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Isolement d'une nouvelle Archaea methanogène "Methanomassiliicoccus luminyensis" à partir du tube digestif humainDridi, Bédis 06 July 2011 (has links)
Les Archaea methanogènes sont des organismes environnementaux ayant été également détectés dans certaines flores associées aux muqueuses des mammifères. Chez l’homme ces microorganismes ont été associés avec les muqueuses intestinale, vaginale et orale. Ces organismes sont des procaryotes anaérobies stricts et leurs conditions de culture restent fastidieuses et très mal connues. En effet, uniquement trois Archaea methanogènes ont été cultivées à partir de prélèvements humains, Methanobrevibater smithii et Methanosphaera stadtmanae à partir des selles puis Methanobrevibater oralis à partir de la plaque dentaire. Récemment l’ADN d’autres Archaea methanogènes et d’Archaea non-methanogènes a été détecté dans des selles humaines, y compris des séquences indiquant la présence d’espèces appartenant à un nouvel ordre de méthanogènes n’ayant aucun représentant cultivé. La connaissance actuelle sur la diversité de ces methanogènes chez l’homme et sur leurs effets potentiels sur la santé humaine est en grande partie basée sur les techniques de détection de l’ADN par PCR et métagénomique. Ces techniques fondées sur la détection de l’ADN ribosomal 16S et du gène mcrA codant la sous-unité alpha du methyl-coenzyme M reductase, une enzyme clé dans le processus de méthanogenèse, ont montré dans un premier temps que M. smithii était détecté chez moins de 50% des individus et M. stadtmanae chez 0-20 % seulement. Ces résultats étaient contradictoires avec le rôle de la méthanogenèse dans l’élimination des acides et d’autres produits du processus digestion, et nous avons émis l’hypothèse que ces résultats pouvaient ne pas refléter la quantité réelle des méthanogènes dans le tube digestif humain, suggérant la mise au point de nouvelles méthodes de détection moléculaire et de culture adaptées aux caractéristiques de ces organismes fastidieux. Dans ce travail, nous nous sommes fixés comme premier objectif de mettre au point une méthode moléculaire permettant de détecter M. smithii chez tous les individus testés et nous avons mis au point un protocole d’extraction et de détection d’ADN d’Archaea à partir des selles en se basant sur les génomes séquencés de M. smithii et M. stadtmanae. Ce protocole nous a permis de détecter M. smithii chez 95,5% des individus et M. stadtmanae chez 29,4% des individus. En ce basant sur ce protocole et moyennant une approche moléculaire basée sur une PCR universelle de l’ADN ribosomal 16S des méthanogènes, le séquençage et le clonage, nous avons également détecté chez 4% de la population, une séquence correspondant à un phylotype (FJ823135) ayant déjà été rapporté comme représentant un nouvel ordre de méthanogènes. A partir de là, nous avons choisi un prélèvement de selle susceptible de contenir le plus fort ratio de FJ823135/ M. smithii et nous avons réussi à isoler et à cultiver une nouvelle Archaea que nous avons nommé Methanomassiliicoccus luminyensis, premier représentant cultivé d’un nouvel ordre de méthanogènes et la quatrième Archaea cultivée chez l’homme. M. luminyensis et M. stadtmanae présentent des métabolismes similaires en réduisant le méthanol en méthane en utilisant l’hydrogène comme donneur d’électrons, cette observation nous a incité à tester l’addition de tungstate de sélénium, requis pour la croissance de M. luminyensis, dans une culture M. stadtmanae, et nous avons observé une accélération de la vitesse de croissance de M. stadtmanae par un facteur 3. Nous avons ensuite étudié la sensibilité des méthanogènes isolés chez l’homme aux antibiotiques et établi qu’ils sont seulement sensibles à des molécules efficaces contre les bactéries et les eucaryotes, ceci étant en accord avec leur position phylogénétique en tant qu’un des quatre domaines de la vie. [...] / Methanogenic Archaea are environmental organisms which have also been associated to mammals mucosa. In humans these microorganisms have been detected in the vaginal, intestinal and oral mucosa. These organisms are strict anaerobes and their culture conditions remains fastidious and poorly known. In fact only three methanogens have been isolated from human samples, both Methanobrevibater smithii and Methanosphaera stadtmanae from stool and Methanobrevibater oralis from dental plaque. Current knowledge on the diversity of methanogens in humans and their potential effects on human health were largely based on DNA detection methods as PCR and metagenomics. These techniques based on 16S rDNA and mcrA gene (encoding the alpha subunit of methyl coenzyme-M-reductase, a key enzyme in methanogenesis process) detection, showed that M. smithii was the most present in man and that the presence of M. stadtmanae was transient. Recently, the DNA of other methanogenic and non- methanogenic Archaea, has been detected in human feces, including sequences indicating the presence of non-cultured species belonging to potential new order of methanogens with no cultured representative. However, these studies detected M. smithii with variable prevalence in less than half of the tested individuals and no M. stadtmanae; such results does not confirm the paramount role of methanogenesis in preventing the accumulation of acids and other reaction end products during the digestion process, and can not reflect the actual amount of these two methanogens in the human digestive tract because of their specific association with the intestinal mucosa. Therefore, these studies pointed that the diversity of methanogens in humans has been underestimated suggesting the development of new molecular detection methods and cultural approaches adapted these fastidious organisms. In this work, we preset as first criteria, the detection of M. smithii in all tested individuals, therefore we developed an improved protocol for archaeal DNA extraction and detection from stool based on sequenced genomes of M. smithii and M. stadtmanae, this protocol allowed us to detect the first one DNA in 95.5% tested individuals and the second in a prevalence of 29.4%. Based on this protocol and through molecular approach based on universal amplification of methanogenic 16S rDNA, sequencing and cloning, we detected in 4% of the tested population, a sequence corresponding to a new phylotype (FJ823135) that has been previously reported and proposed as a representative of a new order of methanogens. From there, we chose one stool specimen susceptible to contain the highest amount of FJ823135 and successfully isolated Methanomassiliicoccus luminyensis B10T clone, the first cultured representative of a new order of methanogens and the fourth Archaea cultured in humans.This archaeon exhibited a similar type of metabolism to that of M. stadtmanae by oxidizing H2 and reducing methanol to methane but require tungstate-selenite, an element essential for its growth, this fact prompted us testing tungstate-selenite addition on M. stadtmanae growth and establishing that it was strongly stimulatory with a growth rate three times faster. We have thereafter studied the sensitivity of methanogens isolated from humans to antibiotics and established that they are susceptible only to molecules also effective against both Bacteria and Eucarya, in agreement with their phylogenetic location as a unique domain of life. The aim of the latter part of this work was to test the effectiveness of MALDI-TOF mass spectrometry identification of environmental and host-associated Archaea. The obtained data indicated that that MALDI-TOF-MS protein profiling is an efficient first-line step for the rapid phenotypic identification of cultured Archaea organisms including host-associated ones. [...]
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Utvärdering av icke-invasiva metoder för diagnostik av Helicobacter pylori-infektion : En systematisk litteraturstudieGonzalez Elfwing, Olivia, Nilsson, Elin January 2020 (has links)
Helicobacter pylori-infektion är en av de ledande orsakerna till utvecklingen av maligniteter i ventrikeln. Tillämpning av pålitliga analytiska metoder är därför väsentlig för en korrekt diagnostik och behandling av infektionen. Syftet med studien var att ge en översikt av icke-invasiva metoder som tillämpas för påvisning av H. pylori och utvärdera vilken metod som är bäst lämpad, med avseende på metodens prestandaegenskaper och det kliniska tillståndet hos patienten. En systematisk litteraturöversikt utfördes, genom sökning efter vetenskapliga artiklar med inklusions- och exklusionskriterier i databaserna PubMed och CINAHL. Utvalda artiklar kvalitetsgranskades och 20 studier inkluderades i resultatet. Sammanställt hade fecesantigentester en sensitivitet och specificitet på 92,64% respektive 91,47%, antikroppstester hade 97,20% respektive 81,59%, urea utandningstester hade 91,40% respektive 91,70% och polymeraskedjereaktionen hade 75,45% respektive 98,30%. Därutöver hade kliniska tillstånd såsom atrofisk gastrit, intestinal metaplasi och gastrointestinal blödning en negativ påverkan på metodernas diagnostiska tillförlitlighet. Studien konstaterade att beträffande metodens prestanda är fecesantigentester mest lämpliga för påvisning av H. pylori- infektion. Vid allvarligare kliniska åkommor bör minst två icke-invasiva diagnostiska metoder tillämpas för att säkerställa pålitliga resultat. / Helicobacter pylori infection is one of the leading causes of ventricular pathologies. Reliable analytic methods are therefore crucial for the correct diagnosis and treatment of the infection. The aim of this study was to provide an overview of non-invasive diagnostic methods used for the detection of H. pylori and to evaluate which method is most suitable, considering its performance and the clinical condition of the patient. A systematic literature review was conducted, searching peer-reviewed research articles with inclusion and exclusion criteria on the databases PubMed and CINAHL. An assessment of the selected articles quality resulted in the inclusion of 20 articles. Overall, stool antigen tests had a sensitivity and specificity of 92,64% and 91,47% respectively, antibody tests 97,20% and 81,59% respectively, urea breath tests 91,40% and 91,70% respectively, and the polymerase chain reaction 75,45% and 98,30% respectively. Furthermore, conditions such as atrophic gastritis, intestinal metaplasia and gastrointestinal bleeding had a negative impact on the diagnostic accuracy of the methods. This study concluded that, regarding the methods performance, stool antigen tests are more suitable for detecting a H. pylori infection. With the mentioned clinical conditions, at least two non- invasive diagnostic methods should be used to ensure reliable results.
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Ancillary Ligand Effects On The Anticancer Activity Of Ruthenium(II) Piano Stool ComplexesDas, Sangeeta 09 1900 (has links)
The thesis “Ancillary Ligand Effects on the Anticancer Activity of Ruthenium (II) Piano Stool Complexes” is an effort to design better antitumor metallodrugs based on ruthenium(II) complexes with various H-bond donor/acceptor ligands and to understand their mechanism of action.
Chapter 1 presents a brief review of metallodrugs and their mechanism of action. Different classes of metallodrugs are discussed. A short discussion on ruthenium based anticancer drugs and their established mechanism of action is also included in this chapter.
Chapter 2 deals with the synthesis, characterization and anticancer activity of Ru(II) complexes with P(III) and P(V) ligands. The effect of a strong hydrogen bond acceptor on the cytotoxicity of the complexes has been investigated which allows comparison of complexes with ligands possessing a strong hydrogen bond donor or hydrogen bond acceptor. Partial oxidation of the tertiary phosphine ligands leads to a decrease in cytotoxicity of the ligand, while coordination to ruthenium resulted in a significant increase in the cytotoxicity. A molecular mechanism of action for these complexes was suggested on the basis of various biophysical studies. These complexes bind DNA through non-intercalative interactions which lead to the destabilization of the double helix of the DNA and also unwinding of the negatively supercoiled DNA. Results show that the presence of a hydrogen bond acceptor on the ligand is not capable of enhancing interactions with DNA in comparison with hydrogen bond donor groups. Cellular studies of these complexes showed that inhibition of DNA synthesis and apoptosis occur on treatment with these complexes. Interestingly, these complexes are found to be not only cytotoxic but also antimetastatic.
Chapter 3 deals with the synthesis, characterization and anticancer activity of Ru(II) complexes with biologically active S containing heterocyclic ligands and their mechanistic study. Complexation of ruthenium with mercaptobenzothiazole (MBT) gave the most cytotoxic complex (H3) in the series. Heterocyclic Ru(II) complexes behave differently as evidenced by cellular and biophysical studies. Unlike phosphine complexes, H3 shows biphasic melting of DNA at higher concentrations which suggests two different types of interaction with DNA.
Chapter 4 deals with synthesis and characterization of water soluble multiruthenated hydrophilic ruthenium(II) complexes with urotropine. An increase in cytotoxicity and binding affinity has been observed with increase in the number of ruthenium atoms per molecule. The complex with three ruthenium atoms showed the best activity. However cytotoxicity of the complexes decreases with decrease in the lipophilicity of the complexes.
Chapter 5 describes studies on the interaction of Ru complexes with water, ss-DNA, AMP, GMP and GSH by various spectroscopic techniques. Hydrolysis of Ru-Cl bond in the complexes correlates with the cytotoxicity.
Chapter 6 reports the summary of the observations of the thesis and the future prospects of metallodrugs.
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Nana Yaa Asantewaa, The Queen Mother of Ejisu: The Unsung Heroine of Feminism in GhanaWiafe Mensah, Nana Pokua 01 January 2011 (has links)
This thesis examines the life story of Nana Yaa Asantewaa and its pedagogical implications for schooling and education in Ghana and Canada. Leadership role among women has been a topic in many debates for a long period. For many uninformed writers about the feminist struggles in Africa, Indigenous African women are docile bodies with little or no agencies and resistance power. However, the life history of Nana Yaa Asantewaa questions the legitimacy and accuracy of this misrepresentation of Indigenous African women. In 1900, Yaa Asantewaa led the Ashanti community in a war against the British imperial powers in Ghana. The role Yaa Asantewaa played in the war has made her the legend in history of Ghana and the feminist movement in Ghana. This dissertation examines the traits of Yaa Asantewaa and the pedagogic challenges of teaching Yaa Asantewaa in the public schools in Ghana and Canada.
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Nana Yaa Asantewaa, The Queen Mother of Ejisu: The Unsung Heroine of Feminism in GhanaWiafe Mensah, Nana Pokua 01 January 2011 (has links)
This thesis examines the life story of Nana Yaa Asantewaa and its pedagogical implications for schooling and education in Ghana and Canada. Leadership role among women has been a topic in many debates for a long period. For many uninformed writers about the feminist struggles in Africa, Indigenous African women are docile bodies with little or no agencies and resistance power. However, the life history of Nana Yaa Asantewaa questions the legitimacy and accuracy of this misrepresentation of Indigenous African women. In 1900, Yaa Asantewaa led the Ashanti community in a war against the British imperial powers in Ghana. The role Yaa Asantewaa played in the war has made her the legend in history of Ghana and the feminist movement in Ghana. This dissertation examines the traits of Yaa Asantewaa and the pedagogic challenges of teaching Yaa Asantewaa in the public schools in Ghana and Canada.
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Tvorba biofilmu u probiotických kultur a možnosti jeho využití ve farmacii / Biofilm formation in probiotic cultures and its application in pharmacyRyšávka, Petr January 2021 (has links)
The work was comprehensively focused on the development of adhesive forms of probiotics in the form of a biofilm on combined carriers with a prebiotic component. The second part dealed with the influence of food on the multiplication and survival of selected types of probiotic bacteria. Subsequently, the effect of individualized probiotic supplements on changes in the human intestinal microbiome was monitored. Suitable adherent probiotic strains for biofilm formation were selected and tested. Methods have been introduced and different variants of carriers for culturing and binding bacteria have been tested. In vitro experiments verified the stability of biofilm stucture and its resistance to low pH, bile and antibiotics in comparison with the planktonic cell form. The antimicrobial effect of probiotic strains in the form of a biofilm was studied. The cultivation of the multispecies biofilm on the combined carrier was optimized and the stability of the biofilm and the final viability of probiotic bacteria were confirmed. Furthermore, the influence of various foods and beverages on the viability of probiotic bacteria was evaluated with emphasis on the simulation of passage through the gastrointestinal tract. Both models, solutions with standardised concentrations of alcohol, sugar, salts, proteins or different pH and different types of real foods and beverages were tested. The effect of food and beverages was tested on monocultures of Lactobacillus acidophilus, Bifidobacterium breve and on probiotic capsules containing a mixed culture of probiotic microorganisms. The survival of probiotics in various food matrices in the simulated gastrointestinal tract was quantitatively different. We managed to define foods suitable for supporting the multiplication of probiotic bacteria. A separate part of the work was focused on the targeted modulation of the intestinal microbiome by individualized probiotics that were prepared on the basis of molecular biological analyzes of the intestinal microbiome aimed at detecting the percentage of lactobacilli, bifidobacteria and phylum Firmicutes and Bacteroidetes. Personalized probiotic supplementation confirmed the positive effect of this approach on microbiome changes, especially on the increase of the content of lactobacilli, bifidobacteria and the overall diversity of the microbiome.
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Preventing fatal effects of overworking : Product design solutionAdawi, Rahim January 2018 (has links)
“Overworking to death” is a phenomenon that has been noticeable in developing countries. The cause of death is mainly through ischemic strokes. While the victims’ occupations differed, they all shared a common characteristic, being positioned in a sedentary work, ranging from IT workers to doctors. This project’s aim was to develop a product that prevented or decreased the strokes that derived from sedentary overwork. This was mainly tackled by preventing one of the three causes of developing blood props, slowed blood flow. In order to gather rich data of the phenomenon, a qualitative study was conducted in China, during two months. By doing an extensive structured sampling, information rich data could be gathered during a short period of time. Data were derived from observations, questionnaires and an interview, which then was interpreted to customer needs and the final product specification. The final product became a trouser with an in built dynamic compression mechanic, that can compress the veins mostly during sitting activities, in order to prevent blood stasis. The compression mechanic works like the Chinese finger trap; compressing the calves while sitting and stretching the legs forward. It is made only out of polysaccharides fibres; cotton and corn. / "Guolaosi" eller död från överarbete är ett fenomen som i regel uppkommer bland utvecklingsländer. Dödsorsaken är huvudsakligen genom stroke. Offrens yrken varierar allt från professorer, IT-arbetare till läkare. De delar dock en sak gemensamt; att arbeta under långa perioder stillasittande. Projektets mål var att utveckla en produkt som minskar dödliga följderna av sedentära överarbete, genom att förebygga en av de tre orsakerna för att utveckla blodproppar; saktad blodström. Målgruppen var då kineser av de yrken som hade tidigare drabbats av fenomenet. För att samla informationsrika data om fenomenet genomfördes en kvalitativ studie i Kina under två månader. Genom att göra en omfattande strukturerad provtagning kunde informationsrika data samlas under en kort tidsperiod. Fältstudien bestod av observationer, frågeformulär och en intervju, som då tolkades till kundbehov och eventuellt produktspecifikationen. Den slutliga produkten kom att bli ett par byxor med en inbyggd dynamisk komprimeringsmekanism, som kan komprimera venerna under sittande aktiviteter, för att förhindra saktad blodström. Kompressionsmekanismen fungerar som den kinesiska fingerfällan. Den komprimerar blodkärlen medan personen sitter och sträcker benen framåt. Produkten är konstruerad på så sätt att den kan tillverkas endast av polysackariders tråd, från bomull och majs. Vilket är lämpligt för Kinas lokala resurser.
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