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Application of Mass Spectrometry in Biology and PhysiologyGong, Jiawei 02 June 2016 (has links)
No description available.
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Functional analysis of Pso2 reveals a novel DNA hairpin endonuclease activity: Implications for interstrand crosslink repairTiefenbach, Tracy E. 10 1900 (has links)
<p>DNA interstrand crosslinks provide a challenge for repair machinery given that both strands contain the lesion. Cells have evolved a sophisticated mechanism to overcome this, by recruiting proteins from several repair pathways. One protein thought to function solely in interstrand-crosslinking repair is Pso2. Pso2 deficient cells display sensitivity towards ICL agents and accumulate DNA double strand breaks upon exposure. However, Pso2 is not required for repair of DNA double strand breaks generated by other means, suggesting that these particular breaks are unique requiring Pso2 processing for successful repair. To identify what characteristics these breaks possess and what role Pso2 plays in processing theses breaks, a thorough <em>in vivo</em> and <em>in vitro </em>characterization of Pso2 was conducted.</p> <p>Pso2 was found to be a 5’-exonuclease independent of DNA structure and length but completely dependent on a 5’-phosphate. Pso2 also displayed structure-specific DNA hairpin-opening activity at the 3’ end two nucleotides from the apex. This activity was required for repair of genomic DNA capped by hairpin structures in the absence of ICL inducing agents as well those generated in response to ICL damage. The constitutively active DNA hairpin endonuclease β-CASP domain of Artemis was able to partially restore the DNA hairpin-opening deficiency and suppress the ICL defect in a <em>pso2 </em>null strain. This suggests that Pso2 acts as an endonuclease in ICL repair and that DNA hairpins may be an encountered intermediate, leading to further understanding of how this unique protein function in ICL repair as well as the repair mechanism itself.</p> / Doctor of Science (PhD)
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The Effects of Dietary Lipids on Bone Chemical, Mechanical and Histological Properties in Japanese Quail (Coturnix C. Japonica)Liu, Dongmin 12 July 2000 (has links)
Japanese quail were used as animal models in four experiments to evaluate the effects of supplementing diets with different lipids on bone chemical, mechanical, and histological properties. In Exp. 1, laying hens were fed a basal diet containing either 5% soybean oil (SBO), hydrogenated soybean oil (HSBO), chicken fat (CF), or menhaden fish oil (FO). The addition of SBO in the maternal diet increased the levels of total n-6 fatty acids and arachidonic acid (AA, 20:4n-6) in yolk and tibial bones of newly hatched progeny (P<0.01), whereas the maternal FO diet elevated the concentrations of total n-3 fatty acids, eicosapentaenoic acid (EPA, 22:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and total saturated acid, but greatly decreased the amount of AA in both egg and progeny tibiae (P<0.01). The maternal HSBO diet resulted in the accumulation of trans-18:1 fatty acid in egg yolks and tibiae at hatch. The addition of FO or HSBO to the maternal diet significantly lowered the ex vivo PGE2 production of tibiae in newly hatched quail compared to those from hens given the SBO or CF diets (P<0.01). In Exp. 2, the addition of different lipids in the maternal diets did not affect growth, tibial length, diameter or collagen content of the progeny. However, supplementing the maternal diet with 5 % FO or HSBO increased the percent bone ash , increased bone pyridinium crosslinks of collagen, enlarged the cartilaginous proliferative and hypertrophied zones, increased diaphyseal cortical thickness of the tibiae in embryos (P<0.05), and subsequently increased tibial shear force, stiffness (P<0.05) and improved cortical thickness, density and trabecular density in early growth and development of progeny compared to those from hens consuming the SBO or CF diets (P<0.05). In Exp. 3, male quail at one month of age were fed a basal diet containing either 5% SBO, HSBO, CF or FO for seven months. Long-term supplementation in the diets of different lipids did not affect body weight, food intake, tibial length or diameter, but the FO group had the highest tibial percent ash, and both FO and HSBO increased tibial mineral content in aged quail compared to those fed the SBO or CF diets (P<0.05). At 8 months of age, quail fed FO had the highest concentrations of (n-3) fatty acids (20:5n-3, 22:5n-3, 22:6n-3) but the lowest amounts of 20:4n-6 in lipids from tibial cortical bone, whereas the SBO and CF diets greatly elevated (n-6) fatty acids and 20:4n-6 levels. The HSBO diet which contains t18:1 fatty acid resulted in t18:1 accumulation in bone. Long-term supplementation with FO or HSBO increased tibial shear force, stiffness and shear stress, as well as improved cortical thickness and density compared with the SBO or CF diets
( P<.05). In Exp. 4, the addition of SBO or CF to the diet for seven months decreased tibial mineral content compared to the FO diet (P<0.05). Quail fed SBO increased collagen concentration in the tibiae (P<0.05), but the level of collagen crosslinks was higher in quail fed FO or HSBO compared to those given the SBO or CF diets (P<0.05). The PGE2 production in bone organ culture and marrow was greatly increased in quail maintained on the SBO or CF diets (P<0.05). PGE2 production in the bone microenvironment was negatively correlated with the tibial percent ash and collagen crosslinks but had a positive correlation with tibial collagen concentration. The results of these studies demonstrate that either supplementing the maternal diets with or long-term exposure to different lipids alters the chemical composition and metabolism of skeletal tissue in both embryos and aged quail. Maternal dietary SBO or CF had an adverse effect on bone growth and development in embryos. Likewise, long-term exposure to SBO or CF diet impaired bone metabolism and remodeling. In contrast, the FO or HSBO diet had beneficial effects on bone modeling in embryos and remodeling in adult quail. / Ph. D.
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Etude des dommages de l'ADN impliquant des pontages ADN-protéines et ADN-polyamines / Study of DNA damages involving DNA-proteins and DNA-polyamines crosslinksSilerme, Stéphanie 31 October 2014 (has links)
Un pontage ADN-protéine se forme lorsqu'une protéine se lie de façon covalente à l'ADN, ce qui a pour conséquence de bloquer certains processus biologiques tels que la réplication, la transcription, la réparation ou la recombinaison. Ces travaux de thèse consistent en l'étude des pontages se produisant lors d'une oxydation à un électron de l'ADN. La guanine possède le potentiel d'ionisation le plus bas parmi les composants de l'ADN; elle est donc facilement oxydée pour former un radical cation, lui-même impliqué dans la formation de nombreuses lésions oxydatives. Des travaux antérieurs ont permis de mettre en évidence la formation d'un adduit entre la guanine et la lysine, résultant de l'oxydation à un électron d'un oligonucléotide TGT en présence d'un peptide trilysine. Le mécanisme de cette réaction est une addition nucléophile de l'acide aminé central par le groupement amine ε, en position C8 du radical cation de la guanine. L'objectif de cette thèse a été de caractériser l'adduit guanine-lysine, de le quantifier dans l'ADN isolé puis dans l'ADN cellulaire, et d'étudier son implication dans la formation des pontages ADN-protéines. Différentes espèces nucléophiles sont capables de s'additionner sur le radical cation de la guanine. Nous nous sommes intéressés au cas des polyamines endogènes, qui sont des cations organiques présents en particulier dans le noyau des cellules. Ces molécules interviennent dans la stabilisation et la condensation de l'ADN, mais elles participent également à de nombreux processus cellulaires. Le lien entre polyamines et cancer a été largement décrit. Cependant le mécanisme par lequel la perturbation de leur métabolisme est impliquée dans le processus cancérogenèse reste à ce jour peu connu.Dans un premier temps, ces lésions ont été synthétisées chimiquement, sous la forme de nucléosides modifiés, afin de les caractériser. Par la suite des méthodes de quantification de ces dommages par chromatographie liquide haute performance couplée à la spectrométrie de masse en tandem ont été développées. Ces méthodes analytiques nous ont permis de démontrer que les adduits guanine-lysine et guanine-polyamines pouvaient se former dans l'ADN isolé suite à une oxydation à un électron. Des pontages entre guanine et lysine ont été mis en évidence dans l'ADN extrait de cellules THP1 irradiées par impulsions laser à 266 nm. Nous avons ensuite développé différents modèles de pontages entre un peptide et un oligonucléotide, afin d'étudier la structure chimique du pontage, et de déterminer si celui-ci pouvait se produire entre la guanine et la lysine. Des adduits guanine-polyamines ont également été détectés dans de l'ADN extrait de spermatozoïdes. Ces résultats ouvrent de nouvelles perspectives dans la compréhension du rôle physiologique des polyamines ainsi que de leur implication dans la fertilité masculine. / A DNA-protein crosslink (DPC) occurs when a protein becomes covalently bound to DNA. This kind of lesions seems to affect several metabolic processes, including DNA replication, transcription, repair and recombination. This PhD work deals with crosslinks which are formed through a one-electron oxidation of DNA. Guanine exhibits the lowest ionization potential among DNA components, therefore it is readily oxidized leading to the formation of a radical cation, which is involved in the formation of numerous oxidative DNA lesions. In a previous study, a crosslink between guanine moiety and a lysine residue, generated subsequently to a one electron oxidation of a TGT oligonucleotide in the presence of a trilysine peptide, has been described. The mechanism of formation of this adduct relies on the nucleophylic addition of the ε amino group of lysine onto the C8 position of the guanine radical cation. The aim of the present work was to characterize the guanine-lysine adduct and to quantify this lesion in isolated DNA and then in cellular DNA, and to investigate their implication in DNA-protein crosslinks. Several nucleophylic species are able to react with the guanine radical cation. We focused on polyamines, which are organic cations localized in the nucleus of cells at millimolar concentration ranges. These molecules are involved in stabilization and condensation of DNA, and participate also in numerous cellular processes. The relation between polyamine and cancer has been widely described. The mechanism by which dysregulation in their metabolism is related to carcinogenesis is still unknown.In the first part of this project, we focused on the synthesis and the characterization of these lesions as modified nucleosides. Subsequently, we have developed and optimized methods of quantification of these damages, using HPLC coupled with tandem mass spectrometry. Thanks to these analytical methods, we have demonstrated that guanine-lysine and guanine-polyamines adducts could be formed in isolated DNA following a one electron oxidation. Crosslinks between guanine and lysine have been highlighted in DNA extracted from THP1 cells exposed to laser pulses at 266 nm. We have then developed several crosslinks models between a peptide and an oligonucleotide, in order to investigate the chemical structure of the crosslink and determine whether it could occur between guanine and lysine. Guanine-polyamines adducts have also been detected in DNA extracted from sperm cells. These results open new prospects in the understanding of the physiological role of polyamines as well as their involvement in male fertility.
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Comparaison des effets précoces d’un agent anti-résorbeur et d’un agent anabolique sur le remodelage osseux et la microarchitecture chez la brebis âgée / Comparison of the early effects of an anti-resorptive agent and an anabolic agent on the bone remodeling and the microarchitecture in the aged ewePortero-Muzy, Nathalie 30 October 2012 (has links)
Les effets des agents anti-ostéoporotiques sur le tissu osseux sont évalués au niveau de la crête iliaque (CI) mais les réponses aux traitements peuvent varier selon le site osseux. Le but de cette étude était de comprarer les effets de l’acide zolérodronique (ZOL) et du tériparatide (TPTD) au niveau de la crête iliaque et de la vertèbre lombaire L1 (VL1) chez la brebis âgée. Le ZOL a induit une forte diminution du remodelage osseux et une augmentation des microendommagements au niveau des deux sites et une modification des crosslinks du collagène surtout au niveau de l’os cortical de la CI. Trois mois de TPTD ont augmenté le remodelage osseux uniquement au niveau de la VL1. En conclusion, les délais et les amplitudes de réponses au ZOL ou au TPTD diffèrent entre la CI et la VL1 chez la brebis. Ces résultats montrent l’importance de prendre en compte le site osseux pour évaluer les effets des agents anti-ostéoporotiques / The effects of anti-osteoporotic agents on bone tissue are evaluated on iliac crest (IC) but the answers to treatments may vary according to the skeletal site. The purpose of this study was to compare the effect of zoledronic acid (ZOL) and teriparatide (TPTD) on IC and lumbar vertebrae (LV1) in ewes. ZOL has induced a high decrease of bone remodeling, an increase in microdamages in both sites and a modification of collagen crosslinks mainly in cortical bone of IC. Three months of TPTD has increased the bone remodeling only in LV1. In conclusion, the delays and the magnitudes of responses to ZOL or to TPTD differ between IC and LV1 in ewes. These results show that the distinction of bone sites to study the early effects of antiosteoporotic therapies appears meaningful
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Molecular Basis of Anticlastogenic Potential of Vanadium in Vivo During the Early Stages of Diethylnitrosamine-Induced Hepatocarcinogenesis in RatsChakraborty, Tridib, Pandey, Nirupama, Chatterjee, Amrita, Ghosh, Balaram, Rana, Basabi, Chatterjee, Malay 30 October 2006 (has links)
Carcinogen-induced DNA base modification and subsequent DNA lesions are the critical events for the expression of premalignant phenotype of the cell. We have therefore investigated the chemopreventive efficacy of a vanadium salt against diethylnitrosamine (DEN)-induced early DNA and chromosomal damages in rat liver. Hepatocarcinogenesis was induced in male Sprague-Dawley rats with a single, necrogenic, intraperitoneal injection of DEN (200 mg/kg body weight). 8-Hydroxy-2′-deoxyguanosines (8-OHdGs), strand-breaks and DNA-protein crosslinks (DPCs) were measured by HPLC, comet assay and spectrofluorimetry, respectively. There was a significant and steady elevation of modified bases 8-OHdGs along with substantial increments of the extent of single-strand-breaks (SSBs), DPCs and chromosomal aberrations (CAs) following DEN exposure. Supplementation of vanadium as ammonium metavanadate (NH4VO3, +V oxidation state) at a dose of 0.5 ppm in terms of the salt weight throughout the experiment abated the formations of 8-OHdGs (P < 0.0001; 79.54%), tailed DNA (P < 0.05; 31.55%) and length:width of DNA mass (P < 0.02; 61.25%) in preneoplastic rat liver. Vanadium treatment also inhibited DPCs (P < 0.0001; 58.47%) and CAs (P < 0.001; 45.17%) studied at various time points. The results indicate that the anticlastogenic potential of vanadium in vivo might be due to the observed reductions in liver-specific 8-OHdGs, SSBs and/or DPCs by this trace metal. We conclude that, vanadium plays a significant role in limiting DEN-induced genotoxicity and clastogenicity during the early stages of hepatocarcinogenesis in rats.
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Cellular Analyses of the RAD51-related Homologous Recombination Repair ProteinsGruver, Aaron Matthew 19 September 2005 (has links)
No description available.
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Processing of Cisplatin Interstrand crosslinks (ICLs) by DNA repair proteinsDangeti, Venkata Srinivas Mohan Nimai January 2012 (has links)
No description available.
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Polymer networks with mobile force-applying crosslinksMateyisi, Mohau Jacob 03 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: We construct and study a simple model for an active gel of
exible polymer filaments
crosslinked by a molecular motor cluster that perform reversible work while translating
along the filaments. The filament end points are crosslinked to an elastic background.
In this sense we employ a simplified model for motor clusters that act as slipping links
that exert force while moving along the strands. Using the framework of replica theory,
quenched averages are taken over the disorder which originates from permanent random
crosslinking of network end points to the background. We investigate how a small motor
force contributes to the elastic properties of the network. We learn that in addition to
the normal elastic response for the network there is an extra contribution to the network
elasticity from the motor activity. This depends on the ratio of the entropic spring constant
for the linked bio-polymerchain to the spring constant of the tether of the motor. / AFRIKAANSE OPSOMMING: Ons konstrueer en bestudeer 'n eenvoudige model vir 'n aktiewe netwerk van
eksieble
polimeerfilamente wat deur grosse van molekulere motors aan mekaar verbind word wat
omkeerbare werk doen terwyl dit langs die filamente transleer. Die eindpunte van die filamente
is aan 'n elastiese agtergrond verbind. In hierdie sin benut ons 'n eenvoudige model
vir motorclusters wat as verskuifbare verbindings krag op die filamente tydens beweging
kan uitoefen. Nie-termiese wanorde gemiddeldes word geneem oor die wanorde wat deur
die lukrake permanente verbindings van netwerk eindpunte aan die agtergrond veroorsaak
word. Ons ondersoek hoe 'n klein motorkrag tot die elastiese eienskappe van die netwerk
bydra. Ons leer dat daar bo en behalwe die gewone elastiese respons vir die netwerk 'n
elastiese bydrae as gevolg van die motors se aktiwiteit voorkom. Dit hang af van die verhouding
van die entropiese veerkonstante van die biopolimerketting tot die veerkonstante
van die anker van die motor.
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Topologie und Regulation der Manduca sexta V-ATPaseReineke, Stephan 22 November 2002 (has links)
Topologie und Regulation der Manduca sexta V-ATPase
Die V-ATPase im Mitteldarm der Tabakschwärmerraupen von Manduca sexta besteht aus zwölf Untereinheiten, von denen vier den membranständigen Vo- und acht den cytosolischen V1-Komplex bilden. Das Enzym energetisiert unter ATP-Verbrauch eine Protonentranslokation über die Gobletzellapikalmembran, wobei eine Potentialdifferenz von etwa 250 mV aufgebaut wird. Durch diese Spannung wird ein elektrogener K+/2H+-Antiport getrieben und indirekt ein K+/Aminosäure-Symport, wodurch die Nährstoffversorgung der Raupen gesichert wird. Da die V-ATPase sehr viel ATP verbraucht, erscheint es ökonomisch, diese in Hungerperioden durch die Dissoziation des Enzyms in den cytosolische V1- und den Vo-Komplex, abzuschalten.
In der vorliegenden Arbeit wurde untersucht, ob auch die Biosynthese der V-ATPase Untereinheiten in Hungerperioden reduziert wird und was eventuellen Änderungen von Transkriptionsraten zugrunde liegt. Mit Ausnahme der Untereinheit D waren die Transkriptmengen aller V-ATPase Untereinheiten in Hungerperioden erniedrigt. Am stärksten betraf dies die Untereinheit G, was auch für den Anstieg der Transkriptmengen nach erneuter Futterzufuhr galt. Da Hungerperioden auch in der Entwicklung von Raupen während der Häutungen vorkommen, wurde exemplarisch die Biosynthese von drei, die verschiedenen Bereiche der V-ATPase (V1-Kopf: Untereinheit B, V1-Stiel: Untereinheit G, Vo-Komplex: Untereinheit d) repräsentierenden Untereinheiten, untersucht.
In allen drei Fällen konnte eine Reduktion der Transkriptmengen in der Mitte der Häutungsphase festgestellt werden, welche zu ihrem Ende hin wieder aufgehoben wurde. Der Abfall und Anstieg der mRNA-Mengen korrelierte mit den Titern der beiden Häutungshormone der Insekten: negativ mit dem Titer des Ecdysons und positiv mit dem des Juvenilhormons. Die Injektion von 20-Hydroxyecdyson in fressende Raupen hatte die Reduktion der Transkriptmengen zur Folge, während Juvenilhormon III fast keinen Einfluss ausübte. Darüberhinaus war zu beobachten, dass sich nach Injektion von 20-Hydroxyecdyson die V1-Komplexe von den apikalen Gobletzellmembranen ablösten.
Um auch den Einfluss der Häutungshormone auf die Promotoraktivität zu untersuchen, und dadurch auf Unterschiede in der RNA-Stabilität zu schließen, wurden ca. 1 kb lange 5`-Bereiche stromaufwärts vom Startcodon der drei verwendeten Gene mvB, mvG und mvd in Reportergenassays getestet. Hierbei wurde als Reportergen eine Luciferase verwendet, die unter der Kontrolle der jeweiligen 5`-Region der V-ATPase Untereinheit stand. Nach Transfektion von Sf21-Zellen konnte wie auch in den vorangegangenen Experimenten gezeigt werden, dass 20-Hydroxyecdyson die Promotoraktivität aller drei V-ATPase-Gene nach einem kurzzeitigen Anstieg bei den Genen mvB und mvG über einen längeren Zeitraum negativ beeinflusst und nach 48 h zu einer Reduktion auf 30-50% gegenüber der Kontrolle führt.Im Gegensatz dazu führte die Anwesenheit von Juvenilhormon III zur Aktivitätssteigerung von mvG um den Faktor 3, während die Aktivität der anderen 5`-Bereiche nicht signifikant verändert wurde.Zusammen mit den Daten der Transkriptmengen unter Juvenilhormon III Einfluss könnte dies der erste Hinweis auf eine reduzierte Stabilität der mRNA der Untereinheit G sein.
Des Weiteren wurde in der vorliegenden Arbeit die Nukleotidsequenz der bei der Insekten V-ATPase lange Zeit nicht nachweisbaren Untereinheit a des Vo-Komplexes aufgeklärt. Neben einer ubiquitär vorkommenden Isoform konnte auch eine Teilsequenz einer Malpighigefäß spezifischen Isoform nachgewiesen werden. Antikörper gegen den in dieser Arbeit exprimierten cytoplasmatischen N-Terminus wurden eingesetzt, um die Untereinheit in der Immunhistochemie sowie in den gebildeten Komplexen der Vernetzungsexperimente nachzuweisen. Die durch Kupfer(II)-chlorid induzierte Vernetzung von Cysteinresten der Untereinheiten des V1Vo-Holoenzyms führte zu der Identifizierung von drei Banden, wobei diese wahrscheinlich aus verschiedenen Subkomplexen der Untereinheiten a, A, B, C, E und G aufgebaut waren. Durch diese Ergebnisse und den Daten aus dem Verdau des V1Vo-Holoenzyms mit Trypsin konnte ein neues Modell der V-ATPase erstellt werden, dass sich erheblich von den bisherigen Modellen unterscheidet, insbesondere in der Lokalisation der Untereinheiten des Stators.
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