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1
Tyler, Simon Nicholas George. "Asymmetric synthesis using enantiopure dihydro-2H-1,4-oxazin-2-one templates". Thesis, University of Reading, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266800.
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McConnell, Matthew S. "Nickel catalyzed formation of 1,2-cis-2-amino sugars to access important biomolecules". Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/1881.
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The stereoselective formation of 1,2-cis-2-amino glycosides remains a challenging obstacle for researchers seeking to study glycan function in nature. A variety of techniques to form α-linked C(2)-aminoglycosides are examined herein. The most prominent of these techniques is the nickel catalyzed stereoselective coupling of C(2)-N-benzylidine protected trichloroacetimidates to form 1,2-cis-2-amino sugars. This protocol demonstrates excellent α-selectivity and is applicable to a large structural variety of C(2)-aminoglycosyl donors and acceptors.
The application of the nickel catalyzed stereoselective coupling of C(2)-N-benzylidine protected trichloroacetimidates toward the synthesis of pseudosaccharides of glycosylphosphatidyl inositol (GPI) anchors and mycothiol (MSH) in good yield and with excellent α-selectivity was also examined. In stark contrast, employing conventional Lewis acids to activate trichloroacetimidate donors provided the desired pseudodisaccharides with poor α-selectivity. Additionally, the facile synthesis of both C(1)- and C(6)-hydroxyl myo-inositols bearing differentiated protecting groups from a common and easily attainable intermediate allows access to a wide variety of GPI anchor and MSH pseudosaccharides.
The highly α-selective and scalable synthesis of the Fmoc-protected GalNAc-threonine amino acid and TN antigen in large quantities is also described. The challenging 1,2-cis-2-amino glycosidic bond is addressed through a coupling of threonine residues with C(2)-N-ortho-(trifluoromethyl)benzylidenamino trihaloacetimidates mediated by Ni(4-F-PhCN)4(OTf)2. The desired 1,2-cis-2-amino glycoside was obtained in large quantities with α-only selectivity and subsequently transformed into the Fmoc-protected GalNAc-threonine and TN antigen.
With the establishment of 1,2-cis-selective synthesis of heparan disaccharides, we sought to develop multivalent inhibitors of heparanase. A model study of protein/glycan interactions, in which various macromolecular architectures were examined, was developed using Concanavalin A as the model protein. Preparations of the highly-ordered monoantennary, homofunctional diantennary, and heterofunctional diantennary glycopolymers of α-mannose and beta-glucose were achieved via ring opening metathesis polymerization. Isothermal titration calorimetry measurements of these synthetic glycopolymers with Concanavalin A, which has been reported to bind strongly to α-mannose unit, revealed that heterofunctional diantennary architectures bearing both α-mannose and non-binding beta-glucose residues, glucose units, enhanced binding affinity.
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Hughes, Juanita Maree. "A novel identification method for ultra trace detection of biomolecules using functionalised Surface Enhanced Raman Spectroscopy (SERS)". Thesis, Queensland University of Technology, 2014. https://eprints.qut.edu.au/72864/2/Juanita_Hughes_Thesis.pdf.
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This thesis developed a new method for measuring extremely low amounts of organic and biological molecules, using Surface enhanced Raman Spectroscopy. This method has many potential applications, e.g. medical diagnosis, public health, food provenance, antidoping, forensics and homeland security. The method development used caffeine as the small molecule example, and erythropoietin (EPO) as the large molecule. This method is much more sensitive and specific than currently used methods; rapid, simple and cost effective. The method can be used to detect target molecules in beverages and biological fluids without the usual preparation steps.
4
Li, Aixiao. "Molecular modeling of non-bonding interactions in biomolecules-ligand systems". Paris 7, 2009. http://www.theses.fr/2009PA077032.
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Ce travail est consacré à la modélisation des interactions entre des inhibiteurs et des molécules impliquées dans la cancérisation, dans le but notamment d'établir de manière précise les modes d'interaction biomolécules-ligand. Dans la famille des CDK (cyclin dépendant kinases) nous nous sommes intéressés à la sélectivité que présente un nouvel inhibiteur (2PU) vis-à-vis de CDK4 par rapport à CDK2. Les méthodes employées : dynamique moléculaire, calculs d'énergies d'interaction, docking et méthodes mixtes du type ONIOM ont permis d'établir les raisons précises de la sélectivité en mettant en évidence des interactions privilégiées (notamment des liaisons H) entre l'inhibiteur et CDK4. Sur le plan méthodologique la méthode ONIOM (à deux ou trois couches) a fait l'objet d'une étude minutieuse et originale quant à la procédure de définition de la partition du système. Une nouvelle approche est proposée. La stabilisation du DNA G-quadruplex par un nouveau ligand (TQMP) a également été étudiée par dynamique moléculaire, ce qui a permis de préciser les modes d'interaction et de démontrer la sélectivité de l'un des deux sites possibles d'interactionThis work is devoted to modelling the interactions between some inhibitors and molecules involved in cancer development and aims at precisely establishing the interactions modes between the ligands and the biomolecules. In the CDK (cyclin dependant kinases) family we have examined the selectivity of a new inhibitor (2PU) towards CDK4 as compared to CDK2. The techniques we have used : molecular dynamics interaction energies calculation, molecular docking and mixed methods of the ONIOM type allowed us to establish the precise causes of this selectivity, showing the existence of specific interactions (H bonds, among others) between the inhibitor and CDK4. From a methodological point of view, the ONIOM method (with 2 or 3 layers) has been carefully examined with respect to the System partitioning procedure. A new approach is proposed. The stabilisation of G-quadruplex DNA by a new ligand (TQMP) has also been studied with molecular dynamics, which allowed establishing the interaction modes and show the selectivity of one of the 2 possible interaction sites
5
Zhang, Xiaochun. "Design and characterization of biomolecule/semiconductor interfaces". Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 221 p, 2009. http://proquest.umi.com/pqdweb?did=1833646481&sid=4&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Perazzolo, Chiara. "Internal motions in biomolecules studied by NMR spectroscopy : an application to major urinary protein-1 and its complex with 2-methoxy-3-isobutylpyrazine /". [S.l.] : [s.n.], 2006. http://library.epfl.ch/theses/?nr=3489.
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Brandani, Giovanni Bruno. "Molecular dynamics simulations of protein adsorption at interfaces". Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/20415.
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Proteins can often adsorb irreversibly at fluid/fluid interfaces; the understanding of the adsorption mechanism has relevance across a variety of industrial (e.g. the creation of stable emulsions) and biological (e.g. biofilm formation) processes. I performed molecular dynamics simulations of two surfactant proteins as they interact with air/water and oil/water interfaces, describing the origin of the surface activity, the adsorption dynamics and the conformational changes that these proteins undergo at the interface. BslA is an amphiphilic protein that forms a highly hydrophobic coat around B. subtilis biofilms, shielding the bacterial community from an external aqueous solution. By investigating the behaviour of BslA variants at oil/water interfaces via coarse-grained molecular dynamics, I show that BslA represents a biological example of an ellipsoidal Janus nanoparticle, whose surface interactions are controlled by a local conformational change. All-atom molecular dynamics simulations then reveal the details of the conformational change of the protein upon adsorption, and the self-assembly into a two-dimensional interfacial crystal. Ranaspumin-2 is one of the main components of the tungara frog foam nest. Contrary to most surfactant proteins, its structure lacks any sign of amphiphilicity. All-atom simulations show that the adsorption proceeds via a two-step mechanism where firstly the protein binds to the interface through its flexible N-terminal tail and then it undergoes a large conformational change in which the hydrophobic core becomes exposed to the oil phase. I then developed a simple structure-based coarse-grained model that highlights the same adsorption mechanism observed in all-atom simulations, and I used it to compare the dynamics of adsorption and the underlying free energy landscape of several mutants. These results agree with and are used to rationalise the observations from Langmuir trough and pendant drop experiments. Colloids can often be considered simpler versions of proteins that lack conformational changes. I performed coarse-grained simulations of the compression of interfacial monolayers formed by rod-like particles. These simulations show a rich behaviour characterised by the flipping of adsorbed rods, nematic ordering and bilayer formation. I report the series of transitions that take place as the rod aspect ratio is increased from 3 to 15.
8
Araiz, Caroline. "Rôle des protéines 14-3-3 dans la régulation de la longévité par la voie DAF-2/Insuline/IGF-1 chez Caenorhabditis elegans". Phd thesis, Université Montpellier II - Sciences et Techniques du Languedoc, 2008. http://tel.archives-ouvertes.fr/tel-00347920.
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Les protéines 14-3-3 sont des protéines ubiquitaires conservées chez tous les eucaryotes. Elles interagissent avec de multiples partenaires, dont elles modulent la fonction et la localisation, et interviennent de ce fait dans de nombreux processus cellulaires. FTT-1 et FTT-2 sont les deux orthologues de 14-3-3 présents chez Caenorhabditis elegans. En utilisant l'approche de RNA interférence, nous avons montré que les protéines FTT sont impliquées dans la voie DAF-2/Insuline/IGF-1, régissant la longévité, la résistance au stress et le métabolisme chez C. elegans, et dont l'effecteur majeur est le facteur de transcription DAF-16, orthologue de Forkhead chez les mammifères. Ce travail de thèse a permis de caractériser les fonctions de chacune des protéines FTT dans la régulation des différents phénotypes engendrés par cette voie et de mettre en évidence le rôle prépondérant de FTT-2. D'autre part, nos données ont révélé la contribution supplémentaire de FTT-2 dans la régulation de la longévité et du métabolisme lipidique et de FTT-1 et FTT-2 dans la résistance au stress, à travers un mécanisme parallèle à la voie DAF-2/Ins/IGF-1 et ne mettant pas en jeu DAF-16. Les résultats de notre étude soulignent la complexité des fonctions des protéines FTT et démontrent leur participation à plusieurs processus importants pour la survie de C. elegans lors d'une exposition à un stress mais aussi lors du vieillissement physiologique du nématode.
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Vejux, Anne. "Caractérisation des figures myéliniques associées à l'accumulation de lipides polaires induites par différents oxystérols cytotoxiques identifiés dans les lésions athéromateuses : étude des relations entre apoptose et métabolisme des lipides". Phd thesis, Université de Bourgogne, 2006. http://tel.archives-ouvertes.fr/tel-00168283.
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L'athérosclérose est une pathologie artérielle complexe à évolution lente définie par un remodelage de la paroi vasculaire, associée à des réactions inflammatoires, à des processus de prolifération, de mort cellulaire et à une accumulation de lipides oxydés tels que les oxystérols (dérivés oxydés du cholestérol) dont le rôle est fortement suspecté dans le développement de l'athérome.Le travail réalisé a été effectué sur des cellules monocytaires humaines U937 et THP-1, aortiques de rat A7R5 et carcinomateuses humaines MCF-7 (déficientes en caspase-3). Différents oxystérols, présents en quantité importante dans les lésions athéromateuses, ont été utilisés (7-cétocholestérol (7KC), 7Β-hydroxycholestérol, 25-hydroxycholestérol, cholestérol-5Α, 6Α-epoxide, cholestérol-5Β, 6Β-epoxide) ainsi que du cholestérol.
La première partie du travail a montré que la mort cellulaire induite par le 7KC est un phénomène complexe présentant des caractéristiques apoptotiques accompagnées d'une synthèse de structures multilamellaires cytoplasmiques appelées corps myéliniques visualisés par microscopie électronique à transmission. La synthèse rapide de ces structures précède les effets cytotoxiques : chute du potentiel membranaire mitochondrial, augmentation de la perméabilité à l'iodure de propidium et modifications de la morphologie nucléaire (condensation, fragmentation, gonflement des noyaux). L'isolement de ces structures par ultracentrifugation après coloration à la monodansylcadavérine (fluorochrome lysosomotropique acide) ou au Nile Red (fluorescence jaune en présence de lipides neutres et rouge en présence de lipides polaires) a permis de montrer qu'elles sont riches en lipides polaires (sphingomyéline, phosphatidylcholine) et en cholestérol et qu'elles accumulent le 7KC. Au sein de ces corps myéliniques, une co-localisation du 7KC avec les lipides polaires a été démontrée par la technique de FRET réalisée par microscopie confocale mono- et bi-photonique. Les caractéristiques morphologiques et biochimiques des figures myéliniques ont permis d'établir que le 7-cétocholestérol est un puissant inducteur de phospholipidose.
Compte tenu des modifications lipidiques spatiotemporelles, quantitatives et qualitatives, induites par le 7KC et révélé par le Nile Red, la seconde partie du travail a précisé les relations entre la mort cellulaire, la synthèse de corps myéliniques, l'accumulation de lipides polaires et l'activité caspase. Avec les différents oxystérols étudiés, les corps myéliniques ne sont observés qu'avec des composés cytotoxiques (7KC, 7Β-hydroxycholestérol, cholestérol-5Β, 6Β-epoxide) et leur synthèse est indépendante d'activité caspase. En revanche, l'accumulation de lipides polaires induite par le 7KC est inhibée en présence de z-VAD-fmk (inhibiteurs de caspases large spectre) et de z-VDVAD-fmk (inhibiteur de caspase-2). Certaines caspases et en particulier la caspase-2 pourraient contribuer à l'accumulation de lipides polaires.
La troisième partie du travail a conduit à étudier les effets de la Vitamine-E (VitE) sur la mort cellulaire induite par le 7KC en raison de ses propriétés anti-apoptotiques et anti-oxydantes. La VitE protège de la mort cellulaire induite par le 7KC. Les effets protecteurs pourraient en partie être dus à la capacité de la VitE à maintenir fonctionnelle la voie PI3-K/c-Akt en s'opposant aux déphosphorylations de PDK-1 et de c-Akt, et en préservant l'activité PI3-K. Par ailleurs, la VitE s'oppose aux modifications lipidiques au niveau de la membrane cytoplasmique et à l'accumulation de lipides polaires. La VitE réduit aussi la dégradation de la pro-forme de la caspase-2L et augmente les taux d'ARNm correspondants.
Ces travaux montrent des relations entre la mort cellulaire induite par des oxystérols et le métabolisme des lipides. Ils révèlent aussi que la VitE protège de la mort induite par le 7KC en agissant au niveau de la voie PI3-K/c-Akt. Par ailleurs, la VitE s'oppose aux modifications lipidiques membranaires et cytoplasmiques associées à la mort cellulaire induite par le 7KC.
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Galian, Barrueco Carmen. "Caractérisation de 2 transporteurs ABC (“ATP-Binding Cassette”) bactériens de fonction inconnue : YheI/YheH de Bacillus subtilis et Rv1747 de Mycobacterium tuberculosis". Phd thesis, Grenoble 1, 2008. http://tel.archives-ouvertes.fr/tel-00369447.
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L'émergence du phénotype MDR (« multidrug resistance») des cellules cancéreuses est souvent corrélée à la surexpression de protéines membranaires appartenant à la superfamille ABC (« ATP binding cassette »). Ces protéines couplent l'hydrolyse de l'ATP au transport d'agents chimiothérapeutiques vers l'extérieur des cellules. Chez les bactéries, des transporteurs homologues ont été impliqués dans certains cas de résistance aux antibiotiques.Deux nouveaux transporteurs ABC bactériens, Rv1747 de Mycobacterium tuberculosis et YheI/YheH de Bacillus subtilis, potentiellement impliqués dans la résistance aux antibiotiques, ont été étudiés ici en réalisant une expression hétérologue chez Escherichia coli et en isolant des vésicules de membrane inversées. Ce système s'est avéré inapproprié pour l'étude du transporteur Rv1747, à cause vraisemblablement des différences entre E. coli et M. tuberculosis dans l'usage des codons. En revanche, nous avons obtenu un degré important de surexpression de YheI/YheH qui nous a permis de caractériser son activité de transport et d'hydrolyse de l'ATP. Nous avons ainsi montré que les deux protéines, YheI et YheH, s'associent pour former un exportateur hétérodimérique capable de transporter de multiples drogues, et que le rôle des deux sous-unités n'est pas identique dans le mécanisme catalytique du transporteur. Enfin, nous avons réussi à purifier le transporteur YheI/YheH avec un rendement élevé et dans un état fonctionnel stable, permettant d'approfondir sa caractérisation biochimique ainsi que d'obtenir des cristaux bidimensionnels pour une étude structurale par microscopie électronique.
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Debrand, Nicolas. "Caractérisation et étude d'un élément régulateur du gène codant pour le récepteur à la vasopressine de type 2". Phd thesis, Université Pierre et Marie Curie - Paris VI, 2008. http://tel.archives-ouvertes.fr/tel-00485725.
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Le contrôle de la transcription constitue le principal niveau de la régulation de l'expression des gènes dans les cellules eucaryotes. Le laboratoire a identifié 6 familles indépendantes avec un diabète insipide néphrogénique (DIN) lié à l'X portant de grandes délétions en amont du gène de l'AVPR2. Dans chacune de ces familles, les gènes AVPR2 et AQP2 sont intacts et les hommes sont atteints de DIN lié à l'X dans sa forme rénale « classique ». Le séquençage et l'analyse de 61 kilobases en amont et en aval de l'AVPR2 ont permis l'identification de 6 zones délétées chez 6 familles indépendantes, dont 5 zones de taille supérieure à 7 kilo bases, et une zone, de 102 paires de bases, commune à l'ensemble des délétions. Chez le patient porteur de cette délétion, les récepteurs V2 ne sont pas exprimés dans le tubule collecteur mais le sont au niveau des cellules endothéliales. Notre travail est de tenter de comprendre les mécanismes régulateurs du locus de l'AVPR2, et d'étudier l'expression « tissu spécifique » de ce gène. Les études réalisées dans le système Hprt, confirment le rôle activateur de la séquence de 102 pb. Les expériences in vitro indiquent que cet effet dépende du contexte extracellulaire, de la nature des cellules, ainsi que du promoteur de l'AVPR2. Les protéines liant potentiellement l'une des extrémités de la délétion a révélé la présence, soit de protéines régulatrices, soit de séquences inconnues, toutes exprimées dans le rein. À terme, ces études, ainsi que celles en découlant, permettront de positionner l'AVPR2 comme une cible de choix dans le traitement des diabètes insipides, centraux et néphrogéniques, par thérapie génique.
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Colas, Romain. "Syndrome métabolique et diabète chez l'Homme : composition lipidique et oxydation des lipoprotéines de basse densité (LDL) plasmatiques en relation avec l'activation des plaquettes sanguines". Phd thesis, INSA de Lyon, 2010. http://tel.archives-ouvertes.fr/tel-00587355.
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Le diabète de type-2 et le syndrome métabolique sont associés à une augmentation du stress oxydant et du risque cardiovasculaire. L'hyperactivation plaquettaire et les dyslipoprotéinémies sont deux causes majeures de l'athérothrombose. Nous avons montré que des lipoprotéines de basse densité (LDL) issues du plasma de diabétiques de type-2 activent les plaquettes sanguines. L'objectif principal de notre étude a été d'établir le profil en lipides et peroxydes lipidiques de LDL provenant de volontaires ayant un syndrome métabolique (SM), un diabète de type-1 (DT-1) ou de type-2 (DT-2), comparativement à celui de volontaires sains (V). Un autre objectif a été de déterminer leur impact sur l'activation plaquettaire. Seules les LDL des groupes SM et DT-2 ont des anomalies lipidiques telles que : augmentation des triacylglycérols, diminution des esters de cholestérol et de l'acide linoléique. Les LDL des groupes SM, DT-1 et DT-2 présentent un stress oxydant, démontré par l'augmentation des produits de peroxydation lipidique comme les acides gras hydroxylés et le dialdéhyde malonique, ainsi que par la diminution des plasmalogènes (sous-classe de phospholipides à éthanolamine). Comparativement aux plaquettes incubées avec les LDL de V, les plaquettes incubées avec les LDL des autres groupes sont activées comme le montre une exacerbation de la cascade de l'acide arachidonique (p38 MAPK, phospholipase A2 cytosolique, thromboxane A2). Ainsi, dans les états pré-diabétique et diabétique de type-2, les LDL subissent des modifications lipidiques et oxydatives, puis activent les plaquettes. Nos résultats suggèrent que les peroxydes lipidiques des LDL induisent l'hyperactivation plaquettaire.
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Huang, Suxian. "Biomolecular nanopatterning and single molecule detection". Diss., Restricted to subscribing institutions, 2009. http://proquest.umi.com/pqdweb?did=1835449111&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Sousa, Rui Pedro Meireles de. "A expressão da COX-2 em patologias da glândula mamária da gata". Master's thesis, Universidade de Aveiro, 2010. http://hdl.handle.net/10773/3159.
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Mestrado em Métodos BiomolecularesPelo menos 80-85% dos tumores da glândula mamária dos felídeos são malignos. Estes tumores ocorrem em média entre os 10-11 anos de idade e são o terceiro tumor mais frequente nesta espécie, precedidos dos tumores hematopoiéticos e cutâneos. A incidência destes tumores nos felídeos é inferior em cerca de metade aos dos canídeos e humanos. Contudo, as neoplasias mamárias correspondem a 17% de todos os tumores que ocorrem na gata, que metastizam com frequência, principalmente para o pulmão e gânglios linfáticos. Com este projecto foi utilizada a ciclooxigenase-2 (COX-2), que é uma proteína enzimática que medeia a produção de prostoglandinas e tromboxano a partir do ácido araquidónico, estando a COX-2 envolvida no processo inflamatório, de forma a estudar a sua expressão no epitélio luminal de várias patologias da glândula mamária da gata. Há evidências que a COX-2 tem um papel importante na tumorogénese através da estimulação da proliferação celular, inibição da apoptose, indução da angiogénese, invasão celular, imunosupressão e aumento de factores mutagénicos. Está também descrito que a expressão aumentada da COX-2 em linhas celulares de cancro da mama da mulher aumenta a migração e invasão celular. Posto isto, foi avaliado o comportamento da COX-2 em várias neoplasias mamárias da gata, do arquivo de lâminas de 1998 a 2009 do laboratório de Patologia Veterinária do Instituto de Ciências Biomédicas Abel Salazar, desde mama sem qualquer alteração, passando por hiperplasias e displasias e neoplasias benignas e malignas. Foram feitas correlações entre o indice proliferativo, para o qual foi utilizado o Ki-67 (MIB) que é detectado em várias fases de proliferação celular (G1,S,G2 e Mitose). Com a Caderina Epitelial (E-Cad) também foram realizadas correlações visto esta proteína estar presente nas junções das células, sendo que a falta desta entre os espaços inter-celulares poderá promover a migração celular provocando metastização. Para este trabalho foi utilizado o método de imunohistoquímica em tecidos parafinados utilizando anticorpos anti-COX-2, anti-Ki67 e anti-E-Cad. Foi verificado neste estudo, que a E-Cad, proteína de adesão celular, apresenta uma grande escassez de imunoreactividade em neoplasias malignas, nomeadamente em Carcinomas Sólidos, contudo o Carcinoma Tubulopapilar apresentou uma conservação desta proteína devido a necessidade deste carcinoma se organizar em estruturas tubulosas e papilares conferindo assim uma conservação da E-Cad. Foi observado que algumas neoplasias apresentaram uma localização aberrante da E-Cad no Citoplasma, em carcinomas sólidos, carcinomas tubulopapilares e de salientar a neoplasia benigna, o fibroadenoma papilar. A relação obtida desta proteína com a COX-2 e o Ki-67, quando há uma perda de expressão da Caderina Epitelial, há um aumento da imunomarcação tanto da COX-2 como da expressão do Ki-67, revelando ser um mau prognóstico. No Ki-67 foi verificado que o Carcinoma Sólido apresentava um maior índice proliferativo, demonstrando que pode ser uma característica de pior prognóstico. Quanto à COX-2 foi observado uma maior intensidade em neoplasias malignas, relativamente a estas, as neoplasias benignas apresentaram uma menor percentagem e intensidade, o que pode demonstrar uma evolução no processo neoplásico, revelando assim que COX-2 poderá estar envolvida na carcinogénese da mama de gata. ABSTRACT: At least 80-85% of tumors of the mammary gland of cats are malignant. These tumors occur on average between 10-11 years of age and are the third most common tumor in this species, followed by hematopoietic and skin tumors. The incidence of these tumors in cats is lower by about half those of canines and humans. However, this type of tumors represents a 17% of all tumors that occur in the cat that frequently metastasize, and in a short time, especially for lung and lymph nodes. With this project we analyze the expression of cyclooxygenase-2 (COX-2) which is a protein enzyme that mediates the production of prostoglandines and thromboxane from arachidonic acid, COX-2 is known to be involved in the inflammatory process. There is evidence that COX-2 has an important role in tumorigenesis by stimulating cell proliferation, inhibition of apoptosis, induction of angiogenesis, cell invasion, immunosuppression and increase of mutagenic factors. Is also described that the increased expression of COX-2 in cell lines of breast cancer in women increases migration and cell invasion. We evaluated the behavior of COX-2 in several mammary tumors of the cat, with slides archive since 1998 to 2009 from the Laboratory of Veterinary Pathology at the Institute of Biomedical Sciences Abel Salazar, with normal breast tissue, through hyperplasia, dysplasia, benign and malignant neoplasms. For proliferative index was used Ki-67 (MIB) that is detected at various stages of cell proliferation (G1, S, G2 and Mitosis). With Epithelial Cadherin (E-Cad) were also performed comparisons, since this protein is present at the junctions of the cells, and lack thereof between the inter-cellular spaces can promote cell migration causing metastasis. For this work we used the method of immunohistochemistry in paraffin embedded tissues using anti-COX-2, anti-Ki67 and anti-E-Cad. It was considered that E-Cad, cell adhesion protein, showed a lack of immunoreactivity in malignant neoplasms, particularly in Solid Carcinomas, however tubulo papillary carcinoma showed a conservation of this protein due to carcinoma organization in tubular and papillary structures giving thus conservation of E-Cad in 68.42%. It was observed that some tumors showed an aberrant localization of E-Cad in the cytoplasm, in solid carcinomas, carcinomas tubulopapilares and to emphasize the papillary fibroadenoma a benign tumor. The comparison of this protein with COX-2 and Ki-67, when a loss of expression of Epithelial Cadherin, there is an increase in both immunostaining of COX-2 as the expression of Ki-67, proving to be a poor prognosis. In Ki-67 was found that the solid carcinoma had a higher proliferative index, indicating that it can be a feature of poor prognosis. As for COX-2 was observed a greater intensity in malignant neoplasms, for these, benign tumors had a lower percentage and intensity, which can demonstrate an evolution in the neoplastic process, thus revealing that COX-2 may be involved in carcinogenesis of female cat breast tumors.
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Ferreira, Ana Sofia Costa. "Efeito de 5-aza-2'-desoxicitidina na expressão proteica de células LNCaP". Master's thesis, Universidade de Aveiro, 2010. http://hdl.handle.net/10773/3155.
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Mestrado em Métodos BiomolecularesNo sentido de contribuir para uma melhor compreensão dos fenómenos moleculares subjacentes ao cancro da próstata e do efeito de um inibidor de DNAmetiltransferases com potencial acção terapêutica, neste estudo efectuou-se a análise do proteoma nuclear e a identificação de PTMs em histonas usando a linha celular LNCaP. Assim, as células LNCaP foram tratadas com diferentes concentrações de 5-aza-2’- desoxicitidina (DAC). As proteínas presentes no núcleo foram identificadas por 2D-PAGE-MS/MS, e as histonas foram analisadas por LC-MS/MS com o intuito de (i) estabelecer o perfil proteico do núcleo das células LNCaP, (ii) comparar, qualitativa e quantitativamente o proteoma nuclear das células LNCaP não tratadas e tratadas com diferentes concentrações de DAC (1mM e 5mM); (iii) identificar as PTMs (metilações e acetilações) nas várias classes de histonas; e (iv) avaliar o efeito de diferentes concentrações de DAC (0mM, 1mM e 5mM) no perfil de PTMs das histonas. Os resultados sugerem que o DAC induz a alteração da expressão de proteínas relacionadas com a transcrição e tradução e, em particular, a administração de 1mM de DAC induz o aumento da expressão de proteínas associadas à apoptose. A análise de PTMs evidenciou a predominância da modificação K96m2 na histona H2A, e a administração de 1DAC induz um aumento do número de modificações, particularmente na classe de histonas H1, com predominância da metilação, tanto na cauda como na componente globular das histonas. O aumento de PTMs em histonas de células tratadas com 1DAC parece estar relacionado com a alteração da expressão de algumas proteínas envolvidas nos processos de transcrição e tradução. ABSTRACT: In order to contribute to a better comprehension of the molecular mechanisms underlying prostate cancer and the effect of the inhibitor DNAmetiltransferases with potential therapeutic role, in this study we analysed the nuclear proteomics and identified PTMs on histones using the cell line LNCaP. Thus, LNCaP cells were treated with different concentrations of 5-aza-2'-deoxycytidine (DAC). The proteins present in the nucleus were identified by 2D-PAGE-MS/MS, and histones were analyzed by LC-MS/MS with the aim of (i) establish the nuclear protein profile of LNCaP cells, (ii) compare qualitative and quantitatively the nuclear proteome of LNCaP cells untreated and treated with different concentrations of DAC (1mM and 5mM), (iii) identify the PTMs (acetylations and methylations) in the various classes of histones, and (iv) evaluate the effect of different concentrations DAC (0mM, 1mM and 5mM) in the profile of PTMs on histones. The results suggest that DAC induces changes in the expression of proteins related with transcription and translation and, in particular, the administration of 1mM DAC induces the up-regulation of proteins associated with apoptosis. The analysis of PTMs showed the predominance of K96m2 in the histone H2A, and administration of 1DAC induces an increase in the number of modifications, particularly in the class of histone H1, with a predominance of methylation in both the tail and the globular component of the histones. The increase of PTMs on histones in cells treated with 1DAC seems to be related with changes in the expression of proteins involved in transcription and translation.
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Kearns, Gregory Justin. "Engineering interfaces at the micro- and nanoscale for biomolecular and nanoparticle self-assembled devices /". view abstract or download file of text, 2007. http://proquest.umi.com/pqdweb?did=1417810561&sid=2&Fmt=2&clientId=11238&RQT=309&VName=PQD.
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Thesis (Ph. D.)--University of Oregon, 2007.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 158-174). Also available for download via the World Wide Web; free to University of Oregon users.
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Porter, Jason Robert. "SPLIT-PROTEIN REASSEMBLY METHODS FOR THE DETECTION AND INTERROGATION OF BIOMOLECULAR INTERACTIONS AND MODULATORS THEREOF". Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/194359.
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The interactions between protein-protein, protein-nucleic acid, and protein-small molecules are central to biological processes and are key for the design of new therapeutics. Rapid and easy to implement methodologies are needed that enable the interrogation of these interactions in a complex cellular context. Towards this goal, I have utilized the concept of split-protein reassembly, also called protein complementation, for the creation of a variety of sensor architectures that enable the interrogation of protein-nucleic acid, protein-protein, and protein-small molecule interactions. Utilizing the enzymatic split-reporter β-lactamase and existing zinc finger design strategies we applied our recently developed technology termed SEquence-Enabled Reassembly (SEER) towards the creation of a sensor capable of the specific detection of the CryIA transgene. Additionally, the split β-lactamase reporter was utilized for the sitespecific determination of DNA methylation at cytosine residues that is involved in epigenetic regulation. This method, dubbed mCpG-SEER, enabled the direct detection of femtomole levels of dsDNA methylation in sequence specific manner. In a separate endeavor, we have developed and optimized the first cell-free split-reporter systems for GFP, split β-lactamase, and firefly luciferase for the successful dsDNA-dependent reassembly of the various reporters. Our cell free in vitro translation systems eliminates previous bottlenecks encountered in split-reporter technologies such as laborious transfection/cell culture or protein purification. Capitalizing on the ease of use and speed afforded by this new technology we describe the sensitive detection of protein-protein, protein-nucleic acid, and protein-small molecule interactions and inhibitors thereof. In a related area, we have applied this rapid cell-free split-firefly luciferase assay to the specific interrogation of a large class of helix-receptor protein-protein interactions. We have built a panel consisting of the clinically relevant Bcl-2 family of proteins, hDM2, hDM4, and p53 and interrogated the specificity of helix-receptor interactions as well as the specificity of peptide and small-molecule inhibitors of these interactions. Finally, we describe the further applications of our cell-free technology to the development of a large number of general split-firefly luciferase sensors for the detection of ssRNA sequences, the detection of native proteins, the evaluation of protease activity, and interrogation of enzyme-inhibitor interactions. The new methodologies provided in this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.
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Lin, Xiu-Mei [Verfasser], Volker Akademischer Betreuer] Deckert i Wolfgang [Akademischer Betreuer] [Weigand. "Tip-enhanced Raman Spectroscopy (TERS) for Biomolecular Analyses at Nanometer / Xiu-Mei Lin. Gutachter: Volker Deckert ; Wolfgang Weigand". Jena : Thüringer Universitäts- und Landesbibliothek Jena, 2014. http://nbn-resolving.de/urn:nbn:de:gbv:27-20140210-112607-2.
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Mills, Landon C. "IMPACT OF CONFORMATIONAL CHANGE, SOLVATION ENVIRONMENT, AND POST-TRANSLATIONAL MODIFICATION ON DESULFURIZATION ENZYME 2'-HYDROXYBIPHENYL-2-SULFINATE DESULFINASE (DSZB) STABILITY AND ACTIVITY". UKnowledge, 2019. https://uknowledge.uky.edu/cme_etds/105.
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Naturally occurring enzymatic pathways enable highly specific, rapid thiophenic sulfur cleavage occurring at ambient temperature and pressure, which may be harnessed for the desulfurization of petroleum-based fuel. One pathway found in bacteria is a four-step catabolic pathway (the 4S pathway) converting dibenzothiophene (DBT), a common crude oil contaminant, into 2-hydroxybiphenyl (HBP) without disrupting the carbon-carbon bonds. 2’-Hydroxybiphenyl-2-sulfinate desulfinase (DszB), the rate-limiting enzyme in the enzyme cascade, is capable of selectively cleaving carbon-sulfur bonds. Accordingly, understanding the molecular mechanisms of DszB activity may enable development of the cascade as industrial biotechnology. Based on crystallographic evidence, we hypothesized that DszB undergoes an active site conformational change associated with the catalytic mechanism. Moreover, we anticipated this conformational change is responsible, in part, for enhancing product inhibition. Rhodococcus erythropolis IGTS8 DszB was recombinantly produced in Escherichia coli BL21 and purified to test these hypotheses. Activity and the resulting conformational change of DszB in the presence of HBP were evaluated. The activity of recombinant DszB was comparable to the natively expressed enzyme and was competitively inhibited by the product, HBP. Using circular dichroism, global changes in DszB conformation were monitored in response to HBP concentration, which indicated that both product and substrate produced similar structural changes. Molecular dynamics (MD) simulations and free energy perturbation with Hamiltonian replica exchange molecular dynamics (FEP/λ-REMD) calculations were used to investigate the molecular-level phenomena underlying the connection between conformation change and kinetic inhibition. In addition to the HBP, MD simulations of DszB bound to common, yet structurally diverse, crude oil contaminates 2’2-biphenol (BIPH), 1,8-naphthosultam (NTAM), 2-biphenyl carboxylic acid (BCA), and 1,8-naphthosultone (NAPO) were performed. Analysis of the simulation trajectories, including root mean square fluctuation (RMSF), center of mass (COM) distances, and strength of nonbonded interactions, when compared with FEP/λ-REMD calculations of ligand binding free energy, showed excellent agreement with experimentally determined inhibition constants. Together, the results show that a combination of a molecule’s hydrophobicity and nonspecific interactions with nearby functional groups contribute to a competitively inhibitive mechanism that locks DszB in a closed conformation and precludes substrate access to the active site.
Limitations in DszB’s potential applications in industrial sulfur fixation are not limited to turnover rate. To better characterize DszB stability and to gain insight into ways by which to extend lifetime, as well as to pave the way for future studies in inhibition regulation, we evaluated the basic thermal and kinetic stability of DszB in a variety of solvation environments. Thermal stability of DszB was measured in a wide range of different commercially available buffer additives using differential scanning fluorimetry (DSF) to quickly identify favorable changes in protein melting point. Additionally, a fluorescent kinetic assay was employed to investigate DszB reaction rate over a 48 hr time period in a more focused group of buffer to link thermal stability to DszB life-time. Results indicate a concerningly poor short-term stability of DszB, with an extreme preference for select osmolyte buffer additives that only moderately curbed this effect. This necessitates a means of stability improvement beyond alteration of solvation environment. To this end, a more general investigation of glycosylation and its impact on protein stability was performed.
Post-translational modification of proteins occurs in organisms from all kingdoms life, with glycosylation being among the most prevalent of amendments. The types of glycans attached differ greatly by organism but can be generally described as protein-attached carbohydrate chains of variable lengths and degrees of branching. With great diversity in structure, glycosylation serves numerous biological functions, including signaling, recognition, folding, and stability. While it is understood that glycans fulfill a variety of important roles, structural and biochemical characterization of even common motifs and preferred rotamers is incomplete. To better understand glycan structure, particularly their relevance to protein stability, we modeled and computed the solvation free energy of 13 common N- and O-linked glycans in a variety of conformations using thermodynamic integration. N-linked glycans were modeled in the β-1,4-linked conformation, attached to an asparagine analog, while O-linked glycans were each modeled in both the α-1,4 and β-1,4-linked conformations attached to both serine and threonine analogs. Results indicate a strong preference for the β conformation and show a synergistic effect of branching on glycan solubility. Our results serve as a library of solvation free energies for fundamental glycan building blocks to enhance understanding of more complex protein-carbohydrate structures moving forward.
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Zanvettor, Paulo Henrique. "Estudo de fatores prognósticos clínicos e biomoleculares de pacientes portadores de câncer de vulva submetidos a tratamento cirúrgico". Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-04042014-123422/.
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INTRODUÇÃO: O câncer de vulva representa entre 3 a 5% dos cânceres do trato genital feminino e aproximadamente 0,6% dos cânceres da mulher. Existem poucos estudos na literatura sobre fatores prognósticos incluindo avaliação molecular, relacionados a esta doença. Este estudo foi realizado para avaliar fatores prognósticos clínicos, epidemiológicos, patológicos e moleculares de pacientes portadores de câncer de vulva submetidos a tratamento cirúrgico. Avaliamos também uma classificação de prognóstico com base na pontuação do risco relativo dos fatores encontrados. MÉTODOS: Foram selecionadas as pacientes portadoras de carcinoma escamocelular de vulva, submetidas a tratamento cirúrgico no Departamento de Cirurgia Pélvica e Serviço de Ginecologia do Hospital Aristides Maltez entre o período de Junho de 1993 e Junho de 2011. Avaliamos as características clínicas, epidemiológicas, de anatomia patológica e molecular, em relação ao prognóstico das pacientes. A avaliação molecular estudou a expressão da proteína p53 e metaloproteinase 2 de matriz por exame de imunohistoquímica pela técnica de arranjo em matriz de amostras teciduais (TMA). Com base no banco de dados realizado através de consulta aos prontuários e com os resultados de anatomia patológica e testes imunohistoquímicos realizamos análise estatística em relação à sobrevida global das pacientes.RESULTADOS: Setenta e cinco pacientes foram elegíveis para o estudo. A análise univariada mostra como fatores relacionados à sobrevida foram a presença de linfonodo suspeito de metástase ao exame clínico (p=0,004), o tamanho maior de 4 centímetros (p=0,001), a presença de linfonodo com metástase (p=0,026), o estadiamento por grupos (p=0,001), a localização mediana (p=0,011), a profundidade de infiltração maior de 2 milímetros (p=0,012), a expressão da MMP 2 em mais de 50% das células (p=0,009). Na análise multivariada os fatores relacionados à sobrevida foram o tamanho maior de 4 centímetros (p=0,014), a profundidade de invasão maior de 2 milímetros (p=0,023) e a expressão de MMP2 em mais de 50% das células (p=0,046). De acordo ao risco relativo dos fatores identificados como relacionados ao prognóstico pela análise multivariada, foi elaborada a contagem de pontos para uma classificação prognóstica. Essa pontuação classifica em três categorias conforme a presença ou ausência dos fatores identificados na análise multivariada e pode corresponder a três expectativas de sobrevida. CONCLUSÕES: Os fatores tamanho maior de 4 centímetros, profundidade de invasão maior de 2 milímetros e expressão em mais de 50% das células tumorais de metaloproteinase 2 parecem estar relacionados a menor taxa de sobrevida global de pacientes portadores de câncer de vulva submetidas a tratamento cirúrgico. Pode ser realizada uma classificação para o prognóstico de acordo com uma contagem de pontos atribuídos ao risco relativo destes fatoresINTRODUCTION: Vulvar cancer accounts for 3-5% of all cancers of the female genital tract and approximately 0.6% of women\'s cancers. There are few studies on prognostic factors including molecular evaluation related to this disease. This study was conducted to evaluate clinical, epidemiological, pathological and molecular prognostic factors of patients with vulvar cancer, undergoing surgical treatment. A classification based on prognostic risk score related to the factors found was also evaluated. METHODS: Patients were selected with squamous cell carcinoma of the vulva who underwent surgical treatment at the Department of Pelvic Surgery and Department of Gynecology, Hospital Aristides Maltez between the period of June 1993 and June 2011. Clinical, epidemiological, and molecular and pathological anatomy characteristics were evaluated in relation to the prognosis of patients. Molecular assessment studied the expression of p53 protein and metalloproteinase 2 by immunohistochemical examination through the technical arrangement on matrix tissue samples (TMA). Based on the database performed on medical records held and the results of pathological anatomy and immunohistochemical tests, the statistical analysis was performed with respect to overall survival of patients. RESULTS: Seventy-five patients were eligible for the study. Univariate analysis shows how factors related to survival were the presence of lymph node metastasis suspected on clinical examination (p = 0.004), the larger size of 4 cm (p = 0.001), presence of lymph node metastasis (p = 0.026), staging groups (p = 0.001), the location median (p = 0.011), the depth of infiltration larger than 2 millimeters (p = 0.012), the expression of MMP 2 in more than 50% of the cells (p = 0.009). In multivariate analysis, factors related to survival were larger size than 4 cm (p = 0.014), depth of invasion greater than 2 mm (p = 0.023) and MMP2 expression in more than 50% of cells (p = 0.046). According to the relative risk of factors identified as related to prognosis by multivariate analysis, a score was developed for a prognostic classification. This score classifies into three categories, according to the presence or absence of the factors identified in the multivariate analysis and may correspond to three survival expectations. CONCLUSIONS: Prognostic factors as the size larger than 4 centimeters, depth of invasion greater than 2 mm and expression in more than 50% of tumor cells of metalloproteinase 2 may be related to lower overall survival rate of patients with cancer of the vulva undergoing surgery. Classification may be performed for the prognostic according to a count point attributed to the relative risk of these factors
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Neto, Evandro Carneiro Martins. "Estudo comparativo da fixação e integração de enxertos ósseos \"onlay\" com uso de N-butil-2-cianoacrilato, parafuso de titânio ou lag screw. Estudo histológico, microtomográfico e biomolecular em coelhos". Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/58/58136/tde-17072014-104337/.
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Alguns trabalhos sobre a resposta do tecido ósseo ao Cianocrilato podem ser encontrados na literatura, embora nenhum deles avalie a resposta histológica, micro-tomográfica e biomolecular na fixação de enxertos ósseos onlay com o N-Butil-2-Cianocrilato (IndermilTM). O objetivo do estudo proposto foi comparar o processo de incorporação, remodelação, revascularização e manutenção do volume de enxertos fixados com parafuso ou adesivo, além de mapear os eventos biomoleculares nos quais os enxertos ósseos onlay possam estar envolvidos. Oitenta e oito coelhos adultos foram envolvidos nesse estudo. Dois blocos ósseos provenientes da calvária dos coelhos foram transplantados para a mandíbula, em que de cada lado do leito receptor o osso autógeno foi fixado com parafuso de osteossíntese de forma aposicional, lag screw, cianoacrilato ou parafuso e cianoacrilato. O sacrifício dos animais ocorreu após 3, 7, 20 e 40 dias do procedimento cirúrgico inicial, quando então os animais foram submetidos às análises micro-tomográficas, histológicas e biomoleculares. Cortes histológicos das áreas enxertadas foram preparados para se avaliar o reparo dos enxertos ósseos no sítio receptor. Os resultados biomoleculares mostraram que o método de fixação utilizando o composto N-Butil-2-Cianocrilato (NB-Cn) apresentou um maior potencial anti-inflamatório, revascularizador e de formação óssea, bem como, uma menor reabsorção óssea. Na avaliação histológica, observou-se que embora o NB-Cn tenha impedido a formação de novo osso na área onde foi aplicado, a estabilidade promovida pela cola permitiu que a revascularização e incorporação do enxerto ocorresse de forma semelhante aos demais grupos. Esses resultados indicam que o NB-Cn se comportou de forma semelhante ao parafuso como material de osteossínte.Some experimental studies on the bone tissue responses to cyanoacrylate can be found in the literature, none of them evaluate the histological response, micro-tomographic and biomolecular on fixation of onlay bone grafts with N-butyl-2-cyanoacrylate (IndermilTM). The aim of the proposed study was to compare the process of incorporation, remodeling, and volume maintenance of grafts set with screw or adhesive and map biomolecular events in which onlay bone grafts may be involved. Eighty-eight adult rabbits were submitted to calvaria onlay bone graft on both sides of the mandible. The grafts were fixated on each side of mandible by a fixation screw appositionally, lag screw, cyanoacrilate or both. The animals were sacrificed on 3rd, 7th, 20th and 40th day after the initial surgical procedure, then they were subjected to micro-computed tomography, histological and biomolecular analysis. Histological sections of the grafted areas were prepared to evaluate the healing of bone grafts in the receptor site. The biomolecular analysis showed that fixation method using N-butyl-2-cyanoacrylate (NB-Cn) presented a greater potential on anti-inflammatory, revascularization and bone formation properties, as well as reduced bone resorption. Histological evaluation observed that although the NB-Cn has prevented the formation of new bone in the area where it was applied, the stability achieved by Nb-Cn allowed revascularization and graft incorporation just like other groups. These results indicate that NB- Cn behaved similarly to the screw as osteosynthesys material.
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Talebi, M. "New stationary phases for high‐performance liquid chromatography of biomolecules". Thesis, 2013. https://eprints.utas.edu.au/17162/2/Whole-Talebi-thesis-2013.pdf.
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This work presents a study on the preparation and application of polymer monoliths for the liquid chromatography of biomolecules with a focus on the ion‐exchange (IEX) mode.
As one important application of polymer monoliths in bioanalysis, charge heterogeneity profiling of monoclonal antibodies (mAbs) in different biopharmaceuticals was performed by developing an elution approach based on shallow pH gradient, generated using single component buffer systems as eluents through cation‐exchange (CEX) monoliths as stationary phases. A useful selection of small molecule buffer species is described that can be used within very narrow pH ranges (typically 1 pH unit) defined by their buffering capacity for producing controlled and smooth pH profiles when used together with porous polymer monoliths. The results obtained appeared to be consistent with those obtained by imaged capillary isoelectric focusing (iCE) in terms of both resolution and separation profile. The retention mechanism based on the trends observed for proteins at pH values higher than the electrophoretic pI, as well as the high resolution gains, were discussed using applicable theories. Very low ionic strength eluents also enabled direct coupling of the ion‐exchange chromatography (IEC) to mass spectrometer for further characterisations of mAbs. Although there are few reports of IEC‐MS technique for small proteins in which the IEX column is directly interfaced to the mass spectrometer, the employment of a linear pH gradient elution scheme directly interfaced to mass spectrometer for the analysis of large proteins such as mAbs is also unique in the present work.
New polymer monoliths were prepared in 100 μm i.d. capillaries by thermally‐initiated co‐polymerisation of glycidyl methacrylate as reacting monomer and pentaerythritol triacrylate as a hydrophilic crosslinker. The monolith recipe and polymerisation conditions were optimised to obtain a homogeneous monolith with good mechanical stability and characteristics suitable for separation of biomacromolecules. Nevertheless, shrinkage of the material prevented making monoliths in a column with conventional dimensions. Post‐polymerisation modification of the monolith was performed via optimised reaction conditions in order to incorporate weak cation‐exchange (WCX) or strong cation‐exchange (SCX) functionalities using amine reagents respectively containing phosphoric acid or sulforic acid groups. Dynamic binding capacities up to 15.1 mg/mL were measured using lysozyme as a standard probe, which is comparable or greater from some of the commercially available columns. Compared to monoliths reported previously for the same purpose, the developed monoliths also demonstrated negligible hydrophobicity with separation efficiency of approximately 55,000 plates/m in isocratic separation of sample proteins.
A versatile epoxy‐based monolith was synthesised in 100 μm i.d. capillaries by polycondensation polymerisation of glycidyl ether 100 with ethylenediamine using a porogenic system consisting of polyethylene glycol, MW = 1000, and 1‐decanol. Polymerisation was performed at 80 °C for 22 h. The resultant monolith possessed hydrophilic properties originating from the incorporation of hetero‐atoms in the monolith skeleton which was further strengthened by simple acid hydrolysis of residual epoxides, resulting in a mixed diol‐amino chemistry. The modified column was used successfully for hydrophilic interaction liquid chromatography (HILIC) of small molecule probes, such as nucleic acid bases and nucleosides, benzoic acid derivatives, as well as for peptides released from a tryptic digest of cytochrome c. The mixed mode chemistry allowed both hydrophilic partitioning and IEX interactions to contribute to the separation, providing flexibility in selectivity control. Residual epoxide groups were also exploited for incorporating a mixed IEX chemistry. Alternatively, the surface chemistry of the monolith pore surface rendered hydrophobic via grafting of a co‐polymerised hydrophobic hydrogel. The inherent hydrophilicity of the monolith scaffold also enabled high performance separation of proteins under IEX and hydrophobic interaction (HIC) modes and in the absence of nonspecific interactions.
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Blagojevic, Voislav. "Ions, biomolecules and catalysis : SIFTing for the origins of life /". 2005. http://proquest.umi.com/pqdweb?index=0&did=1163224921&SrchMode=1&sid=7&Fmt=2&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1195066319&clientId=5220.
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Thesis (Ph.D.)--York University, 2005. Graduate Programme in Chemistry.Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://proquest.umi.com/pqdweb?index=0&did=1163224921&SrchMode=1&sid=7&Fmt=2&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1195066319&clientId=5220
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Ray, Jyotirmoy. "Theoretical Studies on Excited-State Photodynamics: N-H Dissociation in Aniline and Intermolecular H atom Transfer in the 2-Amino Pyridine Dimer". Thesis, 2021. https://etd.iisc.ac.in/handle/2005/5618.
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DNA is composed of light-absorbing molecular units like adenine, guanine, thymine, cytosine,
etc. It is well-known that DNA as a whole is photostable upon exposure to sunlight.
The photostability of biomolecules suggests that the relaxation process from the electronically
excited state to the ground state (GS) is very efficient, and the excited state lifetime is
very short. Several experimental and theoretical studies in the past couple of decades have
investigated such excited state dynamics. Early studies by Domcke, Sobolewski and coworkers
explored excited-state processes in prototypical molecules like phenol, pyrrole and indole
and have proposed that one of the dominant processes on the excited state is the 1πσ∗ state
mediated N-H/O-H dissociation via 1ππ∗/1πσ∗ and 1πσ∗/GS conical intersections (CIs) 1.
They also carried out computational calculations to understand the underlying reason for the
photostability of biomolecules 2. It is now understood that they can undergo a charge transfer
(CT) state-mediated intermolecular proton transfer on the excited state and relax to the
ground electronic state through CT/GS CI very efficiently on an ultrafast timescale. The study
of excited-state dynamics of prototypical molecules to the biomolecules is essential to understand
photodamage and photostability of complex biomolecules. The present thesis explores
these aspects in aniline and the 2-amino pyridine dimer, respectively
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El, Ghazouani Abdelnasser. "Etude biochimique et fonctionnelle de la protéine PerR de Bacillus subtilis : un senseur bactérien du peroxyde d'hydrogène". Phd thesis, 2007. http://tel.archives-ouvertes.fr/tel-00259140.
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La métalloprotéine PerR (Peroxyde resistance Regulator), homodimère de 2 fois 16,5 kDa, est un facteur de la transcription. PerR est le senseur du peroxyde d'hydrogène chez plusieurs microorganismes. PerR de Bacillus subtilis a été isolée sous sa forme apo avec la présence d'un ion Zn2+ par monomère. Dans des conditions de culture non contrôlées, PerR est en partie oxydée de manière aléatoire. La spectrométrie de masse et la spectroscopie RPE nous ont permis de relier la capacité de fixation du métal régulateur (Fe2+ ou Mn2+) et l'oxydation de PerR. Notre travail a permis de mettre au point un protocole d'expression conduisant de manière reproductible à une forme totalement réduite. Ceci a permis la détermination précise de l'affinité de la protéine pour le métal régulateur et l'ADN. La forme apo-PerR-Zn a été cristallisée et cette structure révèle un site structural à zinc avec une coordination tétraédrique par les quatre cystéines de la protéine (motif Zn(Cys)4). Ce site verrouille le domaine de dimérisation, favorisant ainsi la stabilité du dimère. Le rôle structural de ce site a été confirmé par des expériences biochimiques qui mettent en évidence une réactivité faible vis-à-vis d'H2O2. Des études en spectroscopie de fluorescence ont montré des changements conformationnels de PerR en solution après métallation et fixation à l'ADN. La formation du complexe ADN-PerR a été étudiée de manière détaillée et a permis de mettre en évidence deux nucléotides et trois acides aminés essentiels dans cette interaction. Enfin, nos travaux sur la forme oxydée de PerR ont mis en évidence que le résidu 2-oxo-histidine est le marqueur biologique de l'oxydation de PerR.
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Jan, Fu-Min, i 詹富閔. "Characterization of Essential Elements and As-containing Biomolecules in Human Serum by Size-Exclusion coupled with On-line Isotope Dilution Method and 2-D SEC/RPLC Chromatographic Techniques". Thesis, 2002. http://ndltd.ncl.edu.tw/handle/95497952113628761622.
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碩士國立清華大學
原子科學系
90
Abstract The speciation of trace element, in clinical and biological samples has become a focusing discipline, which provides significant information for biochemist to explore the mechanism of availability, cycling, transformation, bioaccumulation and final fate of these “biometals”. In view of the important role of bio-molecules played in the biological systems, development of reliable analytical method aiming at elucidating the structural and quantitative information about these materials is highly demanded. For the purpose of characterizing biometals which bind with various bio-molecules, a hyphenation technique coupling size exclusion chromatographic separation and ICP-MS detection was explored. Various parameters of SEC, such as mobile phase flow rate and sample volume on the effect of separation efficiency were investigated. Following the optimized separation condition, trace amount of Cu, Zn, Cd, Pb and Co in human serum were found bound with the biomolecule of M.W.~6,500 Da and a broad As-containing peak corresponding to M.W.~1,500 Da was observed on SEC-ICPMS chromatograph. The identification of the species was prelimernarily investigated with a molecular specific ESI-MS technique. In this study an on-line SEC-ID-ICPMS system for the quantitative determination of binding and non-binding species of Cu in human serum has also been developed. The accuracy of this hyphenation system for the determination of total concentration of Cu in human serum was verified with the use of NIST SRM 1598 and 311089. With this SEC-ID-ICP-MS system, the presence of the Cu-biomolecule of molecular weight of about 60,000 Da, was found to be about 90% of the total Cu concentration, which is in good agreement with that reported in the literature.
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Renaud, Emilie. "L'hétérotrimère XPC/Rad23B/centrine 2 : un complexe multifonctionnel dans la réponse cellulaire humaine aux agents génotoxiques". Phd thesis, 2008. http://tel.archives-ouvertes.fr/tel-00432991.
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La réponse cellulaire humaine aux agents génotoxiques est essentielle pour le maintien de l'intégrité génétique. Elle implique une régulation coordonnée de nombreuses voies métaboliques qui déclencheront un arrêt du cycle cellulaire et la réparation des macromolécules endommagées, l'inflammation et l'apoptose. La Réparation Globale du Génome par Excision de Nucléotides (GG-NER) est une voie majeure de réparation de l'ADN pour la prévention de la cancérogenèse car elle élimine une grande variété de lésions induites par les rayonnements ultraviolets, des carcinogènes chimiques et d'autres facteurs de notre environnement. Ces lésions sont reconnues par XPC, qui est le premier facteur protéique impliqué dans cette voie de réparation. XPC forme un complexe avec Rad23B. Nous avons montré qu'in vivo le complexe XPC/Rad23B comportait également la centrine 2. Le mode d'interaction de XPC avec la centrine 2 est très conservé de la levure à l'homme indiquant une participation de ce complexe à un processus biologique général à tous les eucaryotes. La centrine 2 est impliquée dans la division cellulaire en régulant la duplication du centrosome. Rad23B intervient dans le contrôle stabilité/dégradation des protéines par le protéasome 26S. Nous avons montré que l'existence de ce complexe ancré à la chromatine permettait son accumulation immédiate sur les sites des lésions induites par les UV ou après l'impact d'un laser à 405 nm. Cette localisation dépend uniquement de la présence de XPC. Nous avons montré que XPC régulait le niveau basal et l'équilibre de la centrine 2 entre le noyau et le centrosome. Ceci pourrait être primordial pour coordonner la réparation de l'ADN et la division cellulaire. De plus, nous avons observé que Rad23B promouvait la survie cellulaire en stabilisant XPC après une irradiation aux UVC. Enfin, nos résultats montrent que la présence de XPC est requise pour l'accumulation des transcrits centrine 2 et Rad23B suite à une irradiation aux UV. Ceci conforte l'idée que XPC pourrait faire partie d'un système de signalisation qui induit l'expression de gènes après la reconnaissance des dommages de l'ADN. Cet hétérotrimère regroupe donc des protéines aux fonctions distinctes qui sont localisées précocement sur les dommages de l'ADN. Nous proposons que ce complexe coordonne différents processus biologiques immédiatement après la reconnaissance des lésions par XPC : la régulation du cycle cellulaire par la centrine 2, le contrôle stabilité/dégradation de protéines, dont XPC, par Rad23B et le déclenchement de la réparation et l'induction de l'expression de gènes par XPC. Des études récentes montrent que XPC serait également impliquée dans d'autres voies de réparation comme la réparation par excision de bases et la réparation des cassures double-brin. L'ensemble de ces observations suggère que ce complexe multifonctionnel pourrait avoir un rôle global dans la réponse cellulaire aux agents génotoxiques.
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(11250960), Guangping Dong. "PRODUCT SPECIFICITY AND INHIBITION OF PROTEIN N-TERMINAL METHYLTRANSFERASE 1/2". Thesis, 2021.
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Protein N-terminal methyltransferases (NTMTs) are a family of enzymes that methylate the α-N-terminus of a variety of protein substrates. Both NTMT1 and NTMT2 recognize a unique N-terminal X-P-K/R motif (X represents any amino acid other than D/E) to install 1-3 methyl group(s) on the substrates. NTMT1 plays important roles in mitosis regulation, chromatin interactions, and DNA damage repair. Another member NTMT2 shares ~50% sequence similarity and the same substrate recognition motif although NTMT2 was initially characterized as a mono-methyltransferase. To understand the molecular mechanism of NTMT2, we obtained the first co-crystal structure of NTMT2 in complex with its peptide substrate. After an extensive investigation of substrate recognition and methylated products of NTMT1/2, we found out that NTMT2 can fully methylate G/P-PKRIA peptides despite a predominant mono-methyltransferase. Moreover, we identified a gatekeeper N89 in NTMT2 that controls the substrate entry and the product specificity of NTMT2.
To elucidate the biological functions of NTMT1/2-catalyzed N-terminal methylation, we applied two different strategies to discover cell-potent inhibitors. Guided by the co-crystal structures of NTMT1 in complex with previously reported inhibitors, we designed and synthesized a series of new peptidomimetic inhibitors. By introducing more hydrophobic groups, the most cell-potent peptidomimetic inhibitor GD562 (IC50 = 0.93 ± 0.04 µM) exhibited over 2-fold increased inhibition on cellular N-terminal methylation levels with an IC50 value of ~50 µM compared to previously reported peptidomimetic inhibitor DC541. Meanwhile, we also discovered the first potent small molecule inhibitor Genz-682452 (IC50 = 0.5 ± 0.04 µM) after screening ~58,000 compounds. Subsequent structural modifications led to the discovery of GD433 (IC50 = 27 ± 0.5 nM) with a 20-fold increased potency compared to the initial hit Genz-682452. Inhibition mechanism indicated both inhibitors bind to peptide-binding pocket and co-crystal structures of both Genz-682452 and GD433 with NTMT1 confirmed their binding modes. Furthermore, GD433 shows over 7-fold selectivity over other major 40 protein methyltransferases and DNA methyltransferase and exhibits improved selectivity for NTMT1 over glucosylceramide synthase (GCS). GD433 significantly decreases the cellular N-terminal methylation level of NTMT1 substrates RCC1 and SET at 10 nM in both HEK293 and HCT116 cells, providing a valuable probe for cell-based studies in the future.
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Tseng, Po-Jung, i 曾柏榕. "(1)Newly Designed Cycloplatinated Polymer Dots as Photocatalysts for Visible Light–driven Hydrogen Evolution(2)Modification Polymer Dots with PEG Moieties to Reduce Non-specific Biomolecular Adsorption". Thesis, 2018. http://ndltd.ncl.edu.tw/handle/rws683.
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碩士國立中山大學
化學系研究所
106
(1) Newly Designed Cycloplatinated Polymer Dots as Photocatalysts for Visible Light–driven Hydrogen Evolution Overuse of fossil fuels is intensifying air pollution and greenhouse effect. Thus, developing a clean, renewable energy is a matter of utmost urgency. Hydrogen has been identified as a potential energy carrier because of its high energy capacity and environmental friendliness. However, hydrogen does not exist naturally on earth; we have to make it before use it. Nowadays, there are two main pathways to produce hydrogen that is steam methane reforming and water electrolysis. Among these pathways, water electrolysis is considered as a sustainable way to produce hydrogen because its feedstock is water. However, water splitting is an uphill reaction, requiring the energy supplied from an external resource. If this energy can be obtained from a renewable energy source such as solar energy, hydrogen can then be considered as a green energy totally. In this research, we provide a series of cycloplatinated polymer dots as photocatalyst, in which the platinum complex unit is used as a co-monomer and then linked to a conjugated polymer through Suzuki coupling polymerization. After optimizing the ratio of the Pt complexes, the hydrogen evolution rate (HER) of the cycloplatinated Pdots can be enhanced 9-times higher than the pristine Pdots under the same conditions. Furthermore, the enhancement of the reaction time and the stability are observed by introducing the cycloplatinated Pdots as photocatalysts. Based on the outstanding performance, our newly designed Pdots systems are promised to be a new type of photocatalysts for visible light–driven hydrogen evolution. Keywords: Semiconducting polymers, Polymer dots, Photocatalysts, Visible light, Hydrogen evolution (2) Modification Polymer Dots with PEG Moieties to Reduce Non-specific Biomolecular Adsorption Lately, semiconducting polymer nanoparticles with small sizes (< 30 nm) have been new highly fluorescent probes in optical imaging techniques because of their outstanding fluorescence brightness, good photostability, and minimal toxicity to biosystems. Due to hydrophobic polymer composition, some challenges limit Pdots development to the clinic like uptake by the reticuloendothelial system (RES), nonspecific binding, and entrapment in the live. The research shows that the addition of poly-ethylene glycol (PEG) into nanoparticles reduces RES uptake and increases circulation time in vivo versus uncoated one. Pdots formation is driven by hydrophobic interactions, means polymers in the Pdots are physically associated with each other. In many cases, the functional molecules may fall off from the nanoparticles due to the weak non-covalent interactions. In this research, a molecular with PEG moieties was first synthesized and then covalently linked to a conjugated polymer through Suzuki coupling. Based on this covalent linking strategy, we hope the PEGylated pdots could load more PEGylated molecular and diminish non-specific effect in biological environment. Keywords: PEGylation、Polymer dots、Non-specific effect、Bioimaging、FRET。