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1
BENDER, Cecilia. « Study of Human T-Lymphotropic Virus type 2 mRNA kinetics of expression and identification of a novel splicing site ». Doctoral thesis, Università degli Studi di Verona, 2010. http://hdl.handle.net/11562/343864.
Texte intégralRésumé :
L’analisi di espressione dei retrovirus Human T-cell lymphotropic viruses (HTLV-1 e 2) durante il processo di infezione è essenziale per capire l’influenza che i prodotti dei geni virali hanno sui processi di proliferazione, ciclo e signalling cellulari.
Studi recenti condotti su cellule infettate con HTLV-1 hanno ermesso di delineare la cinetica di espressione dei diversi trascritti virali. Per quanto concerne HTLV-2, il profilo di espressione degli mRNA virali in cellule infettate risulta ancora poco studiato e le relative cinetiche non sono state ancora delineate.
Scopo di questo studio è stato quindi quello di analizzare l’espressione dei diversi trascritti di HTLV-2 mediante la messa a punto di analisi real time RT-PCR per la loro determinazione quantitativa. In particolare sono state studiate e quantificate le
cinetiche di espressione dei trascritti di HTLV-2 in linee cellulari infettate e in linfociti da sangue periferico (PBMC) provenienti da pazienti infetti.
In modo analogo ad altri retrovirus, HTLV-2 esprime diversi prodotti genici dalla stessa regione codificante, attraverso diverse strategie che sottointendono un complesso pattern di splicing alternativo. L’espressione di HTLV-2 è basata su tre principali classi di mRNA: a) “unspliced” mRNA, che codifica per le proteine Gag, Pol e Proteasi; b) “singly” spliced mRNA che codifica per Env e per le proteine accessorie p28 e p22/p20; c) “doubly spliced” mRNA che codifica per le proteine Tax, Rex e le
proteine accessorie p10, p11 ed 1-2-B. Recentemente inoltre è stato descritto un nuovo prodotto proteico, APH-2, che viene codificato dallo strand negativo di HTLV-2 e sembra essere coinvolto nei processi di silenziamento trascrizionale del virus in cellule infettate. Il profilo di espressione e le cinetiche dei diversi trascritti virali sono stati quantificati mediante la metodica di Real Time RT-PCR, utilizzando sonde e primers specifici per le giunzioni di splicing. Il profilo temporale di ogni trascritto di HTLV-2 è stato studiato utilizzando tre diversi sistemi cellulari: linee stabilmente infettate, cellule trasfettate in modo transiente, e infine PBMC provenienti da soggetti infettati da HTLV-
2B messi in colturea e stimolati con IL-2.
I risultati ottenuti in questo studio hanno portato alla quantificazione di tutti i trascritti virali di HTLV-2. Sono stati inizialmente riscontrati diversi livelli di espressione di env nei due sottotipi HTLV-2 A e B. La diversa espressione del trascritto
env in linee cellulari stabilente infettate, ha indotto ad approfondire il complesso meccanismo di splicing tra gli esoni 1 e 2, portando all’identificazione di un nuovo sito accettore di splicing (3’SS). Si è potuto così determinare che questo nuovo sito
accettore viene utilizzato nell’espressione del trascritto env “singly splicing” e dei trascritti bicistronici tax/rex, p10/p11 e 1-2-B “doubly splicing” derivati da splicing alternativo all’interno della regione genica pX di HTLV-2. I risultati sperimentali hanno
quindi permesso di evidenziare che il nuovo sito 3’SS è presente in entrambi i sottotipi A e B di HTLV-2 e che la nuova isoforma di env, chiamata env 1-2b, è preferenzialmente espressa nel sottotipo 2B mentre nel sottotipo 2A l’espressione di env
utilizza più efficientemente il sito canonico di splicing.
L’analisi della cinetica di espressione del mRNA totale, ha mostrato un andamento caratterizzato da una bassa trascrizione iniziale, seguita da un aumento con un progressivo picco di espressione dopo 21-24 ore dall’inizio della coltura.
Il trascritto “full lenght” gag/pol risulta essere il più espresso durante il periodo analizzato, comportandosi come un gene tardivo e raggiungendo quindi un picco di espressione dopo 21-48 ore. L’env canonico è espresso a livelli molto bassi nelle linee
stabilmente infettate e non e’ rilevabile in PBMC di pazienti infettati da HTLV-2B. Al contrario, l’isoforma env 1-2b viene efficientemente espressa, sia in linee cellulari che in PBMC ex-vivo e presenta un profilo di espressione tardivo seguito da un graduale e costante aumento, raggiungendo il picco massimo di espressione tra 21 e 48 ore. Infine il trascritto di regolazione tax/rex viene espresso precocemente da HTLV-2B sia in linee
stabilmente infettate che in ex-vivo PBMC.
Le cinetiche del trascritto 1-2-B indicano che questo gene viene espresso ad alti livelli negli intervalli di tempo precoci mentre il mRNA per le proteine accessorie p10 e p11 risulta essere poco espresso o inferiore alla soglia di detezione. Tra gli mRNA che
subiscono “singly splicing” e che codificano per le proteine accessorie, l’isoforma p28,p22/p20-II è maggiormente espressa nei due sottotipi HTLV-2A e B rispetto all’isoforma p28, p22/p20-I.
Il trascritto APH-2 viene espresso ad alti livelli sia in cellule stabilmente infettate che in cellule trasfettate e presenta un andamento tardivo in questi sistemi cellulari. Tuttavia nei PBMC ottenuti da pazienti infetti non e' stato possibile determinarne un chiaro pattern di espressione in quanto questo trascritto risulta essere molto variabile.
Questo studio ha messo in evidenza che i trascritti di HTLV-2 presentano cinetiche di espressione specifiche e diversificate. I risultati ottenuti hanno permesso di descrivere un preciso pattern di produzione temporale degli mRNA, con geni precoci quali tax/rex e 1-2-B e geni tardivi quali gag/pol, env e 28,p22/p20-II. L’espressione precoce di tax/rex indica che questa proteina è necessaria nelle fasi iniziali del ciclo virale al fine di transattivare e regolare i geni virali e cellulari. I geni strutturali gag/pol
e env sono invece espressi tardivamente quando i livelli d’espressione dei geni precoci sono in fase di dimunuizione, il che indica quindi un chiaro “switch” temporale tra geni precoci e tardivi. Questo studio ha inoltre evidenziato un nuovo sito accettore di splicing alternativo nella regione pX di HTLV-2.
In conclusione, questo studio ha permesso di stabilire che i livelli dei geni di HTLV-2 e le loro cinetiche di espressione sono strettamente regolati. Inoltre, queste ricerche hanno messo in evidenza che l’utilizzo di nuovi siti accettori gioca un ruolo
fondamentale nell’ espressione preferenziale di proteine virali specifiche alle diverse fasi del ciclo virale di HTLV-2.The analysis of HTLV expression during the infection process is essential to understand the influence of viral gene products on proliferation, cell cycle and signalling. Recent studies carried out on HTLV-1-infected cells have provided information on the relative abundance and timing of expression of different viral transcripts. In the case of HTLV-2, very little information has been so far obtained on the profile of viral gene expression in infected cells, and the kinetics of transcript expression have not yet been investigated. The aim of the study was to further investigate HTLV-2 transcripts expression, by developing new quantitative analyses to measure the levels of expression of different HTLV-2 mRNAs. In particular the kinetics of HTLV-2 transcripts at different stages of virus gene expression and their quantitation in infected cell lines and in PBMCs from infected subjects, have been evaluated. Similarly to other retroviruses, HTLV-2 expresses multiple gene products from the same coding region by choosing different strategies, including a complex pattern of alternative splicing. HTLV-2 expression produces three major classes of mRNAs: unspliced mRNA for Gag, Protease and Pol proteins; singly spliced mRNAs for Env and accessory proteins p28 and p22/p20; and doubly spliced mRNAs for the regulatory Tax, Rex and the accessory p10, p11 and 1-2-B proteins. More recently, the new APH-2 protein coded by the negative strand of HTLV-2, and possibly involved in the transcriptional silencing of the virus in infected cells was described. The expression profile and kinetics of the different transcripts were analysed by RT-PCR assays using a series of splice-junction-specific primers and probes. The time courses of each HTLV-2 transcript in three different cellular systems, namely stably infected cells lines, transiently transfected cells and ex-vivo IL-2 stimulated PBMCs from infected subjects were investigated. The results obtained led to the quantitation of all known HTLV-2 mRNAs. Preliminary data showed different levels of env transcript in HTLV-2 subtypes A and B. Evidence for a differential env expression in HTLV-2B stably infected cells, prompted further investigation on the complex pattern of splicing between exons 1 and 2 and allowed the identification of a novel 3’ acceptor site (SS) of splicing. This novel 3’SS is used to express alternative spliced isoforms within the pX terminal region of HTLV-2, including the singly spliced env and the three doubly spliced bicistronic tax/rex, p10/p11 and 1-2-B transcripts. Results demonstrated that the novel 3’SS was utilised in both HTLV-2 subtypes, and that the novel env isoform, named env 1-2b, was preferentially expressed in 2B subtypes, while 2A subtypes used more efficiently the canonical splice site. The kinetics of total mRNA HTLV-2 expression in these three cellular systems presented a general pattern characterised by an initial low transcript level and a subsequent increase to reach a peak of expression after 21-24 hours in culture. The full length gag/pol mRNA was the most abundant one during the time course, and behaved as a late gene that peaked after 21 to 48 hours. The canonical env transcript was expressed at very low rates in stably infected cells, and it was undetectable in PBMCs from infected subjects and in cells transfected with the HTLV-2 proviral clone. By contrast, env 1-2b isoform was efficiently expressed in stably infected cells as well as in ex-vivo infected PBMCs, behaving as a late gene showing a gradual and steady increase in copy number and reaching maximum expression at 21 to 48 hours. The regulatory tax/rex gene was expressed early and with a high/intermediate transcription rate in HTLV-2B subtypes in both stably infected cells and ex-vivo PBMCs. In this study the kinetics of expression of the yet unkwnon 1-2-B gene was investigated and found to be expressed at high levels at early time points, whereas the doubly spliced mRNA of accessory p10/p11 genes were poorly expressed or under detection limit. Among the singly spliced mRNAs coding for other accessory proteins, the p28, p22/p20-II isoform was found to be highly expressed in both HTLV-2 A and B subtypes as compared to its alternative p28, p22/p20-I form. The APH-2 negative transcript was efficiently expressed at high levels in both stably infected and transfected cells and behaved as a late gene. However, in ex-vivo PBMCs its expression level and kinetics pattern appeared to be variable and a clear pattern of expression was not assessed. In conclusion, this study demonstrated that HTLV-2 transcripts of both A and B subtypes are differentially expressed. A temporal pattern of mRNA production, with early, tax/rex and 1-2-B and late, gag/pol, env, p28,p22/p20-II genes was established. The tax/rex early expression indicates that this protein is necessary at the beginning of the viral cycle to transactivate and regulate viral and cellular genes. Most structural genes were expressed late when the transcription of early genes was already decreasing, thus showing that a temporal switch was occurring between early to late genes production. This study also provided new clues on the selective use of alternative 3’SS within the pX region of HTLV-2 for both subtypes A and B. Overall these findings indicate that the control of HTLV-2 viral gene expression is highly regulated both in its kinetics and expression. Moreover, it suggests that the use of multiple acceptor sites might play an important role on the preferential expression of specific proteins in the different phases of the viral cycle.
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Taylor, David C. « SELEX targeting mRNAs : the hunt for novel riboregulators / ». free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3013032.
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Macdonald, Murdo. « Expression of mRNAs encoding FMRFamide-related peptides in adult and embryo Helix aspersa ». Thesis, University of St Andrews, 1993. http://hdl.handle.net/10023/14454.
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The gastropod mollusc Helix aspersa is known to contain at least seven FMRFamide-related peptides (FaRPs), neuropeptides which fall into two broad classes, distinguished by their primary structure and their physiological actions. We have sought to use the techniques available to us through molecular biology to study the structure and expression of the nucleic acids (RNA and DNA) which encode these peptides in this organism. The two classes of peptide, tetra- and heptapeptides, were found by us to be apparently separated by the stage of mRNA generation: the precursor polypeptides encoded by these mRNAs were also found to have differing structures. Expression of mRNAs specific for the FaRPs were studied during embryogenesis, where there appears to be regulated expression of these mRNAs. In situ hybridization analyses of the central nervous system of adult Helix revealed expression of FaRP-specific mRNAs to be limited to a small number of discrete neurons: it was again observed that there was an apparent distinction between the tetra and extended FaRPs, no ceils being identified in our studies to express both types of mRNA. Confocal scanning microscopy indicated that the distribution of the mRNA, which appeared to be limited to the cytoplasm of cell bodies expressing the peptides, was non-uniform, probably reflecting a functional characteristic of the cells concerned.
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Drummond, D. R. « The stability, movement and expression of natural and synthetic mRNAs injected into Xenopus oocytes ». Thesis, University of Warwick, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.373064.
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Chang, Chih Chien Anne. « Developmental expression of GABAA[subscript]/BZ receptor subunit mRNAs in olivocerebellar circuitry of the mouse / ». The Ohio State University, 1994. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487848891512785.
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Rende, Francesca. « Kinetics and regulation of HTLV-1 gene expression ». Doctoral thesis, Università degli studi di Padova, 2011. http://hdl.handle.net/11577/3421976.
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ABSTRACT
Human T-Lymphotropic virus type 1 (HTLV-1) is the causative agent of two distinct pathologies, adult T-cell leukemia/lymphoma (ATLL), an aggressive malignancy of mature CD4+ T-cells, and tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM), a demyelinating neurodegenerative disease.
The HTLV-1 expression strategy is characterized by the production of plus- and minus-strand transcripts, alternative splicing and polycistronic translation. This strategy greatly increases the coding potential of the virus, resulting in expression of several regulatory and accessory genes (Tax, Rex, p12, p13, p21rex, p30tof and HBZ) in addition to the structural proteins and virion-associated enzymes common to all retroviruses (Gag, Pro, Pol and Env).
In spite of over 30 years of studies, several key features of the HTLV-1 life cycle and pathogenicity remain obscure. In particular, it is still unclear whether HTLV-1 gene expression is characterized by latency patterns, whether the different viral genes follow distinct kinetics of expression and, if this is the case, which molecular mechanisms control these phenomena.
The work described in the present thesis was aimed at understanding these aspects of HTLV-1 regulation. To this end we optimized a Real Time RT-PCR method using splice-site-specific primers to quantitate the different HTLV-1 transcripts and their kinetics of expression in peripheral blood mononuclear cells (PBMCs) isolated from HTLV-1-infected individuals and in cells transfected with HTLV-1 molecular clones. Results indicated that expression of HTLV-1 mRNAs follows a distinct timing upon reactivation of viral expression, with the mRNA coding for the Tax and Rex regulatory proteins acting as an early "master" transcript preceding expression of the other viral transcripts.
Although it is commonly accepted that Rex acts at a post-transcriptional level controlling the nuclear export and stability of viral mRNAs coding for the virion-associated proteins, the Rex-dependency of tax/rex, p12, p13, p21rex, p30tof and hbz transcripts has not been investigated so far. To test if the kinetics of HTLV-1 gene expression might be dependent on Rex function and to determine the Rex-dependence of individual HTLV-1 mRNAs, we generated a Rex knock-out HTLV-1 molecular clone and analyzed the nucleo-cytoplasmic compartmentalization of the viral mRNAs. Results demonstrated the strict Rex-dependency of the “two-phase” kinetics and revealed strong nuclear retention of hbz mRNAs, supporting their function as non-coding transcripts. Furthermore our results revealed that the Rex-responsiveness of the different HTLV-1 mRNAs is determined by a novel 72-nucleotides cis-acting regulatory sequence located upstream of exon 3.
Mathematical modelling underscored the importance of a temporal delay between the Tax and Rex functions, which was supported by experimental evidence of a delayed accumulation and longer half-life of Rex compared to Tax.
These data provide evidence for a temporal pattern of HTLV-1 gene expression, reveal major differences in the intracellular compartmentalization of HTLV-1 transcripts and, importantly, provide clues to a long-standing paradox of HTLV-1 regulation, i.e. the different Rex-dependence of viral transcripts in spite of the presence of the Rex-responsive element (RxRE) in the 3' untranslated region of all viral mRNAs.RIASSUNTO Il virus T-linfotropico umano di tipo 1 (HTLV-1) è l’agente eziologico di due distinte patologie, la leucemia/linfoma a cellule T dell’adulto (ATLL, adult T-cell leukemia/lymphoma), un'aggressiva neoplasia a carico dei linfociti T CD4+ maturi, e della paraparesi spastica tropicale/mielopatia associata ad HTLV-1 (TSP/HAM, tropical spastic paraparesis/HTLV-1-associated myelopathy), una patologia degenerativa del sistema nervoso centrale. La strategia di espressione genica di HTLV-1, caratterizzata dalla produzione di trascritti a partire da promotori localizzati sia nel filamento positivo che in quello negativo del genoma virale, da splicing alternativo e da traduzione bicistronica, incrementa notevolmente la capacità codificante di HTLV-1, con la conseguente espressione di numerosi geni regolatori ed accessori (Tax, Rex, p12, p13, p21rex, p30tof e HBZ) in aggiunta alle proteine strutturali e agli enzimi associati al virione, comuni a tutti i retrovirus (Gag, Pro, Pol ed Env). Nonostante oltre 30 anni di studi, diversi aspetti chiave del ciclo vitale di HTLV-1 e della sua patogenicità rimangono tutt'oggi non noti. In particolare, non è ancora chiaro se l'espressione genica di HTLV-1 sia caratterizzata da stadi di latenza, se i diversi geni virali presentino cinetiche di espressione distinte e quali meccanismi molecolari possano controllare questi fenomeni. Gli studi descritti nella presente tesi sono stati mirati a comprendere questi aspetti della regolazione genica di HTLV-1. A questo scopo abbiamo sviluppato un protocollo di Real Time RT-PCR associato all'impiego di primer specifici per le diverse giunzioni di splicing al fine di quantificare i diversi trascritti codificati da HTLV-1 e di analizzarne le cinetiche di espressione sia in cellule mononucleate di sangue periferico isolate da individui infettati con HTLV-1, che in cellule trasfettate con cloni molecolari di HTLV-1. I risultati ottenuti indicano che l'espressione degli mRNA codificati da HTLV-1 segue una precisa cinetica dopo riattivazione dell'espressione virale: l'mRNA codificante le proteine regolatrici Tax e Rex agisce come trascritto precoce che precede l'espressione degli altri geni virali. Sebbene sia comunemente accettato che Rex eserciti la sua funzione a livello post-trascrizionale controllando l'esporto nucleare e la stabilità degli mRNA che codificano le proteine associate al virione, fino ad oggi non è mai stata investigata la Rex-dipendenza dei trascritti p12, p13, p21rex, p30tof e hbz. Al fine di testare se le cinetiche di espressione genica di HTLV-1 osservate potessero dipendere dalla funzione di Rex e al fine di determinare la Rex-dipendenza dei singoli mRNA virali, abbiamo generato un clone molecolare di HTLV-1 knock-out per Rex e analizzato la compartimentalizzazione nucleo-citoplasmatica dei trascritti virali. I risultati ottenuti hanno dimostrato la stretta Rex-dipendenza delle cinetiche di espressione a "due fasi" ed hanno rivelato una forte ritenzione nucleare degli mRNA codificanti HBZ, supportando la loro funzione come trascritti non codificanti. Inoltre, i risultati ottenuti hanno dimostrato che la responsività a Rex dei differenti mRNA virali potrebbe essere determinata dalla presenza di una sequenza regolatoria di 72 nucleotidi che agisce in cis, localizzata a monte dell'esone 3. Infine, analisi matematiche hanno sottolineato l'importanza di un ritardo temporale tra le funzioni di Tax e di Rex, supportata dall'evidenza sperimentale di un ritardo nell'accumulo e di un'emivita più prolungata di Rex rispetto a Tax. I dati ottenuti in questo studio forniscono l'evidenza di una regolazione temporale dell'espressione genica di HTLV-1, rivelano una differente compartimentalizzazione degli mRNA virali e offrono una possibile spiegazione di un paradosso ancora irrisolto della regolazione di HTLV-1, ovvero la differente Rex-dipendenza dei trascritti virali, nonostante la presenza della sequenza responsiva a Rex (RxRE, Rex-responsive element) nella regione 3' non tradotta di tutti i trascritti virali.
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Crutchfield, Gerald L. « Kruppel-Like Transcription Factor 6 & ; 7 mRNAs (KLF6 & ; KLF7) Expression in the Developing Zebrafish ». University of Akron / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=akron1572200378181869.
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Guo, Fang. « Regulation of expression of SSTR1 and SSTR2 somatostatin receptor mRNAs in the arcuate nucleus by growth hormone ». Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1995. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/MQ37318.pdf.
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Ohmori, Sachiko, Kazumi Kanda, Hirohito Mitsuyama, Takashi Miyazaki, Xia Cao, Fukushi Kambe et Hisao Seo. « Tail-suspension Induces Altered Expression of GAPDH and ZAKI-4β mRNAs in Rat Hindlimbs : Implication for Disuse Muscle Atrophy ». Research Institute of Environmental Medicine, Nagoya University, 2003. http://hdl.handle.net/2237/7561.
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Parkin, Neil T. « Regulation of gene expression by the 5' untranslated region of eukaryotic mRNAS : c-myc and HIV-1 as examples ». Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74327.
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The 5$ sp prime$ untranslated region (UTR) of c-myc and human immunodeficiency virus type 1 (HIV-1) mRNAs were used as models in a variety of in vitro and in vivo systems in order to study the role of this region in the control of eukaryotic gene expression. Using an ultraviolet light-induced crosslinking assay, a 55 kilodalton protein was identified in extracts of HeLa, mouse erythroleukemia, and other cell lines, which interacts specifically with a purine-rich RNA sequence in the 5$ sp prime$ UTR of c-myc. The function of this protein in control of c-myc expression is not known, but may be implicated in the process of transcriptional elongation. The 5$ sp prime$ UTR of HIV-1 mRNAs was shown to inhibit strongly the translation of a heterologous mRNA; this inhibition was dependent on the secondary structure predicted to form in this region, and on the accessibility of the cap structure to initiation factors. The structural requirements in the HIV-1 5$ sp prime$ UTR for trans-activation by the viral tat gene product were examined by mutagenesis studies; the base-pairing in the stem-loop structure, the sequence of the loop, and the presence of a three nucleotide bulge were found to be critical features necessary for complete trans-activation. These findings indicate that the 5$ sp prime$ UTR can have important effects on the expression of eukaryotic genes.
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Donovan, Benjamin Thomas. « Nucleosome Regulation of Transcription Factor Binding Kinetics : Implications for Gene Expression ». The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574774626880568.
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Pignata, Luiz Fernando Martins. « Pipeline para Análise In Sílico de Dados de Expressão de miRNAs e mRNAs em Células de Mamíferos ». Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-14062012-132754/.
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Os microRNAs estão envolvidos no processo de regulação da expressão gênica da célula, onde a molécula de microRNA se liga com o RNA mensageiro interrompendo, assim, a expressão do respectivo gene pela interrupção da tradução. A bioinformática tem auxiliado na identificação de vários genes codificadores de microRNAs em plantas e animais, incluindo mamíferos, por meio de analises de dados de microarray; assim como na predição de estruturas. Os dados de expressão de microRNAs e RNAs mensageiros foram obtidos por meio de cooperação firmada entre o Laboratório de Bioinformática do Departamento de Genética da Faculdade de Medicina de Ribeirão Preto - USP, coordenado pela orientadora desse projeto, e o Laboratório de Imunogenética Molecular do mesmo departamento, coordenado pelo Professor Doutor Geraldo A. S. Passos. Durante o desenvolvimento e os testes realizados, foram utilizados dados (valores numéricos de dados de expressão coletados por microarrays) provenientes da comparação da expressão de microRNAs e RNAs do timo de camundongos non obese diabetic que reproduzem diabetes melitus do tipo 1, e dados provenientes da comparação da expressão de microRNAs e RNAs de outros experimentos. O presente projeto teve como objetivo o desenvolvimento de um pipeline para a análise in silico de dados de expressão gênica de microRNAs e mRNAs obtidos por microarray. Com base em dados de expressão de microRNAs e RNAs mensageiros, foi possível a análise de diversas ferramentas e o desenvolvimento e ajuste de scripts para que seja possível a análise sequencial de tais dados. Dessa forma, o pipeline desenvolvido inclui a quantificação dos dados de expressão gênica a partir das lâminas de microarray, a normalização dos dados, as análises estatísticas das sequências diferencialmente expressas utilizando o Multi Experiment Viewer, a construção de redes de interação microRNAs-RNAs mensageiros e a busca de alvos de microRNAs baseada nesta rede, ambos pelo GenMir++. O pipeline desenvolvido é executado com facilidade e possibilitou a correta análise dos dados, evitando desperdício de tempo em análises de bancada. A partir dos resultados obtidos, novos alvos de miRNA foram encontrados com o uso do pipeline e comprovados em bancada. Tais resultados apresentados no 55º Congresso Brasileiro de Genética com o resumo intitulado MicroRNA-mRNA Network Controlling the Promiscuous Gene Expression in the Thymus of NOD (Non Obese Diabetic) Mice: Implications in the Emergence of Type 1 Diabetes Mellitus.The microRNAs are involved in the regulation of gene expression of the cell. The miRNA molecule binds to the messenger RNA and interrupts the gene expression by disrupting the translation. Through microarray data analysis, bioinformatics is a valuable aid for the identification of several genes that encode miRNAs in plants and animals, including mammals. It is also very useful for predicting structures. Data of miRNA and mRNA expression were obtained by the collaboration the Bioinformatics Laboratory and the Molecular Immunogenetics Laboratory of the Department of Genetics of the Faculty of Medicine of Ribeirão Preto - USP, coordinated by professors Silvana Giuliatti and Geraldo A. S. Passos, respectively. During the development and tests of the research, microarrays data (numerical values os the expression) were obtained from the comparison between the expression of miRNA and mRNA of the thymus of non obese diabetic mice with diabetes mellitus type 1, as well as from comparisons of their expression in other experiments. The present study is aimed at the development of a pipeline for in silico analysis of the data of miRNAs and mRNA gene expression obtained by microarray. Based on miRNAs and mRNA expression, it was possible to analyze several tools, develop and adjust scripts that allowed the sequential analysis of such data. The pipeline includes the quantification of gene expression data from microarray, the normalization of the data, the statistical analysis of differentially expressed sequences using Multi Experiment Viewer, the construction of networks of interaction of miRNA-mRNAs, and the search for targets of miRNAs based on such network using GenMir++. The pipeline was performed easily and allowed the correct analysis of the data, avoiding waste of time in bench analysis. From the results, new targets of miRNA were found using the pipeline and were verified further in bench analysis. The results were presented in the 55 th Brazilian Genetics Congress in the paper entitled \"MicroRNA-mRNA Network Controlling the Promiscuous Gene Expression in the Thymus of NOD (Non Obese Diabetic) Mice: Implications in the Emergence of Type 1 Diabetes Mellitus\".
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Fleeger, Melissa. « Expression kinetics of the quinic acid (qa) gene cluster in Neurospora crassa ». Youngstown State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1299165877.
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Oliveira, Gabriella Borba de. « Integrative analysis of microRNAs and mRNAs involved in regulation of intramuscular fat deposition in Nelore cattle ». Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/11/11139/tde-26072017-103507/.
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The amount of intramuscular fat can influence the sensory characteristics and nutritional value of beef, thus the selection of animals with adequate fat content for consumer becomes important. Intramuscular fat is a complex trait that is difficult to measure and there is growing knowledge about the genes and pathways that control the biological processes involved in fat deposition in muscle. MicroRNAs (miRNAs) are well conserved class of non-coding small RNAs that modulate gene expression of a range of functions in animal development and physiology. This study aimed to identify differentially expressed (DE) miRNAs, regulatory candidate genes and co-expression networks using mRNAs and miRNAs expression data from the Longissimus dorsi muscle of 30 Nelore steers with extreme genomic estimated breeding values (GEBV) for intramuscular fat (IMF) content. The differential expression analysis between the miRNA data from animals with extreme GEBV values for IMF identified six DE miRNAs. Functional annotation of target genes for these microRNAs indicates that PPARs signaling pathway is involved with IMF deposition. Regulatory candidate genes such as SDHAF4, FBXO17, ALDOA and PKM were identified by partial correlation with information theory (PCIT), phenotypic impact factor (PIF) and regulatory impact factor (RIF) approaches from integrated miRNAs-mRNAs expression data. Two DE miRNAs, bta-miR-143 and bta-miR-146b, upregulated in Low IMF group, were also correlated with regulatory candidate genes, which were functionally enriched for GO terms for fatty acids oxidation. Co-expression networks identified several modules related to immune system, protein metabolism, energy metabolism and glucose catabolism by weighted correlation network analysis (WGCNA), which showed possible interaction and regulation between mRNAs and miRNAs. This study contributes to our understanding of regulatory mechanisms of gene signaling networks involved in fat deposition process. Glucose metabolism and inflammation process were the main pathways found in integrative mRNAs-miRNAs analysis and showed to influence intramuscular fat content in beef cattle.A quantidade de gordura intramuscular pode influenciar as características sensoriais e o valor nutricional da carne bovina, assim, a seleção de animais com conteúdo de gordura adequado para o consumidor torna-se importante. A gordura intramuscular é uma característica complexa, de difícil medição e há um conhecimento crescente sobre os genes e vias que controlam os processos biológicos envolvidos na deposição de gordura no músculo. MicroRNAs (miRNAs) são uma classe bem conservados de pequenos RNAs não-codificantes, que modulam a expressão gênica de uma gama de funções no desenvolvimento e fisiologia animal. Este estudo objetivou identificar miRNAs diferencialmente expressos (DE), genes reguladores candidatos e redes de co-expressão usando dados de expressão de mRNAs e miRNAs do músculo Longissimus dorsi de 30 novilhos Nelore com valores genéticos genômicos estimados (GEBV) extremos para conteúdo de gordura intramuscular (IMF). A análise de expressão diferencial entre os dados de miRNA de animais com valores extremos de GEBV para o IMF identificou seis miRNAs DE. A anotação funcional de genes alvos destes microRNAs indica que a via de sinalização de PPAR está envolvida com a deposição de IMF. Os genes reguladores candidatos, tais como SDHAF4, FBXO17, ALDOA e PKM foram identificados pelas abordagens de correlação parcial com teoria da informação (PCIT), fator de impacto fenotípico (PIF) e fator de impacto regulatório (RIF) a partir de dados integrados de expressão de mRNAs-miRNAs. Dois miRNAs, bta-miR-143 e bta-miR-146b, com alta expressão no grupo de baixo conteúdo de IMF, também foram correlacionados com genes reguladores candidatos, os quais foram funcionalmente enriquecidos para termos GO relacionados a oxidação de ácidos graxos. As redes de co-expressão identificaram vários módulos relacionados ao sistema imunológico, ao metabolismo das proteínas, ao metabolismo energético e ao catabolismo da glicose através da análise ponderada da rede de correlação (WGCNA), que mostrou possível interação e regulação entre mRNAs e miRNAs. Este estudo contribui com a compreensão dos possíveis mecanismos reguladores das redes de sinalização genética envolvidas no processo de deposição de gordura. O metabolismo da glicose e o processo de inflamação foram as principais vias encontrados na análise integrada de mRNA-miRNA e mostraram estar associadas ao conteúdo de gordura intramuscular em bovinos de corte.
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Yamaguchi, Shun. « Regional Expression and Regulation of Alternative Forms of mRNAs Derived from Two Distinct Transcription Initiation Sites of the Rat mGluR5 Gene ». Kyoto University, 1998. http://hdl.handle.net/2433/182240.
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Yamada, Lixy. « Embryonic expression profiles and conserved localization mechanisms of pem-like mRNAs of two species of ascidian, Ciona intestinalis and Ciona savignyi ». 京都大学 (Kyoto University), 2006. http://hdl.handle.net/2433/144235.
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Jiang, Fei. « Expression of recombinant manganese peroxidase in Pichia pastoris Biosynthesis, stability and kinetics analysis / ». Related electronic resource : Current Research at SU : database of SU dissertations, recent titles available full text, 2006. http://proquest.umi.com/login?COPT=REJTPTU0NWQmSU5UPTAmVkVSPTI=&clientId=3739.
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吳欲勳 et Yuk-fun Ng. « Regulation of mammary tumor cell cycle kinetics and gene expression bydietary fatty acids ». Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1998. http://hub.hku.hk/bib/B31219779.
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Jennings, Natalie A. « Acetylcholinesterase in the sea urchin Lytechinus variegatus characterization and developmental expression in larvae / ». Birmingham, AL : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2008r/jennings.pdf.
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Touleimat, Mohamed Nizar. « Méthodologie d'extraction et d'analyse de réseaux de régulation de gènes : analyse de la réponse transcriptionnelle à l'irradiation chez S. cerevisiæ ». Thesis, Evry-Val d'Essonne, 2008. http://www.theses.fr/2008EVRY0044/document.
Texte intégralRésumé :
La réponse cellulaire aux dommages de l'ADN provoqués par l'irradiation (IR) est relativement bien étudiée mais de nombreuses observations montrent l'implication de l'expression de nombreux gènes. Nous souhaitons identifier les différentes formes de la réponse transcriptionnelle à l'IR et reconstruire un réseau de régulation génique impliqué dans son contrôle. La problématique réside dans l'exploitation de dynamiques d'expression de gènes dans des conditions de perturbations génétiques et dans l'intégration d'informations biologiques systémiques. Nous définissons une approche constituée d'une étape automatisée de déduction de régulations à partir de perturbations et de deux étapes d'induction qui permettent d'analyser la dynamique d'expression des gènes et d'extraire des régulations des données additionnelles. Cela nous a permis d'identifier, chez la levure, une réponse complexe à l'IR et de proposer un modèle de régulation dont certaines relations ont été validées expérimentalementThe cellular response to the DNA damage provoked by irradiation (IR) is relatively well studied, however, many observations show the involvement of the expression of many genes. We propose to identify the different potential patterns of the transcriptional response to IR and to reconstruct a gene regulatory network involved in its control. The first point of this work lies in the exploitation of the gene expression dynamics in conditions of genetic perturbations. The second point lies in the integration of systemic biological informations. We define an approach composed of one step of automated logical deduction of regulations from a strategy of perturbations and two induction steps that allow the analysis of the gene expression dynamics and the extraction of potential regulation from additional data. This approach allowed to identify, for the yeast, a complex response to IR and allowed to propose a regulation model which some relations have been experimentally validated
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Ng, Yuk-fun. « Regulation of mammary tumor cell cycle kinetics and gene expression by dietary fatty acids / ». Hong Kong : University of Hong Kong, 1998. http://sunzi.lib.hku.hk/hkuto/record.jsp?B19471026.
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Lawson, Vannice Rose Marie. « The molecular cloning and developmental expression of two novel mRNAs encoding putative 14 kDa ODF and FS proteins of the rat sperm tail ». Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq28218.pdf.
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Calder, Michele D. « Ovarian cysts in dairy cattle : importance of serum LH concentrations in maintenance of cysts and expression of mRNAs for steroidogenic enzymes and gonadotropin receptors / ». free to MU campus, to others for purchase, 1998. http://wwwlib.umi.com/cr/mo/fullcit?p9924869.
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Aken, Alexander Frans Johan van. « Effects of the expression of alternative oxidase on oxidising pathway kinetics in Schizosaccharomyces pombe mitochondria ». Thesis, University of Sussex, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.439148.
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Delcau, Michael Asher. « Differentiation of Pseudomonas sp. strain ADP biofilms and planktonic cells using methods in gene expression analysis ». Diss., University of Iowa, 2018. https://ir.uiowa.edu/etd/6093.
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Bacterial strain Pseudomonas sp. ADP is capable of degrading atrazine via an enzymatic pathway in six sequential steps to yield carbon dioxide and ammonia. Atrazine is a persistent herbicide that frequently contaminates soil, drinking water, and ground water throughout areas of heavy use in the United States. A biological remediation approach using Pseudomonas sp. APD is considered as an effective, cost-efficient, and environmentally conscious method of decreasing atrazine concentration in areas of high contamination. Each enzyme in the degradation pathway is encoded by a corresponding gene, AtzA-AtzF, and is located on a self-transmissible 108-kb plasmid.
Due to their prevalence in nature, and their unique genetic and physical characteristics, biofilms are of great interest in the field of bioremediation. Biofilms exhibit high tolerance for harsh environmental stressors/conditions, prodigious potential for recalcitrant compound entrapment via an extracellular polymeric matrix, quorum sensing, and increased horizontal gene transfer compared to their planktonic counterparts. Despite frequent genetic and chemical analyses performed on atrazine-degrading genes on planktonic cells of strain Pseudomonas sp. APD, the genetics and degradation potential of Pseudomonas sp. ADP biofilms is relatively unexplored.
Real-time quantitative PCR was used to differentiate the expression of six genes involved in the process of atrazine degradation. Relative expression experiments revealed no statistically significant difference in the expression of atrazine-degrading genes in Pseudomonas sp. ADP cells grown as biofilms relative to Pseudomonas sp. ADP cells grown as planktonic cells. In biofilms alone, the expression of genes AtzDEF was differentiated via temperature of biofilm growth in cells grown at 25, 30, and 37 degrees.
Analytical techniques, including GC-MS and HPLC, were used to elucidate atrazine remediation potential of Pseudomonas sp. ADP biofilms and our previously collected genetic data. Stable decreases in atrazine degradation following a first-order kinetic model have been demonstrated for planktonic cells compared to a complex degradation pattern, including transient increases, observed for corresponding biofilm-mediated cells. This is attributed to the unique structure of the biofilm and the potential of atrazine to be entrapped in the substances of the extracellular polymeric matrix and subsequently released into the effluent. Overall, the biodegradation efficiency was higher for Pseudomonas sp. ADP biofilm-mediated cells compared to their planktonic counterparts.
A novel methodology of using confocal microscopy and in situ reverse transcription was proposed for optimization to visualize the expression of atrazine-degrading genes in fixed Pseudomonas sp. ADP biofilms. The sugar composition of Pseudomonas sp. ADP was evaluated using fluorescent lectin binding analysis and was determined to exhibit a prominent level of diversity and dependent upon growth medium. The results from these experiments will play a role in application of biofilms grown in bioreactors for atrazine remediation throughout areas of persistent and high contamination throughout the US. The new step in methodology development of an in situ visual gene expression technique can be extended to bioremediation of alternate recalcitrant compounds. The results may also be aid progress in alternate biofilm-related studies in medicine & human health, metallurgy, and engineering.
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SATO, Jun, Kazue MIZUMURA, Ken TAKAHASHI, Kimiaki KATANOSAKA et Yasuko KOZAKI. « Increased C-fiber Activities to Cold in Adjuvant-monoarthritic Rats was not Accompanied by Increased Expression of TREK1 and CMR1 mRNAs(RIEM Conference II, 2002) ». Research Institute of Environmental Medicine, Nagoya University, 2002. http://hdl.handle.net/2237/2809.
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Gustafsson, Sofia. « Expression and Purification of Murine Tripeptidyl Peptidase II ». Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-177009.
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Tripeptidyl peptidase II (TPPII) is an exopeptidase which cleaves tripeptides from theN-terminus of peptides. The exact functional role of TPPII is still a matter of investigation. Itis believed that the enzyme is primarily involved in intracellular protein degradation, where itcooperates with the proteasome and other peptidases to degrade proteins into free aminoacids. These amino acids can subsequently be used in the production of new proteins. The aimof this work was to express murine wild type TPPII using E. coli and thereafter purify theenzyme from the bacterial lysate. Methods used for the purification included protein andnucleic acid precipitation, anion exchange chromatography, hydrophobic interactionchromatography and gel filtration. The presence of TPPII was determined using activityassay, western blot and SDS-PAGE. Despite the fact that some modification is still needed,the purification yielded a total of 34μg TPPII with a purity of approximately 60%. Thispurified enzyme can be used for future functional characterization.
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Naito, Hiroshi. « Altered expression of retinoic acid (RA) receptor mRNAs in the fetal mouse secondary palate by all-trans and 13-cis Ras : Implications for RA-induced teratogenesis ». Kyoto University, 1999. http://hdl.handle.net/2433/181747.
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Luscieti, Sara. « The Iron Regulatory Protein/Iron Responsive Element (IRP/IRE) system : functional studies of new target mRNAs and pathological implications for novel IRE mutations ». Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/399593.
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Iron is an essential micronutrient required for many cellular reactions and a tight regulation of its metabolism is therefore crucial for health. Cellular iron homeostasis relies on the coordination of iron uptake, storage and mobilization. These processes are controlled post-transcriptionally by the IRP/IRE regulatory system. The Iron Regulatory Proteins (IRP1 and IRP2) can recognize cis- regulatory mRNA motifs termed IREs (Iron Responsive Elements), conserved RNA elements located in the untranslated regions (UTR) of mRNAs that encode for proteins involved in iron metabolism. IRP/IRE interactions regulate the expression of mRNAs encoding for proteins for iron acquisition, storage, utilization and export in response to cellular iron level itself being the interaction of the IRPs with IRE motifs promoted under iron-deficient conditions and abolished in iron-replete conditions. Depending on the location of the IRE, IRPs binding regulates gene expression differentially: IRPs inhibit translation initiation when bound to IREs at the 5’ UTR, while IRPs association with 3’ IREs stabilizes and protects the mRNA from degradation.
The lack of control of expression of IRE-containing mRNAs is associated in humans with pathological conditions showing the importance of components of the IRP/IRE regulatory system. In the last decades, significant progress has been made in the iron metabolism field, however, post-transcriptional regulation of gene expression by the IRP/IRE regulatory system has been limited to a small subset of known genes. A genome-wide study carried out by our group to characterize the whole repertoire of mRNAs that can interact with the IRPs, identified 35 novel IRP1 and IRP2 candidate target-genes.
This work focused on the validation and functional characterization of one of this candidates: Profilin2 (Pfn2). Pfn2 is an actin-binding protein involved in the control of cytoskeletal dynamics. We identified a conserved IRE in the 3’ UTR of Pfn2 mRNA which is functional in in vitro binding studies with IRP1 and IRP2.
Pfn2 mRNA showed preferentially downregulation under iron-excess condition in cell lines and we demonstrated to be regulated by IRPs-mediated stabilization in vivo, since Pfn2 mRNA levels are reduced in a mouse model with Irp1 and Irp2 gene inactivation.
Moreover, the reduction of cellular free iron levels by Pfn2 overexpression experiments in cell lines, as well as, the misregulation of iron distribution observed in mice knockout for Pfn2 gene, revealed Pfn2 as a previously unrecognized player in iron metabolism.
In addition, we also contributed to the identification of a functional 3’ IRE in human BDH2 mRNA, a protein involved in lipocalin-siderophores iron-trafficking, as well as, in the identification and characterization of two novel L-ferritin IRE mutations (Heidelberg +52G>C and Badalona+36C>U) causative of Hereditary Hyperferritinemia Cataract Syndrome and a novel mutation in ALAS2 IRE demonstrated to be a modifier of clinical severity in a family with Erythropoietic Protoporphyria.El hierro es un micronutriente esencial requerido por múltiples reacciones celulares y una regulación adecuada de su metabolismo es fundamental para la salud. La homeostasis celular del hierro está basada en la coordinación de la absorción, el almacenamiento y la movilización de este elemento. Estos procesos son controlados post-transcripcionalmente por el sistema IRP/IRE. Las proteínas reguladoras del hierro (IRP1 e IRP2) reconocen un motivo estructural presente en el ARNm de algunos genes denominado IRE (Iron Responsive Element). El IRE es un motivo conservado y situado en las regiones no traducidas (UTR) de los ARNm de proteínas implicadas en el metabolismo del hierro. Las interacciones IRP/IRE regulan la expresión de los ARNm que codifican proteínas para la adquisición, el almacenamiento, la utilización y la exportación de hierro en respuesta a los niveles celulares de hierro, porque la interacción de las IRPs con los motivos IRE se ve incrementada a niveles bajos de hierro y disminuida en condiciones altas de hierro. Según la localización de los IRE, las IRPs regulan de manera distinta la expresión de sus dianas: las IRPs inhiben la iniciación de la traducción cuando se unen a IREs situados en el 5' UTR, mientras que su asociación con los IREs del 3' UTR estabiliza y protege dicho ARNm de la degradación. La falta de coordinación de la expresión de genes que contienen IRE está asociada con condiciones patológicas ilustrando la importancia de los componentes del sistema regulador IRP/IRE. En las últimas décadas se han realizado progresos importantes en el campo del metabolismo del hierro. Sin embargo, la regulación post-transcripcional de la expresión génica por el sistema IRP/IRE se ha limitado a un pequeño subconjunto de genes. Un estudio “genome-wide” llevado a cabo en nuestro grupo para la caracterización global del sistema regulador IRP/IRE dio como resultado la identificación de 35 genes diana que se unen a IRP1 e IRP2. La presente tesis tiene como objetivo general validar y caracterizar funcionalmente una de esas dianas: Profilin2 (Pfn2). Pfn2 es una proteína que une actina y que está involucrada en la regulación de la dinámica del citoesqueleto. Hemos identificado un IRE localizado en el 3’ UTR del ARNm de Pfn2; el IRE es funcional en estudios in vitro para la unión a IRP1 e IRP2. El ARNm de Pfn2 viene preferentemente regulado y reducido en condiciones altas de hierro en líneas celulares y hemos demostrado in vivo que es regulado por estabilización mediada de las IRPs, ya que los niveles de ARNm de Pfn2 se encuentran reducidos en ratones a los que los genes de Irp1 y Irp2 han sido inactivados. Así mismo, la sobrexpresión de Pfn2 en líneas celulares asociada a una reducción en los niveles de hierro libre, así como la desregulación de la distribución del hierro encontrada en ratones “knockout” para el gen de Pfn2; convierten a Pfn2 como un actor nuevo en el metabolismo del hierro. Por otro lado hemos contribuido tanto a la identificación de un 3’ IRE funcional en el ARNm humano de BDH2, una proteína involucrada en el tráfico de hierro a través del sistema de lipocalinas-sideróforos, como a la identificación de dos nuevas mutaciones en el IRE de la Ferritina L (Heidelberg +52G>C y Badalona+36C>U) responsables del Síndrome Hereditario de Hiperferritinemia con Cataratas y también a la identificación de una nueva mutación en el IRE de ALAS2 que ha demostrado ser un modificador de la importancia clínica de los síntomas en una familia con Protoporfiria Eritropoyética.
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Omlin, Teye D. « Effects of Hypoxia and Exercise on In Vivo Lactate Kinetics and Expression of Monocarboxylate Transporters in Rainbow Trout ». Thèse, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/30652.
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The current understanding of lactate metabolism in fish is based almost entirely on interpretation of concentration measurements that cannot be used to infer changes in flux. Moreover, the transporters regulating these fluxes have never been characterized in rainbow trout. My goals were: (1) to quantify lactate fluxes in rainbow trout under normoxic resting conditions, during acute hypoxia, and exercise by continuous infusion of [U-14C] lactate; (2) to determine lactate uptake capacity of trout tissues by infusing exogenous lactate in fish rest and during graded exercise, and (3) to clone monocarboxylate transporters (MCTs) and determine the effects of exhausting exercise on their expression. Such information could prove important to understand the mechanisms underlying the classic “lactate retention” seen in trout white muscle after intense exercise. In normoxic resting fish, the rates of appearance (Ra) and disappearance (Rd) of lactate were always matched (~18 to 13 µmol kg-1 min-1), thereby maintaining a low baseline blood lactate concentration (~0.8 mM). In hypoxic fish, Ra lactate increased from baseline to 36.5 µmol kg-1 min-1, and was accompanied by an unexpected 52% increase in Rd reaching 30.3 µmol kg-1 min-1, accounting for a rise in blood lactate to 8.9 mM. In exercising fish, lactate flux was stimulated > 2.4 body lengths per second (BL s-1). As the fish reached critical swimming speed (Ucrit), Ra lactate was more stimulated (+67% to 40.4 μmol kg-1 min-1) than Rd (+41% to 34.7 μmol kg-1 min-1), causing an increase in blood lactate to 5.1mM. Fish infused with exogenous lactate stimulated Rd lactate by 300% (14 to 56 μmol kg-1 min-1) during graded exercise, whereas the Rd in resting fish increased by only 90% (21 to 40 µmol kg-1 min-1). Four MCT isoforms were partially cloned and characterized in rainbow trout: MCT1b was the most abundant in heart, and red muscle, but poorly expressed in gill and brain where MCT1a and MCT2 were prevalent. MCT4 was more expressed in the heart. Transcript levels of MCT2 (+260%; brain), MCT1a (+90%; heart) and MCT1b (+50%; heart) were stimulated by exhausting exercise. This study shows that: (i) the increase in Rd lactate plays a strategic role in reducing the lactate load imposed on the circulation. Without this response, blood lactate accumulation would double; (ii) a high capacity for lactate disposal in rainbow trout tissues is elicited by the increased blood-to-tissue lactate gradient when extra lactate is administered; and (iii) rainbow trout may be unable to release large lactate loads rapidly from white muscle after exhausting exercise (lactate retention) because they poorly express MCT4 in white muscle and fail to upregulate its expression during exercise.
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Rosario, Wenzel Ileana del. « Anatomical localization and kinetics of expression of HSV and MHC antigens during viral encephalitis in a mouse model / ». The Ohio State University, 1993. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487842372895558.
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Tourlet, Sébastien. « Proposition d’une stratégie d’analyse statistique des données de puces à ADN décrivant une cinétique d’expression génique ». Thesis, Tours, 2009. http://www.theses.fr/2009TOUR3134.
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Les résultats d’expériences de microarray furent décriés par le manque de concordance inter-expériences. Les listes immenses de gènes résultant de filtrages statistiques sont difficiles à exploiter. La Food and Drug Administration a montré que le choix d’indicateurs de filtrage de gènes était la source d’une grande disparité entre expériences de microarray issus de laboratoires indépendants. Dans ce contexte, nous avons développé une méthode de sélection basée sur la modélisation de l’allure de la courbe d’expression avec le Log2 du « fold-change » entre les points de cinétique. En effet, des gènes co-régulés au cours d’une cinétique temporelle présentent des courbes d’expression d’allure similaire alors que leur niveau d’expression peut être différent. Nous avons validé la méthode grâce à 2 expériences indépendantes de microar-ray étudiant la différenciation des ovaires d’embryons de souris. Ainsi, nous avons obtenu une liste réduite et pertinente de gènes exprimés. Puis, une analyse de ces résultats dans le cas de la différenciation ovarienne nous a permis d’identifier 9 nouveaux gènes candidats validés in silico et restant à être testés biologiquementMicroarray results were blamed because of their lack of concordance. Moreover, the huge candidate gene lists from statistical filterings are not useful for biologists. FDA proved that the lack of reliability between microarray experiments came from the choice of gene filtering indicators. In this context, a filtering method was developed based on expression curve shape modelling with the use of Log 2 of fold-change between kinetic points. Actually, the co-regulated genes display similar expression shape but with heterogeneous expression level.Our method was developed and validated thanks to two independent microarray experiments (Affymetric®) from mouse embryonic ovaries. Therefore, a short and relevant list of genes was obtained. Thus, a study of results linked to ovarian differentiation permitted to identify nine new candidate genes that were in silico validated. These genes might be biologically tested (i.e. RT PCR) by the scientific community
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Huang, Yehong. « The Kinetics of G2 and M Transitions Regulated by B Cyclins ». Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1386197228.
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Tabata, Sumie. « Distribution and kinetics of SR-PSOX/CXCL16 and CXCR6 expression on human dendritic cell subsets and CD4[+] T cells ». Kyoto University, 2006. http://hdl.handle.net/2433/143841.
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Takahashi, Paula. « Perfis de Expressão Gênica e Possíveis Interações entre microRNAs e mRNAs em Diabetes Mellitus Tipo 1 com Enfoque em Resposta ao Estresse Oxidativo e Reparo do DNA ». Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-01072015-092915/.
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O Diabetes Mellitus tipo 1 (DM1) resulta de um ataque autoimune contra as células pancreáticas, extinguindo a produção de insulina e levando à hiperglicemia. Evidências indicam uma associação entre o estresse oxidativo (que pode causar danos no DNA) e o DM1, sendo que apenas alguns trabalhos da literatura relataram a expressão de genes relacionados à respostas ao estresse oxidativo e reparo do DNA em DM1. Ainda, os microRNAs (reguladores pós-transcricionais da expressão gênica) estão envolvidos em vários processos biológicos e condições patológicas, mas informação sobre a expressão dos microRNAs em DM1 ainda é escassa. A fim de proporcionar um melhor entendimento sobre as vias de regulação de genes participantes de processos biológicos relevantes para o DM1, o presente estudo consistiu em analisar os perfis de expressão gênica (método de microarranjos) de microRNAs e de mRNAs (bem como de algumas proteínas) provenientes de células mononucleares do sangue periférico (PBMCs, do inglês peripheral blood mononuclear cells) de pacientes DM1 (n=19) em comparação com indivíduos sadios não diabéticos (n=11), dando maior enfoque a genes associados à resposta ao estresse oxidativo e reparo do DNA. Os resultados de expressão obtidos pelo método de microarranjos apontaram 44 microRNAs diferencialmente expressos (35 induzidos e nove reprimidos) nos pacientes DM1 e esses microRNAs apresentaram grande especificidade ao estratificar pacientes DM1 dos controles, incluindo hsa-miR-101, hsa-miR148a, hsa-miR-27b e hsa-miR-424, cujos dados de expressão foram confirmados por qRT-PCR. A análise funcional dos genes-alvo dos microRNAs, tanto dos induzidos quanto dos reprimidos, apontou 22 e 12 vias KEGG significativamente enriquecidas, respectivamente, incluindo vias relacionadas ao câncer. Com relação à análise de expressão de mRNAS, 277 genes diferencialmente expressos foram identificados nos pacientes DM1, sendo que 52% deles são potenciais alvos dos microRNAs diferencialmente expressos nos pacientes DM1. Dentre esses alvos foram encontrados genes candidatos ao desenvolvimento da doença, assim como genes implicados nos processos biológicos resposta ao estresse oxidativo e reparo do DNA, como UCP3, PTGS2, ATF3, FOSB, DUSP1 e TNFAIP3, cujos dados de expressão foram confirmados por qRT-PCR. Já a análise de grupos gênicos identificou 49 e 55 grupos gênicos significativamente expressos e enriquecidos em pacientes DM1, respectivamente, destacando-se vias relacionadas à sinalização apoptótica, resposta ao hidroperóxido, reparo do DNA por recombinação homóloga e resposta ao estresse do retículo endoplasmático. Quanto aos dados de expressão proteica (western blotting), PTGS2 e ATF3 não apresentaram níveis de expressão detectáveis em nenhum dos dois grupos estudados, enquanto que para DUSP1 não foi observada diferença estatisticamente significativa entre os grupos, apesar de os três genes se apresentarem induzidos em pacientes DM1. Os resultados do ensaio do gene repórter da luciferase demonstraram a ocorrência da interação entre hsa-miR-148a e DUSP1 em meio celular. Essa evidência aliada aos dados de western blotting, sugerem a possibilidade de hsa-miR-148a atuar na repressão traducional de DUSP1. Em conjunto, os resultados do presente estudo indicaram perfis distintos de expressão de microRNAs e mRNAs em PBMCs de pacientes DM1 comparados a indivíduos sadios, sendo que adicionalmente, dados inéditos relacionados à interação microRNAs-mRNAs em DM1 foram obtidos, principalmente associados à resposta ao estresse oxidativo e reparo do DNA, sugerindo um distúrbio na rede microRNA-alvo em pacientes DM1.Type 1 Diabetes Mellitus (T1DM) results from an autoimmune attack against the pancreatic cells, ceasing insulin production, which causes hyperglycemia. Although associations between oxidative stress, which can cause DNA damage, and T1DM have been demonstrated, only a few studies have reported differential expression of genes associated with response to oxidative stress and DNA repair in T1DM patients. Moreover, microRNAs (post-transcriptional regulators of gene expression) are implicated in many biological processes and pathological conditions; however, only scarce information is available in the literature concerning the expression of microRNAs in T1DM. In order to better understand the regulatory pathways involved in biological processes that are relevant to T1DM, we aimed to investigate the microRNA and mRNA transcriptional expression profiles by microarray analysis (as well as expression of selected proteins) in peripheral blood mononuclear cells (PBMCs) from T1DM patients (n=19) compared with healthy non-diabetic individuals (n=11), emphasizing genes related to response to oxidative stress and DNA repair. Microarray expression results indicated 44 differentially expressed microRNAs (35 up- and nine down-regulated) in T1DM patients, with those microRNAs possessing a discriminatory power to clearly stratify the patients from the controls, including hsa-miR-101, hsa-miR148a, hsa-miR-27b, and hsa-miR-424, whose expression data were confirmed by qRT-PCR. Functional annotation analysis performed on the predicted targets of the differentially expressed microRNAs pointed 22 and 12 annotated KEGG pathways for the overexpressed and repressed microRNAs, respectively, many of them related to cancer. Regarding mRNA microarray results, we detected 277 differentially expressed genes in T1DM patients, with 52% of them being potential targets of the differentially expressed microRNAs in T1DM patients. Among these targets, we identified candidate genes for T1DM as well as genes involved in the biological processes response to oxidative stress and DNA repair, such as UCP3, PTGS2, ATF3, FOSB, DUSP1 and TNFAIP3, whose expression data were confirmed by qRT-PCR. Furthermore, out of the 49 and 55 significantly expressed/enriched gene sets in T1DM patients, respectively, five pathways related to apoptotic signaling, response to hydroperoxide, DNA repair via homologous recombination, and response to endoplasmic reticulum stress were of interest for the present work. Concerning protein expression results (western blotting), PTGS2 and ATF3 expression was not detected for either the patient or the control group, while significant difference in DUSP1 expression was not observed between the two groups, although the corresponding mRNAs of those genes were found induced. Regarding the luciferase assay, our results demonstrated that the interaction between hsa-miR-148a and DUSP1 occurs in the cellular milieu. Therefore, these findings together with those western blotting results suggest that hsa-miR-148a could play a role in DUSP1 translational repression. Altogether, our results indicate distinctive microRNA and mRNA expression profiles in PBMCs from T1DM patients relative to healthy non-diabetic individuals. Furthermore, we have provided novel data regarding microRNA-mRNA interactions in T1DM, in particular involving genes associated with response to oxidative stress and DNA repair, suggesting a perturbation in the microRNA-target network in T1DM patients.
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Fernandez, Christine Cheryl. « Characterisation of cytochrome P450 azole drug-resistant sterol demethylase CYP51B1 and expression of CYP123 and CYP136 from Mycobacterium tuberculosis ». Thesis, University of Manchester, 2011. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:171502.
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Tuberculosis (TB) affects nearly a third of the world’s population and has been termed a ‘Global Emergency’ by the WHO. The emergence of multi/extensively drug resistant (M/XDR) strains of Mycobacterium tuberculosis (Mtb), the causative agent of TB, and the increasing incidences of azole drug resistant sterol demethylases (CYP51) from pathogenic fungi has propelled studies to understand mechanisms of azole drug resistance on the drug target CYP51. Since Mtb is devoid of a sterol biosynthetic pathway, the presence and study of CYP51B1 and 19 other Cytochrome P450s in its genome is important to clarify host-pathogen mechanism of infection and the potential of using azole drugs to treat TB. In this study, CYP51B1 from Mtb was used as the model enzyme to study CYP51 mutants from Candida albicans fluconazole-resistant clinical strains. By protein engineering methods, F89H, L100F, S348F, G388S and R391K CYP51B1 mutants were made and azole drug binding properties were investigated using stopped-flow kinetics and static equilibrium methods. Dissociation constant (Kd) values were derived for a range of commercially available azole drugs by fitting the equilibrium binding data to a hyperbolic equation. Kd values for stopped-flow kinetics were derived by plotting observed binding rates (kobs) across different azole drug concentrations against time, followed by fitting multiple kobs data to a linear equation to derive azole drug de-binding (koff) and binding (kon) rate constants – the Kd was obtained by koff/kon. Extinction coefficient for heme b content in mutants and Wild Type (WT) CYP51B1 were an average of ɛ419 = 96.1 mM-1 cm-1. Biochemical characterisation of the mutants were carried out using established experiments on CYP51 – reduction of Fe(III)-heme to Fe(II)-heme, NO binding to Fe(III)-heme, rates of CO-Fe(II) adduct formation and rates of collapse of the P450 to P420 species in the presence of CO and estriol with redox partners from Mtb. In order to elucidate the effects of the above mutations on the iron-heme catalytic region, electron paramagnetic resonance (EPR) experiments were carried out with and without azole drugs. Circular dichroism (CD), differential scanning calorimetry (DSC) and multi-angled laser light scattering (MALLS) analysis confirmed that F89H, R391K and L100F mutants were stable and homogeneous. Crystallogenesis was successful for the above mentioned mutants and atomic structures were obtained for all mutants and WT CYP51B1 (in ligand-bound and substrate-free forms), except for S348F and G388S mutants which were expressed as inclusion bodies and 60% holoenzyme, respectively. Reconstituted catalytic assays to determine the sterol demethylating propensity of the mutants were carried out using redox partners from Mtb or E. coli, and with lanosterol and dihydrolanosterol as the surrogate substrates. Redox potentiometry showed similar potentials to WT for all mutants except for the G388S mutant which was relatively positive (–102 mV). Redox cycling experiments followed by EPR analysis for mutants and WT resulted in a novel P450 high-spin species at g value 5.84 (80 %) which gradually collapsed to the initial low spin state over 48 h. Expression trials were concurrently carried out on two other Mtb P450 genes – CYP123 (Rv0744c) and CYP136 (Rv3059) products of which may have similar functions to CYP51B1 or may share similar redox partners. CYP123 is located on the same operon as CYP51B1 while CYP136 has a 29% sequence identity to another CYP51 from a marine slime bacterium. Although further work is necessary, in this study CYP123 was expressed totally as inclusion bodies while CYP136 was expressed as soluble apoprotein fused with trigger factor chaperone.
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Beloti, Lilian Luzia 1980. « Estudo funcional de uma epóxido hidrolase de Aspergillus brasiliensis CCT1435 = expressão, purificação e caracterização enzimática = Functional study of an epoxide hydrolase from Aspergillus brasiliensis CCT1435 : expression, purification and enzymatic characterization ». [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316511.
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Orientadores: Anete Pereira de Souza, Valéria Maia MerzelTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Doutorado
Genetica de Microorganismos
Doutora em Genética e Biologia Molecular
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Lindås, Ann-Christin. « Tripeptidyl-Peptidase II : Structure, Function and Gene Regulation ». Doctoral thesis, Uppsala University, Department of Biochemistry, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7345.
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The protein degradation process is of vital importance for the cell to maintain cellular functions. An important enzyme in this process is the multimeric tripeptidyl-peptidase II (TPP II). It removes tripeptides from a free N-terminus of the substrates. TPP II has broad substrate specificity and wide-spread distribution, suggesting that the TPP II gene is a house-keeping gene. However, the levels of both mRNA and TPP II protein varies during different conditions and the TPP II gene promoter was therefore identified and characterized. It is a 215 bp fragment just upstream of the coding sequence. This fragment lacks a TATA-box but contains an initiator, two inverted CCAAT-boxes and an E-box. The CCAAT-boxes and the E-box were found to bind the nuclear factor Y (NF-Y) and upstream stimulatory factor-1 (USF-1) respectively. The CCAAT-boxes appear to be most important for the transcriptional activation. Furthermore, several silencer element were identified further upstream of the 215 bp promoter and the octamer binding factor Oct-1 was found to bind one of these fragments. If Oct-1 is responsible for the inhibition of the transcription of the TPP II gene remains to be investigated. In addition, the substrate specificity was investigated. For this purpose an expression system using Pichia pastoris was developed. The purified recombinant TPP II was found to have the same enzymatic properties as the native enzyme. In order to identify the amino acids involved in the binding of the N-terminus of the substrate, wild-type murine TPP II and four mutants E305Q, E305K, E331Q and E331K were purified. Steady-state kinetic analysis clearly demonstrated that both Glu-305 and Glu-331 are important for this binding as the KMapp is more than 102 higher for the mutants than wild-type. Finally, the pH-dependence for cleavage of two chromogenic substrates was compared for TPP II from different species.
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Marcaud, Hélène. « Etude sur les transitions coopératives et les cinétiques de transconformation associées dans les acides nucléiques ». Paris 6, 1986. http://www.theses.fr/1986PA066514.
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Yalala, Bongani Ndhlovu. « Ion exchange resins an functional fibres :a comparative study for the treatment of brine waste water ». Thesis, University of the Western Cape, 2009. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_8342_1298358875.
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To improve the adsorption capacity of polyacrylonitrile (PAN) fibres, hydrophilic amidoxime fibres were prepared by subsequent conversion of the cyano groups to an amidoxime group by reacting with hydroxylamine at 80°
C at an optimum amidoximation time of 2 hrs. The amidoxime fibre was hydrolyzed/alkali treated in a solution of sodium hydroxide to enhance or improve the adsorption properties. This was followed by characterization of the amidoxime and hydrolyzed fibres using Scanning electron microscopy (SEM)
Fourier transform Infrared Spectroscopy (FTIR) and exchange capacity (cationic and anionic). SEM showed that the hydrolysis process made the surface of Amidoxime fibre rougher than that of Polyacrylonitrile fibre. FTIR revealed that the hydrolyzed Amidoxime fibres contained conjugated imine (-C=N-) sequences. Functionalization enhanced the sorption of amidoxime fibres by an increase of 20 % in the cationic exchange capacity. This was achieved by the part conversion of the cyano groups into the carboxylic acid groups. The fibres showed faster kinetics largely due the available exchange sites on the surface of the fibres hence the equilibration was achieved much quicker.
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Nilsson, Lisa O. « Redesign of Alpha Class Glutathione Transferases to Study Their Catalytic Properties ». Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2001. http://publications.uu.se/theses/91-554-5171-3/.
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Montaño, Inti Doraci Cavalcanti. « Otimização dinâmica do cultivo semi-contínuo de Pichia pastoris recombinante para produção das enzimas heterólogas alfa amilase e penicilina G acilase ». Universidade Federal de São Carlos, 2010. https://repositorio.ufscar.br/handle/ufscar/4051.
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Previous issue date: 2010-03-31Universidade Federal de Minas Gerais
This master's thesis project aims at studying the dynamic optimization of the operation of a bench scale (up to 5L) automated, agitated and aerated bioreactor, where the semi-continuous cultivation of recombinant Pichia pastoris is run. This yeast was cloned using the PGK1 promoter, which precludes the use of methanol as inducer, expressing constitutively the enzyme penicillin G Acylase (PGA) from Bacillus megaterium. While the group of molecular biology of DEQUFSCar is working on cloning the PGA, d P. pastoris expressing the enzyme - amylase from Bacillus subtilis was cultivated. This clone, provided by prof. Fernando Torres, UnB, uses the same construction and, therefore, its kinetics of growth and production should be very similar to the PGA s. Cultivation of recombinant Pichia pastoris was performed in flasks (skaker) using standard culture medium, aiming at obtaining kinetic data, which are the starting point for the escalation to a benchtop bioreactor. Following that, tests were performed in a 5L bioreactor in batch and fed batch operation modes. With the bioreactor data , kinetic parameters of growth, to be further used in the simulations, were estimated, using a hybrid algorithm (which combines the global method Simulated Annealing, with the local one Levenberg- Marquardt). This algorithm, is implemented in Matlab and available in the software library of Ladabio (Laboratory of Development and Automation of Bioprocesses ). From these data, models of microbial growth and of production were developed, following a classic approach (unstructured, non-segregated). Computer simulations using different feeding strategies and employing these models allowed mapping the dynamics of the system. From this information, optimal control strategies were proposed to define optimal feeding profiles. Cellular concentrations of 5.4 g/L (dry weight) were reached in shaker (20h of cultivation, when glucose is exhausted), expressing 218 U/mL of -amylase, compared to 11.4 g/L (dry weight) that were achieved in cultures in a bioreactor in batch simple (10h of cultivation, when glucose is exhausted), expressing 156 U/mL of -amylase In fed-batch cultures, cell concentrations of up to 45 g/L were achieved, expressing up to 260 U/mL of - amylase, with a productivity of 5.2 U/mL/ h. In fed-batch cultures of P. pastoris expressing PGA, cell concentrations of up to 35 g/L were achieved. Enzyme activity was not detected in the culture broth due to the effect of glycosylation. Immunodetection reaction confirmed the expression of the recombinant enzyme. Four specific growth rate equations were adjusted, with different types of inhibition by one product, detected at significant levels by liquid chromatography highperformance, but not yet identified. This metabolite was added as an inhibitor in kinetic models, using the peak areas, normalized as a pseudoconcentration. The best fit to the experimental data were the Monod kinetic model with non-competitive inhibition. Typical values obtained for the maximum specific growth and glucose/ cell conversion factor in bioreactor were max=0,24 h-1 and YX/S = 0,48. Algorithm for optimal control in open loop was developed and successfully implemented, providing a robust profiles of great power, whose validation is proposed as a continuation of this work.
Este mestrado se propoe a estudar a otimizacao dinamica de biorreator automatizado, tipo tanque agitado e aerado, em escala de bancada (ate 5L), onde se processa o cultivo semi-continuo de Pichia pastoris recombinante. Essa levedura foi clonada pelo grupo do prof. Fernando Torres, da UnB, utilizando o promotor PGK1, que dispensa a utilizacao de metanol como indutor, expressando constitutivamente a enzima -amilase de Bacillus subtilis. Durante a execucao deste mestrado, a enzima penicilina G acilase (PGA) de Bacillus megaterium esta sendo clonada pelo grupo de biologia molecular do DEQ-UFSCar usando a mesma construcao e, portanto, a cinetica de crescimento e producao da PGA heterologa devera ser muito semelhante as da -amilase, utilizada como estudo de caso para otimizacao do bioprocesso. Cultivos de Pichia pastoris recombinante foram realizados em frascos agitados, utilizando meio de cultivo padrao, objetivando o levantamento de dados cineticos, ponto de partida para o escalonamento em biorreator de bancada. Posteriormente, foram realizados ensaios em biorreator de 5L, em batelada e batelada alimentada. Com os dados obtidos nos cultivos em biorreator, e utilizando algoritmo hibrido para estimativa de parametros (que combina o metodo global Simulated Annealing, com o local de Levenberg-Marquardt), implementado em MatLab e disponivel no LaDABio (Laboratorio de Desenvolvimento e Automacao de Bioprocessos), foram ajustados parametros cineticos de crescimento, para serem utilizados nas simulacoes dos cultivos em biorreator. A partir dai, foi desenvolvido modelo de crescimento microbiano e de producao, utilizando um enfoque classico (modelo nao-estruturado, nao-segregado) para descrever o sistema. Com isso, torna-se possivel realizar simulacoes em computador usando diferentes estrategias de alimentacao, para mapear a dinamica do sistema. A seguir, foram desenvolvidos algoritmos de controle otimo em malha aberta para definicao de estrategias de alimentacao. Concentracoes celulares de 5,4 g/L (massa seca) foram alcancadas em cultivos em camara rotatoria (20h de cultivo, quando se esgota a glicose), expressando 218 U/mL de -amilase, comparado com 11,4 g/L(massa seca) que foram atingidos em cultivos em biorreator em bateladas simples (10h de cultivo, quando se esgota a glicose), expressando 156 U/mL de -amilase. Em cultivos em batelada alimentada concentracoes celulares de ate 45 g/L foram atingidas, expressando ate 260 U/mL de -amilase, com uma produtividade de 5,2 U/mL/h. Em cultivo em batelada alimentada de P. pastoris expressando PGA, concentracoes celulares de ate 35 g/L foram atingidas. Nao foi detectada atividade enzimatica no caldo de cultivo devido ao efeito da glicosilacao. Reacao de imunodeteccao confirmou a expressao da enzima recombinante. Foram ajustadas quatro equacoes de velocidade especifica de crescimento, com diferentes tipos de inibicao por um produto, detectado em niveis importantes por cromatografia liquida de alto desempenho, mas ainda nao identificado. Esse metabolito foi inserido como inibidor nos modelos cineticos, utilizando as areas dos picos, normalizadas, como uma pseudoconcentracao. Os melhores ajustes aos dados experimentais foram com modelo cinetico de Monod com inibicao nao-competitiva. Valores tipicos obtidos para a velocidade especifica maxima de crescimento e de fator de conversao glicose/celula em biorreator foram max = 0,24 h-1 e YX/S = 0,48. Algoritmo de controle otimo em malha aberta foi desenvolvido e implementado com sucesso, prevendo de forma robusta perfis otimos de alimentacao, cuja validacao fica proposta como continuidade deste trabalho.
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Curien, Gilles. « Activation allostérique de la thréonine synthase d'A. Thaliana par la S-adénosylméthionine : mécanismes moléculaires et aspects régulateurs ». Université Joseph Fourier (Grenoble ; 1971-2015), 1998. http://www.theses.fr/1998GRE10143.
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La threonine synthase de plante est specifiquement activee par la s-adenosylmethionine, produit directement derive de la methionine. Afin de mieux comprendre le mecanisme de cette interaction de la s-adenosylmethionine avec l'enzyme, l'adnc codant la threonine synthase d'arabidopsis a ete isole pour la premiere fois chez les plantes par complementation fonctionnelle d'un mutant bacterien. Dans une seconde etape, l'adnc codant la threonine synthase de plante a ete utilise pour surproduire chez e. Coli la forme nature de la threonine synthase. L'enzyme a ete purifiee a homogeneite et s'est averee etre identique a l'enzyme purifiee a partir de plante. Des experiences de cinetiques a l'etat-stationnaire et de liaison a l'equilibre nous ont permis de deduire que la fixation de la sam a l'enzyme augmente de 8 fois sa vitesse de catalyse et augmente considerablement son affinite apparente pour le substrat o-phosphohomoserine (diminution de 25 fois du k#m). Un modele cinetique a alors ete etabli, permettant de rendre compte a la fois des donnees de cinetique a l'etat stationnaire et des donnees de fixation a l'equilibre de la sam sur l'enzyme libre. Nous suggerons de plus que les modifications considerables des proprietes cinetiques de l'enzyme resultent d'une transition allosterique cooperative induite par la s-adenosylmethionine. Des analyses en cinetiques rapides indiquent que cette transition se deroule a une vitesse plus elevee en presence du substrat. Recemment, des cristaux de l'enzyme diffractant a environ 3 angstroms ont ete obtenus en presence de s-adenosylmethionine. Afin de mieux comprendre la fonction physiologique de l'activation de la threonine synthase par la s-adenosylmethionine, le controle du partage du flux d'o-phosphomoserine entre la threonine synthase et l'enzyme competitrice cystathionine -synthase a ete simule numeriquement par integration des parametres cinetiques de ces enzymes.
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Rabêlo, Flávio Henrique Silveira. « Sulfur supply as a sustainable technology for phytoextraction : its effects on cadmium uptake and detoxification in Panicum maximum Jacq. cv. Massai ». Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/64/64133/tde-07032019-134024/.
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Cadmium (Cd) concentration in the environment has increased in most recent decades, which represents a serious socio-environmental problem, since Cd is toxic to most living beings. Thus, it is important to reduce the concentration of this metal in contaminated environments, and the use of plants properly supplied with sulfur (S) can contribute to this (phytoextraction), since S is a component of metabolites involved in defense responses against stress caused by Cd. Therefore, our aim with this study was to evaluate the effect of S on i) Cd uptake kinetics, ii) root development and nutrient uptake, iii) metabolic profiling and thiol peptides synthesis, and iv) activity of antioxidant and photosynthetic system of Massai grass (Panicum maximum Jacq. cv. Massai) used for Cd phytoextraction. The studies were carried out in a greenhouse conditions using pots of 0.5 and 2.0 L for development of the study about Cd uptake kinetics (treatments represented by combinations of S: 0.1 and 1.9 mmol L-1 and Cd concentrations: 0.1 and 0.5 mmol L-1) and Cd detoxification mechanisms (treatments represented by combinations of S: 0.1, 1.9 and 3.7 mmol L-1 and Cd concentrations: 0.0, 0.1 and 0.5 mmol L-1), respectively. Pots were distributed in randomized blocks, with four replications. Plants used in Cd kinetics study were exposed to treatments for 14 days after they remained in solutions containing only S for 42 days, while plants used in Cd detoxification study were exposed to treatments for 9 days after growing in solutions containing only S for 44 days. At the end of studies, plants used were harvested and sent for analysis. Maximum uptake rate (Vmax) and Cd apoplastic influx of Massai grass exposed to highest Cd concentration was highest when the plants were supplied with highest S concentration. The root development of Massai grass was strongly inhibited when plants were exposed to 0.5 mmol L-1 Cd, but proper (1.9 mmol L-1) S supply allowed highest Cd uptake, while excessive S supply (3.7 mmol L-1) decreased iron plaques formation in the roots of plants. Lysine, cysteine, ornithine, arginine, tryptophan and histidine were accumulated in more than one tissue in plants exposed to Cd, as well as galactinol. Glutathione (GSH), phytochelatins (PCs) and their homologues were strongly induced by Cd, whereas plants supplied with 1.9 and/or 3.7 mmol L-1 S showed the highest concentrations of these peptides. Plants supplied with highest S concentration showed lowest lipid peroxidation and highest photosynthetic rate, which evidences that antioxidant system of these plants was more efficient in mitigating the damages caused by Cd. In view of these results, it is clear that Massai grass properly supplied with S shows greatest Cd tolerance, as well as phytoextraction potentialA concentração de cádmio (Cd) no ambiente aumentou em décadas recentes, o que representa sério problema sócio-ambiental, visto que o Cd é tóxico para a maioria dos seres vivos. Por isso, é importante reduzir a concentração desse metal em ambientes contaminados e o uso de plantas adequadamente supridas com enxofre (S) pode contribuir para isso (fitoextração), uma vez que o S é componente de metabólitos envolvidos no combate ao estresse causado pelo Cd. Assim, o nosso objetivo com esse estudo foi avaliar o efeito do S na i) cinética de absorção do Cd, no ii) desenvolvimento radicular e na absorção de nutrientes, no iii) perfil metabólico e síntese de compostos tiol, e iv) na atividade do sistema antioxidante e fotossintético do capim-massai (Panicum maximum Jacq. cv. Massai), utilizado para fitoextração de Cd. Os estudos foram conduzidos em casa de vegetação utilizando-se vasos de 0,5 e 2,0 L para a condução do estudo de cinética de absorção de Cd (tratamentos representados pelas combinações das doses de S de 0,1 e 1,9 mmol L-1 e Cd de 0,1 e 0,5 mmol L-1) e para o estudo dos mecanismos de detoxificação de Cd (tratamentos representados pelas combinações das doses de S de 0,1; 1,9 e 3,7 mmol L-1 e Cd de 0,0; 0,1 e 0,5 mmol L-1), respectivamente. Os vasos foram distribuídos em blocos ao acaso, com quatro repetições. As plantas utilizadas no estudo de cinética foram expostas aos tratamentos durante 14 dias após as mesmas terem permanecido em soluções contendo apenas S durante 42 dias, enquanto as plantas utilizadas no estudo de detoxificação de Cd foram expostas aos tratamentos pelo período de 9 dias após terem crescido em soluções contendo apenas S por 44 dias. Ao final dos estudos, as plantas utilizadas foram colhidas e encaminhadas para análises. A velocidade máxima de absorção (Vmax) e o influxo apoplástico de Cd do capim-massai exposto à maior dose de Cd foram maiores quando as plantas foram supridas com a maior dose de S. O desenvolvimento radicular do capim-massai foi fortemente inibido quando as plantas foram expostas à dose de Cd de 0,5 mmol L-1, mas o adequado (1,9 mmol L-1) suprimento de S permitiu maior absorção de Cd, enquanto o suprimento excessivo (3,7 mmol L-1) de S diminuiu a formação de placas de ferro nas raízes das plantas. A lisina, cisteína, ornitina, arginina, triptofano e histidina foram acumulados em mais de um tecido nas plantas expostas ao Cd, assim como o galactinol. A glutationa (GSH), as fitoquelatinas (PCs) e seus homólogos foram fortemente induzidos pelo Cd, sendo que as plantas supridas com as doses de S de 1,9 e/ou 3,7 mmol L-1 apresentaram as maiores concentrações desses peptídeos. As plantas supridas com as maiores doses de S apresentaram menor peroxidação lipídica e maior taxa fotossintética, o que demonstra que o sistema antioxidante dessas plantas foi mais eficiente na atenuação dos danos causados pelo Cd. Diante desses resultados, fica claro que o capim-massai suprido adequadamente com S apresenta maior tolerância ao Cd, assim como maior potencial de fitoextração
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Ravanel, Stéphane. « Biosynthèse de la méthionine chez les plantes supérieures : étude biochimique et moléculaire des enzymes de la voie de transsulfuration ». Université Joseph Fourier (Grenoble ; 1971-2015), 1995. http://www.theses.fr/1995GRE10238.
Texte intégralRésumé :
Chez les plantes, l'atome de soufre de la methionine derive de la cysteine par une serie de reactions dites de transsulfuration faisant intervenir la cystathionine et l'homocysteine comme intermediaires. En presence de cysteine et d'o-phosphohomoserine, la cystathionine gamma-synthase catalyse une synthese de cystathionine qui est ensuite clivee en homocysteine, pyruvate et ammoniac par la cystathionine beta-lyase. La mise au point de techniques de dosage tres sensibles de ces activites enzymatiques a permis de demontrer que la cystathionine gamma-synthase et la cystathionine beta-lyase sont presentes uniquement dans le stroma des chloroplastes. Ces enzymes ont ensuite ete purifiees a l'homogeneite a partir de chloroplastes de feuilles d'epinard et leurs proprietes cinetiques et physicochimiques ont ete definies. La cystathionine gamma-synthase et la cystathionine beta-lyase jouent un role primordial dans le metabolisme cellulaire puisque l'inhibition de l'une ou l'autre de ces enzymes par la propargylglycine ou l'aminoethoxyvinylglycine est letale pour la plante. Un adnc codant pour la cystathionine beta-lyase d'arabidopsis thaliana a ensuite ete clone par complementation fonctionnelle d'un mutant d'e. Coli deficient en activite enzymatique endogene. Cette proteine est synthetisee sous la forme d'un precurseur polypeptidique de 50,4 kda qui est importe et clive dans le chloroplaste pour obtenir une sous-unite mature de 46 kda. La cystathionine beta-lyase d'a. Thaliana a finalement ete surproduite dans e. Coli. La purification de la proteine recombinante en grande quantite permet d'initier une etude structurale de l'enzyme ainsi que le developpement d'un test de criblage automatise de molecules chimiques en vue de la mise au point d'herbicides nouveaux et specifiques
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Lencer, Doerthe Adriana. « Expression und Regulation von Zytokinrezeptor-mRNAs in Bronchialbiopsien von Patienten mit chronisch obstruktiver Lungenerkrankung / ». 2005. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=014594483&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Wang, Sihong. « Kinetics study of heat shock protein 70 expression ». 2003. http://wwwlib.umi.com/cr/utexas/fullcit?p3116221.
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Lin, Chiu-Fang, et 林秋芳. « Differential Expression of mRNAs in Epithelia of Tubular Shell Glands of Brown Tsaiya Varying with Eggshell Strength ». Thesis, 2001. http://ndltd.ncl.edu.tw/handle/00736888582993646061.
Texte intégralRésumé :
碩士國立臺灣大學
畜產學研究所
89
Deterioration of eggshell quality associated with many factors causes economic loss due to breakage during the egg transportation and processing. While many approaches including aspects related to nutrition, feeding management, or housing environment etc. have so far been employed years for enhancing the shell strength of chicken eggs, there are approximate 12% of all commercial eggshells broken or cracked between oviposition and consumption. The organic matrix, though it occupies only 1 ~ 2% of the whole eggshell contents, can be one of the most important components involved in eggshell quality. Therefore, in this present study, attempts were made to identify and characterize gene(s) differentially expressed 4.5 h post oviposition in epithelia of tubular shell-glands of brown Tsaiya and it is hopefully that the results will provide a better understanding to the molecular mechanisms related to eggshell formation. To achieve the said purpose, two herds of brown Tsaiya ducks showing significant difference in eggshell strength (ES), the high eggshell strength (HES) with an average ES value over 5.5 kg and the low eggshell strength (LES) with an average ES value less than 3.5 kg, were employed in the present study. Total RNA samples were extracted from epithelial cells of tubular shell gland 4.5 h post-ovipostion. Each RNA sample was subjected to analysis by the method of differential display reverse transcription polymerase chain reaction （DDRT-PCR） and two-step DDRT-PCR with a total of 72 primer sets designed. Comparisons were made to identify specific fragments of cDNA(s) between each sample amplified after the DDRT-PCR and two-step DDRT-PCR analysis. Among the 72 pairs of primer sets tested, only 8 primer-pairs were found to be effective in amplifying specific DNA fragments from the diversified products amplified. A total of 16 differential fragments of cDNA were obtained, including 11 fragments from the method of DDRT-PCR and 5 fragments from the two-step DDRT-PCR method. One of them, named TG36S, was further subjected to quantitative comparisons of its mRNA expressed between herds of HES- and LES-ducks, with the method of Real-Time Quantitive polymerase chain reaction (QPCR). Results appeared that the amount of TG36S mRNA expressed in the tubular shell glands from HES-ducks was 1.83 fold higher than that in the same tissue from LES-ducks. Moreover, to those HES-ducks, it was also noticed that much higher expression of TG36S, with some 7.57, 4.05, and 2.60 folds higher based on the amount of TG36S mRNA expressed, was found in epithelia of shell glands, tubular shell glands, and magnum, respectively, when comparisons were made to that of in epithelia of isthmus. Based on the fact that much higher expression of TG36S found in HES-ducks, particularly in epithelia of shell glands and tubular shell glands, it is worth to further investigate the physiological function of this gene product.
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Hsiao, Yi-Jing, et 蕭乂菁. « Study on the expression profiles of mRNAs and microRNAs in HLJ1-silenced non-small cell lung cancer cells ». Thesis, 2008. http://ndltd.ncl.edu.tw/handle/48388871183461389865.
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碩士國立中興大學
生物醫學研究所
96
Recently, lung cancer has dominantly increased and ranked up to the first place among Taiwanese women died from cancers. Therefore, understanding the tumorigenesis and metastasis in lung cancer are very important events. Previously study in our lab proved HLJ1 is a tumor suppressor. Moreover, HLJ1 expression was also associated with reduced cancer recurrence and improved overall survival in non-small cell lung cancer (NSCLC) patients. Over-expression of HLJ1 in cancer cells suppressed cell growth, invasion and migration. In order to clarify the role of HLJ1 in NSCLC cell, CL1-0, with the high HLJ1 expression but low capability of invasion and metastasis was utilized in this study. After reducing HLJ1 expression with RNA interference, the stable cell lines were successfully selected. It was observed that remolding of fiber actin induced reorganization of cell cytoskeleton, and the metastasis capability would increase significantly in these HLJ1-silenced cells. Furthermore, microarray was used to profile the expressions of genes and microRNAs in HLJ1-silenced cells and analyzed the results by composite regulatory signature database which was developed in our lab. The results revealed that HLJ1 could not only regulate the genes involved in cytoskeleton and cell cycle, but also moderate the signaling pathways such as MAPK, Wnt and epithelial-to-mesenchymal transition (EMT) pathway. In cytoskeleton signaling pathway, Rac1-GTP was prove to be up-regulated in HLJ1-silenced clones by GST pull-down assay. In these pathways, 43 genes were selected for further validation of gene expression patterns by real-time PCR. There are 34 genes with matched expression patterns analyzed by chip and real-time PCR, such as NTS、cyclin D1 and ROCK1 etc. Moreover, there are 15 genes as transcription factors, including PITX2、HOXA1、MEIS2、HOXA5、FOXC1、CITED2、PAX9 and E2F5 those were up-regulated and ATF3、KLF5、ATBF1、HEY1、HOXB5、S100A10 and TAF5 were down-regulated in HLJ1-silenced cells. For example, transcription factor PITX2 and HOXA1 would up-regulate the oncogene cyclin D1. Aadditionally, profiling of the microRNA expressions in HLJ1-silenced cells by microRNA chip was no significantly different with HLJ1-non-silenced cells. (From 0.6 to 1.6 fold) Furthermore, the nuclear aggregates of β-catenin were found in HLJ1-silenced cells. The function of β-catenin in nucleus was proved previously to activate transcription. Those evidences show that HLJ1 may regulate β-catenin localization to activate the transcription of the downstream genes, including PITX2 and cyclin D1 genes. In conclution, HLJ1 mediates the activation of Rac1 and the localization of β-catenin to regulate the expression of downstream genes, and moderate the cell morphology and the abilities of invasion and migration in cancer cells. These studies give a new annotation of HLJ1 in cancer metastasis and invasion mechanism. But in the other signaling pathways, the role of HLJ1 still needs more efforts to research. The combined microarray and bioinformatic anyalsis provides more directions for further investigation. In the future, it is expected that can facilitate on the treatment of cancer.
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Lin, Fang-Yin, et 林芳因. « IL-1 may up-regulate the expression of IL-6 and ColIA1 mRNAs in the cells of dihydropyridine induced gingival ». Thesis, 2009. http://ndltd.ncl.edu.tw/handle/42883920578723368475.
Texte intégralRésumé :
碩士臺北醫學大學
牙醫學系碩博士班
97
Nifedipine is a well known calcium channel blockers for treatment of hypertension and angina. The side effect as nifedipine-induced gingival overgrowth (NIGO) can be frequently found in clinic. NIGO cells, as stimulated by IL-1β, may elicit a stronger IL-6 expression and collagen production. Moreover, Akt phosphorylation was correlated to collagen overproduction. We propose a hypothesis that PI3K/Akt/NFκB pathway is one of the possible mechanisms which may be related to the over expression of IL-6 and overproduction of extra-cellular collagen in dihydropyridine induced gingival overgrowth (DIGO) cells. In this study, we will use western blot technique to detect the expression of Akt. Moreover, real-time PCR is utilized to explore the expression of IL-6 and collagen-I mRNA. Sircol dye assay was used to detect total collagen concentration. Finally, we will interpret the correlation of p-Akt/Akt, IL-6 and collagen by non-parametric Spearman correlation coefficient. Result revealed that by stimulating with IL-1β, the value of p-Akt/Akt ratio, the expression of IL-6 and collagen-I mRNA, and the concentration of IL-6 and collagen in supernatant were all increased. In addition to the expression of IL-6 mRNA, the other three factor described above were higher in DIGO group then in healthy group stimulated by IL-1β. Spearman correlation coefficient shows high relationship between the value of p-Akt/Akt, the expression of IL-6 and collagen-I mRNA, and the concentration of IL-6 in supernatant. The data mentioned above approve that Akt is in connection with gingival overgrowth.