion.
Deletions of the genes encoding the diguanylate cyclases YeaI, YedQ, and YfiN
increased swimming motility and eDNA as expected for low c-di-GMP levels. However,
contrary to the current paradigm, early biofilm formation increased dramatically for the yeaI (30
fold), yedQ (12 fold), and yfiN (18 fold) mutants. Hence, our results suggest that c-di-GMP
levels should be reduced for initial biofilm formation because motility is important for initial
attachment to a surface.
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Beckett, Emma Louise. "Folate and vitamin D: the role of nutritional status and nutrigenetics in predicting levels of extracellular microRNA and circulating DNA methylation status." Thesis, 2016. http://hdl.handle.net/1959.13/1316824.
Full textAbstract:
Research Doctorate - Doctor of Philosophy (PhD)microRNA (miRNA) in systemic circulation are proposed as potential biomarkers for disease diagnosis and prognosis. However, miRNA profiles may also be modulated by other exposures such as nutritional status, and this may have consequences for use of miRNA as biomarkers, particularly in diseases for which diet is a modifiable determinant. Furthermore, little is known about the interactions that exist between these relationships and underlying variance in genes related to the processing of nutrients that may influence these relationships, or how these miRNA interact with other modifiers of gene expression, such as DNA methylation. This thesis focuses on folate and vitamin D, two key micronutrients known to have the potential to influence gene expression. The data presented here investigates the relationships between these micronutrients and related nutrigenetics in predicting levels of extracellular miRNA and circulating DNA methylation status. The studies presented here were designed to capitalise on the availability of two well-characterised human cohorts; a case-control cohort of adenomatous polyp patients and healthy controls (n=263), and an elderly cross-sectional cohort (n=649). These are appropriate cohorts in which to investigate these relationships, as systemic circulating miRNA have been proposed as biomarkers for adenomatous polyps and colorectal cancer (CRC), diseases with known dietary modifiers of risk (including folate and vitamin D) which accumulate over a lifetime of exposures. Four candidate miRNA (let-7a, miR-15a, miR-21 and miR-155) were selected due to a combination of factors; each has known oncogenic or tumour-suppressor properties and each had existing evidence to suggest potential regulation by nutritional factors. The first results chapter (Chapter 2) presents novel observations on the levels of systemic circulating levels of let-7a, miR-15a and miR-155 in adenomatous polyp cases relative to controls. Furthermore, by adding a sex specific level of analysis, it adds to the body of knowledge surrounding these miRNA and miR-21, which is currently proposed as a biomarker for adenomatous polyps. Novel data on the correlations between blood levels of folate and related micronutrients and the candidate miRNA are presented, with key findings including a positive correlation between red blood cell folate levels and all candidate miRNA, regardless of their tumour-suppressor or oncogenic properties. Stepwise regression analyses investigating the correlations between systemic circulating miRNA levels and multiple dietary intakes, including vitamin D, are also presented. Chapter 3 builds upon these results by incorporating common folate and vitamin D related genetic polymorphisms into the analyses. The relationships between these polymorphisms, systemic circulating miRNA levels, and risk for adenomatous polyps were assessed, as well as interactions with nutrient status. Statistically significant relationships were identified between multiple polymorphisms and risk for adenomatous polyps, and miRNA levels, as well as potential interactions between folate status and genotype in predicting miRNA levels. These are the first reported observations of the potential relationships and interactions between miRNA profiles and nutrigenetic variance. As the human cohorts used can only demonstrate correlation and not causation, Chapter 4 contains data obtained from cell culture models. Three CRC cell lines were used to demonstrate that miRNA are differentially expressed intracellularly and extracellularly under folate excess or deficient conditions, and following stimulation with the active vitamin D metabolite. Treatment with a DNA demethylating agent was also used to demonstrate that some of these processes are dependent on DNA methylation. The relationships between vitamin D and DNA methylation were further investigated in Chapter 5. A sub-cohort was used to conduct a pilot study investigating the relationships between vitamin D status, methyl donor-related micronutrients and DNA methylation in genes of vitamin D metabolism. The relationship between methylation status in this pathway and the systemic circulating levels of the candidate miRNA were also assessed, and provides new information demonstrating the potential complexity of the complementary pathways for the regulation of cellular processes and pathways. Together, the data in this thesis constitute a significant contribution to the body of knowledge surrounding the extracellular levels of miRNA, and how this may relate to vitamin D and folate status, related polymorphisms, DNA methylation, and intracellular miRNA expression levels. Relationships were identified between folate status, nutrient intake and systemic circulating levels of multiple candidate miRNA. Relationships identified between polymorphisms in related genes and systemic circulating miRNA levels support these observations, and these observations may link dietary factors to modified risk for disease. This thesis expands our understanding of how nutrition and nutrigenetics can interact to modify nutrigenomics and disease risk. The data presented here for the candidate miRNA and two key nutrients, provides an impetus to investigate these relationships for other nutrients and miRNA, particularly those known to modify disease risk. These results have implications for the use of systemic circulating miRNA as biomarkers, and may also have implications for the future of personalised nutrition and personalised medicine.