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Borodina, Tatiana. "Entwicklung des Ligationsdetektionreaction (LDR)-Taqman Assay eine neue SNP-Genotypisierungsmethode /." [S.l.] : [s.n.], 2005. http://www.diss.fu-berlin.de/2005/298/index.html.
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Armbruster, Katrin. "Entwicklung einer Multiplex-TaqMan-PCR zur Diagnostik der viralen Enzephalitis." [S.l. : s.n.], 2006. http://nbn-resolving.de/urn:nbn:de:bsz:25-opus-49607.
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Savill, Rachel. "Validering av realtids-PCR-metod för Herpes simplex- och Varicella-zoster virus." Thesis, Hälsohögskolan, Högskolan i Jönköping, HHJ, Avd. för naturvetenskap och biomedicin, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:hj:diva-27225.
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Scheer, Carola. "Nachweis von Aspergillus-fumigatus-Infektionen aus dem Respirationstrakt mittels TaqMan-PCR." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2006. http://dx.doi.org/10.18452/15415.
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Die invasive Aspergillose (IA) wird mehr und mehr zu einem lebensbedrohlichen Problem bei immunsupprimierten Patienten. Der Hauptverursacher der IA ist der Aspergillus fumigatus. Es existieren verschiedene DNS-Extraktions-Methoden und PCR Assays, um Aspergillus fumigatus DNS in Bronchiallavagen (BAL) nachzuweisen. Häufig sind diese Methoden langwierig und verdeutlichen den Bedarf an einer klinisch relevanten Methode, mit der der Aspergillus-fumigatus-DNS Nachweis schnell und zuverlässig erbracht werden kann. Wir haben eine schnelle Methode entwickelt, mit der wir quantitative Ergebnisse innerhalb sechs Stunden erhalten können. Es wurde ein Extraktionsverfahren auf mechanischer Basis (FastPrep), in Kombination mit einer real-time-PCR, basierend auf der TaqMan- Technologie, etabliert. Dazu wurde eine Aspergillus-fumigatus-spezifischeSonde entwickelt und ein Aspergillus-fumigatus-spezifisches Primerpaar eingesetzt. Durch den Einsatz einer Standardreihe wurden die Ergebnisse quantifiziert. Es wurden hauptsächlich Bronchiallavagen sowie andere Materialien von 204 Patienten untersucht. Die Patienten wurden hinsichtlich ihres Verdachtes an einer IA erkrankt zu sein, anhand bestimmter Kriterien (EORTC) in bestimmte Gruppen eingeteilt. Die Ergebnisse wurden innerhalb der einzelnen Gruppen analysiert. Bei allen 8 Patienten mit bewiesener IA gelang der Nachweis von Aspergillus-fumigatus-DNS. In dieser Gruppe konnten quantitativ die höchsten Werte gemessen werden. Des Weiteren konnte eine Rückläufigkeit der Werte mit abnehmendem Aspergillose-Verdacht, sowie nach erfolgter antimykotischer Therapie beobachtet werden. Die diagnostische Sensitivität der Methode beträgt 81,3%, die Spezifität liegt bei 93,7%. Der positive prädiktive Wert ist 72,2%, der negative prädiktive Wert beträgt 96,1%. Als zusätzlicher Baustein in einer umfangreichen Gesamtdiagnostik, kann diese Methode einen Beitrag zur Verbesserung molekularer Nachweisverfahren leisten.Invasive aspergillosis (IA), often caused by Aspergillus fumigatus, is an important cause of death of immunocompromised patients. Several DNA-extraction methods and PCR assays are available for detecting Aspergillus fumigatus DNA in bronchoalveolar lavage (BAL) samples of patients with invasive aspergillosis. These methods are often time consuming and emphasize the need to develop a clinical relevant rapid DNA isolation assay that gives reliable results in a short time. We have developed a new and rapid method which yields results within six hours.This was achieved by combining high-speed cell disruption using a mechanical extraction procedure (FastPrep), with a real-time PCR assay based on TaqMan technology.A newly designed Aspergillus-fumigatus-specific probe and Aspergillus-fumigatus-specific primers were established. This combination also produces quantitative results by comparing the results with a DNA serial dilution used in the real-time PCR. BAL fluids and other material from 204 patients were analyzed. According to the risk for an IA, the patients were devided in different groups, using specific criteria published by the EORTC. The results were related to these groups. For all 8 patients with proven IA, the detection of Aspergillus-fumigatus-DNA succeeded. In this group, the highest amount of Aspergillus-fumigatus-DNA was detected. A decraesing quantitiy of Aspergillus-fumigatus-DNA could be detected after antifungal treatment and connected with the decreasing suspicion of an IA in the other groups. The diagnostic sensitivity of this method is 81,3%, the specifity is 93,7%. The positive predictive value is 72,2%, the negative predicitive value 96,1%. Adjunctive use of these method may be helpful in the diagnosis of IA.
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Herrmann, Ilka [Verfasser]. "Quantifizierung frühbesiedelnder oraler Actinomyces spp. mittels neu etablierter TaqMan-PCRs / Ilka Herrmann." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2016. http://d-nb.info/1111038759/34.
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Simecek, Nikol. "Development of a database with web-based user interface for taqman assay design." Thesis, Cranfield University, 2007. http://hdl.handle.net/1826/1773.
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TaqMan RT-PCR (reverse transcription-polymerase chain reaction) is a technique used to measure the relative gene expression in a biological sample and is one of the core technologies used by the Molecular Pathology and Toxicology (MPT) Group at GlaxoSmithKline. Conducting TaqMan experiments is a complex process which involves the design of a TaqMan assay specific to a gene of interest. A wealth of data has been generated during assay design, but systems are not currently available to readily share this data within the MPT group.
There is a need for a central data storage repository so that data associated with assay design can be organised efficiently and rapidly accessed. Experiments are conducted within limited timeframes and resource is often limited so this would be of great benefit to the MPT group.
This thesis describes the development of a database to house data associated with TaqMan assay design, software to populate the database with minimal user interaction and a web based CGI application for members of the MPT group to query and submit data to the database. Finally, the output from testing the software is provided and discussed.
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Camarão, António Alexandre Riachos. "Development and optimisation of a group-specific real-time RT-PCR assay for the broad detection of the Simbu serogroup orthobunyaviruses." Master's thesis, Universidade de Lisboa, Faculdade de Medicina Veterinária, 2018. http://hdl.handle.net/10400.5/15823.
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The Simbu serogroup within the genus Orthobunyavirus belongs to the family Peribunyaviridae and comprises 32 recognised three-segmented negative-sense single-stranded RNA viruses, divided into two phylogenetic clades. Some members of this group of arthropod-borne viruses, cosmopolitan distributed, cause neurologic disease in humans as well as reproductive and neurologic disease in domestic animals, however, definitive diagnosis always requires laboratorial confirmation. Few real-time RT-PCR assays have been developed for the molecular diagnosis of Simbu serogroup orthobunyaviruses. There are two published methods with broad detection capacity, utilising either a SYBR Green based chemistry able to recognise viruses from both clades, which is not absolutely specific, or a TaqMan based chemistry that recognises only clade B viruses. A novel group-specific TaqMan-based real-time RT-PCR assay was developed, optimised and laboratory validated for the broad detection of the Simbu serogroup orthobunyaviruses. The published genomic data of the Simbu serogroup members were evaluated, and a conserved region, situated in the segment encoding the nucleocapsid protein, was selected to design a universal primer set and a pair of differently labelled hydrolysis probes, which allowed for the distinction between the two phylogenetic clades of the Simbu serogroup. Seven prototype Simbu serogroup isolates were used for the development of the assay, namely Akabane orthobunyavirus, Simbu orthobunyavirus, Shuni orthobunyavirus, Sathuperi orthobunyavirus, Shamonda orthobunyavirus, Ingwavuma virus and Sabo virus. The primer and probe concentrations in the reaction were optimised. Amplification efficiency was determined for each one of the viruses: AKAV (99%), SIMV (96%), SHUV (96%), SATV (97%), SHAV (84%), INGV (93%) and SABOV (110%). A panel constituted of genetically related, causative agents of abortion in ruminants and arthropod-borne viruses was selected for in vitro specificity analysis, and in silico analysis was also performed. The assay was shown to be specific, as no cross-reactions were observed either in vitro or in silico, and sensitive, with a 95% limit of detection ranging from 10-0,39 to 10-3,61 TCID50/reaction, for the detection of Simbu serogroup viruses. The repeatability of the assay was evaluated for both probes detection, using the intra- and inter-run standard deviations and coefficient of variation. This work resulted in a manuscript in submission process to a peer-reviewed journal. In addition, a comprehensive review of the viruses of the Simbu serogroup was carried out, including sites of viral isolation and seroconversion, and a map was generated using a geographic information system tool.RESUMO - O serogrupo Simbu pertence ao género Orthobunyavirus, família Peribunyaviridae, e é constituído por 32 vírus de RNA tri-segmentado de cadeia simples e polaridade negativa, divididos em duas clades filogenéticas. Alguns membros deste grupo de vírus transmitidos por artrópodes, com distribuição cosmopolita, causam doença neurológica em humanos bem como doença reprodutiva e neurológica em animais domésticos, no entanto, o diagnóstico definitivo requer sempre confirmação laboratorial. Poucos ensaios de RT-PCR em tempo real têm sido desenvolvidos para o diagnóstico molecular destes vírus, ainda assim, existem dois que se destacam pela capacidade de detecção em largo espectro. Um deles, com sistema de detecção de fluorescência baseado na utilização de SYBR Green, é capaz de detectar vírus das duas clades, mas não é absolutamente específico, e o outro, baseando-se na utilização de sondas TaqMan, só detecta vírus de uma das clades. Um ensaio de RT-PCR em tempo real grupo-específico, inédito, com sondas TaqMan, foi desenvolvido, optimizado e caracterizado em termos laboratoriais, para a detecção em largo espectro dos orthobunyavirus do serogrupo Simbu. Os dados publicados referentes ao genoma dos membros do serogrupo foram analisados, e uma região conservada, situada no segmento codificante da proteína da nucleocápdise, foi eleita para o desenho de um par de primers específicos universal, bem como de duas sondas de hidrólise específicas, distintamente marcadas, e que, portanto, permitem a diferenciação entre as clades filogenéticas. Sete isolados de referência foram utilizados no desenvolvimento do ensaio, nomeadamente Akabane orthobunyavirus, Simbu orthobunyavirus, Shuni orthobunyavirus, Sathuperi orthobunyavirus, Shamonda orthobunyavirus, Ingwavuma virus e Sabo virus e, consequentemente, as concentrações dos primers e sondas na reação foram optimizadas. A eficiência de amplificação foi determinada para cada um dos vírus: AKAV (99%), SIMV (96%), SHUV (96%), SATV (97%), SHAV (84%), INGV (93%) e SABOV (110%). Um painel constituído por vírus geneticamente relacionados, agentes causais de aborto em ruminantes e transmitidos por artrópodes foi seleccionado no sentido de avaliar a especificidade do ensaio in vitro, tendo sido também efectuada uma análise in silico. O ensaio é específico, visto que não foram observadas reacções cruzadas quer in vitro quer in silico, e sensível, com um limite de detecção de 95% entre 10-0,39 a 10-3,61 TCID50/reacção, para a detecção de vírus do serogrupo Simbu. A repetibilidade foi avaliada para a detecção com ambas as sondas, pelo cálculo do desvio padrão intra- e inter-corridas bem como do coeficiente de variação. Este trabalho originou um manuscrito em processo de submissão para uma revista cientifíca. Além disso, foi levada a cabo uma revisão bibliográfica do serogrupo Simbu, incluindo locais de isolamento viral e seroconversão, e um mapa inédito desta distribuição foi gerado.
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Cunty, Amandine. "Caractérisation de Pseudomonas syringae pv. actinidiae l’agent responsable de l’émergence d’une épidémie de chancre bactérien du kiwi en France et description de Pseudomonas syringae pv. actinidifoliorum, agent causal de taches foliaires sur kiwi." Thesis, Angers, 2015. http://www.theses.fr/2015ANGE0018/document.
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La bactérie responsable de chancre sur bois, Pseudomonas syringae pv. actinidiae (Psa), a causé trois épidémies depuis les années 1980 et se décline en trois biovars. La plus récente et dévastatrice (causée par Psa biovar 3), a été détectée pour la première fois en 2008 en Italie et s’est rapidement répandue dans la majorité des pays producteurs de kiwi, dont en France en 2010. Nous avons analysé la diversité de 280 souches de P. syringae isolées de kiwi en France. La caractérisation biologique et l’analyse phylogénétique des souches par MLSA ont révélé que les biovars 1, 2 et 3 appartenaient à une même lignée génétique, groupant également P. s. pv. theae. Les souches de biovar 4 constituent un ensemble de 4 lignées génétiques distinctes qui ont été rassemblées au sein d’un nouveau pathovar (Pseudomonas syringae pv. actinidifoliorum (Psaf)). Ces souches sont caractérisées par une pathogénie réduite (taches foliaires mais pas de chancre). Cette nouvelle classification permet une meilleure gestion des épidémies de chancre bactérien du kiwi. Le développement d’un schéma MLVA composé de 11 VNTRs a permis d’étudier la structuration génétique de populations de Psa biovar 3, de révéler de la diversité au sein de ce pathovar et d’identifier l’origine italienne de l’épidémie en France. Le séquençage du génome de cinq souches de Psaf et la comparaison de ces séquences avec celles d’autres génomes de Psa et Psaf, disponibles sur NCBI, a permis le développement d’un nouvel outil de détection par PCR temps réel, plus spécifique de chaque biovar de Psa et de Psaf. La MLVA et la PCR temps réel développées ici contribueront à l’amélioration de la surveillance de Psa dans le mondeThe causal agent of bacterial canker, Pseudomonas syringae pv. actinidiae (Psa),has been responsible ofthree epidemics since 1980’s.Psa is divided in three biovars. The most recent and severe outbreak (causedby Psa biovar 3) was detected for the first time in Italy in 2008. It has spread very quickly in the main kiwifruit producing countries, as in France in 2010. We analyzed the diversity of 280 strains of P. syringae isolated fromkiwifruit in France. The biological characterization and the phylogenetic analysis of the strains by MLSArevealed that the biovars 1, 2 and 3 belong to the same genetic lineage, which include P. s. pv. theae, as well.The biovar 4 strains, which are structured in 4 distinct genetic lineages, have been grouped in a new pathovar(Pseudomonas syringae pv. actinidifoliorum (Psaf)). These strains are characterized by a low virulence (onlyspots on leaves and no canker on wood). This new classification help with the management of the bacterialkiwifruit canker outbreaks. The development of an MLVA scheme composed of 11 VNTRs allowed to studythe genetic structuration of Psa biovar 3 populations, to reveal the diversity within this pathovar and to identifythe Italian origin of the epidemic in France. The genome sequencing of five Psaf strains and the comparisonbetween these sequences and those of Psa and Psaf genomes already available on NCBI, allowed thedevelopment of a new detection tool by real-time PCR, specific of each Psa biovar and of Psaf. The MLVA andthe real-time PCR based detection technique developed here will contribute to the improvement of the monitoringof kiwifruit bacterial canker around the world
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Kessler, Yvonne. "Quantitative TaqMan® real-time PCR assays for gene expression normalisation in feline tissues /." [S.l.] : [s.n.], 2009. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000292626.
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Steyn, HC, A. Pretorius, and CME McCrindle. "A quantitative real-time PCR assay for Ehrlichia ruminantium using pCS20." Elsevier, 2008. http://encore.tut.ac.za/iii/cpro/DigitalItemViewPage.external?sp=1000379.
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Heartwater is a tick borne disease that affects ruminants and wild animals in Africa south of the Sahara. It is caused by
Ehrlichia ruminantium and transmitted by the tick Amblyomma hebraeum. The protocols currently used to detect heartwater
take several days to complete. Here, we describe the development of a pCS20 quantitative real-time PCRTaqMan probe assay to
detect E. ruminantium in livestock blood and ticks from the field. The assay is based on the conserved pCS20 gene region of E.
ruminantium that contains two overlapping genes, rnc and ctaG [Collins, N.E., Liebenberg, J., De Villiers, E.P., Brayton, K.A.,
Louw, E., Pretorius, A., Faber, F.E., Van Heerden, H., Josemans, A., Van Kleef, M., Steyn, H.C., Van Strijp, M.F., Zweygarth, E.,
Jongejan, F., Maillard, J.C., Berthier, D., Botha, M., Joubert, F., Corton, C.H., Thomson, N.R., Allsopp, M.T., Allsopp, B.A.,
2005. The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy
number. PNAS 102, 838–843]. The pCS20 quantitative real-time PCRTaqMan probe was compared to the currently used pCS20
PCR and PCR/32P-probe test with regards to sensitivity, specificity and the ability to detect DNA in field samples and in blood
from experimentally infected sheep. This investigation showed that the pCS20 quantitative real-time PCRTaqMan probe was the
most sensitive assay detecting seven copies of DNA/ml of cell culture. All three assays, however, cross react with Ehrlichia canis
and Ehrlichia chaffeensis. The pCS20 real-time PCR detected significantly more positive field samples. Both the PCR and
pCS20 real-time PCR could only detect E. ruminantium parasites in the blood of experimentally infected sheep during the febrile
reaction. The PCR/32P-probe assay, however, detected the parasite DNA 1 day before and during the febrile reaction. Thus,
because this new quantitative pCS20 real-time PCRTaqMan probe assay was the most sensitive and can be performed within 2 h
it is an effective assay for epidemiological surveillance and monitoring of infected animals.
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Trouchet, Amandine. "PCR digitale pour la détection et la caractérisation de micro-organismes pathogènes au niveau de la cellule unique." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC170/document.
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Nous avons pour but de développer un système microfluidique en gouttes, capable, à l’échelle de la cellule/bactérie unique, de détecter et de co-localiser plusieurs marqueurs génétiques, en utilisant une version digitale et multiplexée de la réaction de polymérisation en chaîne (PCR). Les systèmes de PCR digitale actuellement commercialisés ne le permettent toujours pas. Un tel prototype garantira la présence de multiples marqueurs à l’intérieur d’un même génome, ce qui permettra l’identification du pathogène avec précision et un taux de faux-positifs proche de zéro. Comme première application, nous démontrerons la possibilité de co-localiser quatre gènes de virulence de la souche O157:H7 d’Escherichia coli, un pathogène majeur, qui est détecté dans des échantillons alimentaires ou provenant de fèces cliniques pouvant aussi contenir des E. coli non pathogènes porteuses d’une partie des gènes de virulence. Avant de procéder à des tests TaqMan multicolores en point final, E. coli sera d’abord encapsulée dans des gouttes micrométriques et lysée par la chaleur in situ. Notre objectif est de démontrer que ce test peut être appliqué avec succès à un petit ensemble d’échantillons cliniques ou alimentairesWe aim to develop a prototype of droplet-based microfluidic system capable of detecting and colocalizing multiple genetic markers at the single cell/bacteria level, using the Polymerase Chain Reaction (PCR) in a digital multiplexed version. This cannot be achieved using current commercial digital PCR systems, and should increase the sensitivity and reliability of the detection of pathogens. Importantly, the system will guarantee the presence of multiple markers within the same genome and enable accurate identification, and bring the false positive rate close to zero. As a first application, we will demonstrate the possibility to co-localize 3 virulence genes in the E.coli strain O157:H7, a major foodborne pathogen, which has to be detected in clinical feces samples or food samples, which may also contain non pathogenic E. coli carrying only a subset of these virulence genes. E. Coli will be encapsulated in micrometric droplets, lysed by heating in situ prior performing a multicolour end-point Taqman assay. Our objective is to demonstrate that this test can be successfully applied to real clinical or food samples
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Yuen, Marcio Seto Yu. "Efeito do laser de Diodo de 808nm como coadjuvante ao tratamento periodontal na redução de periodontopatógenos." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/23/23146/tde-24102009-123122/.
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O objetivo do estudo foi avaliar, o efeito do laser de Diodo 808nm como coadjuvante ao tratamento periodontal na redução de periodontopatógenos Porphyromonas gingivalis, Treponema denticola e Tannerella forsythia pela técnica da Reação em Cadeia da Polimerase em Tempo Real. Foram selecionados vinte e quatro pacientes portadores de periodontite crônica neste estudo de boca dividida, duplo cego e randomizado. Dois sítios uniradiculares de cada paciente foram utilizados e divididos em dois grupos experimentais: TESTE - raspagem alisamento polimento corono radicular (RAPCR) associado à duas aplicações de laser de Diodo de alta potência (comprimento de onda de 808nm, 1,5 Watts, 597,1 W/cm2, durante 20 segundos no modo contínuo). A primeira aplicação foi realizada 24 horas após RAPCR e a segunda após sete dias; CONTROLE foi realizado o mesmo procedimento porém sem a aplicação do laser. O biofilme subgengival foi coletado antes do tratamento e seis semanas após a segunda aplicação do laser. A avaliação microbiológica foi feita através da Reação em Cadeia da Polimerase em tempo real para a quantificação de Porphyromonas gingivalis, Treponema denticola e Tannerella forsythia. A comparação entre os grupos não demonstram diferenças estatisticamente significantes (p<0,05). Concluiu-se que, dentro dos limites deste estudo, a aplicação do laser de Diodo de 808nm como coadjuvante ao tratamento periodontal não reduziu de forma significativa os periodontopatógenos: Porphyromonas gingivalis, Treponema denticola e Tannerella forsythia quando comparado à RAPCR.The aim of this study was to evaluate, by real-time polymerase chain reaction, the effect of Diode laser 808nm as an adjuvant in the reduction of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia in to periodontal treatment. Twenty-four patients with chronic periodontitis the study designs was split-mouth, double blind and ramdomized controlled trial. Two sites from uniradicular teeth of each patient were used and divided in two experimental groups: TEST scalling and root planing (SRP) associated with two high power Diode laser application (wavelength of 808nm, 1,5W at the display (597,1 W/cm2) for 20 seconds in the continuous-wave mode) the first laser application was 24 hours after SRP and the second seven days later; CONTROL a similar procedure without laser application. The subgingival biofilm was colleted before treatment and six weeks after the second laser application. The microbiologic evaluation was done by real-time polymerase chain reaction for the quantification of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia. The comparison between the groups did not show significant differences (p<0,05). Within the limits of this study, it can be concluded that Diode laser 808nm application as an adjuvant in the periodontal treatment did not reduce the periodontal pathogens Porphyromonas gingivalis, Treponema denticola e Tannerella forsythia when compared by scaling root planning.
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Gregorio-King, Claudia C., and mikewood@deakin edu au. "The Identification of novel genes differentially expressed in Haemopoietic progenitor cells." Deakin University. School of Health Sciences, 2001. http://tux.lib.deakin.edu.au./adt-VDU/public/adt-VDU20051111.113037.
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The biochemical and molecular processes that maintain the stem cell pool, and govern the proliferation and differentiation of haemopoietic stem/progenitor cells (HSPCs) have been widely investigated but are incompletely understood. The purpose of this study was to identify and characterise novel genes that may play a part in regulating the mechanisms that control the proliferation, differentiation and self-renewal of human HSPCs.
Reverse transcription differential display polymerase chain reaction (dd-PCR) was used to identify differences in gene expression between a HSPC population defined by expression of the CD34 phenotype, and the more mature CD34 depleted populations. A total of 6 differentially expressed complementary deoxyribonucleic acid (cDNA) sequences were identified. Four of these transcripts were homologous to well characterised genes, while two (band 1 and band 20) were homologous to unknown and uncharacterised partial gene sequences on the GenBank database and were thus chosen for further investigation.
The partial cDNA sequences for band 1 and band 20 were designated ORP-3 and MERP-1 (respectively) due to homologies with other well-characterised gene families. Differential expression of the ORP-3 and MERP-1 genes was confirmed using Taqman™ real-time polymerase chain reaction (PCR) with 3 - 4-fold and 4-10 -fold higher levels in the CD34+ fractions of haemopoietic cells compared to CD34- populations respectively. Additionally, expression of both these genes was down regulated with proliferation and differentiation of CD34+ cells further confirming higher expression in a less differentiated subset of haemopoietic cells.
The full coding sequences of ORP-3 and MERP-1 were elucidated using bioinformatics, rapid amplification of cDNA ends (RACE) and PCR amplification. The MERP-1 cDNA is 2600 nucleotides (nt) long, and localizes by bioinformatics to chromosome 7.. It consists of three exons and 2 introns spanning an entire length of 31.4 kilobases (kb). The MERP-1 open reading frame (ORF) codes for a putative 344 amino acid (aa) type II transmembrane protein with an extracellular C-terminal ependymin like-domain and an intracellular N-terminal sequence with significant homology to the cytoplasmic domains of members of the protocadherin family of transmembrane glycoproteins. Ependymins and protocadherins are well-characterised calcium-dependant cell adhesion glycoproteins. Although the function of MERP-1 remains to be elucidated, it is possible that MERP-1 like its homologues plays a role in calcium dependent cell adhesion. Differential expression of the MERP-1 gene in haemopoietic cells suggests a role in haemopoietic stem cell proliferation and differentiation, however, its broad tissue distribution implies that it may also play a role in many cell types. Characterization of the MERP-1 protein is required to elucidate these possible roles.
The ORP-3 cDNA is 6631nt long, and localizes by bioinformatics to chromosome 7pl5-p21. It consists of 23 exons and 22 introns spanning an entire length of 183.5kb. The ORP-3 ORF codes for a putative 887aa protein which displays the consensus sequence for a highly conserved oxysterol-binding domain. Other well-characterised proteins expressing these domains have been demonstrated to bind oxysterols (OS) in a dose dependant fashion. OS are hydroxylated derivatives of cholesterol Their biological activities include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, including haemopoietic cells. Differential expression of the ORP-3 gene in haemopoietic cells suggests a possible role in the transduction of OS effects on haemopoietic cells, however, its broad tissue distribution implies that it may also play a role in many cell types.
Further investigation of ORP-3 gene expression demonstrates a significant correlation with CD34+ sample purity, and 2-fold higher expression in a population of haemopoietic cells defined by the CD34+38- phenotype compared to more mature CD34+38+ cells. This finding, taken together with the previous observation of down-regulation of ORP-3 expression with proliferation and differentiation of CD34+ cells, indicates that ORP-3 expression may be higher in a less differentiated subset of cells with a higher proliferative capacity. This hypothesis is supported by the observation that expression of the ORP-3 gene is approximately 2-fold lower in differentiated HL60 promyelocytic cells compared to control, undifferentiated cells.
ORP-3 expression in HL60 cells during normal culture conditions was also found to vary with expression positively correlated with cell number. This indicates a possible cell cycle effect on ORP-3 gene expression with levels highest when cell density, and therefore the percentage of cells in G(0)/G(1) phase of the cell cycle is highest. This observation also correlates with the observation of higher ORP-3 expression in CD34+38-cells, and in CD34+ and HL60 cells undergoing OS induced and camptothecin induced apoptosis that is preceded by cell cycle arrest at G(0)/G(1). Expression of the ORP-3 gene in CD34+ HSPCs from UCB was significantly decreased to approximately half the levels observed in control cells after 24 hours incubation in transforming growth factor beta-1 (TGFâl). As ≥90% of these cells are stimulated into cell cycle entry by TGFâl, this observation further supports the hypothesis that ORP-3 expression is highest when cells reside in the G(0)/G(1) phase of the cell cycle. Data obtained from investigation of ORP-3 gene expression in synchronised HL60 cells however does not support nor disprove this hypothesis.
Culture of CD34+ enriched HSPCs and HL60 cells with 25-OHC significantly increased ORP-3 gene expression to approximately 1.5 times control levels. However, as 25-OHC treatment also increased the percentage of apoptotic cells in these experiments, it is not valid to make any conclusions regarding the regulation of ORP-3 gene expression by OS. Indeed, the observation that camptothecin induced apoptosis also increased ORP-3 gene expression in HL60 cells raises the possibility that up-regulation of ORP-3 gene expression is also associated with apoptosis,
Taken together, expression of the ORP-3 gene appears to be regulated by differentiation and apoptosis of haemopoietic progenitors, and may also be positively associated with proliferative and G(0)/G(1) cell cycle status indicating a possible role in all of these processes.
Given the important regulatory role of apoptosis in haemopoiesis and differential expression of the ORP-3 gene in haemopoietic progenitors, final investigations were conducted to examine the effects OS on human HSPCs. Granulocyte/macrophage colony forming units (CFU-GM) generated from human bone marrow (ABM) and umbilical cord blood (UCB) were grown in the presence of varying concentrations of three different OS - 7keto-cholesterol (7K-C), 7beta-hydroxycholesterol (7p-OHC) and 25-hydroxycholesterol (25-OHC). Similarly, the effect of OS on HL60 and CD34+ cells was investigated using annexin-V staining and flow cytometry to measure apoptosis. Reduction of nitroblue tetrazolium (NBT) was used to assess differentiative status of HL60 cells. CFU-GM from ABM and HL60 growth was inhibited by all three OS tested, with 25-OHC being the most potent. 25-OHC inhibited ≥50% of bone marrow CFU-GM and ≥95% of HL60 cell growth at a level of 1 ug/ml. Compared to UCB, CFU-GM derived from ABM were more sensitive to the effects of all OS tested. Only 25-OHC and 7(5-OHC significantly inhibited growth of UCB derived CFU-GM. OS treatment increased the number of annexin-V CD34+ cells and NBT positive HL60 cells indicating that OS inhibition of CFU-GM and HL60 cell growth can be attributed to induction of apoptosis and differentiation.
From these studies, it can be concluded that dd-PCR is an excellent tool for the discovery of novel genes expressed in human HSPCs. Characterisation of the proteins encoded by the novel genes ORP-3 and MERP-1 may reveal a regulatory role for these genes in haemopoiesis. Finally, investigations into the effects of OS on haemopoietic progenitor cells has revealed that OS are a new class of inhibitors of HSPC proliferation of potential relevance in vivo and in vitro.
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Korimbocus, J., David Coates, I. Barker, and N. Boonham. "Improved detection of Sugarcane yellow leaf virus using a real- time fluorescent (TaqMan) RT-PCR assay." Elsevier, 2002. http://hdl.handle.net/10454/4037.
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noYellow leaf syndrome (YLS) of sugarcane has been associated with Sugarcane yellow leaf virus (ScYLV) and has been reported from most sugarcane growing countries around the world. As sugarcane is vegetatively propagated, it is important to use effective and sensitive detection methods to screen new propagating material. Virus detection in symptomatic tissue is currently achieved using enzyme linked immunosorbent assay (ELISA), tissue blot immunoassay (TBIA) or a conventional RT-PCR based assay. This paper reports the development of an improved assay based on multiplex real-time fluorescent RT-PCR. The new assay is 100-fold more sensitive than conventional RT-PCR, and incorporates a novel `RNA specific¿ internal positive control (based around the intron of the caffeic acid 3-o-methyltransferase gene) to guard against false negative results. The paper also describes the comparison of eight RNA extraction methods for sugarcane tissue giving a number of alternatives for different laboratory situations. The sensitivity of this assay has allowed the detection of ScYLV in many samples that were thought to be healthy following conventional testing (RT-PCR, ELISA or TBIA). The detection of ScYLV using this TaqMan assay can be applied to the production of ScYLV-free plants and prevents its spread through the propagation material.
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Abucar, Ramla. "Utvärdering av prestanda vid olika reaktionsvolymer med QuantStudio qPCR samt jämförelse mellan två pipetteringsrobotar." Thesis, Örebro universitet, Institutionen för hälsovetenskaper, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-92903.
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Introduktion: Polymerase chain reaction (PCR) är en biokemisk och molekylärbiologisk laboratorieteknik som används för in vitro amplifiering av specifika gensekvenser. Det finns olika varianter av PCR, en mer utvecklad version är Realtids-PCR, även benämndkvantitativ PCR (qPCR). qPCR mäter fluorescensintensitet i varje qPCR cykel. Metoden delas in i fyra huvudfaser: linjär-, tidig exponentiell-, exponentiell- och platåfas. Syfte: Syftet med projektet var att utvärdera prestanda hos Quantstudio 7 vid varierande reaktionsvolym och plattposition, samt vid singleplex och duplex, för att öka kvaliteten på resultat och göra metoden mer kostnadseffektiv. Material & metod: Syntetisk DNA-sekvens (gBlock) späddes och sattes upp i en standardkurva med sju punkter och användes för 20 µl respektive 10 µl reaktionsvolymen, varje punkt bestod av 4 replikat. För att utvärdera Duplex vs Singelplex förbereddes standardkurva i kombination med en konstant koncentration av en annan assay. För att undersöka intra-plate variation sattes upp identiska reaktioner i samtliga brunnar i PCR-plattan. Resultat: Samtliga experiment gav detekterbara ampliferingsprodukter. Cq-värdet användes för att beräkna medelvärde och standardavvikelse, samt effektivitet och R2-värde Slutsats: Resultatet som erhålls från QS instrumentet visade att reaktionsvolymerna 10 µl och 20 µl är jämförbara. Duplex experimentet visade att gener med låg genuttryck kan duplexas med gener som har 10 000x högre genuttryck. Resultatet från intra-plate variation visade att variationen i SD var högre i den högre sidan av PCR-plattan.Introduction: Polymerase chain reaction (PCR) is a biochemical and molecular laboratory technique that is used for amplification of specific gene sequences. There are different variants of PCR. A more developed version is quantitative PCR (qPCR). In qPCR the fluorescence intensity is measured in realtime during each qPCR cycle. Aim: The purpose of the project is to evaluate whether the reaction volume can be reduced by half, which leads to using less material and thus make the method more cost-effective. Matherial & method: Synthetic DNA sequence (gBlock) was diluted and set up in a standard curve with seven standards and used for 20 μl and 10 μl reaction volume, respectively. Each standard consisted of 4 replicates. To evaluate Duplex vs Singelplex, standard curve was prepared in combination with a constant concentration of another assay. To investigate intra-plate variation, identical reactions were set up in all wells of the PCR-plate. Results: All experiments yielded detectable amplification products. The Cq value was used to calculate the mean and standard deviation, as well as the efficiency and R2 value Conclusion: The obtained results showed that the reaction volumes 10 and 20 µl are comparable. In duplex assay, genes with low gene expression can be analyzed with genes that have 10,000x higher gene expression. In intraplate-assay variation, the variation in the standard deviation increased in the right side of PCR-plate.
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Gameeldien, Hajirah. "The identification of candidate genes using cDNA microarray and the analysis of two SNPs of the reelin gene in a South African austistic population." Thesis, University of the Western Cape, 2009. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_3430_1297755889.
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Autism is a pervasive developmental disorder (PDD) that&rsquo
s incidence is approximately 1 in 158. It is four times more prevalent in males than females and is believed to be caused by both genetic and environmental factors. Research indicates that several genes are involved in autism and it is believed that these genes act together to produce autism. Many genes implicated in this disorder are involved with brain structure formation and brain functioning. Studies have identified the reelin (RELN) gene as necessary for proper formation of brain, which indicates that RELN abnormalities could contribute to the aetiology of several neurogenetic diseases such as schizophrenia, bipolar and autism. The aims of the study were (i) to genotype two SNPs (exonic rs3622691 and intronic rs736707) in the RELN gene using Taqman®
SNP Genotyping assays to detect association with autism in three distinct South African (SA) ethnic groups (Black, Caucasian and Mixed), and (ii) to detect candidate genes that are over and under-expressed in the samples taken from a SA Caucasian autistic group and compare those with samples taken from a healthy Caucasian group using cDNA microarray. The Taqman®
study indicated significant association for the intronic SNP, rs736707, with a p-value of 0.0009 in the total SA group. More so, the Mixed group displayed the highest significance amongst the ethnic groups, with a p-value of 0.00014. The microarray study yielded 21 genes with 95% significance in the Caucasian sample group. Most genes were hypothetical proteins and formed part of the FAM90A family. The LOC83459 showed the highest level of expression in the autistic samples, while the BTNL8 gene was shown to be highly suppressed in the control samples.
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Bernardes, Nara Thiers Cacciatori Galleti. "Desenvolvimento PCR em Tempo-Real em sistema TaqMan® para detecção de rotavírus em amostras clínicas de bovinos." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-26092016-103616/.
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A diarréia neonatal é o principal problema sanitário que acomete os bezerros nas primeiras semanas de vida, causando grandes prejuízos devido à morbidade, mortalidade, custos com tratamento e atraso no desenvolvimento. Os rotavírus são os mais importantes agentes virais causadores de gastroenterites em crianças e diarreia em diferentes espécies animais. Além do seu impacto econômico na produção animal, os bovinos podem atuar como reservatórios da diversidade genética e antigênica para humanos. Assim, o diagnóstico deste agente é fundamental para o desenvolvimento de medidas profiláticas mais especificas visando o seu controle. O objetivo deste trabalho foi desenvolver um método de PCR em Tempo Real para a detecção de rotavírus bovinos em sistema Taqman® tendo como alvo, a proteína não-estrutural 5 (NSP5). Para isso, 113 amostras de fezes bovinas foram coletadas de reabanhos do estado de São Paulo, e previamente testadas por PCR convencional. Para a padronização da PCR em Tempo Real, a estirpe padrão foi clonada, gerando um plasmídeo com 3x1010 cópias/reação. Como controle exógeno foi utilizado β-actina. O limite de detecção foi determinado por diluições seriadas na base 10, detectando até 6x101 cópias/µl. A curva padrão da PCR em Temo Real para a detecção do segmento codificador da proteína NSP5 teve como resultados, uma eficiência de 100,47%; com slope igual a -3,18 e R2 de 1,0. De um total de 113 amostras testadas pela PCR convencional 63 delas (55,7%) foram consideradas positivas para rotavírus. Dessas mesmas amostras testadas, 5 não amplificaram para β-actina e não foram incluídas nas análises posteriores. Para a PCR em Tempo Real o limite de detecção foi considerado o valor de 6x101 cópias/reação, sendo definido o ponto de corte o ciclo (Ct) de número 36 para o teste com a amostra viral a partir do DNA ligado em vetor plasmidial. Considerando-se o ponto de corte de 60 (6x101) cópias/reação, das 108 amostras testadas, 63 (58,3%) foram consideradas positivas ao teste. O valor de concordância obtido através do teste Kappa, a um intervalo de confiança de 95%, a partir dos resultados gerados entre os testes de PCR convencional e PCR em foi de 0.279 (baixa concordância). Os resultados obtidos nesse estudo demonstrou que o teste foi eficiente podendo ser utilizado para um diagnóstico rápido e eficiente do rotavirus do grupo A, aumentando assim o repertório dos testes já estabelecidosNeonatal diarrhea is the main health problem affecting the calves in the first weeks of life, causing major losses due to morbidity, mortality, treatment costs and delayed development. Rotaviruses are the most important causative agents of viral gastroenteritis in children and in different animal species. In addition to its economic impact on livestock, cattle can act as reservoirs for genetic and antigenic diversity for human samples. Therefore, the diagnosis of this agent is critical to the development of more specific preventive measures for their control. The objective of this work is to develop a method of PCR for rotavirus detection in cattle using TaqMan® system targeting the 5 nonstructural protein (NSP5). For this, 113 samples of cattle feces were collected from farms of São Paulo, and previously tested by convencional PCR. To PCR standardization, the standard sample was cloned, generating plasmid 3x1010 copies/reaction. The β-actin was usede as exogenous control. The limit of detection was determined by serial dilutions, to detect 6 x101 copies/µl. The standard curve of PCR to encoding segment detection NSP5 protein had as a result, an efficiency of 108.5%; with slope equal to -3.18 and R2 equal 1.. From a total of 113 samples tested by conventional PCR 63 (55.7%) were positive for rotavirus. From these samples 5 not amplifyed for β-actin gene and were not included in subsequent analyzes. For detection limit of Real-Time PCR was considered the amount of 6x101 copies / reaction, the cut-off being defined cycle (Ct) number 36 to the test with a viral sample from the DNA ligated into plasmid vector. Considering the cut-off 60 (6x101) copies/reaction of the 108 samples tested, 63 (58.3%) were positive to the test. The correlation value obtained by the Kappa test, at a 95% confidence interval, based on results generated between the conventional and PCR testing PCR was 0.279 (low agreement). The results of this study demonstrated that the probe can be efficiently used for a fast and efficient diagnosis of rotavirus of group A, thereby enhancing the repertoire of the established tests
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Luz, Ana Júlia Bretanha. "Quantificação da carga viral do HIV-1 no líquor : comparação entre os ensaios Abbott m2000rt e COBAS TaqMan v2.0." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2017. http://hdl.handle.net/10183/159589.
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Introdução A preocupação crescente com as possíveis consequências da replicação viral no sistema nervoso central mostra a necessidade da detecção do HIV no compartimento cerebral. O teste de PCR em tempo real desenvolvido pela Abbott, o Abbott m2000 RealTime HIV-1 (m2000rt), quantifica a carga viral do HIV em amostras de sangue com um procedimento efetivo e de baixo custo no nosso país, por isso é adotado como método padrão pelo Ministério da Saúde, mas não é utilizado em amostras de líquor. O ensaio produzido pela Roche, o COBAS TaqMan HIV-1, version 2 (COBAS v2.0), é o método de PCR em tempo real que tem sido amplamente utilizado para detectar a carga viral do HIV no compartimento cerebral. No entanto, esse método ainda não foi validado para esse propósito e seu custo pode ser uma limitação em diversas regiões com baixos recursos. Objetivos Considerando que não há uma metodologia padronizada para essa situação específica (detecção do HIV no líquor), nós conduzimos esse estudo a fim comparar os desempenhos dos testes m2000rt e COBAS v2.0, na tentativa de propôr um método alternativo e com baixos custos ao mais utilizado nesse contexto (COBAS v2.0). Métodos O estudo foi realizado no período de maio de 2015 a julho de 2016. O cálculo do tamanho da amostra foi baseado em dados de um estudo piloto que revelaram ser necessário um número mínimo de 37 amostras, para detectar uma diferença de 0,20 log10 na carga viral, com um coeficiente de correlação de 0,979 e um poder de 90%. Essa equação permitiria uma perda de 10%. As amostras de líquor foram coletadas consecutivamente a partir de 37 pacientes HIV positivos atendidos no Hospital de Clínicas de Porto Alegre/RS. Os métodos foram processados de acordo com o proposto pelo fabricante para utilização com amostras de plasma. Pequenas modificações foram necessárias no teste em estudo (m2000rt) para neutralizar qualquer diferença metodológica e evitar vieses de mensuração: o congelamento das amostras foi realizado a -20ºC até o momento da análise. O ensaio COBAS v2.0 foi utilizado como referência, uma vez que é o método mais utilizado. Foram realizadas análises quantitativas com resultados que estavam dentro da faixa linear em ambos os métodos (n = 18). Para tornar os métodos comparáveis, adotou-se o limite de detecção do ensaio m2000rt para ambos (40 cp/mL ou 1,60 log10 cp/mL). Os resultados abaixo do limite de detecção foram apresentados como uma variável categórica, uma vez que não são quantificáveis. O coeficiente de correlação de Pearson foi utilizado para comparar os métodos. A normalidade das variáveis foi então resumida calculando o viés estimado pela diferença média "đ " e o desvio padrão das diferenças realizadas pelo teste t para amostras pareadas. Com base na falta de normalidade dos métodos, o grau de concordância dos resultados das cargas virais de HIV foi analisado pelo índice Kappa. Esse estudo foi aprovado pelo comitê de ética do Hospital de Clínicas de Porto Alegre (RS)/ Brasil, registrado na Plataforma Brasil como sendo CAAE: 35072214.7.0000.532. Conclusão Em conclusão, o teste m2000rt que foi modificado para este ensaio mostrou boa concordância e correlação com o teste mais utilizado nesse contexto e pode ser considerado um método alternativo com resultados semelhantes ao COBAS v2.0 e baixos custos na quantificação da carga viral do HIV no líquor. Sugerimos, principalmente em locais onde este método está prontamente disponível com uma relação custo-benefício aceitável, que o exame m2000rt deva ser realizado.Introduction Growing concern about possible consequences of viral replication in the central nervous system shows the need for HIV detection in the cerebral compartment. The real time PCR test developed by Abbott, Abbott RealTime m2000 HIV-1 (m2000rt) quantifies HIV viral load in blood samples effectively and with low costs Brazil. It is the standard method by the Brazilian Ministry of Health, but it has never been utilized to measure HIV in cerebrospinal fluid samples. The assay produced by Roche, COBAS TaqMan HIV-1, version 2 (COBAS v2.0) is the real-time PCR method that has been widely used to detect HIV viral load in cerebral compartment. However, this method has not yet been validated for this purpose and its cost may be a limitation in several regions in the world with low resources. Objective Taking under consideration that there was no standard methodology for this specific situation (detecting HIV in cerebrospinal fluid), we conducted this study to compare the performances of the m2000rt and COBAS v2.0 assays, to propose an alternative and low-cost method to more used in this context (COBAS v2.0). Methods The study was conducted from May 2015 to July 2016. The sample size calculation was based on data from a pilot trial that revealed that a minimum of 37 samples would be needed to detect a difference of 0.20 log10 in viral load, with a correlation coefficient of 0.979 and a 90% power. This equation would allow a 10% lost. CSF samples were collected consecutively from 37 HIV-positive patients seen at Hospital de Clínicas, Porto Alegre, RS. Methods were processed according to proposed by the manufacturer for utilization with plasma samples. Small modifications were necessary in the study test (m2000rt) to neutralize any methodological differences, thus avoiding measurement bias: the freezing of samples was carried out at -20ºC until the moment of the analysis. The COBAS v2.0 test was used as a reference since it is the most commonly used method. Quantitative analyzes were performed with results that were within the linear range in both methods (n=18). To make the methods comparable, the detection limit of the m2000rt assay for both (40 cp/mL or 1.60 log10 cp/mL) was adopted. The results below the limit of detection were presented as a categorical variable, since they are not quantifiable. The Pearson correlation coefficient was used to compare methods. The normality of the variables was then summarized calculating the estimated bias by the mean difference "đ" and standard deviation of the differences performed by t test for paired samples. Based on the lack of normality of the methods, the degree of agreement of the HIV viral load results was analyzed by the Kappa index. This study was approved by the Hospital de Clínicas of Porto Alegre (southern Brazil) Ethics Review Board, registered in the Brazil Platform as CAAE: 35072214.7.0000.532. Conclusion In conclusion, the m2000rt test that was modified for this trial showed good agreement and correlation with the most used test in this context and can be considered an alternative method with similar results to COBAS v2.0 and low costs in the HIV viral load quantification in cerebrospinal fluid. We suggest, especially in places where this method is readily available with an acceptable cost-benefit ratio, that the m2000rt exam should be performed.
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Schöldström, Degenne Jacob. "Development of a new quantitative PCR analysis method for HIV-1." Thesis, KTH, Proteinvetenskap, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-299883.
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På grund av planerat tillverkningsstopp av instrumenten som används idag på Octapharma AB, så syftar detta projekt till nyutvecklingen av en kvantitativ PCR analysmetod för detektion av HIV-1 i human blodplasma, genom TaqMankemi. Projektet inkluderade design, testning och utvärdering av olika set av primer och probe sekvenser. För att säkerställa specificiteten hos metoden designades primrarna och proberna för att vara komplementära till olika konservativa regioner av HIV-1:s genom. Primer och probe set:en (P/P) testades både individuellt och kombinerat i spädningsserier och genotypspaneler. Analyserna visade att det inte fanns någon korrelation mellan felmatchning av P/P och referenssekvenser hos subtyper, och detektionsnivå. När P/P testades i kombination hittades falsk-positiva signaler i negativa kontroller (4 falsk-positiva signaler; n= 106). Detta åtgärdades genom att utesluta specifika prober(0 falsk-positiva signaler; n= 152)(p = 0,030, Fisher’s exact test). Kombinationen av primer och prober, med någraprober uteslutna, hade en högre detektionsnivå för HIV-1 subtyper än de individuella set:en (29 positiva prover vs 23.5; n= 64), och lyckadesäven detektera åtminstone ett positivt prov hos varje subtyp A, B, C, D, AE, F, G och H. Avsaknaden av korrelation mellan felmatchning av P/P och detektionsnivå, visar på att orsaken av den suboptimala detektionsnivån av subtyper var inte på grund av antalet felmatchningar mellan primer och probe set:en till målsekvenserna. Den högre specificiteten vid kombination av P/P indikerar även att primrar och prober som riktar in sig på olika regioner avHIV-1 genomet ytterligare ökar specificiteten och detektionsnivån hos metoden.As a result of future instrument discontinuation by Octapharma AB’s manufacturers, this project sought to develop a new quantitative PCR analysis method for the detection of HIV-1 in human blood plasma using TaqMan chemistry. The project included the design, testing and statistical analysis of different sets of novel primer and probe sequences. To ensure specificity, the primer and probe sequences were designed to target conserved regions of the HIV-1 genome. The primer and probe sets were tested both individually and in combination in dilution series and genotype tests. Linear regression analyses showed no correlation between mismatches between the primer and probe sets and the subtype reference sequences tested, and detection rate. When the primer and probe setswere tested in combination, false positive signals were obtained in negative control samples (4 false positive signals; n= 106). However, this obstacle was overcome by the omission of certain probes, resulting in no false positive signals (0 false positive signals; n= 152)(p = 0.030, Fisher’s exact test).The combination of primer and probe sets, with certain probes omitted, had an increased HIV-1 subtype detection rate compared to the individual P/P (29 positive samples detected versus 23.5; n= 64), and were also able to detect at least one positive sample from each of the HIV-1 subtypes A, B, C, D, AE, F, G, and H. The absence of correlation between primer and probe set mismatch and detection rate, suggests that the cause of the suboptimal detection rate of the subtypes was not the result of primer and probe set mismatch to the target sequences. The increased subtype detection rate upon combining the P/P also indicates that targeting different region of the HIV-1 genome further improves the detection rate and specificity of the method.
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Cameron, Melissa A. "Evaluation of TaqMan Real-Time PCR for the Detection of Viable Cryptosporidium parvum Oocysts in Environmental Water Samples." [Tampa, Fla.] : University of South Florida, 2007. http://purl.fcla.edu/usf/dc/et/SFE0002027.
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McDonald, Kristina Marie. "The Development of a Dual Target Mycoplasma bovis TaqMan real-time PCR System for the Rapid Analysis of Bovine Semen." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1345047775.
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Scheer, Carola [Verfasser], H. [Gutachter] Einsele, H. [Gutachter] Hof, and M. [Gutachter] Ruhnke. "Nachweis von Aspergillus-fumigatus-Infektionen aus dem Respirationstrakt mittels TaqMan-PCR / Carola Scheer ; Gutachter: H. Einsele, H. Hof, M. Ruhnke." Berlin : Humboldt-Universität zu Berlin, 2006. http://d-nb.info/1208076965/34.
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Björklund, Kristofer. "Evaluation and optimization of four real-time PCRs, using TaqMan-probes, for detection of and discrimination between barley, oat, rye and wheat." Thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9171.
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Coeliac disease is a chronic inflammatory disease treated with a gluten-free diet, excluding barley, rye and wheat. Hence, there is a demand for methods able to detect gluten in foods in order to ensure correct labeling of products. According to the Codex Alimentarius Commission, 20ppm gluten is the maximum amount allowed in food labeled gluten-free.
PCR can detect DNA from cereals in food. Four real-time PCR-systems,
using TaqMan®-probes for detection of barley, oat, rye and wheat were optimized and evaluated. Evaluations were carried out using seeds. Primers were targeted to genes coding for prolamines, seed storage proteins. PCR-systems targeted to barley, oat and wheat were shown to be specific for the cereals corresponding to each system. The system targeted to rye showed cross-reactions with durum wheat and spelt wheat. Detection limits were 50pg, corresponding to <10 haploid genome copies for each cereal. All systems were able to detect 250ppm amounts of DNA, most likely even smaller amounts are detectable. All systems showed an amplification efficiency of ≥95%.
Systems for detection of barley, oat and wheat are ready for further evaluation, using food products as samples. The rye system however, needs to be re-designed before further evaluation can take place.
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Rebollo, González René, and Benitez Jonnathan G. Santillan. "“Validación de la expresión génica de NFκB y 18s empleando sondas TaqMan® en muestras de linfocitos de pacientes con Artritis Reumatoide”." Tesis de Licenciatura, Medicina-Quimica, 2014. http://ri.uaemex.mx/handle/123456789/14457.
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Whiteman, Erin Tuxford. "The development of a real-time PCR multiplex TaqMan™ assay for vertebrate class in non-human DNA samples for forensic science." Thesis, Whiteman, Erin Tuxford (2021) The development of a real-time PCR multiplex TaqMan™ assay for vertebrate class in non-human DNA samples for forensic science. Masters by Research thesis, Murdoch University, 2021. https://researchrepository.murdoch.edu.au/id/eprint/64836/.
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Non-human DNA (e.g. animal DNA) has become an increasingly useful tool to forensic science, aiding in linking suspects, victims, and crime scenes (24). However, due to many complexities (eg. the potential for non-human DNA to be from any species and the challenges of distinguishing DNA mixtures) and limited resources, this kind of DNA often remains unanalysed in investigations. Presumptive identification of blood is not always human specific and current DNA testing usually stops when DNA samples fail human DNA testing protocols. An efficient way to routinely detect and confirm the presence of animal DNA within samples is therefore needed. This research outlines the framework and development of a universal Vertebrate Class assay for the detection of Mammalian (mammal), Aves (bird), Fish, and Reptilia (reptile) DNA. The assay functions as a real-time PCR TaqMan multiplex assay, with an internal positive control. Primers and probes have been designed with Geneious Prime, targeting the mitochondrial genome, specifically regions of the cytochrome b gene (Cyt b) and cytochrome c oxidise subunit 1 gene (COX1) (4, 25-29). Multistage testing was conducted (eg. primer and probe screening and selection, SYBR green testing and TaqMan multiplex testing). Varied success was reported for the identification of Mammalian, Aves and Fish DNA samples across all multistage laboratory testing processes. The Reptilia probe was not able to be tested at this time due to unforeseen delays. With necessary further testing and validation, this will be the first Vertebrate Class assay design with potential capabilities of detecting Mammalian, Aves, Fish, and Reptilia classes simultaneously, providing both universal and specific detection in one convenient and rapid assay.
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Martins, Fábio Tadeu Arrojo 1989. "Otimização de rastreamento simultâneo das principais mutações envolvidas na surdez neurossensorial não-sindrômica utilizando a plataforma TaqMan 'MARCA REGISTRADA' OpenArray 'TRADE MARK' Genotyping." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316700.
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Orientador: Edi Lúcia SartoratoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A perda auditiva é a deficiência sensorial mais frequente em humanos, atingindo aproximadamente 10% de toda a população mundial. A restrição da comunicação pela expressão oral resulta em alterações no desenvolvimento cognitivo e psicológico do indivíduo afetado. Em países desenvolvidos, um a cada 500 indivíduos apresenta perda auditiva neurossensorial bilateral profunda/severa. Já nos casos de indivíduos com até 5 anos, a porcentagem é maior, atingindo 0,27% de 1000 indivíduos, número que se torna maior ainda nos casos em jovens, chegando a 0,35%. Dentre as causas da perda auditiva, mais de 60% dos casos de perda auditiva congênita são genéticos. Até o momento já se tem conhecimento de 150 loci e 103 genes envolvidos com a perda auditiva, sendo que a maioria deles apresenta, no mínimo, 20 alterações (mutações de ponto, deleções, inserções, etc.) que podem causar a perda. O gene que apresenta maior número de alterações é o GJB2, codificador da conexina 26, uma proteína relacionada a trocas iônicas intercelulares, mantendo a homeostase de potássio do sistema auditivo, essencial para a audição. Apenas este gene apresenta mais de 302 alterações confirmadas até o presente momento, sendo o principal gene relacionado aos casos de perda auditiva de origem genética. Tendo em vista a grande heterogeneidade clínica e genética da perda auditiva e a importância do diagnóstico molecular correto dos indivíduos que apresentam perda auditiva hereditária, o presente trabalho propôs padronizar um layout para diagnóstico através de genotipagens utilizando uma tecnologia 'high-throughput' baseada em PCR (Polymerase Chain Reaction) em tempo real denominada TaqMan® OpenArrayTM Genotyping. Com esta, foi desenvolvido um layout das placas de genotipagem de OpenArrayTM, sendo possível analisar 32 alterações de 96 indivíduos simultâneamente por placa. Ao todo, foram analisados 376 indivíduos, sendo 94 deles controles ouvintes, totalizando 4 placas em duplicata. Todas as 31 alterações analisadas estavam presentes nos genes nucleares GJB2, GJB6, CRYL1, TMC1, SLC26A4, miR-96, OTOF e nos genes mitocondriais 12S rRNA e MT-TR1. As reações foram validadas posteriormente por técnicas previamente estabelecidas (sequenciamento direto, PCR Multiplex e RFLP-PCR) nos testes utilizados para o diagnótisco molecular da perda auditiva do Laboratório de Genética Humana do Centro de Biologia Molecular e Engenharia Genética (CBMEG) da Universidade Estadual de Campinas (UNICAMP). Ao total, foram realizadas 11.656 reações de genotipagem. Apenas 353 reações falharam, representando, aproximadamente, 3,03% das reações. Dentre as reações que falharam, estavam as amostras de nove indivíduos que não obedeciam aos requisitos mínimos de concentração, pureza e integridade do DNA para a realização dos experimentos. Com isso, calculou-se o rendimento médio das placas de genotipagem utilizando as placas de OpenArrayTM, que apresentou acurácia de, aproximadamente, 96,97%. Tais resultados comprovam a ótima acurácia, o baixo custo e a fácil reprodutibilidade da técnica, tornando este layout customizado para a plataforma TaqMan® OpenArrayTM Genotyping uma ferramenta ótima e confiável a ser empregada nos teste de diagnóstico molecular da perda auditiva no nosso país
Abstract: Hearing loss is the most common sensory deficit in humans, affecting approximately 10% of the entire world population. The restriction of communication by the oral expression results in changes in cognitive and psychological development of the affected individual. In developed countries, one in every 500 individuals has severe/profound bilateral sensorineural hearing loss. In cases of individuals with up to 5 years, the percentage is higher, reaching 0.27% of 1000 individuals, that number becomes even greater in cases where young people, reaching 0.35%. Among all the causes of hearing loss, more than 60% of congenital hearing loss is genetics. So far already aware of 150 loci and 103 genes involved in hearing loss, and most of them have at least 20 changes (point mutations, deletions, insertions, etc.) which may cause the loss. The gene that has the higher number of changes is the GJB2, encoding connexin 26, a protein related to ion exchange intercellular maintaining homeostasis potassium auditory system, essential for hearing. Only this gene has over 302 changes confirmed so far, being the main gene related to cases of hearing loss with genetic origin. Due to the great clinical and genetic heterogeneity of hearing loss and the importance of correct molecular diagnosis of individuals with hereditary hearing loss, this work proposes standardize a layout to the diagnosis by a genotyping technology using a high-throughput technique based on real-time PCR called TaqMan® OpenArrayTM Genotyping. With this, we customized a layout to the OpenArrayTM genotyping plates, being possible to analyze 32 changes of 96 individuals per plate simultaneously. Were analyzed 376 individuals, being 94 of them controls listeners, totaling 4 plates in duplicate. All 31 changes analyzed were present in the nuclear genes GJB2, GJB6, CRYL1, TMC1, SLC26A4, miR-96, OTOF and in the mitochondrial genes 12S rRNA and MT-TR1. Reactions were subsequently validated by previously established techniques (direct sequencing, multiplex PCR and RFLP-PCR), tests used for the molecular diagnostic of the hearing loss at Human Genetics Laboratory of the Center for Molecular Biology and Genetic Engineering (CBMEG), located at State University Campinas (UNICAMP). In total, 11.656 reactions of genotyping were performed using this platform. Only 353 reactions failed, representing approximately 3.03% of the reactions. Among the reactions that failed, were samples of nine individuals who did not meet the minimum concentration, purity and integrity of the DNA for the experiments. With this, was calculated the average income of the OpenArrayTM genotyping plates, which showed an accuracy of approximately 96.97%. These results and the comparative analysis of the costs among OpenArrayTM platform and the others molecular techniques demonstrated the great accuracy, low cost and easy reproducibility of the technique, making this layout customized for the platform TaqMan® OpenArrayTM Genotyping a good and reliable tool to be used in the molecular diagnostic of hearing loss in our country
Mestrado
Genetica Animal e Evolução
Mestre em Genética e Biologia Molecular
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Egli, Christoph. "Quantitative TaqMan RT-PCR for the detection and differentiation of European and North American strains of porcine reproductive and respiratory syndrome virus (PRRSV) /." [S.l.] : [s.n.], 2001. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Andersson, Eva. "TaqMan® Sample-to-SNP Kit™ : evaluation of kit for low-cost and fast preparing of DNA-samples before genotype analysis." Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-105963.
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Genotyping can be used to link genetic variation among individuals to certain diseases or conditions. Some known disorders and states that are dependent on single nucleotide polymorphism (SNPs) are lactose intolerance, venous thrombosis, hereditary hemochromatosis and the difference in sensibility among people to metabolise drugs. In this project a new kit, TaqManÒ Sample-to-SNP KitÔ for extraction of DNA and preparation of the extract for genotyping with real-time PCR and allelic discrimination, was evaluated. QIAamp® DNA Blood Biorobot® MDx Kit was used as the reference method. The purpose of the comparison was to find a method that makes DNA extraction from blood samples cheaper and faster, but with the same reliability as the reference procedure. The results of the evaluation showed a complete agreement of the genotype results between the methods tested, which means that the new method was as reliable as the reference method. The costs of reagents and material would be reduced with 52% if the new method is adopted, that alone would result in a cost reduction of 144 000SEK a year with a sample volume of 650 samples/month. The time for DNA extraction would also be reduced with the new procedure.
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Stupin, Jens [Verfasser]. "Quantitative mRNA-Bestimmung von Zytokinen aus mononukleären Nabelschnurblutzellen nach Geburten von Schwangeren mit Verdacht auf ein Amnioninfektionssyndrom mittels real-time TaqMan RT-PCR / Jens Stupin." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2010. http://d-nb.info/1024865932/34.
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Haddi, Khalid. "Studies on insecticide resistance in tuta absoluta (Meyrick), with special emphasis on characterisation of two target site mechanisms." Doctoral thesis, Università di Catania, 2012. http://hdl.handle.net/10761/1226.
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Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) is a primary pest of tomato plants and is native to South America. Since the first documented European case in 2006, it has spread throughout the Mediterranean basin and North Africa. Larval stages cause direct feeding damage and reductions to both yield and fruit quality. Chemical insecticides have been the main control tools used against T. absoluta, but decreasing efficacy has been attributed to the development of insecticide resistance.
During this study, leaf-dip bioassays were used to quantify responses of five field strains of T. absoluta to insecticides belonging to different chemical classes. The results showed significant variation in susceptibilities to organophosphates and pyrethroids, which are a major class of neurotoxic insecticides and acaricides used extensively over the last decades.
One important mechanism of resistance to pyrethroids, termed knockdown resistance (kdr), has been shown to arise through alterations (point mutations) in the para-type sodium channel protein leading to reduced sensitivity of the insect nervous system to the pyrethroids. Mutations in the Ace gene have also been reported to cause insensitivity to organophosphates.
A combination of PCR-based molecular methods and biochemical assays was used to investigate possible existence of any mutations in these two insecticides targets in several laboratory strains.
Cloning and sequencing of domains II, III, and IV of the T. absoluta sodium channel gene revealed the presence of several kdr mutations previously reported to confer reduced sensitivity in other arthropod species. These included L1014F, M918T, T929I and L925M mutations. Characterisation and sequencing of Ace gene revealed the existence of a single mutation A201S previously related to organophosphate insensitivity in several insect species.
Diagnostic tools that allow detection in individual larvae and adults were developed and used to screen field samples of diverse geographical origin and assess their distribution in global T. absoluta populations.
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Patuelli, Claudia. "Implementation, set up and validation of multiplex qualitative two-step RT-PCR based on TaqMan® method for the diagnosis of viruses in Vitis vinifera L." Bachelor's thesis, Alma Mater Studiorum - Università di Bologna, 2020.
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La viticoltura si trova ad affrontare il problema dalla lotta contro i patogeni che comporta costi ingenti dovuti anche alla sostituzione di piante infette. Le patologie più gravi sono l’Infezione degenerativa, l’Accartocciamento fogliare e il Complesso del legno riccio, tutti causati da infezioni virali.
L’impresa cilena Concha y Toro ha deciso di studiare la presenza dei virus all’interno dei propri vigneti in modo da evitarne la diffusione, applicando la tecnica analitica della Real Time PCR, strategia SYBR® Green e TaqMan®.
L’obiettivo di questa ricerca è di mettere a punto un protocollo che permetta di rilevare la presenza dei virus applicando una strategia multiplex, al fine di ridurre i costi e i tempi di analisi. Inoltre è possibile inserire un controllo interno che assicuri la veridicità di una risposta negativa alla presenza del virus.
Sono state costruite curve standard per le analisi in singleplex dei virus testati dall’azienda e del gene actina di vite, allo scopo di ottimizzare l’analisi. È seguito lo studio delle analisi in duplex prima con l’introduzione del controllo interno e in seguito con test per analizzare combinazioni di virus. Sono state confrontate le efficienze delle curve standard verificando che il valore rimanesse all’interno di un range stabilito e che non ci fosse differenza significativa tra l’analisi in singleplex e la corrispondente in duplex.
Lo stesso è stato ripetuto per un protocollo in triplex.
Sono state studiate la sensibilità e la specificità di ogni analisi in modo da validare il metodo.
I risultati ottenuti hanno dato la possibilità di ottimizzare i protocolli singleplex e confermato la possibilità di introdurre il gene actina come controllo interno nelle analisi.
Sono stati raggiunti ottimi risultati anche in molte delle combinazioni in duplex tra virus.
In futuro sarà necessario approfondire lo studio delle sensibilità e specificità d’analisi in modo da validare il metodo evitando falsi negativi o falsi positivi.
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Bojolly, Daline. "Développement d'une méthode méthodologie de PCR en temps réel pour l'identification et la quantification de trois espèces de thon (Thunnus obesus, Thunnus albacares et Katsuwonus pelamis) dans les produits appertisés." Thesis, Littoral, 2017. http://www.theses.fr/2017DUNK0445/document.
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Le thon obèse (Thunnus obesus), le thon alabore (Thunnus albacares) et le listao (Katsuwonus pelamis) comptent parmi les espèces de thons les plus utilisées en conserve. Lors de la fabrication de conserves de thon, la substitution d'espèce et/ou le mélange de différentes espèces de thon sont interdits par la réglementation européenne. L'authentification des espèces de thon reste complexe à cause du degré de similitude élevé entre les espèces de thon, ou encore, lorsque les caractéristiques morphologiques externes sont éliminées au cours du filetage et lors de la mise en conserve. Par conséquent, des substitutions involontaires ou frauduleuses peuvent se produire. Dans cette étude, le marqueur mitochondrial du gène de la sous-unité 2 de la NADH déshydrogénase a été utilisé pour identifier le thon obèse et le gène de la sous-unité II de la cytochrome c oxydase a été utilisé pour identifier le thon albacore et le listao en utilisant la PCR en temps réel basée sur la technologie TaqMan. Deux méthodes différentes basées sur la qPCR ont été développées pour quantifier le pourcentage de chair de chaque espèce présente au sein d'une boîte de thon. La première a été basée sur la quantification absolue avec standard externe réalisée avec les deux marqueurs. La seconde a été basée sur la quantification relative avec standard externe avec le gène endogène de l'ARN 12S. Sur la base de ces résultats, nous pouvons conclure que notre méthode peut s'appliquer pour quantifier les deux espèces de thon albacore et obèse génétiquement très proches lorsqu'elles sont utilisées dans un mélange binaire en conserveBigeye tuna (Thunnus obesus), yellowfin tuna (Thunnus albacares) and skipjack tuna (Katsuwonus pelanis) are among the most widely used tuna species for canning purposes. Not only substitution but also mixing of tuna species is prohibited by the European regulation for canned tuna products. However, it can be difficult to authenticate the tuna species, due to their high degree of similarity or even when the external morphological characteristics are removed due to filleting before canning. Consequently, involuntary or fraudulent substitutions may occur during the canning process. In this study, the mitochondrial marker from NADH dehydrogenase subunit 2 gene was used to identify bigeye tuna and the mitochondrial marker cytochrome c oxidase subunit II gene was used to identify yellowfin tuna and skipjack tuna, utilizing TaqMan qPCR methodology. Two different qPCR-based methods were developed to quantify the percentage of flesh of each species used for can processing. The first one was based on absolute quantification using standard curves realized with these two markers ; the second one was founded on relative quantification with the universal 12S rRNA gene as the endogenous gene. On the basis of our results, we conclude that our methodology could be applied to authenticate the two closely related tuna species (bigeye tuna and yellowfin tuna) when used in a binary mix in tuna cans
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Kim, Albert H. "Expression Profiling and Functional Validation of MicroRNAs Involved in Schizophrenia and Bipolar Disorder." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/2907.
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MicroRNAs (miRNAs) are a family of small non-coding RNAs that regulate gene expression at both the mRNA and protein levels. MiRNAs have been shown to affect neuronal differentiation, synaptosomal complex localization and synapse plasticity, all functions thought to be disrupted in schizophrenia. We investigated the expression of 667 miRNAs (miRBase v.13) in the prefrontal cortex of individuals with schizophrenia (SZ, N = 35) and bipolar disorder (BP, N =35) using a real-time PCR-based Taqman Low Density Array (TLDA). After extensive QC steps, 441 miRNAs were included in the final analyses. At a FDR of 10%, 22 miRNAs were identified as differentially expressed between cases and controls, 7 dysregulated in SZ and 15 in BP. Using in silico target gene prediction programs, the 22miRNAs were found to target brain-specific genes contained within networks overrepresented for neurodevelopment, behavior, and SZ and BP disease development. Given that miRNAs can bind to their targets with imperfect complementarity, computational prediction of true miRNA:mRNA interactions has been difficult and therefore, functional validation of miRNA:mRNA interactions has been relatively sparse. Thus, it was the goal of this study to demonstrate biological functionality of miRNAs on their targets by evaluating transcriptional and translational levels of gene expression(real-time PCR, western blot) as well as determining miRNA target-site specificity (luciferase reporter gene assays). We investigated two miRNAs, miR-132 and miR-137, both of which have been shown to regulate neuronal function and development, and are believed to be associated with schizophrenia from two distinct avenues of research, miR-132 from expression studies and miR-137 from genetic studies. We demonstrated miR-132 down-regulates NTF3, DISC1, and GRIK5 at the transcript level and down-regulates GRIK5 at the protein level as well. Furthermore, we demonstrated miR-137 down-regulates TCF4, CACNA1C, CDK6, ANK3, and ZNF804A at the transcript level, and down-regulates TCF4, CACNA1C, and CDK6 at the protein level. Going further, we also demonstrated miR-137 binds specifically to target sites in the 3'-UTR of CACNA1C, TCF4, and CDK6, suggesting repression of these genes is directly mediated by miR-137. In total, this study provides strong evidence that miRNA dysregulation may contribute to schizophrenia pathogenesis.
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Lowe, Chinn-woan. "A Quadruplex Real-Time PCR Assay for the Rapid Detection and Differentiation of the Burkholderia pseudomallei Complex: B. mallei, B. pseudomallei, and B. thailandensis." BYU ScholarsArchive, 2013. https://scholarsarchive.byu.edu/etd/6247.
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Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. Although B. thailandensis is generally avirulent in mammals, this species displays very similar phenotypic characteristics to that of B. pseudomallei. Optimal identification of these species remains problematic, due to the difficulty in developing a sensitive, selective, and accurate assay. To date, no real-time, multiplex PCR assay has been developed that can detect and differentiate between B. pseudomallei, B. mallei, and B. thailandensis in a single tube format. Here, we describe the development of such an assay that detects and differentiates the species of the B. pseudomallei complex. A real-time quadruplex qPCR assay, Bcom, was designed to target unique genomic regions of B. pseudomallei, B. mallei, B. thailandensis, and the B. pseudomallei complex that detects and differentiates the three species. A total of 299 isolates within the B. pseudomallei complex was evaluated in this study, as well as 15 near-neighbors and other bacterial species. The results showed that this quadruplex assay was capable of detecting the respective species in a given sample at a sensitivity between 288 fg and 277 pg of genomic DNA. The B. pseudomallei- and B. pseudomallei complex-specific assays tested negative on two presumed B. pseudomallei isolates. In addition, a third presumed B. pseudomallei isolate tested negative by the B. pseudomallei-specific test, but was detected by the B. thailandensis and B. pseudomallei complex-specific assays. After cultural and biochemical characterization, 16s rRNA sequencing, and multiple loci sequencing, it is proposed that B. pseudomallei 34 is B. thailandensis 82172 (Accession No. DQ388536), B. pseudomallei Darwin 175 is Elizabethkingia meningoseptica, and B. pseudomallei 135 is a new strain of B. ubonensis 135.
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Budnjo, Almir. "Gene expression of MAP2K1 and Cyclin D1 in BDII rat model of Endometrial cancer." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-12869.
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Endometrial adenocarcinoma (EAC) is the most frequently diagnosed gynecological cancer of the female genital tract in the Western world. Research studies in EC is difficult to conduct on human tumor samples due to the complex nature of tumor arousal and genetic heterogeneousness in the human population. Therefore, inbred animal models can be promising tools to use in EC research due to similar histopathology and pathogenesis as humans. Studies performed on MAP2K1 and CCND1 has shown that their altered expression play a crucial role in carcinogenesis. CCND1 has been demonstrated to have oncogenic properties when overexpressed in human neoplasias. The aim of this study is to investigate gene expression levels of MAP2K1 and CCND1 in BDII rat model of endometrial adenocarcinoma cells. Quantitative real-time PCR was used to analyze expression levels of MAP2K1 and CCND1 genes in BDII/Han rat model of endometrial cancer cells using TaqMan approach. The differences in gene expression levels of MAP2K1 and CCND1 between pathologically EAC malignant and nonmalignant cells showed an upregulation of MAP2K1 and CCND1 in EAC malignant cells. The analyzed data presented observable mean differences between MAP2K1 and CCND1 in several endometrial cell lines that were examined. Although no statistical significance was reached, an alteration in gene expression levels in malignant and nonmalignant endometrial cells could be observed. Furthermore, this present study shows observable upregulation of MAP2K1 and CCND1 in endometrial carcinoma cells vs. nonmalignant endometrium cells and encourages further investigation of the role of CCND1 and MAP2K genes in endometrial carcinogenesis.
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Vaccaro, Tamara da Silva. "Identificação de mutações no gene da fenilalanina hidroxilase por PCR em tempo real." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2008. http://hdl.handle.net/10183/14245.
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Fenilcetonúria (PKU) é uma doença autossômica recessiva caracterizada pela deficiência da enzima fenilalanina hidroxilase (PAH). Mutações no gene da PAH são responsáveis por PKU e uma grande heterogeneidade alélica é observada em pacientes em todo o mundo. Até o momento, mais de 500 diferentes alterações no gene PAH são encontradas no PAHdb. A maioria dessas mutações são raras e/ou específicas nas populações estudadas. O espectro de mutações de PKU em pacientes do sul do Brasil foi definido anteriormente e 6 mutações (IVS2+5G>C, p.I65T, p.R261X, p.R261Q, p.R408W e IVS12+1G>A) foram identificadas como responsáveis por 63,6% dos alelos mutantes. Este trabalho apresenta uma estratégia baseada no sistema TaqMan® (Applied Biosystems) para uma identificação rápida e específica das mutações listadas acima. Esta estratégia foi aplicada a uma amostra populacional que anteriormente foi caracterizada por diferentes técnicas. Os testes mostraram-se altamente sensíveis e são potencialmente aplicáveis à identificação de amostras de sangue impregnadas em papel filtro, podendo ser utilizados na triagem neonatal.Phenylketonuria (PKU) is an autossomal recessive disease characterized by phenylalanine hydroxylase (PAH) deficiency. Mutations in the PAH gene are responsible for PKU, and an extensive allelic heterogeneity was observed in patients worldwide. Up to date, more than 500 different alterations can be found in PAHdb, the majority being rare mutations and/or specific to a genetic background. Our group was able to define mutation spectrum in PKU patients in Southern Brazil, and identified six mutations, named IVS2+5G>C, p.I65T, p.R261X, p.R261Q, p.R408W and IVS12+1G>A, that are responsible for 63.6% of mutant alleles. This work introduced a strategy, based in the TaqMan® System (Applied Biosystems), for a fast and specific identification of the above mutations. This strategy was applied to a sample population that was previously characterized by different techniques. Assays were shown to be highly sensitive and are potentially applicable to blood spots in order to be used in neonatal screening.
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Sammito, Mariangela. "I miR-720 e miR-1290 sono significativamente sovraespressi nel siero di pazienti con fenotipo grave di sindrome di Tourette: potenziali implicazioni biomolecolari e cliniche." Doctoral thesis, Università di Catania, 2014. http://hdl.handle.net/10761/1530.
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La sindrome di Gilles de la Tourette (GTS) è un disordine neuropsichiatrico ad esordio infantile, caratterizzato da tic motori e vocali causati da contrazioni muscolari involontarie che producono movimenti stereotipati. Nel 60% dei casi la malattia mostra comorbilità con la sindrome da deficit di attenzione e iperattività (ADHD) e nel 40-60% con il disturbo ossessivo compulsivo (OCD). La combinazione delle tre condizioni (TS, ADHD e OCD) è spesso chiamata triade della sindrome di Tourette ed è molto spesso causa di peggioramento nella qualità di vita del paziente se non identificata correttamente. La diagnosi si basa sui criteri proposti dal DSM-V Diagnostic and Statistical Manual of Mental Disorders, 5th edition. Sulla base della sintomatologia clinica e della comorbilità con altri disordini neuropsichiatrici, si può affermare che esiste uno spettro di fenotipi TS, la cui gravità può variare da una manifestazione severa (Severe) TSS (Tourette Syndrome Severe Phenotype) ad una moderata (mild) TSM (Tourette Syndrome Mild Phenotype).
Negli ultimi anni l identificazione di miRNA circolanti in fluidi biologici (siero, plasma o urine) ha aperto una nuova frontiera nella diagnosi precoce non invasiva di patologie neoplastiche, degenerative e neurodegenerative. Con l'obiettivo di analizzare le basi biomolecolari della TS, il nostro gruppo di ricerca ha caratterizzato il trascrittoma di 754 miRNAs nel siero di pazienti affetti da TSS, tramite TaqMan® Low Density Array (TLDA). Queste ricerche hanno inoltre consentito di individuare potenziali biomarkers diagnostici, da associare alla tradizionale indagine effettuata tramite il DSM-V. I microRNA da noi identificati come differenzialmente espressi sono stati ulteriormente validati in singoli saggi TaqMan assay, sia nella coorte di pazienti TSS precedentemente analizzata, che anche in una coorte di pazienti TSM, allo scopo di identificare uno o più potenziali marcatori utili per la diagnosi differenziale tra pazienti TS con fenotipo clinico di differente gravità (severe oppure mild).
Le nostre indagini sperimentali ci hanno portato all identificazione di due miR, il miR-720 ed il miR-1290, i cui livelli d espressione sono più elevati nei pazienti TS rispetto ai controlli in maniera statisticamente significativa. Inoltre, il miR-720 risulta statisticamente discriminante tra il fenotipo TSS e TSM e dunque è da considerarsi come marcatore molecolare nella discriminazione fenotipica nell'ambito della sindrome di Gilles de la Tourette.
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Hahn, Andreas [Verfasser]. "Analyse der Sensitivität und Spezifität einer kommerziellen und zweier in-house Taqman Real-time-multiplex-PCR-Assays zur Diagnose von enteropathogenen, enterotoxischen und enteroaggregativen Escherichia coli ohne Verwendung eines Goldstandards / Andreas Hahn." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2018. http://d-nb.info/1170876250/34.
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Saracino, Ilaria Maria <1980>. "Metodi biochimici e biomolecolari applicati alla medicina clinica: rendimento della real-time TaqMan PCR per la valutazione della resistenza alla claritromicina in H.pylori e tests fecali nella diagnorstica del carcinoma colo-rettale: M2PK, calprotectina e FOBT." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2011. http://amsdottorato.unibo.it/3499/1/saracino_ilariamaria_tesi.pdf.
Full textAbstract:
1) Background: The most common methods to evaluate clarithromycin resistance is the E-Test, but is time consuming. Resistance of Hp to clarithromycin is due to point mutations in the 23S rRNA.
Eight different point mutations have been related to CH resistance, but the large majority of the clarithromycin resistance depends on three point mutations (A2142C, A2142G and A2143G).
A novel PCR-based clarithromycin resistance assays, even on paraffin-embedded biopsy specimens, have been proposed.
Aims: to assess clarithromycin resistance detecting these point mutation (E-Test as a reference method);secondly, to investigate relation with MIC values.
Methods: Paraffin-embedded biopsies of patients Hp-positive were retrieved. The A2142C, A2142G and A2143G point mutations were detected by molecular analysis after DNA extraction by using a TaqMan real-time PCR.
Results: The study enrolled 86 patients: 46 resistant and 40 sensible to CH. The Hp status was evaluated at endoscopy, by rapid urease test (RUT), histology and hp culture. According to real-time PCR, 37 specimens were susceptible to clarithromycin (wild type dna) whilst the remaining 49 specimens (57%) were resistant. A2143G is the most frequent mutation. A2142C always express a resistant phenotype and A2142G leads to a resitant phenotype only if homozigous.
2) Background: Colonoscopy work-load for endoscopy services is increasing due to colorectal cancer prevention. We tested a combination of faecal tests to improve accuracy and prioritize the access to colonoscopy.
Methods: we tested a combination of fecal tests (FOBT, M2-PK and calprotectin) in a group of 280 patients requiring colonoscopy.
Results: 47 patients had CRC and 85 had advanced adenoma/s at colonoscopy/histology. In case of single test, for CRC detection FOBT was the test with the highest specificity and PPV, M2-PK had the highest sensitivity and higher NPV.
Combination was more interesting in term of PPV. And the best combination of tests was i-FOBT + M2-PK.
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Saracino, Ilaria Maria <1980>. "Metodi biochimici e biomolecolari applicati alla medicina clinica: rendimento della real-time TaqMan PCR per la valutazione della resistenza alla claritromicina in H.pylori e tests fecali nella diagnorstica del carcinoma colo-rettale: M2PK, calprotectina e FOBT." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2011. http://amsdottorato.unibo.it/3499/.
Full textAbstract:
1) Background: The most common methods to evaluate clarithromycin resistance is the E-Test, but is time consuming. Resistance of Hp to clarithromycin is due to point mutations in the 23S rRNA.
Eight different point mutations have been related to CH resistance, but the large majority of the clarithromycin resistance depends on three point mutations (A2142C, A2142G and A2143G).
A novel PCR-based clarithromycin resistance assays, even on paraffin-embedded biopsy specimens, have been proposed.
Aims: to assess clarithromycin resistance detecting these point mutation (E-Test as a reference method);secondly, to investigate relation with MIC values.
Methods: Paraffin-embedded biopsies of patients Hp-positive were retrieved. The A2142C, A2142G and A2143G point mutations were detected by molecular analysis after DNA extraction by using a TaqMan real-time PCR.
Results: The study enrolled 86 patients: 46 resistant and 40 sensible to CH. The Hp status was evaluated at endoscopy, by rapid urease test (RUT), histology and hp culture. According to real-time PCR, 37 specimens were susceptible to clarithromycin (wild type dna) whilst the remaining 49 specimens (57%) were resistant. A2143G is the most frequent mutation. A2142C always express a resistant phenotype and A2142G leads to a resitant phenotype only if homozigous.
2) Background: Colonoscopy work-load for endoscopy services is increasing due to colorectal cancer prevention. We tested a combination of faecal tests to improve accuracy and prioritize the access to colonoscopy.
Methods: we tested a combination of fecal tests (FOBT, M2-PK and calprotectin) in a group of 280 patients requiring colonoscopy.
Results: 47 patients had CRC and 85 had advanced adenoma/s at colonoscopy/histology. In case of single test, for CRC detection FOBT was the test with the highest specificity and PPV, M2-PK had the highest sensitivity and higher NPV.
Combination was more interesting in term of PPV. And the best combination of tests was i-FOBT + M2-PK.
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Vasconcellos, Jaíra Ferreira de. "Análise da expressão do oncogene PML-RARalfa por PCR quantitativa em pacientes com leucemia aguda promielocítica." Universidade Federal de Pernambuco, 2006. https://repositorio.ufpe.br/handle/123456789/6478.
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Previous issue date: 2006O gene de fusão PML-RARα é o mais freqüente marcador molecular da Leucemia Aguda Promielocítica (LAP). Com o objetivo de comparar os métodos moleculares qualitativo e quantivo, avaliar os níveis de expressão do gene correlacionar com características biológicas foram de pacientes com LAP ao diagnostico e pós-consolidação. O RNA total foi extraído a partir de e o cDNA sintetizado por RT-PCR. O gene ABL foi utilizado como controle constitutivo e a análise quantitativa realizada por curva padrão. Ao diagnóstico houve concordância dos métodos na detecção do gene PML-RARα e o coeficiente normalizado de expressão foi 55,3. Após a consolidação do tratamento apenas um paciente apresentou a expressão do gene PML-RARα que foi detectado pela PCR quantitativa. Não foram encontradas diferenças significantes na análise de associação dos níveis de expressão do gene PML-RARα com carocteristicas biológicas. Os resultados permitiram estimar o nível de expressão do gene PML-RARα no grupo estudado e o estabelecimento de parâmetros para o seguimento de pacientes com LAP
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Gómez, Marco Francesc. "Integrated Pest Management of Aphis spiraecola (Hemiptera: Aphididae) in clementines: enhancing its biological control." Doctoral thesis, Universitat Politècnica de València, 2015. http://hdl.handle.net/10251/53732.
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[EN] Aphis spiraecola Patch. (Hemiptera: Aphididae) is a key pest of clementines. Biological control of A. spiraecola is still poorly known and efforts were based on the use and conservation of parasitoids but it did not success. With all this said, the aims of this thesis were: i) to disentangle the reasons behind the low parasitism of A. spiraecola; ii) to determine when and how predators can control A. spiraecola populations; and, finally, iii) to evaluate whether a ground cover of Poaceae plants can enhance the biological control of this aphid in clementines by improving the establishment of its predators.
In the first objective we sampled four orchards and determine the parasitoid complex and parasitism (and hyper-) rates weekly. Binodoxys angelicae Haliday (Hymenoptera: Braconidae) was the unique primary parasitoid emerged from mummies of A. spiraecola. At least six hymenopteran hyperparasitoid species were identified attacking this primary parasitoid: Syrphophagus aphidivorus (Mayr) (Encyrtidae), Alloxysta sp. (Forster) (Figitidae), Asaphes sp. (Walker) (Pteromalidae), Pachyneuron aphidis (Bouché) (Pteromalidae), Dendrocerus sp. (Ratzeburg) (Megaspilidae) and Phaenoglyphis villosa (Hartig) (Figitidae). We developed a DNA-based approach to untangle the structure of the aphid-parasitoid food web in citrus. This methodology confirmed that all six species hyperparasitized B. angelicae And they dominated this food web and they were abundant from the beginning of the season. Thus, hyperparasitism probably explains the low impact of B. angelicae on A. spiraecola populations.
For the second objective we sampled three clementine orchards to determine the effect of aphid predators on A. spiraecola colonies over a three-year period. Life parameters of A. spiraecola colonies varied among the orchards over the three years. The maximum number of aphids and the longevity of A. spiraecola colonies were negatively correlated with the time of first attack by predators. More importantly, the percentage of shoots occupied by A. spiraecola (damages) remained below or close to the intervention threshold when colonies were attacked prior to ~200 degree days (DD) since the beginning of the aphid colonization. These results suggest that: i) the presence of predators at the beginning of the season should be considered to develop new intervention thresholds and ii) biological control programs should promote the early presence of predators in clementine orchards.
To promote the early presence of predators in clementine orchards, in the third objective we evaluated ground cover management. This ground cover management may provide alternative preys to natural enemies. The effect of a sown ground cover (based on Poaceae plants) on the biological control of A. spiraecola was evaluated in four orchards with ground cover management compared with four orchards with bare soil management. This sown Poaceae cover coexists with a complex of wild plants that might also affect biological control of A. spiraecola. Finally, we compared the presence of A. spiraecola and its natural enemies in these orchards. While Poaceae plants represented ~66% of the ground cover, the rest of the cover comprised mainly Malva sp. (13%), Oxalis sp. (5%) and Sonchus sp. (2%). Poaceae plants harbored aphids which appeared sooner in the system than citrus aphids. These aphids serve as alternative prey/hosts for natural enemies. By contrast, Malva sp. and Sonchus sp. harbored aphids with potential to become citrus pest. Although these wild plants may act as reservoirs for A. spiraecola as well as other aphid species that can disrupt the biocontrol services of natural enemies, overall, the sown cover was effective in terms of biological control of A. spiraecola in the citrus canopy. It promoted the early presence of predators in citrus canopies. These attacks resulted in satisfactory aphid control, because citrus orchards with ground cover never exceeded the aphid economic threshold.[ES] Aphis spiraecola Patch. (Hemiptera: Aphididae) es una de las plagas claves en el cultivo de clementinos. Los esfuerzos realizados hasta la fecha se han centrado en el uso y conservación de parasitoides aunque se desconocen las causas de su baja eficacia. Por todo ello, los objetivos de esta tesis han sido i) desentrañar las razones por las que se dan bajos niveles de parasitismo de A. spiraecola ii) determinar cuándo y cómo los depredadores pueden controlar las poblaciones de A. spiraecola y finalmente iii) determinar si una cubierta de poáceas puede mejorar el control biológico de este pulgón en clementinos mediante la mejora en el establecimiento de sus depredadores. En el primer objetivo se muestrearon semanalmente cuatro parcelas y se identificó el complejo de parasitoides y las tasas de parasitismo (e hiperparasitismo). Los porcentajes de parasitismo fueron bajos (~menos del 5%) y Binodoxys angelicae Haliday (Hymenoptera: Braconidae) fue el único parasitoide primario emergido de las momias de A. spiraecola. Se identificaron al menos seis especies de hiperparasitoides atacando este parasitoide primario: Syrphophagus aphidivorus (Mayr) (Encyrtidae), Alloxysta sp. (Forster) (Figitidae), Asaphes sp. (Walker) (Pteromalidae), Pachyneuron aphidis (Bouché) (Pteromalidae), Dendrocerus sp. (Ratzeburg) (Megaspilidae) y Phaenoglyphis villosa (Hartig) (Figitidae). Se desarrolló un método basado en la detección de ADN con el cual se confirmó que todas las especies de hiperparasitoides hiperparasitan B. angelicae. Los hiperparasitoides dominaron esta red trófica y fueron abundantes desde el inicio de la estación. De este modo, el hiperparasitismo probablemente explica el bajo impacto que B. angelicae tiene sobre las poblaciones de A. spiraecola. Para el segundo objetivo se muestrearon tres campos de clementinos donde se determinó el efecto de los depredadores en las colonias de A. spiraecola. Los parámetros de vida de las colonias de A. spiraecola variaron entre los diferentes cultivos los tres años. El máximo número de pulgones y la longevidad de las colonias de A. spiraecola se correlacionaron negativamente con el momento del primer ataque del depredador a la colonia. Cabe destacar que el porcentaje de brotes ocupados por A. spiraecola permaneció por debajo o cerca del umbral de tratamiento cuando las colonias fueron atacadas antes de los 200 grados días (GD) desde el inicio de formación de la colonia. Estos resultados sugieren: i) la presencia de depredadores al inicio de la temporada de pulgón debes ser considerado para el desarrollo de nuevos umbrales de tratamiento y ii) los programas de control biológico deben promover el adelanto de la presencia de depredadores en los campos de clementinos. Para promover la presencia anticipada de depredadores en los campos de clementinos, como tercer objetivo se evaluó el manejo de cubiertas vegetales a base de poáceas. Con este manejo se persigue aportar presas alternativas para los enemigos naturales de A. spiraecola. Para ello, se compararon cuatro campos de cítricos con cubierta vegetal frente a cuatro con suelo desnudo. En los campos con cubierta sembrada apareció además de las poáceas sembradas, un complejo de plantas salvajes que podrían afectar también el control biológico de A. spiraecola. Las poáceas representaron un 66% de la cubierta vegetal. Las poáceas y Oxalis sp. albergaron respectivamente pulgones estenófagos de plantas poáceas y Macrosiphum euphorbiae Thomas (Hemiptera: Aphididae). Estas especies de pulgones aparecieron más pronto en el ecosistema que los pulgones de cítricos y sirvieron como presas/hospederos alternativos para los enemigos naturales. Al contrario, Malva sp. y Sonchus sp. albergaron especies de pulgón que podrían ser potenciales plagas de cítricos. El efecto total de la cubierta sembrada resultó positivo para el control de A. spiraecola. Por lo tanto, las parcelas de cítricos con cubierta vegetal tendieron a no
[CAT] Aphis spiraecola Patch. (Hemiptera: Aphididae) és una de les plagues clau en el cultiu de clementins. Els esforços realitzats fins ara s'han centrat en el us i conservació de parasitoids encara que es desconeix les causes de la seua baixa eficàcia. Tenint en compte estos antecedents, els objectius d'esta tesis foren: i) desentrampar les raons per les quals els parasitoids no són efectius; ii) determinar quan i com els depredadors poden controlar les poblacions d'A. spiraecola; i finalment iii) determinar si una coberta de poàcies pot millorar el control biològic d'este àfid en clementins mitjançant la millora en el establiment del seus depredadors. En el primer objectiu es van mostrejar setmanalment quatre parcel·les i s'identificà el complex de parasitoids i les taxes de parasitisme (i hiperparasitisme). Binodoxys angelicae Haliday (Hymenoptera: Braconidae) va ser l'únic parasitoid primari emergit de les mòmies d'A. spiraecola. Se van identificar al menys sis especies d' hiperparasitoids atacant este parasitoid primari: Syrphophagus aphidivorus (Mayr) (Encyrtidae), Alloxysta sp. (Forster) (Figitidae), Asaphes sp. (Walker) (Pteromalidae), Pachyneuron aphidis (Bouché) (Pteromalidae), Dendrocerus sp. (Ratzeburg) (Megaspilidae) i Phaenoglyphis villosa (Hartig) (Figitidae). Se desenvolupà un mètode basat en la detecció de DNA amb el que es confirmà que totes les especies d'hiperparasitoids hiperparasiten B. angelicae. Els hiperparasitoids dominaren aquesta xarxa tròfica i foren abundants a l'inici de l'estació. Per tant, l'hiperparasitisme podria explicar el baix impacte que B. angelicae té sobre les poblacions d'A. spiraecola. Per al segon objectiu es mostrejaren tres camps de clementins on es determinà l'efecte dels depredadors en les colònies d'A. spiraecola. Els paràmetres de vida de les colònies d'A. spiraecola variaren entre les tres parcel·les els tres anys. El màxim número d'àfids i la longevitat de les colònies d'A. spiraecola es correlacionaren negativament amb el moment del primer atac del depredador a la colònia. Caldria destacar que el percentatge de brots ocupats per A. spiraecola es mantingué per baix o prop del llindar de tractament quan les colònies foren atacades abans dels ~200 graus dia (GD) des de l'inici de formació de la colònia. Estos resultats sugereixen que: i) la presència de depredadors a l'inici de la estació de l'àfid podria ser considerada per al desenvolupament de nous llindars de tractament i ii) els programes de control biològic deurien promoure l'avançament de la presència de depredadors en els camps de clementins. Per promoure la l'avançament de la presència de depredadors en els camps de clementins, com tercer objectiu s'avaluà el maneig de cobertes vegetals basades en poàcies. Amb aquest maneig es persegueix aportar preses alternatives per als enemics naturals d'A. spiraecola. Se compararen quatre camps de clementins amb coberta vegetal front a quatre amb sol nu. En els camps amb coberta sembrada creix, junt a les Poáceas sembrades, un conjunt de plantes salvatges que podrien afectar també el control biològic d'A. spiraecola. S'investigà quines especies de plantes componien la coberta vegetal així com les especies d'àfids que les habitaven. Les poàcies representaren un 66% de la coberta vegetal, sent les plantes salvatges més abundants Malva sp. (13%), Oxalis sp. (5%) i Sonchus sp. (2%). Les poàcies hostejaren àfids estenòfags de poàcies. Estes espècies d'àfid aparegueren més prompte en l'ecosistema que aquells àfids associats a cítrics. Al contrari, Malva sp. i Sonchus sp. hostejaren especies d'àfids que podrien ser potencials plagues de cítrics. Este últim grup pot atenuar l'atac dels enemics naturals a les poblacions d'A. spiraecola que habiten les copes. Encara així, l'efecte total de la coberta sembrada a base de poàcies va resultar positiu per al control d'A. spiraecola. En conseqüència les parcel·les de cítrics am
Gómez Marco, F. (2015). Integrated Pest Management of Aphis spiraecola (Hemiptera: Aphididae) in clementines: enhancing its biological control [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/53732
TESIS
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Theriault, Mylene A. "Development and Validation of Quantitative PCR Assays for DNA-Based Newborn Screening of 22q11.2 Deletion Syndrome, Spinal Muscular Atrophy, Severe Combined Immunodeficiency and Congenital Cytomegalovirus Infection." Thesis, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/30318.
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The development of new high throughput technologies able to multiplex disease biomarkers as well as advances in medical treatments has lead to the recent expansion of the newborn screening panel to include DNA-based targets. Four rare disorders; deletion 22q11.2 syndrome and Spinal Muscular Atrophy (SMA), Severe Combined Immunodeficiency (SCID) and Congenital Cytomegalovirus (CMV), are potential candidates for inclusion to the newborn screening panel within the next few years. The major focus of this study was to determine whether 5’-hydrolysis assays developed for the four distinct disorders with specific detection needs and analytical ranges could be combined on the OpenArray system and in multiplexed qPCR reactions. SNP detection of homozygous SMN1 deletions in SMA, CNV detection in the 22q11.2 critical region, and quantification of the SCID biomarker, T-cell receptor excision circles (TRECs) and CMV were all required for disease confirmation. SMA and 22q11.2 gene deletions were accurately detected using the OpenArray system, a first for the technology. The medium density deletion 22q11.2 multiplex successfully identified deletion carriers having either the larger 3 Mb deletion or the smaller 1.5 Mb deletions. Both TREC and CMV targets were detected but with a decrease in sensitivity when compared to their singleplex counterparts. Lastly, copy number detection of the TBX1 was performed when multiplexed with the TREC assay, without a decrease in detection limit of either assay. Here, we provide proof of principal that qPCR multiplexing technologies are amenable to implementation with a newborn screening laboratory.
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Pinto, Renata Machado. "Estudo de associação entre obesidade na infância e os SNPs TaqIA C32806T do gene DRD2 e G308A do gene TNF-a." Pontifícia Universidade Católica de Goiás, 2014. http://tede2.pucgoias.edu.br:8080/handle/tede/3581.
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The worldwide prevalence of obesity has increased at an alarming rate in all age groups. Although it has been described some rare obesity syndromes caused by mutations in single genes, the highest proportion of obesity results from an interaction between variants in multiple genes (susceptible genotype) and a favorable environment (plenty of calories and lack of physical activity). Classically, it has been studied genes involved in the control of intake, energy expenditure and the metabolism of glucose and lipids. In the last decade the discovery of numerous substances secreted by adipocytes among them hormones and inflammatory cytokines opened a new research front. The “Reward Deficient Syndrome” (RDS) is characterized as a hypodopaminegic state that predisposes to obsessive-compulsive behavior and impulsivity. Obesity is part of the RDS, as the individual overeats as a manner to compensate de dopamine defect. In this context, this study was designed to investigate the relationship between childhood onset obesity and polymorphisms of the tumor necrosis factor alpha (TNF alpha) and the Dopamine D2 Receptor (DRD2). Our research is a case control study that included 105 children, being 55 obese and 50 normal weight (control group). Peripheral blood samples were collected for the determination of lipid profile, glucose and insulin levels, and for the evaluation of the genetic polymorphisms of TNF- gene (G308A) and DRD2 (Taq1 - C32806T), by ARMS-PCR and RFLP-PCR, respectively. Genetic variants of the SNP G308A TNF- failed to confirm a participation in weight gain in children. The A1 allele (T) of the Taq1 polymorphism in the DRD2 gene was associated with an increase in childhood obesity and higher BMI of the parents, corroborating some data of the literature. Our results showed for the first time that the A1 allele is associated with Total Cholesterol 70 mg/dl, minor levels of triglycerides and a relative risk of 1.5 for HOMA ß secretion. Understanding how genetic variations affect the tendency to become or remain obese is an important step in understanding the mechanisms that lead to obesity.
A prevalência mundial da obesidade tem aumentado a um ritmo alarmante em todos os grupos etários. Embora tenham sido descritas algumas síndromes raras de obesidade causadas por mutações em genes individuais, a maior proporção de obesidade resulta de uma interação entre variantes em vários genes (genótipo suscetível) e um ambiente favorável (abundância de calorias e falta de atividade física). Classicamente, tem-se estudado genes envolvidos no controle da ingestão, do gasto de energia e do metabolismo da glicose e lipídios. Na última década a descoberta de numerosas substâncias secretadas pelos adipócitos, entre elas hormônios e citocinas inflamatórias abriu uma nova frente de pesquisa. A "Síndrome de Deficiência de Recompensa" (RDS) é caracterizada como um estado hipodopaminérgico que predispõe a comportamentos obsessivo-compulsivos e impulsividade. A obesidade é parte da RDS, já que o indivíduo come demais como uma forma de compensar o defeito nos níveis de dopamina. Neste contexto, o presente estudo foi desenhado para investigar a relação entre a obesidade de início na infância e polimorfismos do fator de necrose tumoral alfa (TNF- ) e do Receptor D2 de dopamina (DRD2). Nossa pesquisa é um estudo de caso-controle que incluiu 105 crianças, sendo 55 obesas e 50 eutróficas (grupo controle). Amostras de sangue periférico foram colhidas para a determinação do perfil de lipídios, glicemia e os níveis de insulina, e para a avaliação dos polimorfismos do gene do TNF - (G308A) e DRD2 - C32806T), por ARMS - PCR e PCR - RFLP, respectivamente. Variantes genéticas do SNP G308A do TNF- não conseguiu confirmar uma participação no ganho de peso em crianças. O alelo A1 (T) do polimorfismo Taq1A do gene DRD2 foi associado a um aumento da obesidade infantil e IMC superior dos pais, corroborando alguns dados da literatura. Os nossos resultados mostram pela primeira vez que o alelo A1 está associado a colesterol total , a menores níveis de triglicérides, e confere um risco relativo de 1,5 para HOMA ß proteção contra a secreção de insulina prejudicada. A compreensão de como variações genéticas afetam a tendência de tornar-se ou permanecer obeso é um passo importante na compreensão dos mecanismos que levam à obesidade.
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Pinto, Renata Machado. "Estudo de associação entre obesidade na infância e os SNPs TaqIA C32806T do gene DRD2 e G308A do gene TNF-α." Pontifícia Universidade Católica de Goiás, 2014. http://localhost:8080/tede/handle/tede/2375.
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Previous issue date: 2014-03-26The worldwide prevalence of obesity has increased at an alarming rate in all age groups. Although it has been described some rare obesity syndromes caused by mutations in single genes, the highest proportion of obesity results from an interaction between variants in multiple genes (susceptible genotype) and a favorable environment (plenty of calories and lack of physical activity). Classically, it has been studied genes involved in the control of intake, energy expenditure and the metabolism of glucose and lipids. In the last decade the discovery of numerous substances secreted by adipocytes among them hormones and inflammatory cytokines opened a new research front. The Reward Deficient Syndrome (RDS) is characterized as a hypodopaminegic state that predisposes to obsessive-compulsive behavior and impulsivity. Obesity is part of the RDS, as the individual overeats as a manner to compensate de dopamine defect. In this context, this study was designed to investigate the relationship between childhood onset obesity and polymorphisms of the tumor necrosis factor alpha (TNF alpha) and the Dopamine D2 Receptor (DRD2). Our research is a case control study that included 105 children, being 55 obese and 50 normal weight (control group). Peripheral blood samples were collected for the determination of lipid profile, glucose and insulin levels, and for the evaluation of the genetic polymorphisms of TNF- gene (G308A) and DRD2 (Taq1 - C32806T), by ARMS-PCR and RFLP-PCR, respectively. Genetic variants of the SNP G308A TNF- failed to confirm a participation in weight gain in children. The A1 allele (T) of the Taq1 polymorphism in the DRD2 gene was associated with an increase in childhood obesity and higher BMI of the parents, corroborating some data of the literature. Our results showed for the first time that the A1 allele is associated with Total Cholesterol 70 mg/dl, minor levels of triglycerides and a relative risk of 1.5 for HOMA ß secretion. Understanding how genetic variations affect the tendency to become or remain obese is an important step in understanding the mechanisms that lead to obesity.
A prevalência mundial da obesidade tem aumentado a um ritmo alarmante em todos os grupos etários. Embora tenham sido descritas algumas síndromes raras de obesidade causadas por mutações em genes individuais, a maior proporção de obesidade resulta de uma interação entre variantes em vários genes (genótipo suscetível) e um ambiente favorável (abundância de calorias e falta de atividade física). Classicamente, tem-se estudado genes envolvidos no controle da ingestão, do gasto de energia e do metabolismo da glicose e lipídios. Na última década a descoberta de numerosas substâncias secretadas pelos adipócitos, entre elas hormônios e citocinas inflamatórias abriu uma nova frente de pesquisa. A "Síndrome de Deficiência de Recompensa" (RDS) é caracterizada como um estado hipodopaminérgico que predispõe a comportamentos obsessivo-compulsivos e impulsividade. A obesidade é parte da RDS, já que o indivíduo come demais como uma forma de compensar o defeito nos níveis de dopamina. Neste contexto, o presente estudo foi desenhado para investigar a relação entre a obesidade de início na infância e polimorfismos do fator de necrose tumoral alfa (TNF- ) e do Receptor D2 de dopamina (DRD2). Nossa pesquisa é um estudo de caso-controle que incluiu 105 crianças, sendo 55 obesas e 50 eutróficas (grupo controle). Amostras de sangue periférico foram colhidas para a determinação do perfil de lipídios, glicemia e os níveis de insulina, e para a avaliação dos polimorfismos do gene do TNF - (G308A) e DRD2 - C32806T), por ARMS - PCR e PCR - RFLP, respectivamente. Variantes genéticas do SNP G308A do TNF- não conseguiu confirmar uma participação no ganho de peso em crianças. O alelo A1 (T) do polimorfismo Taq1A do gene DRD2 foi associado a um aumento da obesidade infantil e IMC superior dos pais, corroborando alguns dados da literatura. Os nossos resultados mostram pela primeira vez que o alelo A1 está associado a colesterol total , a menores níveis de triglicérides, e confere um risco relativo de 1,5 para HOMA ß proteção contra a secreção de insulina prejudicada. A compreensão de como variações genéticas afetam a tendência de tornar-se ou permanecer obeso é um passo importante na compreensão dos mecanismos que levam à obesidade.
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Heslop, Karen Ruth. "Binocular rivalry and visuospatial ability in individuals with schizophrenia." Thesis, Queensland University of Technology, 2012. https://eprints.qut.edu.au/59610/1/Karen_Heslop_Thesis.pdf.
Full textAbstract:
Visual abnormalities, both at the sensory input and the higher interpretive levels, have been associated with many of the symptoms of schizophrenia. Individuals with schizophrenia typically experience distortions of sensory perception, resulting in perceptual hallucinations and delusions that are related to the observed visual deficits. Disorganised speech, thinking and behaviour are commonly experienced by sufferers of the disorder, and have also been attributed to perceptual disturbances associated with anomalies in visual processing. Compounding these issues are marked deficits in cognitive functioning that are observed in approximately 80% of those with schizophrenia. Cognitive impairments associated with schizophrenia include: difficulty with concentration and memory (i.e. working, visual and verbal), an impaired ability to process complex information, response inhibition and deficits in speed of processing, visual and verbal learning. Deficits in sustained attention or vigilance, poor executive functioning such as poor reasoning, problem solving, and social cognition, are all influenced by impaired visual processing. These symptoms impact on the internal perceptual world of those with schizophrenia, and hamper their ability to navigate their external environment. Visual processing abnormalities in schizophrenia are likely to worsen personal, social and occupational functioning.
Binocular rivalry provides a unique opportunity to investigate the processes involved in visual awareness and visual perception. Binocular rivalry is the alternation of perceptual images that occurs when conflicting visual stimuli are presented to each eye in the same retinal location. The observer perceives the opposing images in an alternating fashion, despite the sensory input to each eye remaining constant. Binocular rivalry tasks have been developed to investigate specific parts of the visual system. The research presented in this Thesis provides an explorative investigation into binocular rivalry in schizophrenia, using the method of Pettigrew and Miller (1998) and comparing individuals with schizophrenia to healthy controls. This method allows manipulations to the spatial and temporal frequency, luminance contrast and chromaticity of the visual stimuli. Manipulations to the rival stimuli affect the rate of binocular rivalry alternations and the time spent perceiving each image (dominance duration). Binocular rivalry rate and dominance durations provide useful measures to investigate aspects of visual neural processing that lead to the perceptual disturbances and cognitive dysfunction attributed to schizophrenia. However, despite this promise the binocular rivalry phenomenon has not been extensively explored in schizophrenia to date.
Following a review of the literature, the research in this Thesis examined individual variation in binocular rivalry. The initial study (Chapter 2) explored the effect of systematically altering the properties of the stimuli (i.e. spatial and temporal frequency, luminance contrast and chromaticity) on binocular rivalry rate and dominance durations in healthy individuals (n=20). The findings showed that altering the stimuli with respect to temporal frequency and luminance contrast significantly affected rate. This is significant as processing of temporal frequency and luminance contrast have consistently been demonstrated to be abnormal in schizophrenia.
The current research then explored binocular rivalry in schizophrenia. The primary research question was, "Are binocular rivalry rates and dominance durations recorded in participants with schizophrenia different to those of the controls?" In this second study binocular rivalry data that were collected using low- and highstrength binocular rivalry were compared to alternations recorded during a monocular rivalry task, the Necker Cube task to replicate and advance the work of Miller et al., (2003). Participants with schizophrenia (n=20) recorded fewer alternations (i.e. slower alternation rates) than control participants (n=20) on both binocular rivalry tasks, however no difference was observed between the groups on the Necker cube task.
Magnocellular and parvocellular visual pathways, thought to be abnormal in schizophrenia, were also investigated in binocular rivalry. The binocular rivalry stimuli used in this third study (Chapter 4) were altered to bias the task for one of these two pathways. Participants with schizophrenia recorded slower binocular rivalry rates than controls in both binocular rivalry tasks. Using a ‘within subject design’, binocular rivalry data were compared to data collected from a backwardmasking task widely accepted to bias both these pathways. Based on these data, a model of binocular rivalry, based on the magnocellular and parvocellular pathways that contribute to the dorsal and ventral visual streams, was developed.
Binocular rivalry rates were compared with performance on the Benton’s Judgment of Line Orientation task, in individuals with schizophrenia compared to healthy controls (Chapter 5). The Benton’s Judgment of Line Orientation task is widely accepted to be processed within the right cerebral hemisphere, making it an appropriate task to investigate the role of the cerebral hemispheres in binocular rivalry, and to investigate the inter-hemispheric switching hypothesis of binocular rivalry proposed by Pettigrew and Miller (1998, 2003). The data were suggestive of intra-hemispheric rather than an inter-hemispheric visual processing in binocular rivalry.
Neurotransmitter involvement in binocular rivalry, backward masking and Judgment of Line Orientation in schizophrenia were investigated using a genetic indicator of dopamine receptor distribution and functioning; the presence of the Taq1 allele of the dopamine D2 receptor (DRD2) receptor gene. This final study (Chapter 6) explored whether the presence of the Taq1 allele of the DRD2 receptor gene, and thus, by inference the distribution of dopamine receptors and dopamine function, accounted for the large individual variation in binocular rivalry. The presence of the Taq1 allele was associated with slower binocular rivalry rates or poorer performance in the backward masking and Judgment of Line Orientation tasks seen in the group with schizophrenia.
This Thesis has contributed to what is known about binocular rivalry in schizophrenia. Consistently slower binocular rivalry rates were observed in participants with schizophrenia, indicating abnormally-slow visual processing in this group. These data support previous studies reporting visual processing abnormalities in schizophrenia and suggest that a slow binocular rivalry rate is not a feature specific to bipolar disorder, but may be a feature of disorders with psychotic features generally.
The contributions of the magnocellular or dorsal pathways and parvocellular or ventral pathways to binocular rivalry, and therefore to perceptual awareness, were investigated. The data presented supported the view that the magnocellular system initiates perceptual awareness of an image and the parvocellular system maintains the perception of the image, making it available to higher level processing occurring within the cortical hemispheres. Abnormal magnocellular and parvocellular processing may both contribute to perceptual disturbances that ultimately contribute to the cognitive dysfunction associated with schizophrenia. An alternative model of binocular rivalry based on these observations was proposed.
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Brito, Rodrigo Bernini de. "Relação entre a indução ao ganho de peso decorrente do uso crônico de olanzapina e os SNPs TaqIA no gene DRD2 e G-308A no gene TNF-α." Pontifícia Universidade Católica de Goiás, 2011. http://tede2.pucgoias.edu.br:8080/handle/tede/3579.
Full textAbstract:
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Rodrigo Bernini de Brito.pdf: 1595621 bytes, checksum: eef53307cc2b9e3d872ec9478aeef786 (MD5)Made available in DSpace on 2017-01-31T11:34:35Z (GMT). No. of bitstreams: 1 Rodrigo Bernini de Brito.pdf: 1595621 bytes, checksum: eef53307cc2b9e3d872ec9478aeef786 (MD5) Previous issue date: 2011-12-20
Olanzapine is a second generation antipsychotic that show low incidence of extrapyramidal side effects and has been recommended as the first line drug for the treatment of schizophrenia and is also used in the treatment of bipolar disorder. But it has the weight gain as a side effect which is common in the chronic use of this medicine. A comprehensive review of the literature revealed that olanzapine induces more weight gain than most other antipsychotics, except clozapine. The incidence of weight gain induced by olanzapine and associated diseases such as diabetes and cardiovascular diseases is higher in this group of patients than in the general population. These unwanted side effects have decreased patients' adherence to treatment. Many clinical observations and studies have attempted to elucidate the possible mechanism involved. However, to date, the mechanism underlying the weight gain induced by olanzapine remains unclear. This present study retrospective evaluates 21 patients using olanzapine for a period of 20 to 119 months, compared within the sample, patients who lost weight or remained stable (<7% gain in relation to BMI) group which gained weight at a moderate or severe way during the use of olanzapine (> 7% gain in relation to BMI). We also evaluated the levels of glucose and plasma lipids of all patients. For the group of patients were also analyzed genetic polymorphisms of TaqIA DRD2 gene and G-308A TNF-α gene by PCR-RFL and ARMS-PCR, respectively. TaqIA of genetic polymorphism (C32806T) in the DRD2 gene was correlated with prolonged use of olanzapine with relevant statistical significance in relation to weight gain and biochemical changes observed in plasma of patients. Regarding the genetic variants of the SNP G-308A TNF-α gene, the findings of this study failed to corroborate or refute the findings of other studies.
A olanzapina é um antipsicótico de segunda geração que exibe uma baixa incidência de efeitos colaterais extrapiramidais e tem sido recomendada como fármaco de primeira linha para o tratamento da esquizofrenia e também é utilizada no tratamento do transtorno bipolar, mas tem o ganho de peso como efeito colateral comum no uso crônico deste medicamento. Uma análise abrangente da literatura revelou que a olanzapina induz maior ganho de peso do que a maioria dos outros antipsicóticos, com exceção da clozapina. A incidência de ganho de peso induzido pela olanzapina e doenças associadas, como diabetes e doenças cardiovasculares, é maior entre o grupo de pacientes do que a da população em geral. Estes efeitos secundários indesejados têm diminuído a adesão dos pacientes ao tratamento. Muitas observações clínicas e estudos têm tentado elucidar o possível mecanismo envolvido. No entanto, até o momento, o mecanismo subjacente ao ganho de peso induzido pela olanzapina permanece obscuro. No presente estudo foi realizada uma investigação retrospectiva, que avaliou 21 pacientes em uso de olanzapina por um período de 20 a 119 meses, comparando dentro da amostra, pacientes que perderam peso ou ficaram estáveis (< 7% ganho em relação ao IMC) ao grupo que ganhou peso de forma moderada ou grave durante o uso da olanzapina (>7% ganho em relação ao IMC). Também foram avaliados os níveis de glicose e lipídeos plasmáticos de todos os pacientes. Para o grupo de pacientes ainda foram analisados os polimorfismos genéticos de TaqIAno gene DRD2 e G-308A do gene TNF-α por PCR-RFL e ARMS-PCR, respectivamente. O polimorfismo genéticos da TaqIA (C32806T) no gene DRD2 apresentou relação com o uso prolongado de olanzapina com relevante significância estatística em relação ao ganho de peso e às alterações bioquímicas observadas no plasma dos pacientes. Em relação as variantes genética do SNP G-308A no gene TNF-α, os achados do presente estudo não permitiram corroborar ou refutar as conclusões de outros estudos.
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Brito, Rodrigo Bernini de. "Relação entre a indução ao ganho de peso decorrente do uso crônico de olanzapina e os SNPs TaqIA no gene DRD2 e G-308A no gene TNF-α." Pontifícia Universidade Católica de Goiás, 2012. http://localhost:8080/tede/handle/tede/2354.
Full textAbstract:
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Previous issue date: 2012-12-20Olanzapine is a second generation antipsychotic that show low incidence of extrapyramidal side effects and has been recommended as the first line drug for the treatment of schizophrenia and is also used in the treatment of bipolar disorder. But it has the weight gain as a side effect which is common in the chronic use of this medicine. A comprehensive review of the literature revealed that olanzapine induces more weight gain than most other antipsychotics, except clozapine. The incidence of weight gain induced by olanzapine and associated diseases such as diabetes and cardiovascular diseases is higher in this group of patients than in the general population. These unwanted side effects have decreased patients' adherence to treatment. Many clinical observations and studies have attempted to elucidate the possible mechanism involved. However, to date, the mechanism underlying the weight gain induced by olanzapine remains unclear. This present study retrospective evaluates 21 patients using olanzapine for a period of 20 to 119 months, compared within the sample, patients who lost weight or remained stable (<7% gain in relation to BMI) group which gained weight at a moderate or severe way during the use of olanzapine (> 7% gain in relation to BMI). We also evaluated the levels of glucose and plasma lipids of all patients. For the group of patients were also analyzed genetic polymorphisms of TaqIA DRD2 gene and G-308A TNF-α gene by PCR-RFL and ARMS-PCR, respectively. TaqIA of genetic polymorphism (C32806T) in the DRD2 gene was correlated with prolonged use of olanzapine with relevant statistical significance in relation to weight gain and biochemical changes observed in plasma of patients. Regarding the genetic variants of the SNP G-308A TNF-α gene, the findings of this study failed to corroborate or refute the findings of other studies.
A olanzapina é um antipsicótico de segunda geração que exibe uma baixa incidência de efeitos colaterais extrapiramidais e tem sido recomendada como fármaco de primeira linha para o tratamento da esquizofrenia e também é utilizada no tratamento do transtorno bipolar, mas tem o ganho de peso como efeito colateral comum no uso crônico deste medicamento. Uma análise abrangente da literatura revelou que a olanzapina induz maior ganho de peso do que a maioria dos outros antipsicóticos, com exceção da clozapina. A incidência de ganho de peso induzido pela olanzapina e doenças associadas, como diabetes e doenças cardiovasculares, é maior entre o grupo de pacientes do que a da população em geral. Estes efeitos secundários indesejados têm diminuído a adesão dos pacientes ao tratamento. Muitas observações clínicas e estudos têm tentado elucidar o possível mecanismo envolvido. No entanto, até o momento, o mecanismo subjacente ao ganho de peso induzido pela olanzapina permanece obscuro. No presente estudo foi realizada uma investigação retrospectiva, que avaliou 21 pacientes em uso de olanzapina por um período de 20 a 119 meses, comparando dentro da amostra, pacientes que perderam peso ou ficaram estáveis (< 7% ganho em relação ao IMC) ao grupo que ganhou peso de forma moderada ou grave durante o uso da olanzapina (>7% ganho em relação ao IMC). Também foram avaliados os níveis de glicose e lipídeos plasmáticos de todos os pacientes. Para o grupo de pacientes ainda foram analisados os polimorfismos genéticos de TaqIAno gene DRD2 e G-308A do gene TNF-α por PCR-RFL e ARMS-PCR, respectivamente. O polimorfismo genéticos da TaqIA (C32806T) no gene DRD2 apresentou relação com o uso prolongado de olanzapina com relevante significância estatística em relação ao ganho de peso e às alterações bioquímicas observadas no plasma dos pacientes. Em relação as variantes genética do SNP G-308A no gene TNF-α, os achados do presente estudo não permitiram corroborar ou refutar as conclusões de outros estudos.
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White, Melanie Jade. "Understanding impulsivity : molecular genetic and environmental influences." Thesis, Queensland University of Technology, 2008. https://eprints.qut.edu.au/16578/1/Melanie_J._White_Thesis.pdf.
Full textAbstract:
Features of impulsivity underlie multiple psychological disorders. The body of work examining impulsivity has largely focussed on self-report measurement and has incorporated psychological constructs without reference to the broader biological factors that may influence impulsive behaviour. Two studies were conducted to examine whether environmental stress and genetic status associated with dopaminergic and serotonergic function (DRD2, ANKK1 and 5HT2AR genotypes) were predictive of dimensions of impulsivity and risky behaviour (alcohol use). The two studies used a multi-method approach in a non-clinical community sample of young adults (aged 17-25 years). Dopamine is integral to the two leading theories of impulsive personality, Gray's Reinforcement Sensitivity Theory and Cloninger's Psychobiological model of personality. Dopamine plays a crucial role in reward reinforcement circuits in the brain. The A1 allele of the ANKK1 gene (also referred to as TaqIA of the DRD2 gene region) and the CC genotype of the C957T polymorphism of the DRD2 gene have both been associated with reduced D2 dopamine receptor density in key structures linked to brain reward. In addition, a strong body of evidence implicates their involvement in a number of clinical disorders associated with impulsivity. Serotonin function has also been associated with impulsivity in Cloninger's theory and there is also evidence of associations of two polymorphisms of the 2A serotonin receptor gene (5HT2AR T102C and -1438A/G SNPs) with impulsivity. Acute and chronic forms of stress are also important correlates of impulsive behaviour and the two studies directly examined the relationship between genotype, stress and impulsivity.
Study 1 (N=180) utilised a cross-sectional design and examined interactions between these polymorphisms and chronic stress exposure on key impulsivity dimensions of reward sensitivity, Novelty Seeking and rash impulsiveness. Participants completed psychological questionnaires measuring chronic stress, dimensions of impulsivity, mood and substance use and provided mouth swab samples of buccal mucosal cells for DNA analysis. The study confirmed the association between A1 and CC allelic status and chronic stress being associated with harm avoidance and sensitivity to punishment. This suggests a role for both dopamine and background stress in impulsive behaviour. Study 2 (N=73) built upon this questionnaire research in the laboratory by utilising experimental psychological paradigms of impulsive behaviour and experimentally manipulating acute stress. Study 2 employed a mixed experimental design with a sub-sample of those studied in the cross-sectional sample. These behavioural paradigms included pre- and post- stress induction administration of the Card Arranging Reward Responsiveness Objective Test (capturing behavioural approach in the presence of reward cues, presumed to reflect reward sensitivity) and post-induction delay discounting and response inhibition measures. Study 2 confirmed the role of one of the two dopamine-related polymorphisms, with those with A1+ allelic status demonstrating lower reward responsiveness prior to rest or stress induction, which was overcome in the second administration of this task, independent of environment. A1+ allelic individuals also demonstrated significantly poorer response inhibition independent of stress, further confirming the association between A1+ allelic status and impulsivity. Those with CC allelic status showed an increase in reward responsiveness only in the stress induction condition.
Together, results from the two studies inform the development of a multidimensional model of impulsivity that captures gene-environment influences on discrete aspects of impulsive personality and behaviour. Further refinement of this model may lead to the development of more effective customised prevention and treatment interventions for clinically disordered impulsivity. The implications of dopaminergic systems and stress in understanding disorders such as ADHD and substance dependence are discussed.
50
White, Melanie Jade. "Understanding impulsivity : molecular genetic and environmental influences." Queensland University of Technology, 2008. http://eprints.qut.edu.au/16578/.
Full textAbstract:
Features of impulsivity underlie multiple psychological disorders. The body of work examining impulsivity has largely focussed on self-report measurement and has incorporated psychological constructs without reference to the broader biological factors that may influence impulsive behaviour. Two studies were conducted to examine whether environmental stress and genetic status associated with dopaminergic and serotonergic function (DRD2, ANKK1 and 5HT2AR genotypes) were predictive of dimensions of impulsivity and risky behaviour (alcohol use). The two studies used a multi-method approach in a non-clinical community sample of young adults (aged 17-25 years). Dopamine is integral to the two leading theories of impulsive personality, Gray's Reinforcement Sensitivity Theory and Cloninger's Psychobiological model of personality. Dopamine plays a crucial role in reward reinforcement circuits in the brain. The A1 allele of the ANKK1 gene (also referred to as TaqIA of the DRD2 gene region) and the CC genotype of the C957T polymorphism of the DRD2 gene have both been associated with reduced D2 dopamine receptor density in key structures linked to brain reward. In addition, a strong body of evidence implicates their involvement in a number of clinical disorders associated with impulsivity. Serotonin function has also been associated with impulsivity in Cloninger's theory and there is also evidence of associations of two polymorphisms of the 2A serotonin receptor gene (5HT2AR T102C and -1438A/G SNPs) with impulsivity. Acute and chronic forms of stress are also important correlates of impulsive behaviour and the two studies directly examined the relationship between genotype, stress and impulsivity. Study 1 (N=180) utilised a cross-sectional design and examined interactions between these polymorphisms and chronic stress exposure on key impulsivity dimensions of reward sensitivity, Novelty Seeking and rash impulsiveness. Participants completed psychological questionnaires measuring chronic stress, dimensions of impulsivity, mood and substance use and provided mouth swab samples of buccal mucosal cells for DNA analysis. The study confirmed the association between A1 and CC allelic status and chronic stress being associated with harm avoidance and sensitivity to punishment. This suggests a role for both dopamine and background stress in impulsive behaviour. Study 2 (N=73) built upon this questionnaire research in the laboratory by utilising experimental psychological paradigms of impulsive behaviour and experimentally manipulating acute stress. Study 2 employed a mixed experimental design with a sub-sample of those studied in the cross-sectional sample. These behavioural paradigms included pre- and post- stress induction administration of the Card Arranging Reward Responsiveness Objective Test (capturing behavioural approach in the presence of reward cues, presumed to reflect reward sensitivity) and post-induction delay discounting and response inhibition measures. Study 2 confirmed the role of one of the two dopamine-related polymorphisms, with those with A1+ allelic status demonstrating lower reward responsiveness prior to rest or stress induction, which was overcome in the second administration of this task, independent of environment. A1+ allelic individuals also demonstrated significantly poorer response inhibition independent of stress, further confirming the association between A1+ allelic status and impulsivity. Those with CC allelic status showed an increase in reward responsiveness only in the stress induction condition. Together, results from the two studies inform the development of a multidimensional model of impulsivity that captures gene-environment influences on discrete aspects of impulsive personality and behaviour. Further refinement of this model may lead to the development of more effective customised prevention and treatment interventions for clinically disordered impulsivity. The implications of dopaminergic systems and stress in understanding disorders such as ADHD and substance dependence are discussed.