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1
Nielsen, Torsten. "Human origins of DNA replication : identification, analysis and application." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40411.
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While replication origins, cis-acting sequences directing the initiation of DNA synthesis, have been well-characterized in many model organisms, the multiple sequence and protein components present at the chromosomal origins of higher eukaryotic organisms have not yet been fully defined. Genetic assays that identify origin function in cloned DNA fragments would provide a useful approach for the isolation and analysis of mammalian DNA replication origins.In this thesis, (1) cloned fragments from a known mammalian origin, the ori$ beta$ of the hamster 3$ sp prime$ DHFR region, are demonstrated to replicate autonomously, both following transfection into human cells, and when used as templates in an in vitro replication system based on human cell extracts; (2) larger scale versions of these two assay methodologies are used to isolate over 40 novel putative origins of DNA replication from anticruciform purified human genomic DNA libraries; (3) transfection and in vitro autonomous replication assays are applied to demonstrate the potential origin function of a mitochondrial DNA sequence implicated in the insertional mutagenesis of a human genomic locus; (4) an origin mapping strategy based on the in vitro assay is used to provide evidence for the existence of a replication origin in a cloned and sequenced portion of the human 15q11q13 chromosomal subdomain, a region associated with allele-specific replication timing, genomic imprinting, and genetic disease; and (5) some of these autonomously replicating origins are cloned into a selectable YAC vector and are shown to permit the long term episomal maintenance, in human cells, of the transfected plasmid constructs.
These results consistently demonstrate that short mammalian genomic DNA fragments can replicate autonomously, supporting the applicability of the replicon model in humans, and could be extended to the search for an origin core consensus element, to the investigation of higher order organization and temporal control of human DNA replication origins, and to the construction of a complete human artificial chromosome.
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Esmaeil, Shalaby A. A. "Molecular analysis of chordomas and identification of therapeutic targets." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/20213/.
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Chordoma is a rare malignant bone tumour, showing notochordal differentiation, which occurs in the axial skeleton. Brachyury, a molecule involved in notochordal development, is a highly specific and sensitive marker for chordoma. It is hypothesised that brachyury or genes involved in its activation are implicated in the pathogenesis of chordoma. As there is currently no effective drug therapy for chordoma the aim of this study was to identify genetic events involved in chordoma pathogenesis with a view to identifying potential therapeutic targets. One hundred chordomas (50 skull-based, 50 non-skull based) were studied. Immunohistochemistry showed that the PI3K/AKT/TSC/mTOR pathway was activated in 65% of chordomas, thereby providing a rationale for testing mTOR inhibitors for the treatment of selected cases. DNA sequencing revealed no mutations in PI3KCA or RAS homologue enriched in brain (Rheb) in 23 tumours. Immunohistochemistry and Western blotting showed activation of the fibroblastic growth factor receptor (FGFR)/RAS/RAF/MEK/ERK/ETS2/brachyury pathway in more than 90% of cases, but no mutations were found in the genes analysed (FGFRs, KRAS, BRAF and brachyury) in 23 tumours. Three percent of cases revealed brachyury amplification but nearly half of the cases showed chromosomal abnormalities involving the brachyury locus. Knockdown of brachyury was achieved in the U-CH1 chordoma cell line using shRNA and resulted in premature cell senescence. These findings demonstrate that brachyury plays an important role in chordoma pathology. FISH analysis showed EGFR copy number gain in 45% of chordomas, including 6% with amplification and 39% with high level polysomy. The EGFR inhibitor, tyrphostin (AG1478) significantly inhibited growth of the chordoma cell line, and Western blotting showed this was associated with reduced phosphorylation of EGFR in a dose dependent manner. This study provides evidence for the first time that selected chordomas may be susceptible to treatment with EGFR inhibitors.
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Pourahmad, Fazel. "Molecular detection and identification of aquatic mycobacteria." Thesis, University of Stirling, 2007. http://hdl.handle.net/1893/355.
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Mycobacteriosis (fish tuberculosis) is a progressive disease of a wide range of wild and captive marine and freshwater fish species. While Mycobacterium marinum, M. fortuitum and M. chelonae are the most frequently reported species to be involved in the disease, several new mycobacteria species have also recently been implicated. Conventional detection / identification of fish mycobacteria is based on histopathology, culture and biochemical characteristics. In this study complementary molecular approaches were developed to assist in Mycobacterium identification. First, a highly specific and sensitive multiplex PCR-based assay, targeting two genes (hsp65 and 16S RNA), was established to simultaneously detect the genus Mycobacterium and identify M. marinum, M. fortuitum or M. chelonae from culture or infected fish tissue, based on presence / absence of specific amplicons. In addition, PCR-restriction enzyme analysis (PRA) and DNA sequence analysis of the 16S-23S internal transcribed spacer (ITS) region and a 441 bp fragment of the hsp65 gene demonstrated the limitations of multiplex PCR (and commercial line probe assays) to differentiate among the species of the M. fortuitum complex. However DNA sequence analysis of the hsp65 gene fragment was found to reliably identify M. fortuitum from closely related species, M. conceptionense and M. senegalense. Reliable identification of novel species (or very similar species) of aquatic mycobacteria requires more extensive DNA sequence comparisons. Thus, multigene (polygenetic) analyses, as used here, provide rapid, accurate and reliable species identification of aquatic mycobacteria. Furthermore, a number of novel species of aquatic mycobacteria, M. stomatepiae, ‘M. angelicum’, ‘M. aemonae’ and M. salmoniphilum were discovered using the polygenetic analysis approach. Correct identification of Mycobacterium species by DNA sequence comparisons relies on accurate database information. Difficulties in this study in assigning M. marine and M. gordonae to their correct taxa suggest errors in the current public sequence repositories. The above methods were successfully applied to detect and identify mycobacteria in field samples including formalin-fixed, paraffin-embedded (FFPE) fish tissue, water and frozen fish tissue.
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Chandrashaker, Akhila. "Systems analysis of early endosome motility through identification of molecular motors." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2010. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-61594.
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Endocytosis is an evolutionary conserved process of internalization of cargo from the extracellular environment, be they ligands, nutritional and signaling or pathogens into cells. Following their entry, cargo is received into vesiculo-tubular network of early endosomal compartments from where they are sorted and routed to appropriate cellular destinations through transport along the endocytic network. Recycling cargo is sorted away from other cargo resident in early endosomes through tubulation resulting in fission of recycling vesicles, while those to be degraded are progressively concentrated in early endosomes to be degraded in lysosomes.
Early endosomes are dynamic organelles that have been shown to move centripetally following the internalization of cargo into at the cell periphery. Their motility from the cell periphery to the juxtanuclear location of the cell involves convoluted trajectories that include directed motility, bi-directional switches, saltatory behavior and stalls. This complex motility presumably contributes toward the cargo sorting, duration of cargo residence and spatio-temporal signaling by early endosomes. How the different regimes of motility, and nature and number of molecular motors involved in early endosome motility contribute toward endosome function is not understood.
The aim of this study was to probe into the regulation of endosome motility and understand how transport organizes early endosome network. Towards this end, live cell time-lapse movies of Rab5 endosomes were analyzed to derive motility properties contributing to organization of early endosomes. Consistent and significant bias toward the cell centre (minus end motility) in kinetic parameters such as speed, displacement and duration of motility contribute to centripetal flux of Rab5 early endosomes.
A phenomenological property of early endosome motility is its saltatory behavior that produces saturation curves in Mean Square Displacement (MSD) plots. This phase of motility is descriptive, with no understanding of its mechanism or function. Live cell candidate RNAi screen and cytoskeletal perturbation analysis were performed to identify molecules regulating saltatory motility. To this end, cellular microtubule perturbation and RNAi knock down of several Kinesin motor candidates showed a loss in saturation behavior. Potential candidates identified have to be tested for their effect on endosome function through cargo sorting and kinetic assays to gain insights into the role of saltatory motility in endosome function.
Molecular motors mediate Rab5 motility. Therefore, understanding regulation of motility requires identifying number and nature of molecular motors involved in their transport. Towards this end, a functional cargo (LDL) degradation RNAi screen targeting molecular motors was performed. The Ambion Select technology was used with 3 siRNAs targeting every gene in the library. Analysis of screen produced by lack of phenotype consistency between the multiple siRNAs targeting the same gene. Hence, a search for technology with better target specificity was initiated. Technologies tested were Ambion Select, Ambion Silencer Select, Dharmacon ON-TARGET Plus, esiRNA and Invitrogen Stealth. Invitrogen Stealth technology was found to produce the least off-targets and was most specific in terms of consistency of phenotypes produced by multiple siRNAs silencing the same target gene. Assay conditions were also found to influence the silencing specificities to a significant extent. Hence, a systematic assay optimization exercise was performed in terms of the concentration of siRNA used for transfection and time window of assay to maximize specificity of siRNA silencing. Insights obtained from methodologies developed herein not only provide invaluable guidelines in choosing RNAi commercial libraries for screens, but also underscore the importance of establishing optimal assay conditions to minimize off-targets and improve specificity of silencing target genes.
The motor screen was repeated with RNAi library from Invitrogen Stealth. Several potentially interesting candidates have been identified. Also, correlation analyses of phenotypes produced in the screen have indicated toward potential regulatory motor complexes, all of which await biochemical validation.
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Neels, Jacobus Gerardus. "LDL receptor-related protein molecular analysis and identification of new ligands /." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2001. http://dare.uva.nl/document/60201.
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de, Sousa Ines Girao Meireles. "Molecular genetics of autism : Identification and analysis of autism susceptibility genes." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526435.
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Hoang, Tiffany Truc. "Speciation and identification of low molecular weight organoselenium metabolites in human urine." Diss., Georgia Institute of Technology, 2003. http://hdl.handle.net/1853/30671.
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Amagase, Yoko. "Identification and molecular analysis of novel splice variants of the SUR1 gene." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619509.
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Yu, Xuejie. "Molecular identification of rickettsial bacteria by using monoclonal antibodies and genetic analysis." Aix-Marseille 2, 1993. http://www.theses.fr/1993AIX22058.
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Nous avons mis au point une methode d'analyse par restriction enzymatique (rflp) de fragments d'adn rickettsien amplifies (pcr) pour differencier les rickettsies du groupe boutonneux. Nous avons isole et identifie deux souches de rickettsies du groupe boutonneux, dont l'une est nouvelle. Nous avons produit des anticorps monoclonaux specifiques d'espece reagissant contre des bacteries rickettsiennes recemment caracterisees, telles qu'afipia felis ehrlichia chaffeensis et rochalimaea henselae, pour le diagnostic d'infections provoquees par ces organismes. Nous avons demontre qu'il n'y a pas de correlation entre souches de coxiella burnetii et les deux formes de fievre q, c'est-a-dire la forme aigue et les endocardites, en utilisant des anticorps monoclonaux diriges specifiquement contre la souche priscilla, consideree comme etant le representant des isolats obtenus de patients souffrant d'endocardite
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Pyz, Elwira. "Identification of rat NKT cells and molecular analysis of their surface receptor mediated activation." Doctoral thesis, [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972705112.
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Stagni, Camilla. "Genomic analysis in cutaneous melanoma: a tool for predictive biomarker identification and molecular classification." Doctoral thesis, Università degli studi di Padova, 2017. http://hdl.handle.net/11577/3426683.
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Project 1. Identification of molecular signatures associated with response to MAPK inhibitors.
BRAF V600-mutated melanoma benefits from MAPK inhibitors-based therapy. Yet, the onset of resistance impacts long-term efficacy and can even be immediate. In this study, we examined the genetic alterations characterizing melanoma progression to identify predictive factors of response to MAPK inhibitors (MAPKi). Specifically, we evaluated BRAF copy number variation (CNV), BRAF mutant (BRAFmut) allele frequency, PTEN loss or mutations and TERT promoter mutations in pre-treatment melanoma specimens from MAPKi-treated patients (pts) and we analyzed their association with progression free survival (PFS). We also applied a comprehensive unbiased approach, using genome-wide CNV analysis, to identify additional genomic aberrations potentially associated with response to therapy.
We found that 65% pts displayed BRAF gains, often supported by chromosome 7 polysomy. In addition, we observed that 64% pts had a balanced BRAF mutant/wild-type allele ratio, while 14% and 23% pts had low and high BRAFmut allele frequency, respectively. Notably, a significantly higher risk of progression was observed in pts with a diploid BRAF status vs. those with BRAF gains (HR = 2.86; 95% CI 1.29‒6.35; p = 0.01) and in pts with low vs. those with a balanced BRAFmut allele percentage (HR = 4.54, 95% CI 1.33‒15.53; p = 0.016).
We identified PTEN gene mutations affecting the catalitic and C2 domains in 27% pts. Moreover, we observed a complete PTEN loss in 42% pts, partial loss in 35% pts and no loss in 23% pts. Of note, we found PTEN loss also in pre-treatment samples from pts with long PFS.
Sequencing of TERT promoter gene disclosed mutations in 78% pts. The -124C>T and the -146C>T mutations were equally frequent (36%) while the -138-139CC>TT was present only in 5% pts. Fifty-one % pts carried also the neighboring polymorphism rs2853669, which reportedly counteracts the activating effect of the above-mentioned mutations on TERT expression. Upon stratification of the TERT promoter mutant cohort based on presence/absence of the polymorphism, TERTmutant/SNPcarrier pts showed a trend toward better PFS (median PFS 11.5 mo., 95% CI 3.12‒19.88) compared to TERTmutant/SNPnon-carrier pts (median PFS 7 mo., 95% CI 4.27‒9.72). When stratifying based on mutation type, the -146C>T mutation correlated with shorter PFS (median PFS 5.45 mo., 95% CI 2.80‒9.20) compared to the -124C>T one (median PFS 15.2 mo., 95% CI 5.57‒).
Genome-wide CNV analysis pointed at chr3p24, chr3p21.2 and chr17p13.1, which are differently alterated between pts with long and short time to disease progression, as regions of potential interest to identify new genes involved in therapeutic resistance.
Our data suggest that quantitative analysis of the BRAF gene and sequencing of the TERT promoter gene could be useful to select the melanoma pts who are most likely to benefit from MAPKi therapy. In addition, chromosome 3 and 17 could be regions that warrant further investigation. Conversely, because PTEN loss was present in pre-treatment samples from pts with both short and long PFS, the assessment of PTEN gene status does not seem to provide information about patient responsiveness to treatment.
Project 2. Research of molecular biomarkers to classify the acral melanoma.
Acral lentiginous melanoma (ALM) is a rare subtype of cutaneous melanoma with specific morphological, epidemiological, and genetic features. Since the genomic landscape of ALM is still incompletely described, we used whole genome CNV analysis to characterize ALM and detail the genomic signatures that differentiate ALM from non-acral melanoma (NAM).
We observed that the most strikingly different copy number aberrations were a higher frequency of losses of chromosome 16q24.2-16q24.3 in ALM than in NAM (64.7% vs. 10%) and a lower frequency of gains of chromosome 7q21.2-7q33 in ALM than in NAM (26.5% vs.79.5%). We observed also that ALM more often (than NAM) harbored clusters of breakpoints and isochromosomes. Moreover, in ALM we identified focal amplification of TERT, CCND1, MDM2 and MITF. In NAM, instead, we found only two focal amplifications, involving BRAF and MITF. Focal homozygous copy losses affected especially the CDKN2A and PTEN genes, both in ALM and in NAM, even though they were more frequent in the latter group.
In keeping with previous observations that led to classify ALM as a distinct molecular subtype of melanoma, we observed a peculiar genomic landscape in ALM (vs. NAM). Our study provides insights into the molecular characteristics of ALM, which is key to full elucidation of its pathogenesis.Progetto 1: identificazione di signatures molecolari associate alla risposta al trattamento con inibitori del MAPK pathway. I melanomi portatori di una mutazione nel codone V600 del gene BRAF rispondono agli inibitori del MAPK pathway, ma l’efficacia a lungo termine di questa terapia è limitata dallo sviluppo di resistenza, talvolta immediata. In questo studio, abbiamo esaminato le alterazioni molecolari caratterizzanti la progressione del melanoma al fine di identificare fattori predittivi di risposta/resistenza ai MAPK-inibitori. Nello specifico, su una serie di campioni pretrattamento di pazienti affetti da melanoma, trattati con MAPK-inibitori, abbiamo valutato numero di copie del gene BRAF e percentuale di allele V600-mutato, delezione e mutazioni di PTEN, alterazioni del promotore di TERT, e ne abbiamo analizzato l’associazione con la risposta dei pazienti alla terapia. Inoltre, abbiamo determinato il copy number variation dell’intero genoma dei nostri campioni per individuare ulteriori aberrazioni non note potenzialmente associate con la risposta alla terapia. Abbiamo identificato un numero aumentato di copie (gain) del gene BRAF, spesso dovuto a polisomia del cromosoma 7, nel 65% dei pazienti; l’allele mutato è stato trovato in una percentuale compresa tra il 35% e il 65% nel 64% dei pazienti, inferiore al 35% nel 14% dei pazienti e superiore al 65% nel 23% dei pazienti. Dall’analisi di sopravvivenza, è risultato che i pazienti con BRAF diploide o una percentuale di allele mutato inferiore al 35% presentano un più alto rischio di progressione rispetto a coloro che presentano gain di BRAF (HR=2.86; 95% CI 1.29-6.35; p=0.01) o tra il 35% e il 65% di allele mutato (HR=4.54,CI 1.33-15.53; p=0.016), rispettivamente. L’analisi di PTEN ha rivelato la presenza di mutazioni nel 27% dei pazienti, localizzate a livello dei domini catalitico e C2 della proteina codificata; inoltre, il 42% dei casi valutati mostrava una delezione completa del gene, il 35% una delezione parziale, mentre nel 23% dei pazienti non è stata individuata alcuna aberrazione di PTEN. Da notare, delezioni di PTEN sono emerse sia nei casi di melanoma resistente alla terapia, che in quelli che avevano risposto a lungo. Il sequenziamento del promotore del gene TERT ha permesso l’identificazione di mutazioni nel 78% dei pazienti. Le mutazioni -124C>T e -146C>T mostravano la stessa frequenza (36%) nella nostra coorte, mentre la -138-139CC>TT è stata individuata solo nel 5% dei casi. Il 51% dei pazienti presentava inoltre lo SNP rs2853669, noto per contrastare l’effetto attivante delle mutazioni sull’espressione di TERT. Stratificando la coorte di pazienti mutati in base alla presenza/assenza del polimorfismo, i pazienti TERT mutati/SNP carriers mostravano un trend verso una migliore PFS (PFS mediana 11.5 mesi, 95% CI 3.12-19.88) rispetto ai TERT mutati/SNP non-carriers (PFS mediana 7 mesi, 95% CI 4.27-9.72). La mutazione -146C>T, inoltre, correlava con PFS più breve (PFS mediana 5.45 mesi, 95% CI 2.80-9.20) rispetto alla -124C>T (PFS mediana 15.2 mesi, 95% CI 5.57-). Dall’analisi del copy number variation (CNV) sull’intero genoma, le regioni chr3p24, chr3p21.2 e chr17p13.1 hanno mostrato pattern di alterazioni diverse in pazienti responsivi vs. non-responsivi alle terapie; risultano pertanto regioni di potenziale interesse per l’individuazione di nuovi geni coinvolti nella resistenza alla terapia. I nostri dati suggeriscono dunque che l’analisi quantitativa del gene BRAF e il sequenziamento del promotore di TERT costituiscono un utile strumento di selezione dei pazienti con maggiore probabilità di rispondere alla terapia con MAPK-inibitori, contrariamente alla valutazione dello status di PTEN. L’analisi genome-wide, invece, indica di approfondire lo studio dei cromosomi 3 e 17. Progetto 2: ricerca di marcatori biomolecolari per la classificazione del melanoma acrale. Il melanoma acrale lentigginoso è un raro sottotipo di melanoma cutaneo con specifiche caratteristiche morfologiche, epidemiologiche e genetiche. Poiché il genoma del melanoma acrale non è ancora stato pienamente caratterizzato, ne abbiamo analizzato il CNV per individuare quei caratteri genomici peculiari che lo differenziano dal melanoma non acrale. La nostra analisi genome-wide ha evidenziato una maggiore frequenza di delezioni della regione 16q24.2-16q24.3, gains meno frequenti nella regione 7q21.2-7q33, una più accentuata frammentazione genomica e numerosi isocromosomi come caratteri che distinguono il melanoma acrale dal non acrale. Abbiamo inoltre identificato amplificazioni focali nei geni TERT, CCND1, MDM2 e MITF, più rare nei non acrali, laddove interessavano altri geni, come BRAF e MITF. Delezioni focali sono state individuate soprattutto nei geni CDKN2A e PTEN in entrambi i sottotipi di melanoma, anche se più frequenti nei non acrali. I nostri dati, in accordo con il classificare il melanoma acrale come tipo distinto di melanoma, hanno consentito di delinearne alcune delle peculiarità genomiche, chiave per elucidarne anche la patogenesi.
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Al, Masalma Mouhamad. "Molecular and cultural analysis of the bacterial flora associated with brain abscesses." Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX20663/document.
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Les abcès cérébraux sont des infections potentiellement mortelles, entraînant souvent des séquelles graves. La prise en charge médicale en reste empirique en raison d’un manque de connaissance approfondie des microorganismes responsables de cette condition. Dans la plupart des laboratoires microbiologiques, le diagnostic d’abcès cérébral est basé sur la culture du pus recueilli chirurgicalement. Malheureusement, cette procédure a de nombreuses limites et ne permet l’identification que d’une petite partie de la population microbienne en cause. L’amplification par PCR et le séquençage du gène codant la fraction 16S de l’ADN ribosomal ont récemment été utilisées pour surmonter les limites de la culture, et ont été démontré leur efficacité dans la documentation des infections bactériennes. Malheureusement, cette procédure présente un degré de discrimination limité en cas d’infection polymicrobienne. Des études métagénomiques de flores complexes de l’homme, basées sur une combinaison de PCR, clonage et séquençage des produits de PCR se sont avérées utiles pour évaluer la diversité bactérienne des flores dentaires, vaginales et intestinales. Nous avons appliqué cette technique à des échantillons d’abcès cérébral pour étudier la flore associée à cette maladie. Dans une première étape, nous avons réalisé une enquête en utilisant la culture et les techniques moléculaires. Le but de cette étude était d’analyser et d’évaluer les bactéries de la flore responsable des abcès cérébraux, en comparant la culture à trois techniques moléculaires basées sur le gène 16S rDNA, incluant le séquençage direct, le clonage suivi de séquençage par méthode de Sanger, et le séquençage direct des produits de PCR par pyroséquençage. Cette enquête a déterminé que la variété des espèces bactériennes associée aux abcès cérébraux est beaucoup plus grande que précédemment décrite, et inclut de nombreuses bactéries anaérobies et des bactéries incultivables de la flore buccale. Cette étude préliminaire a identifié 49 agents bactériens différents, et a permis l’identification de 27 bactéries jamais détectées auparavant dans des abcès du cérébraux, dont 15 n’avaient jamais été cultivées. Un tel nombre d’espèces bactériennes impliquées dans les abcès cérébraux a motivé l’étude de 51 nouveaux spécimens dans le but de décrire plus en détail la flore associée aux abcès cérébraux en fonction de leurs étiologies. Ainsi, nous avons effectué une analyse métagénomique, basé sur le gène 16S rDNA, de 51 patients ayant développé un abcès cérébral. Notre stratégie a été beaucoup plus discriminatoire et a permis à l’identification d’un plus grand nombre de bactéries que la culture et l’amplification et le séquençage direct de l’ANRr 16S. La combinaison des données de 71 patients (20 de la première étude et 51 de la deuxième étude) a permis l’identification de plusieurs associations à l’aide de la méthode de data mining.En outre, notre étude a permis l’identification de deux nouvelles bactéries, la première étant une nouvelle espèce de genre Staphylococcus (Staphylococcus massiliensis) et la seconde étant une bactérie anaérobie qui représente une nouvelle espèce dans un nouveau genre au sein du phylum des Bacteroidetes (Phocaeicola abscesses). En outre, nous avons décrit deux cas inhabituels d’abcès du cerveau, à Mycoplasma hominis après curetage utérin, et à Nocardia carnea chez un greffé rénal. Malgré les limites inhérentes à la procédure de clonage, nos résultats suggèrent que le clonage et le séquençage de gène DNAr 16S est une méthode très performante pour identifier les agents bactériens associés aux abcès cérébrauxBrain abscess is a life-threatening infection with frequent serious sequelae. The medical management remains empirical due to a lack of comprehensive knowledge of the microorganisms responsible for this condition. In most microbiology laboratories the diagnosis of brain abscess is based on culture from pus collected surgically. Unfortunately, this procedure has many limitations and reveals only a small portion of the true microbial population. PCR-amplified 16S rDNA sequencing has recently been used to overcome the limitations of culture-based bacterial detection in brain abscess pus, and it was demonstrated to be effective in the documentation of monomicrobial infections. Unfortunately, this procedure failed to discriminate among polymicrobial floras.Metagenomic studies of complex human floras using a combination of 16S rDNA PCR and cloning-sequencing of PCR products proved useful to evaluate the bacterial diversity of dental, vaginal and intestinal floras. Thus, we applied this technique to brain abscess samples to study the flora associated with this condition. In a first step, we performed an investigation using culture and molecular techniques. The purpose of this investigation was to analyze and evaluate the bacterial flora responsible for brain abscess by comparing standard culture technique to three techniques using 16S rDNA amplification, that is, direct sequencing, multiple sequencing following cloning, and multiple sequencing via high throughput pyrosequencing. This investigation has determined that the variety of brain abscess-associated bacterial species is much larger than previously reported, and it includes many anaerobes and uncultured bacteria from the oral cavity flora. This preliminary study identified 49 distinct brain abscess bacterial agents, and enabled the identification of 27 bacteria never detected before in brain abscess, 15 of which were uncultured.Such a high number of bacterial species involved in brain abscess prompted the study of 51 new specimens in an effort to describe further the flora associated with brain abscesses and their etiologies. Thus, we performed a 16S rDNA-based metagenomic analysis of cerebral abscesses from 51 patients. Our strategy was significantly more discriminatory and enabled the identification of greater number of bacterial taxa, than culture and conventional 16S rDNA PCR/sequencing, respectively. The combination of data from 71 patients (20 from the first study and 51 from the second study) enabled the identification of several associations using the data mining analysis. Also, these studies permitted the identification of two novel bacteria, the first being a novel Staphylococcus species (Staphylococcus massiliensis) and the second being a novel anaerobic bacterium that represents a novel species in a new genus within the phylum Bacteroidetes (Phocaeicola abscesses). In addition, we reported tow unusual cases of brain abscess, the first case was a Mycoplasma hominis brain abscess following uterus curettage and the second case was a Nocardia carnea infection in a kidney transplant recipient patient.Despite limitations inherent to the cloning procedure, our results suggest that cloning and sequencing of PCR-amplified 16S rDNA is a highly valuable method to identify bacterial agents of brain abscesses
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McLaughlin, Sara Koenig. "Identification and Analysis of a New Tumor and Metastasis Suppressor Gene, RASAL2." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10756.
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RAS is one of the most commonly mutated genes in human cancer; its aberrant activation drives tumor cell proliferation and survival. However, RAS mutations are rare in some cancers, including breast cancer, even though the Ras pathway is hyperactivated, suggesting that alternative mechanisms deregulate Ras signaling in these settings. The RasGAPs are negative regulators of Ras and, as such, are poised to function as tumor suppressors whose loss might contribute to Ras pathway hyperactivation in cancer. However, the RasGAPs remain an understudied family of genes whose role in cancer has not been fully explored. In this Dissertation I identify a previously uncharacterized RasGAP, RASAL2, as the newest tumor suppressor in this gene family.
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Paré, Chantal. "Genetic analysis of localization of a Bic-D::GFP fusion protein and identification of novel subcellular domains." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0028/MQ50851.pdf.
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Niederwieser, Igor. ""Leishmania infantum" : molecular analysis for identification of potential virulence factors and genes of diagnostic use /." Basel : [s.n.], 2004. http://edoc.unibas.ch/diss/DissB_7571.
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MACCESI, Martina. "In silico protein modelling applied to the identification of new therapeutic agents." Doctoral thesis, Università degli studi di Ferrara, 2019. http://hdl.handle.net/11392/2488106.
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This thesis collects the results of three different research projects carried out in collaboration with the University of Parma. In chapter 2 the identification and characterization of antimicrobial compounds active as protein-protein interaction inhibitors of bacterial RNA polymerase subunit β' and transcription initiation factor σ is described. This peculiar protein-protein interaction has been identified as a promising target for antibacterial research and some small molecules were identified as disruptors of this interaction. In this project, a virtual screening campaign, aimed to the identification of novel antimicrobial molecules acting disrupting β'-σ interaction, was carried out. The newly identified compounds were experimentally tested in different assays, confirming their activity as PPI disruptors and highlighting their antimicrobial potential. A pharmacophore model was built for the most promising compounds to identify common patterns of interactions which were necessary for the activity and a binding hypothesis was proposed for each active compound that could be exploited as starting point for further optimization and for the discovery of new molecules.
In the third chapter modelling studies were applied to NAPE-PLD, an enzyme responsible for the synthesis of bioactive lipids involved in the regulation of several physiological and pathological conditions. Modulation of NAPE-PLD activity could represent a promising therapeutic strategy for a wide range of diseases but, despite the important role played by this enzyme, no new active molecules have been reported so far. This work describes the identification and characterization of the first small molecule inhibitor of NAPE-PLD. Docking studies were performed, thanks to the availability of NAPE-PLD crystal structure, highlighting the binding pose of the compound and they were confirmed by SAR studies and mutagenesis experiments. Despite its low potency, this molecule represents a molecular probe which can help in better characterize and investigate NAPE-PLD mechanisms and its role in pathologic and physiological processes.
NAPE-PLD together with other lipases, is characterized by a mechanism called “interfacial activation” which affects enzyme conformation and its catalytic machinery. The understanding of this phenomenon could be crucial to gain insight into enzyme’s behavior highlighting aspects of the catalytic mechanism that couldn’t be revealed by the X-ray structure alone. Molecular dynamics simulations were thus performed on the apo form of NAPE-PLD to better understand the conformational changes of the enzyme in both aqueous and membrane environment. Results highlights that the membrane environment stabilizes the open conformation, which is compatible with substrate recruitment, while water stabilizes a closed that block the access of the substrate to the active site, confirming the interfacial activation phenomenon.
The last chapter of this thesis describes the statistical analysis that was performed on a large database of activity data collected by testing 400 compounds against Schistosoma mansoni, a parasite responsible for schistosomiasis, a tropical neglected disease. Three organizations, the University of California San Diego (UCSD), the Swiss Tropical and Public Health Institute (STPH) and Fiocruz (Fundação Oswaldo Cruz) Foundation in Brazil have experimentally tested the Pathogen Box, a collection of molecules active against different pathogens, monitoring the effects on the helminth. Different assays were performed and the results were collected using different numerical ranges, thus a mathematical transformation of the data was applied to the dataset to obtain activity values in the same scale. This was necessary to perform a statistical analysis to evaluate coherence of the collected data and to identify the most promising compounds.Questa tesi di dottorato raccoglie i risultati di tre diversi progetti di ricerca che ho svolto in collaborazione con l’Università di Parma. In particolare, il capitolo 2 descrive l’identificazione e la caratterizzazione di composti con attività antimicrobica che agiscono come inibitori dell’interazione proteina-proteina tra subunitá β' e fattore di trascrizione σ nell’RNA polimerasi batterica. Evidenze sperimentali hanno dimostrato che l’interfaccia tra le due proteine rappresenta un sito di legame per piccole molecole e composti che legano questa regione sono in grado di inibire l’interazione proteina-proteina arrestando la trascrizione e mostrando un’attività antibatterica. L’obiettivo di questo lavoro è stato identificare, tramite un protocollo di virtual-screening, all’interno di una libreria di composti, un sottoinsieme di molecole attive come inibitori dell’interazione β'-σ. Test sperimentali sono poi stati eseguiti sui composti più promettenti confermando l’attività per alcuni e provando la loro efficacia come composti antibatterici. Un modello farmacoforico è stato poi costruito per razionalizzare la relazione tra struttura e attività e si è inoltre ipotizzata una modalità di legame per i composti attivi con la subunità target β' che potrà essere utilizzata come punto di partenza per lo screening di nuove librerie o per la progettazione di nuovi composti. Nel capitolo 3 vengono invece descritti studi di modellistica molecolare effettuati sull’enzima NAPE-PLD. Questa lipasi di membrana è responsabile della produzione di lipidi bioattivi coinvolti in diversi processi fisiologici e patologici e la sua modulazione risulta essere particolarmente importante nel trattamento di diverse patologie. La disponibilità della struttura cristallografica dell’enzima umano ha reso possibile effettuare studi di docking per ipotizzare le modalità di legame della prima molecola capace di inibire NAPE-PLD, identificata mediante HTS. La posa di docking ottenuta risulta compatibile con i dati SAR in nostro possesso ed è inoltre confermata da studi di mutagenesi. Nonostante l’attività inibitoria limitata del composto esso può essere considerato il punto di partenza per sviluppare nuovi inibitori. Sono poi state effettuate simulazioni di dinamica molecolare per valutare i cambiamenti conformazionali dell’enzima in due ambienti differenti: in presenza di solvente acquoso oppure in presenza di membrana cellulare per testare l’ipotesi di “attivazione interfacciale”, un fenomeno caratteristico delle lipasi. Questo meccanismo è caratterizzato dall’equilibrio conformazionale tra uno stato definito “aperto”, compatibile con l’accesso del substrato e uno stato “chiuso” in cui l’accesso del substrato è bloccato. Durante il tempo di una simulazione di dinamica molecolare si sono potuti evidenziare cambiamenti conformazionali della proteina compatibili con questa teoria permettendoci di ipotizzare un meccanismo di “recruitment” del substrato quando l’enzima si trova in presenza della membrana cellulare. L’ultimo capitolo descrive il progetto di ricerca che ho seguito presso la University of California San Diego: qui ho svolto un lavoro di analisi di dati di attività biologica ottenuti testando 400 composti di una libreria chiamata Pathogen Box sul platelminta Schistosoma mansoni per identificare composti attivi contro la schistosomiasi, una parassitosi comune nei paesi sottosviluppati. Tre organizzazioni, la University of California San Diego (UCSD), il Swiss Tropical and Public Health Institute di Basilea (STPH) e la fondazione brasiliana Fiocruz (Fundação Oswaldo Cruz) hanno eseguito diversi saggi collezionando una grande quantità di dati di attività che ho successivamente analizzato per verificare diversi aspetti tra cui l’identificazione dei composti più potenti e la coerenza dei dati raccolti tra le diverse organizzazioni.
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Rodrigues, Tania 1979. "Identification and analysis of new mutations that suppress the slow defecation phenotype of clk-1(qm30) mutants." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98781.
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Mutations in the clk-1 gene of Caenorhabditis elegans result in an average slowing down and deregulation of a variety of developmental and physiological processes. In addition, clk-1 mutants are defective in responding to temperature changes. For example, wild-type worms adjust their defecation cycle length after a temperature shift whereas the defecation cycle length of clk-1 mutants is unaffected by such a shift. To understand the basis of the clk-1 phenotype, a number of genetic screens have been carried out to isolate feature-specific suppressor mutations. dsc-3(qm179) and dsc-4(qm182) were isolated in this manner. It has previously been found that dsc-3(qm179) and dsc-4(qm182) strongly suppress the slow defecation phenotype of clk-1 mutants at 20°C, as well as after a temperature shift to 25°C. Molecular analysis of dsc-4, which encodes the microsomal triglyceride transfer protein, suggests that dsc genes affect lipid metabolism. We carried out a genetic screen for additional mutations that can suppress the slow defecation of clk-1 mutants after a temperature shift to 25°C and isolated two new suppressor mutations. Complementation tests as well as linkage analysis and mapping indicates that dsc-6(qm192) and dsc-7(qm193) define new dsc genes. We analyzed the phenotype of the new suppressor strains and have found that, like dsc-4(qm182), dsc-6(qm192) and dsc-7(qm193) can suppress a variety of clk-1 phenotypes. Based on additional phenotypic analyses of the new suppressor strains, including the determination of their sensitivity to exogenous cholesterol, we believe that, like dsc-4, dsc-6 and dsc-7 encode activities that affect lipid metabolism in worms.
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Ahmed, Firdous. "Identification of potential biomarkers in lung cancer as possible diagnostic agents using bioinformatics and molecular approaches." University of the Western Cape, 2015. http://hdl.handle.net/11394/4862.
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>Magister Scientiae - MScLung cancer remains the leading cause of cancer deaths worldwide, with the majority of cases attributed to non-small cell lung carcinomas. At the time of diagnosis, a large percentage of patients present with advanced stage of disease, ultimately resulting in a poor prognosis. The identification circulatory markers, overexpressed by the tumour tissue, could facilitate the discovery of an early, specific, non-invasive diagnostic tool as well as improving prognosis and treatment protocols. The aim was to analyse gene expression data from both microarray and RNA sequencing platforms, using bioinformatics and statistical analysis tools. Enrichment analysis sought to identify genes, which were differentially expressed (p < 0.05, FC > 2) and had the potential to be secreted into the extracellular circulation, by using Gene Ontology terms of the Cellular Component. Results identified 1 657 statically significant genes between normal and early lung cancer tissue, with only 1 gene differentially expressed (DE) between the early and late stage disease. Following statistical analysis, 171 DE genes selected as potential early stage biomarkers. The overall sensitivity of RNAseq, in comparison to arrays enabled the identification of 57 potential serum markers. These genes of interest were all downregulated in the tumour tissue, and while they did not facilitate the discovery of an ideal diagnostic marker based on the set criteria in this study, their roles in disease initiation and progression require further analysis.
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Henryka, Gawrzak Sylwia. "Identification and functional analysis of molecular mechanisms involved in the latency of ER positive breast cancer." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/402624.
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Breast cancer is the most frequently diagnosed cancer and remains the second leading cause of death among women in Europe and United States. In this malignancy, metastasis remains to be an incurable condition, and therefore the major cause of death. Metastatic lesions can appear within a wide time ranging from months to years or decades after primary tumor resection. In particular, in the estrogen receptor (ER) positive breast cancer subgroup metastatic latency continues to be a major challenge for the researchers, clinicians and patients.
This thesis reports the identification and functional analysis of molecular mechanisms involved in the latency of ER positive breast cancer. For that purpose we based our research on a comprehensive approach that relies on genetically engineered human breast cancer cells, experimental mouse models, unbiased whole- genome screen and clinical data. The first part of the thesis describes a novel mouse model of breast cancer dormancy. We showed that metastatic cells home the bone and enter the latency phase as micrometastatic lesions where tumor growth is restricted mainly due to the equilibrated ratios of cell proliferation and cell death. This experimental mouse model was used to identify genes relevant for long- latent relapse. To this end, we performed in vivo loss-of- function shRNA screening. In the screening we challenged a whole-genome library of shRNA to uncover genes whose depletion negatively regulates dormancy. Among the candidate genes revealed by the screen we focused on MSK1 as a long-latent metastasis regulator. The in vivo and in vitro validation results indicate that MSK1 plays a role in homing and differentiation of metastatic cells. We showed that MSK1 promotes the expression luminal transcription factors - FOXA1 and GATA-3. Therefore, MSK1 depletion is beneficial for metastatic cells leading to a partial phenotype shift towards a more aggressive and poorly differentiated basal population. Furthermore, our data suggest that MSK1 may be involved in metastatic cell plasticity by remodeling the chromatin. Importantly, low MSK1 gene expression levels associate with early metastasis in ER positive breast cancer.El cáncer de mama es el tipo de cáncer más frecuentemente diagnosticado, siendo la segunda causa de muerte entre las mujeres de Europa y Estados Unidos. En esta enfermedad, la metástasis sigue siendo incurable, y por ello es la principal causa de muerte. Las lesiones metastásicas pueden aparecer dentro de un amplio periodo de tiempo que va desde meses hasta años o incluso décadas después de la extirpación del tumor primario. Concretamente, en el subgrupo de cáncer de mama RE positivo, este largo periodo de latencia es el principal desafío para investigadores, médicos y pacientes. En esta tesis se muestra la identificación y el análisis funcional de mecanismos moleculares implicados en la latencia del cáncer de mama RE positivo. Para este propósito, nuestros estudios se han llevado a cabo mediante una estrategia experimental basada en líneas celulares de cáncer de mama genéticamente modificadas, modelos experimentales de ratón, análisis global del genoma y datos clínicos. La primera parte de la tesis describe un novedoso modelo de ratón de dormancia de cáncer de mama. Observamos que, en nuestro modelo, las células metastásicas llegan al hueso y entran en una fase de latencia en forma de lesiones micrometastásicas en la que el crecimiento del tumor se ve impedido, principalmente debido a que la tasa de proliferación celular se iguala a la tasa de muerte celular. Este modelo experimental de ratón se usó para identificar genes relevantes en el proceso de latencia y por tanto en la recurrencia a largo plazo. Para ello, llevamos a cabo un análisis in vivo de pérdida de función con shRNA. En este análisis utilizamos una amplia librería de shRNA para descubrir genes cuya eliminación regula la dormancia de manera negativa. Entre los genes candidatos identificados en este análisis nos focalizamos en MSK1 como un regulador de la metástasis latente. La validación in vitro e in vivo indica que MSK1 juega un papel en el anidamiento y la diferenciación de las células metastásicas.
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Guo, Xiaoxiao. "Identification, molecular and functional analysis of bioactive peptides from the venom of the scorpion, Tityus serrulatus." Thesis, Queen's University Belfast, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.601648.
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Scorpion venoms are rich sources of bioactive polypeptides. They are classified into two major groups: peptides with or without disulfide bridges. Toxins with disulfide bridges are responsible for toxicity of venom by targeting ion channels on cell membranes, while peptides without disulfide bridges are diverse in both structures and bioactivities, rendering them interesting to study. This project aimed to discover novel bioactive peptides from the venom of a South American scorpion, Tityus serrulatus. There are four highlights in this study. Firstly, two putative antimicrobial peptides (TsAP-l and TsAP-2) were identified in scorpion venom, by shotgun molecular cloning and RP-HPLC fractionation of venom. Their synthetic replicates were subjected to a series of bioactivity assays. TsAP- l exhibited low potency against all three test organisms, whereas TsAP-2 exhibited high potency against Gram-positive bacterium, Staphylococcus aureus, and the yeast, Candida albicans. Meanwhile, haemolytic and anti-proliferative activities of TsAP-l were low, while those of TsAP-2 were considerably higher. Secondly, two analogous peptides (TsAP-Sl and TsAP-S2), in which positive net charges and a-helicity increased, were designed by site substitutions in natural antimicrobial peptides. They both exhibited dramatic enhancement in antimicrobial, haemolytic and anti-proliferative activities, indicating the significance of cationicity and a-helical structure in bioactivities of antimicrobial peptides. Thirdly, two putative bradykinin-potentiating peptides (TsHpt-I and QUB 1397) were also identified in venom, and synthesised. In vitro pharmacological assays revealed that they could enhance bradykinin-induced hypotension on rat vascular tissues at micromole levels, although neither of them produced any significant effects on the same tissue. Finally, six cDNAs encoding putative classical disulphide-bridged neurotoxin precursors were also cloned from the same venom-derived cDNA library and were structurally-analysed. These extend knowledge of scorpion venom neurotoxins. All these findings suggest the value of scorpion venom in searching and designing potential drug candidates.
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Jones, Brande. "Identification, isolation, expression analysis and molecular characterization of nine genes key to late embryogenesis in Loblolly pine." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/43598.
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A basic understanding of the molecular events occurring during zygotic embryogenesis is required to fully understand how and why only a very small percentage of somatic embryos develop past the late embryogeny phase of embryogenesis. In this work, we have identified genes that have been demonstrated to be required for late embryonic development in the model plant system Arabidopsis thaliana.
These genes were subsequently isolated and cloned from Loblolly pine embryos. These isolated clones were sequenced and analyzed to reveal significant homology to the known Arabidopsis ABA responsive genes ABI3, ABI4, and ABI5. Expression analyses of all three genes were completed, and compared to reported data of ABA accumulation, as well as, expression of other ABA responsive genes during the same stages of embryogenesis.
Six putative root development genes were isolated and cloned from Loblolly pine embryos. These isolated clones were sequenced and analyzed to reveal significant homology to the known Arabidopsis root development genes WOODENLEG, SHORT ROOT, SCARECROW, HOBBIT, BODENLOS, and MONOPTEROS. Full-length cDNAs were isolated and cloned for WOODENLEG, SHORT ROOT, SCARECROW and BODENLOS. Expression analyses of all six genes were completed throughout mid to late embryogenesis in Loblolly pine.
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Markljung, Ellen. "QTL Analysis in the Pig : From the Identification of Quantitative Trait Loci to the Understanding of Molecular Mechanisms." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9556.
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Al_griw, Huda Hm. "Molecular detection of bloodstream pathogens in critical illness." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/molecular-detection-of-bloodstream-pathogens-in-critical-illness(5f143a31-3694-454c-8940-5ae434f1eb31).html.
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Background: Critically ill patients are at particular risk of developing bloodstream infection. Such infections are associated with the development of sepsis, leading to a marked increase in mortality rate. Early detection of the causative organism and appropriate antibiotic treatment are therefore critical for optimum outcome of patients with nosocomial infection. Current infection diagnosis is based on standard blood culture techniques. However, microbiological culture has a number of limitations, not least that it takes several days to confirm infection and is therefore not useful in directing the early treatment with antibiotics. New techniques based on the detection of pathogen DNA using real-time polymerase chain reaction (PCR) technology have the potential to address these limitations but their clinical utility is still to be proved. Objectives: Develop and evaluate novel PCR-based approaches to bloodstream infection diagnosis in critical illness based on detection and identification of bacterial and fungal DNA in blood. Methods: A range of commercial and 'in-house' PCR-based assays for detection of bacterial and fungal DNA were developed and/or optimised for use in clinical blood samples. These included LightCycler SeptiFast, a CE-marked multi-pathogen assay for common bloodstream pathogens, BactScreen and GramScreen, broad spectrum bacterial assays based on 16S rRNA gene and real-time PCR assays developed to detect a range of clinically important fungal pathogens. Novel approaches to speciation of pathogen DNA using melting temperature (Tm) profiling and high resolution melting analysis (HRMA) were developed. Clinical evaluation of assays was either on blinded clinical isolates or blood samples from critically ill patients with clinical suspicion of bloodstream infection against conventional microbiological culture. Several techniques aimed at improving extraction of pathogen DNA from blood were also investigated. Results: The CE-marked commercial assay SeptiFast showed analytical sensitivity and specificity of 79% and 83% respectively. Concordance with positive culture results was good but high levels of 'false positives' were detected possibly attributed to detection of free pathogen DNA not associated with viable pathogens. The predictive value of a negative SeptiFast test was 98% suggesting that absence of pathogen DNA is a strong indicator of absence of infection. Further studies were aimed at detailed optimisation and validation of 16S rRNA gene real-time PCR assays for bacterial DNA. BactScreen and GramScreen were able to detect a broad range of clinically important bacteria down to <50 CFU/ml blood. A preliminary comparative evaluation against SeptiFast showed BactScreen gave excellent concordance with blood culture results with minimal false positive results compared to SeptiFast. Efficient extraction of pathogen DNA was shown to be a key factor in determining analytical sensitivity and several protocols were evaluated. Low cost approaches to speciation of bacterial DNA were developed by combining broad range real-time PCR with HRMA. A novel HRMA method based on Tm profiling was shown to identify 89% and 96% of blinded clinical isolates at species or genus level respectively. Real-time PCR/HRMA approaches were also successfully developed for detection and identification of fungal pathogens including a range of Candida and Aspergillus species associated with bloodstream fungal infection. Conclusions: These studies have highlighted some of the key factors that need to be considered when developing and validating PCR based assays for pathogen DNA detection in blood. A set of novel tools have been developed for rapid detection and identification of bacterial and fungal pathogens that could address the challenges of infection diagnosis based on pathogen DNA detection. Further work is required, not least in development of more efficient pathogen DNA extraction and detailed clinical validation but the tools described here have the potential to provide cost effective solutions to aid infection diagnosis that would be complementary to current culture-based methods. The provision of time critical information could have a positive impact on clinical decision-making leading to more effective management and treatment of patients with suspected bloodstream infection.
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Blasingame, Eric M. "Molecular identification of silk proteins in the gumfooted lines and attachment discs of the black widow spider, latrodectus hesperus." Scholarly Commons, 2009. https://scholarlycommons.pacific.edu/uop_etds/731.
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Silks from araneoid spiders have become an active area of research for material scientists, biochemists, and molecular biologists. Mechanical properties of spider silk such as elasticity, tensile strength, and toughness make the manufacturing of silk for medical sutures, body armor, ropes and other synthetic material applications great possibilities. The difficulties of having a black widow spider farm to harvest silk, due to their cannibalistic nature, make recombinant expression of silk proteins a fundamental goal of spider silk research. In order to express silk fibers, cDNAs encoding the corresponding silk fiber products must first be isolated and identified. One of the first steps in gene identification relies on the identification of the proteins in the silk fibers.
No previous study has demonstrated the molecular constituents of gumfooted lines. In the course of this research, the core fibroins in the gumfooted lines were identified to be members of the Major Ampullate Spidroin family (MaSp), using mass spectrometry. This research was the first to identify the core fibroins of the gumfooted lines.
Novel peptide fragments from solubilized gumfooted lines were acquired from manual de novo MSIMS sequencing after in-gel tryptic digestion. These peptide fragments showed post-translational modifications consistent with glycosylation, which aligns with the reported chemical properties of glue proteins.
Novel peptide sequences were also acquired from the attachment discs as well as novel scanning electron microscopy images and reveal, for the first time, the physical attributes and molecular properties of threads attached to the surface of an immobilized structure. This study was the first to identify the molecular constituents of the attachment discs.
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De, Brabandere Heidi. "Organic Phosphorus Compounds in Aquatic Sediments : Towards Molecular Identification with Mass Spectrometry." Doctoral thesis, Uppsala universitet, Institutionen för fysikalisk och analytisk kemi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9319.
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Phosphorus (P) regulates trophic status in most aquatic systems. However, only bioavailable P contributes to primary production. In most lakes and shallow seas, mineralisation of sediment P into its bioavailable form and its release to the water column is important for maintaining primary production. Sediment organic P forms a substantial proportion of this P to be mineralised and can originate from different sources on land (farmland, forests, etc.) or from primary production in the lake. These organic P forms can thus be expected to have differing composition, degradability and recyclable P content. Knowledge of the chemical structure of sediment organic P compounds is scarce, mainly due to lack of appropriate analytical techniques. The commonly used 31P-nuclear magnetic resonance (31P-NMR) technique, only identifies P binding groups, so a mass spectrometric (MS) analysis method was developed that allows individual sediment organic P compounds to be identified. EDTA as pre-extractant resulted in the highest P yield in subsequent NaOH extraction. Extracted organic P compound groups were identified using 31P-NMR. For identification of specific P compounds with MS, a sample preparation method prior to electrospray tandem mass spectrometry (ESI-MS/MS) analysis was developed. Liquid chromatography (LC) with porous graphitic carbon prior to ESI-MS/MS enhanced sensitivity and selectivity, enabling several of the ions detected to be identified as nucleotides. 31P-NMR analysis showed P monoesters to be the most stabile P compounds throughout a lake sediment profile. The developed LC-ESI-MS/MS analysis method revealed that some monoester-P (nucleotides) were labile, while other P compounds increased in concentration with Baltic Sea sediment depth and were therefore considered stabile. Differences in patterns of P compounds detected were also shown depending on catchment characteristics in relation to Baltic Sea sediment age. For cost-effective management of eutrophication, knowledge of the sources of degradable organic P forms, contributing to internal loading, is needed. This thesis showed the developed LC-ESI-MS/MS analysis method to be a powerful analytical tool for this purpose.
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Beligala, Dilshan Harshajith. "Identification of Rhizobial Symbionts Associated with Lupinus SPP." Bowling Green State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1435855654.
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Fooyontphanich, Kim. "Identification and analysis of the molecular components involved in the oil palm (Elaeis guineensis) fruit abscission processhuile (Elaeis guineensis)." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTS197/document.
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L’abscission des organes chez les végétaux est hautement régulée au cours du développement. Ce processus physiologique qui consiste à diminuer l’adhésion entre deux cellules adjacentes dépend de l’environnement, du stress, de l’attaque de pathogènes ou encore de l’état physiologique de la plante. L’abscission du fruit et de la graine jouent un rôle déterminant dans le cycle de vie de la plante et en particulier, un rôle central dans la dispersion des graines. C’est également un caractère commun de domestication avec des conséquences économiques pour la plus part des espèces cultivées. Le palmier à huile (Elaeis guineensis) est largement cultivé dans toutes les zones tropicales et l’huile de palme représente aujourd’hui plus d’un tiers des huiles végétales produites dans le monde. La maturation des fruits au sein des régimes est asynchrone. Ainsi, les fruits les plus murs tombent avant le murissement complet du régime, entrainant une baisse du rendement d’une part et rendant leur collecte manuelle fastidieuse et couteuse d’autre part. Dans ce context, le contrôle ou la réduction de la chute des fruits permettrait une meilleure gestion de la récolte à des couts réduits. Dans le cadre de cette étude, un protocole de phénotypage du processus d’abscission du fruit du palmier à huile a été développé et utilisé pour identifier des génotypes à faible ou retard d’abscision des fruits arrivés à maturité. En parallèle, des analyses comparatives de transcriptomes et de protéomes issus de la zone d’abscission (ZA) du fruit ont été conduites tout au long du processus de séparation cellulaire, déclenché au laboratoire par un traitement à l’éthylène ou bien de manière naturelle au champ. Au total 1957 gènes présentent une expression différentielle significative dans la ZA du fruit au cours du processus d’abscission induit par l’éthylène. Parmi ces gènes, 64 sont spécifiquement (ou majoritairement) exprimés dans la ZA des fruits arrivés à maturité par comparaison avec les tissus où le processus de séparation cellulaire n’est pas observé (pédicelle et mésocarpe des fruits murs ; ZA non fonctionnelle des fruits immatures). Le profil d’expression de ces 64 gènes candidats a été également analysé dans la ZA des fruits mûrs prélevés au champ, afin de conforter leur rôle potentiel au cours de l’abscission déclenchée naturellement. Ainsi, en utilisant les nouvelles technologies de séquençage du transcriptome et du protéome, couplées à une analyse biochimique et cellulaire des modifications de la paroi dans la ZA, ce travail a permis de mettre en évidence la conservation de certains processus moléculaires associés à l’abscission des organes chez les monocotylédones par comparaison avec les espèces modèles dicotylédones, telles que la tomate et Arabidopsis. Par exemple, l’identification de gènes codant des polygalacturonases très proches de celles qui sont impliquées dans l’abscission de la fleur chez Arabidopsis suggère la conservation de leur fonction dans l’hydrolyse de la pectine des cellules des ZA, malgré la divergence phylogénétique entre les espèces. Enfin, ce travail a permis également d’identifier de nouveaux régulateurs associés au processus de séparation cellulaire et fournir une liste de gènes associés à des processus biologiques étroitement liés à la fonction de la ZA chez le fruit du palmier à huilePlant organ abscission is a complex developmental process that involves cell separation regulated by the environment, stress, pathogens and the physiological status of the plant. In particular, seed and fruit abscission play a central role in seed dispersion and plant reproductive success, and are common domestication traits with important agronomic consequences for many crop species. Oil palm (Elaeis guineensis) is cultivated throughout the tropical regions as one of the most economically important oil crop species in the world. The unsynchronized ripening of the oil palm fruit bunch leads to the abscission of the ripest fruit and consequently high labor cost for harvest and loss of yield. In this context, the control of oil palm ripe fruit abscission is an important agricultural concern for the cultivation of oil palm in a sustainable and cost effective way. In the present study, a protocol to phenotype the oil palm fruit abscission process was developed and used to identify a tree in the field that does not undergo ripe fruit abscission. In parallel, transcriptome and proteome analyses of the oil palm ripe fruit abscission zone (AZ) during abscission induced experimentally by ethylene compared to the AZ undergoing natural abscission in the field was performed. A total of 1,957 candidate genes were identified statistically as differentially expressed in the ripe fruit AZ during ethylene-induced abscission. Furthermore, a total of 64 of these differentially abundant candidates were statistically specific or enriched at least during one time point of the ethylene induced abscission, compared to their profiles in the AZ of immature fruit and the pedicel of ripe fruit, where cell separation is not observed. The profiles of these gene candidates were examined in the ripe fruit AZ undergoing natural abscission in the field to validate their potential role during abscission. Finally, the profiles of selected candidate genes were then examined in the AZ of the tree observed not to undergo fruit abscission in the field. The combined approaches provide evidence of wide scale conservation of the molecular components involved in organ abscission of this monocot compared with the model dicot plants tomato and Arabidopsis. For example, the identification of polygalacturonases very similar to those that function during Arabidopsis floral organ abscission suggests a conservation of the components for pectin disassembly despite the phylogenetic distance between these species. In addition, the data from the global analysis and complementary molecular, cellular and biochemical approaches suggest novel components and provide a robust list of genes and processes important for AZ function during ripe fruit abscission of this important monocot crop species
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Grawenda, Anna Maria. "The identification and analysis of molecular biomarkers in the p53 tumour suppressor pathway that affect cancer progression in humans." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:5a76b7ca-22f6-4f49-b715-5ad43f916984.
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The tumour suppressor p53 is at the centre of the signalling pathway that controls cellular processes crucial in tumourogenesis, cancer progression and tumour clearance. Alterations in the p53 pathway that lead to cancer progression can be good candidates for molecular biomarkers that would assist in the identification of patients with different prognoses, but also serve as good predictors of appropriate targeted therapies. Patient cohorts and cancer cell panels are utilised to seek associations with the attenuation of the p53 pathway and cancer progression. Firstly, the alternatively spliced transcript of the p53 inhibitor HDMX, which is frequently found in tumours with poor prognosis, is studied. The high ratio of the alternatively spliced HDMX-S transcript over the full-length HDMX-FL transcript (HDMX-S/FL) is demonstrated to associate with p53 pathway attenuation in cancer cells and breast carcinomas, and with faster metastatic progression of osteosarcoma and breast cancer patients. Secondly, inherited polymorphism in the HDMX gene is investigated and demonstrated as a unique and highly reproducible eQTL, which identifies patients with different prognoses for metastatic disease in breast cancer and melanoma cohorts. Lastly, a screening approach to identify novel inherited polymorphisms in the p53 pathway genes that associate with metastatic progression of melanoma is developed and implemented, and subsequently in silico and in vitro functional analyses are performed to investigate a mechanism behind the FOXO3 SNP, identified as the strongest candidate, whereby the experimental evidence demonstrate that the causal SNP in the FOXO3 haplotype is controlled by the GATA3 transcription factor. Together, the work presented in this thesis provides strong support for the role of the p53 pathway in the metastatic progression of cancer, and suggests that molecular biomarkers that can detect changes in the activity of p53 pathway genes could offer a robust set of biomarkers for cancer progression applicable to different types of cancer.
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Zhang, Jibin. "Identification of Important Cell Cycle Regulators and Novel Genes in Specific Tissues using Microarray Analysis, Bioinformatics and Molecular Tools." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1429637289.
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Chung, Peter. "Identification and molecular analysis of a modular gene in Cryptosporidium parvum encoding two putative proteins involved in amylopectin synthesis /." Search for this dissertation online, 2004. http://wwwlib.umi.com/cr/ksu/main.
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Bacich, Dean J. "Identification, characterisation, and molecular analysis of the alternatively spliced forms of the Luteinizing hormone receptor on the ovine ovary." Title page, contents and summary only, 1996. http://web4.library.adelaide.edu.au/theses/09PH/09phb125.pdf.
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Errata posted onto front end paper. Bibliography: leaves 264-330. Provides evidence that the alternatively spliced LHR transcripts are translated in vivo and consequently suggests the alternatively spliced products may play an important role in ovarian function.
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Raghavan, Bindu. "Analysis of the Human Cytomegalovirus Transcriptome and Identification and Characterization of a HCMV gene involved in disruption of Interferon Signaling." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1218473086.
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PADOAN, ANDREA. "Statistical methods for mass spectrometry data analysis and identification of prostaste cancer biomarkers." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2014. http://hdl.handle.net/10281/50248.
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BACKGROUND
Prostate Cancer (PCa) is the most common cancer among males in Europe. Patients developing early PCa sometimes refer non-specific symptoms, namely lower urinary tract symptoms (LUTS), and they usually undergo medical investigations based on Prostate Specific Antigen (PSA) and Digital Rectal Examination (DRE). Suspicious results of one or both testings are prerequisite to Prostate Biopsy. However, due to PSA low sensitivity/specificity in predicting positive prostate biopsy, the identification of new PCa biomarkers is actually a real need. MALDI-TOF/MS protein profiling could be a valuable technology for biomarkers identification. However, up to now its use is laden with lack of reproducibility that confounds scientific inferences and limits its broader use.
AIMS
Goal of this study is to analyze urine collected after prostatic massage in patients referring LUTS, to identify candidate biomarker for PCa, by using MALDI-TOF/MS. We considered important aspects of MALDI-TOF/MS label-free proteomic profiling, in order to assess features reproducibility and to propose appropriate strategy to handle both measurement error and limit of detection (LOD) problems. The study results should aid in reducing the number of worthless first-biopsied and assist Urologists on differential diagnosis of PCa.
METHODS
In a cross-sectional study, we collected urine obtained after DRE from 205 patients that referred LUTS to consultants at the Urological Unit at University of Padova. All patients undergone to prostate biopsy for suspicious PCa. Urines were dialyzed and analyzed by MALDI-TOF/MS in reflectron mode. For the MALDI-TOF/MS reproducibility evaluation, we analyzed a urine pooled from 10 reference samples, spiked with 12.58 pmol of a 1589.9 m/z internal standard (IS) peptide. For the inter-run variability assessment, 14 aliquots were dialyzed by MALDI-TOF/MS. For the intra-run study, an aliquot was divided into 26 separate sub-aliquots and analyzed by MALDI- TOF/MS. To estimate the signal detection limit (sLOD), serial dilution up to 1/256 of a urine pool were analyzed in triplicate. We evaluated the sLOD and adjusted the data appropriately to reduce its variability. We investigated six data normalization approaches - the mean, median, internal standard, relative intensity, total ion current and linear rescaling normalization. Between-spectrum and the overall spectra variability were evaluated by the coefficient of variation (CV). An optimized signal detection strategy was also evaluated to overcome peak detection algorithms errors. Measurement errors and with-in subject variances were evaluated by an external dataset, made of urine repeatedly collected from 20 reference subjects. Intra class correlation coefficient (ICC), Regression Calibration (RCAL) and SIMEX analyses were used to estimate unbiased logistic regression coefficients relating MALDI-TOF/MS features with Patients biopsy outcome. Monte Carlo simulations were used to estimate influence of different LOD adjustment methods on ICC and RCAL.
RESULTS
Initially, we evaluated the intra- and inter-run on data obtained from automatic peak detection. Normalization methods performed almost similarly in both studies, except IS, which resulted in an increased CV. Calculated sLOD varied with spectra m/z. After sLOD adjustment, raw and normalized data showed a reduction in CVs, while median and mean normalizations performed better, especially in the intra-assay study. However, by optimizing the peak signal detection, the overall features variability drastically decreased. Median normalization with sLOD correction remained the preferable choice for further analyses. Evaluating the external dataset, we found that most of the MALDI-TOF/MS variability is intrinsic to the biological matrix. By using substitution of below LOD values by LOD/2, simulation studies showed that ICC estimations were poorly affected by LOD, when measurement error σ is less that 0.36 and values below LOD are less that 50%. Comparing results from naïve logistic regression, RCAL and SIMEX, measurement error appeared to cause a "bias toward the null". However, SIMEX estimations seemed to correct for a smaller amount of bias than RCAL. Overall, we found eight MALDI-TOF/MS features associated with positive biopsy results.
CONCLUSION
Findings from the reproducibility study showed that the major contributing factor for MALDI-TOF/MS profiling variability is the peak detection process. So, a new algorithm suited for MALDI-TOF reflectron mode is desirable for its applications in profiling studies. However, normalization strategies aid in increasing MALDI-TOF/MS label-free data reproducibility, especially with sLOD correction. Despite urine does not seem to be a promising biological fluid for proteomic biomarker discovers, RCAL and SIMEX appeared valuable approaches to obtain regression coefficients adjusted for biological and instrumental errors on MALDI-TOF/MS features.
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Dieterich, Mabin Molly E. "Effects of conservation biological control practices on predatory arthropod assemblages and molecular identification of cucumber beetle biological control agents." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1492531428052099.
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Goel, Gautam. "Dynamic flux estimation a novel framework for metabolic pathway analysis /." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/31769.
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Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2010.Committee Chair: Voit, Eberhard O.; Committee Member: Butera, Robert; Committee Member: Chen, Rachel; Committee Member: Kemp, Melissa; Committee Member: Neves, Ana Rute. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Zhou, Xiao. "Identification of Plant SUN Domain-Interacting Tail Proteins and Analysis of Their Function in Nuclear Positioning." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1385480937.
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Taylor, Richard M. I. "Identification of M-CSF independent mechanisms of human osteoclast formation and analysis of the cellular and molecular mechanisms involved in tumour osteolysis." Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.669949.
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Richter, Suzanne C. "New approaches to molecular diagnostics, identification of differentially expressed genes in breast cancer cell lines using cDNA aray hybridization and sequence analysis." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape11/PQDD_0009/MQ40773.pdf.
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Raj, Sharad K. "Chemical Information Based Elastic Network Model: A Novel Way To Identification Of Vibration Frequencies In Proteins." Amherst, Mass. : University of Massachusetts Amherst, 2009. http://scholarworks.umass.edu/theses/261/.
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Penkler, David Lawrence. "In silico analysis of human Hsp90 for the identification of novel anti-cancer drug target sites and natural compound inhibitors." Thesis, Rhodes University, 2015. http://hdl.handle.net/10962/d1018938.
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The 90-KDa heat shock protein (Hsp90) is part of the molecular chaperone family, and as such it is involved in the regulation of protein homeostasis within cells. Specifically, Hsp90 aids in the folding of nascent proteins and re-folding of denatured proteins. It also plays an important role in the prevention of protein aggregation. Hsp90’s functionality is attributed to its several staged, multi-conformational ATPase cycle, in which associated client proteins are bound and released. Hsp90 is known to be associated with a wide array of client proteins, some of which are thought to be involved in multiple oncogenic processes. Indeed Hsp90 is known to be directly involved in perpetuating the stability and function of multiple mutated, chimeric and over-expressed signalling proteins that are known to promote the growth and survival of cancer cells. Hsp90 inhibitors are thus thought to be promising therapeutic agents for cancer treatment. A lack of a 3D structure of human Hsp90 however has restricted Hsp90 inhibitor development in large to in vivo investigations. This study, aims to investigate and calculate hypothetical homology models of the full human Hsp90 protein, and to probe these structural models for novel drug target sites using several in silico techniques. A multi-template homology modelling methodology was developed and in conjunction with protein-protein docking techniques, two functionally important human Hsp90 structural models were calculated; the nucleotide free “v-like” open and nucleotide bound closed conformations. Based on the conservation of ligand binding, virtual screening experiments conducted on both models using 316 natural compounds indigenous to South Africa, revealed three novel putative target sites. Two binding pockets in close association with important Hsp90-Hop interaction residues and a single binding pocket on the dimerization interface in the C-terminal domain. Targeted molecular docking experiments at these sites revealed two compounds (721395-11-5 and 264624-39-7) as putative inhibitors, both showing strong binding affinities for at least one of the three investigated target sites. Furthermore both compounds were found to only violate one Lipinski’s rules, suggesting their potential as candidates for further drug development. The combined work described here provides a putative platform for the development of next generation inhibitors of human Hsp90.
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Josey, Michelle. "Molecular-Genetic Methods for Predicting Bio-Geographical Ancestry From Bone Specimens to Aid in Forensic Identification." Honors in the Major Thesis, University of Central Florida, 2007. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/1036.
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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.Bachelors
Sciences
Forensic Science
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Paine, Melissa A. "A Molecular Technique for Specific Identification of Western Atlantic Ocean Scombrids and an Analysis of a Larval Scombrid Assemblage off the Kona Coast of Hawaii." W&M ScholarWorks, 2006. https://scholarworks.wm.edu/etd/1539617849.
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Nunez-Valdez, Maria Eugenia. "Identification and analysis of the virulence factors in Serratia entomophila causing amber disease to the grass grub Costelytra zealandica : A molecular genetics approach." Thesis, University of Canterbury. Molecular Genetics, 1994. http://hdl.handle.net/10092/6807.
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Amber disease caused by Serratia entomophila to larvae of Costelytra zealandica (Coleoptera: Scarabaeidae), is characterized by the production of two symptoms: anti-feeding effect (AFE) and amber coloration (AC). This study was aimed to identify and characterize the virulence factors involved in the disease. Three factors were identified: i) MRE-HA fimbriae; ii) an extracellular protease and, iii) an anti-feeding toxin.
i) Fimbriae type 1, 3 and MRE-HA were identified and characterized in S. entomophila by haemagglutination tests and electron microscopy. Analysis of nonpathogenic mutants suggested that the MRE-HA fimbriae were associated with pathogenicity.
ii) The locus coding for the extracellular protease of S. entomophila was identified and cloned. Examination and complementation assays of pathogenic and nonpathogenic strains showed that the protease is not directly involved, but it might potentiate the disease. It was suggested that the protease might be linked with pathogenicity by a common regulator factor.
iii) A locus named amb2 was identified, isolated and cloned. Genetic evidence and complementation assays with nonpathogenic mutants demonstrated that amb2 is responsible for the AFE. SDS-PAGE analysis of the amb2 gene products expressed in minicells showed the synthesis of two proteins of 21 and 25 kDa, named AnfA and AnfB. The genes encoding these proteins were mapped by deletion analysis and lacZ-gene fusions. DNA sequencing of the anfA gene revealed that another protein of ~12 kDa (AnfA2) was also encoded by amb2. Consensus sequences with homology to the binding sites of the bacterial regulators CAP, Fur and ToxR were identified in the promoter regions. Homology of 50% was found between a hydrophobic motif of the δ-endotoxin of Bacillus thuringiensis and AnfA1, The results suggest that AnfA1 , AnfA2 and AnfB might be subunits of a toxin causing the AFE.
It was concluded that virulence determinants in S. entomophila including the MRE-HA fimbriae, the extracellular protease and the anti-feeding toxin act in collaboration to produce amber disease.
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Tuppen, Helen. "Gene expression analysis in prostate cancer : characterisation of the molecular basis of progression to androgen-independence and identification of novel androgen-responsive genes." Thesis, University of Newcastle Upon Tyne, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.427319.
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GRATTON, ROSSELLA. "Hidradenitis suppurativa: identification of the main cellular and molecular pathways involved in immune and cutaneous cell biology." Doctoral thesis, Università degli Studi di Trieste, 2021. http://hdl.handle.net/11368/2997561.
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L’idrosadenite suppurativa (HS) è una patologia infiammatoria cutanea altamente debilitante caratterizzata dalla presenza di lesioni profonde, ascessi, noduli infiammati, lesioni purulente e cicatrici nelle regioni inverse del corpo ricche di ghiandole apocrine.
La patogenesi dell’HS è complessa e multifattoriale che prevede una stretta interazione tra fattori genetici, immunologici, infettivi e ormonali, attraverso meccanismi molecolari non ancora completamente definiti.
Un ulteriore livello di complessità si ritrova nel fatto che spesso i casi HS manifestano simultaneamente varie comorbidità che coinvolgono più sistemi d’organi, dando origine a delle varianti sindromiche in cui l’HS costituisce un segno distintivo. In questo lavoro l’attenzione è stata focalizzata su una variante sindromica nota come sindrome PASH.
Lo scopo di questo lavoro è stato quello di identificare varianti genetiche in pazienti affetti da HS e PASH, cercando di definire il loro ruolo nell’insorgenza, progressione e severità di queste patologie. A tal fine, nel presente studio sono state reclutate e sottoposte ad analisi genetica una famiglia con un paziente PASH e due famiglie che presentavano una forma famigliare di HS. Queste indagini hanno consentito l’identificazione di varianti geniche specifiche in ciascuna famiglia, che sono state ulteriormente caratterizzate in seguito all’allestimento di modelli in vitro al fine di comprendere il loro effetto sul fenotipo PASH e HS.
In un paziente PASH è stata identificata una variante missenso in omozigosi nel gene DSP che codifica per la desmoplachina (DSP), una componente obbligatoria e la più abbondante dei desmosomi, mentre la stessa è presente in eterozigosi nei sani. Sono state eseguite delle procedure sperimentali su biopsie di pelle, in silico e in un modello genetico in vitro ottenuto in una linea cellulare di cheratinociti spontaneamente immortalizzati (HaCaT) ingegnerizzate con la tecnologia di CRISPR-Cas9 per farle esprimere la variante d’interesse. Complessivamente i risultati hanno confermato un potenziale effetto dannoso della variante nella pelle in quanto è stato osservato che essa impatta la struttura della proteina, presumibilmente contribuisce ad un reclutamento insufficiente di DSP sui desmosomi, determina livelli elevati di perdita di acqua attraverso lo strato corneo della pelle e non influenza la proliferazione e il differenziamento dei cheratinociti. È stato proposto che questo effetto dannoso alteri le proprietà meccaniche di adesione dei desmosomi e ad una perdita dell’integrità strutturale del tessuto che sembra influenzare le caratteristiche di permeabilità del distretto cutaneo.
Nel caso della famiglia sarda, è stata identificata una rara variante in eterozigosi presente negli individui affetti sul gene ZNF318, che codifica per una proteina zinc-finger associata alla pathway di segnalazione degli androgeni, che non è stata associata a nessuna patologia. È stato sviluppato un modello genetico in vitro sulle HaCaT mediante la tecnologia di CRISPR-Cas9 allo scopo di generare cloni che esprimessero la variante d’interesse. Saggi in vitro che mirano a rilevare cambiamenti nella trasduzione del segnale del recettore degli androgeni sono attualmente in fase di esecuzione.
Nella famiglia friulana è stata trovata una frameshift insertion nel gene DCD, che codifica per il peptide antimicrobico dermocidina, in eterozigosi negli individui affetti della famiglia ma assente nei sani. Verranno effettuati ulteriori studi volti a valutare l’attività antimicrobica del peptide mutato unitamente alla quantificazione dei suoi livelli nel sudore di tutti gli individui della famiglia.
In questo studio, sono state ottenute nuove informazioni relative alla base genetica di HS e PASH che hanno permesso di mettere in luce nuovi scenari potenzialmente coinvolti nell’insorgenza e nella progressione di tali patologie.HS is a highly debilitating inflammatory skin disease presenting deep-seated lesions, abscesses, inflamed nodules, pus-discharging tunnels and scars, typically occurring in intertiginous apocrine gland-bearing regions. The pathogenesis of HS is complex and multifactorial in which a strict interplay between genetic, immunologic, infectious and hormonal factors have been identified, though the precise molecular mechanisms underlying these aspects have not yet been fully characterised. A further level of complexity is given by the fact that commonly HS may also occur in combination with other comorbidities involving multiple organ systems giving rise to syndromic variants. In this work the attention was focused on a syndromic variant of HS, PASH syndrome. The aim of this work was to identify genetic changes in HS and PASH patients, trying to understand their role in these diseases. To this aim, in the present study one family with one PASH patient and two families presenting a familiar form of HS were recruited and subjected to genetic analysis. These assays allowed the identification of specific variants in each family that were further characterised in in vitro studies in order to define their effect on PASH and HS phenotype. In a PASH patient a missense variant in DSP gene, encoding for desmoplakin (DSP) that is an obligate and the most abundant component of desmosomes, was identified in homozygosis while the variant was carried in heterozygosis by his healthy parents. Experimental procedures were performed on skin sample biopsies, in silico and in an in vitro genetic model obtained in spontaneously immortalised human keratinocyte cells line (HaCaT cells) engineered with CRISPR-Cas9 technology to carry the mutation of interest. Results confirmed a potential damaging effect of the identified single nucleotide variation (SNV) in the skin since it has been seen to: impact protein structure; potentially contribute to an insufficient recruitment of DSP on desmosomes; determine higher levels of water loss through the stratum corneum of the epidermis; do not influence keratinocytes proliferation and differentiation. This damaging effect has been proposed to alter the mechanical adhesion properties of desmosomes and lead to the disruption of tissue integrity by affecting permeability properties. In the case of the Sardinian family, a rare mutation that has never been previously associated to any disease was found in affected family members in heterozygosis on ZNF318 gene, encoding for a zinc finger protein associated with androgen receptor signalling. An in vitro genetic model was assessed in HaCaT cells through CRISPR-Cas9 technology in order to retrieve cellular clones carrying the mutation of interest. In vitro assays are currently ongoing and are aimed at characterising any variation in androgen receptor signal transduction pathway.In a Friulian family, a frameshift insertion in the DCD gene, encoding for the antimicrobial peptide dermcidin, was found in heterozygosis in affected family members but not in the healthy control. Further studies aimed at assessing the antimicrobial activity of the mutated peptide together with the quantification of its levels in the sweat of all family members will be performed. In this study, new findings on the genetics of HS and PASH syndrome were obtained that collectively allowed to shed light into novel scenarios potentially involved in the onset and progression of these disorders.
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Hopitzan, Alexander A. "Expression of the Ank3 gene in rat skeletal muscle : gene structure, splice pattern, phylogenetic analysis, molecular identification and subcellular targeting of encoded ankyrins-G." Paris 6, 2005. http://www.theses.fr/2005PA066311.
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Brettell, Rhea C. "The final masquerade : a molecular-based approach to the identification of resinous plant exudates in Roman mortuary contexts in Britain and evaluation of their significance." Thesis, University of Bradford, 2016. http://hdl.handle.net/10454/15886.
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This study provides chemical confirmation for the use of resinous plant exudates in mortuary contexts in Roman Britain. Analysis of amorphous masses, adhering residues and grave deposits using gas chromatography-mass spectrometry has revealed terpenoid biomarkers in sixteen inhumation and two cremation burials. The natural products characterized include European Pinaceae (conifer) resins, Pistacia spp. (mastic/terebinth) resins from the Mediterranean or the Levant and Boswellia spp. (frankincense) gum-resins from southern Arabia or eastern Africa. In addition, traces of a balsamic resin, probably Liquidambar orientalis, have been identified. A correlation between the use of these exotic exudates and interment in substantial, often multiple, containers with high-quality textiles and grave goods was observed. Theoretical consideration of this imported rite illuminates the multiplicity of roles played by resins/gum-resins in the mortuary sphere. The material properties of these highly scented substances speak to the biological reality of the decomposing body and to the socially constructed identity of the individual. On a practical level, they acted as temporary preservatives and masked the odour of decay. As social signifiers, they denoted the status of the deceased and promoted remembrance through conspicuous consumption and sensory impact. Encoded with ritual meaning, they purified the body and facilitated the final rite of passage to the afterlife. The recovery of these resinous traces provides us with new insights into the treatment of the body in the Roman period and establishes fresh links between the remote province of Britannia and the remainder of the Empire.
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Sathanantham, Preethi, Shiva K. Devaiah, and Cecelia A. McIntosh. "Structure-Function Analysis of Grapefruit Glucosyltransferase Protein – Identification of Key Amino Acid Residues for its Rigid Substrate Specificity." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/352.
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Flavonoids are an important class of secondary metabolites widely distributed in plants. The majority of naturally occurring flavonoids are found in glucosylated form. Glucosyltransferases are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor flavonoid substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi (Cp3OGT) has strict substrate and regiospecificity. In this study, amino acid residues that could potentially alter the rigidity observed in this enzyme were mutated to position equivalent residues of a putative anthocyanin specific glucosyltransferase from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling followed by site directed mutagenesis to identify candidate regions, three double mutations were made. To test the basis of substrate specificity, biochemical analysis of the three recombinant mutant proteins was carried out. Recombinant protein with mutation S20G+T21S revealed that the enzyme retained activity similar to the wildtype (Cp3OGT) (WT- Km app-104.8 µM; Vmax = 24.6 pmol/min/µg, Mutant- Km app-136.42 µM; Vmax -25pmol/min/µg) but the mutant was more thermostable compared to the WT. The (S290C+S319A) mutant protein retained 40% activity relative to wildtype and has an optimum pH shifted towards the acidic side (pH 6) (Km app-8.27 µM; Vmax-90.9 pmol/min/µg). Mutation of Glutamine87 and Histine154 (H154Y+Q87I) have rendered this recombinant protein inactive with every class of flavonoid tested. Interestingly, the single point mutations H154Y and Q871I had significant activity, slightly greater than that of wildtype enzyme. The two active recombinant proteins will further be analyzed to determine whether the mutations have altered regiospecificity of the original enzyme. Product identification is being conducted using HPLC.
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Couto, Manuela Soares. "Molecular identification of clinical and strains environmental Burkholderia pseudomallei, from the State of CearÃ: based on analysis regions 16S and 16S-23S ribosomal DNA nuclear." Universidade Federal do CearÃ, 2009. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=10501.
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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel SuperiorMelioidosis is a potentially fatal disease caused by the bacterium Burkholderia pseudomallei, considered emerging in Brazil since the first cases were reported in 2003, on State of CearÃ. This study aimed to perform the molecular identification of 31 isolates of B. pseudomallei (26 clinical and 5 environmental) maintained in the culture collection of CEMM (Specialized Center for Medical Mycology), based on sequences 16S and 16S-23S rRNA. The DNA of these samples was extracted with the kit Wizard  Genomic DNA Purification (Promega), quantified by spectrophotometry and stored at 4ÂC. The amplification of a fragment of 302 bp of 16S-23S rRNA specific to B. pseudomallei was performed by PCR reaction with primers Bp1 and Bp4. The sequencing of 16S and 16S-23S rRNA was performed by using of the kit DYEnamicTM ET terminators cycle sequencing (GE Healthcare). The phylogenetic tree of 16S rRNA and the sequence identity matrix and sequence difference count matrix based on the 16S-23S rRNA were generated by the program MEGA4, version 4.1. The results confirmed the identification of 15 strains of B. pseudomallei (5 clinical and 10 environmental), which represents 48.4% of the isolates analyzed in this study. The phylogenetic tree based on 16S rRNA shows that the clinical and environmental isolates of B. pseudomallei of State of Cearà are evolutionarily clustered with the strains B. pseudomallei MSHR346 (Australia), B. pseudomallei 1106a (Thailand), B. pseudomallei K96243 (Thailand), B. pseudomallei 1710b (Thailand) and B. pseudomallei 668 (Australia). Using the same extraction kit was possible to extract DNA from B. pseudomallei directly from clinical specimen (bronchoalveolar lavage), confirming a new case of melioidosis in Ubajara/CE. In this study, the use of PCR for amplification of a fragment of 302 bp of 16S-23S rRNA identified correctly B. pseudomallei, and to confirm the discrimination between B. pseudomallei and B. mallei, the sequencing of the 16S and 16S-23S rRNA genes was performed. The technique of PCR coupled with sequencing of 16S and 16S-23S rRNA resulted in a high sensitivity and specificity of detection of B. pseudomallei in this study.
A melioidose à uma doenÃa potencialmente fatal causada pela bactÃria Burkholderia pseudomallei, sendo considerada emergente no Brasil desde que os primeiros casos foram reportados em 2003, no Estado do CearÃ. Este estudo pretendeu realizar a identificaÃÃo molecular de 31 isolados de B. pseudomallei (cinco clÃnicos e 26 ambientais) mantidos na coleÃÃo de culturas do CEMM (Centro Especializado em Micologia MÃdica), com base nas sequÃncias 16S e 16S-23S DNAr. O DNA destas amostras foi extraÃdo com o kit Wizard Genomic DNA Purification (Promega), quantificado por espectrofotometria e armazenado a 4ÂC. A amplificaÃÃo de um fragmento de 302 pb da regiÃo espaÃadora 16S-23S DNAr especÃfico para B. pseudomallei foi realizada por meio de reaÃÃo de PCR com os primers Bp1 e Bp4. O sequenciamento das regiÃes 16S e 16S-23S DNAr foi realizado pelo mÃtodo da terminaÃÃo da cadeia pelo didesoxinucleotÃdeo, usando-se o kit DYEnamicTM ET terminators cycle sequencing (GE Healthcare). A Ãrvore filogenÃtica da regiÃo 16S DNAr e as matrizes sequÃncia identidade e contagem de diferenÃas baseadas na regiÃo 16S-23S DNAr foram geradas pelo programa MEGA4, versÃo 4.1. Os resultados confirmaram a identificaÃÃo de 15 cepas de B. pseudomallei (cinco clÃnicas e dez ambientais), o que corresponde a 48.4% dos isolados em estudo. A Ãrvore filogenÃtica baseada na regiÃo 16S DNAr demonstra que os isolados clÃnicos e ambientais de B. pseudomallei do Estado do Cearà sÃo evolutivamente agrupados com as cepas B. pseudomallei MSHR346 (AustrÃlia), B. pseudomallei 1106a (TailÃndia), B. pseudomallei K96243 (TailÃndia), B. pseudomallei 1710b (TailÃndia) e B. pseudomallei 668 (AustrÃlia). Com a utilizaÃÃo do mesmo kit de extraÃÃo tambÃm foi possÃvel extrair DNA de B. pseudomallei diretamente de espÃcime clÃnico (lavado brÃnquico), confirmando um novo caso de melioidose no MunicÃpio de Ubajara/CE. Em nosso estudo, o uso da PCR para a amplificaÃÃo de um fragmento de 302 pb da regiÃo 16S-23S DNAr identificou corretamente B. pseudomallei, sendo que para confirmar a discriminaÃÃo entre B. pseudomallei e B. mallei, o sequenciamento das regiÃes 16S e 16S-23S DNAr foi realizado. A tÃcnica de PCR aliada ao sequenciamento das regiÃes 16S e 16S-23S do DNA ribossÃmico nuclear resultaram em uma elevada sensibilidade e especificidade de detecÃÃo de B. pseudomallei neste estudo.
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