Dissertations / Theses: 'Multiplex amplification'

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Zabre, Sidkièta. "Amplification non-linéaire d’un multiplex de porteuses modulées à fort facteur de crête." Rennes 1, 2007. http://www.theses.fr/2007REN1S014.

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Au nombre des difficultés de mise en œuvre de la Radio Logicielle (RL) réside le problème de l’amplification non linéaire des signaux à fort variation de puissance. Il est connu, que les amplificateurs de puissance présentent des caractéristiques non linéaires; et si tôt qu'un signal radio fréquence à fortes fluctuations d'enveloppe doit être amplifié, des distorsions néfastes viennent perturber de façon non négligeable les données utiles entraînant une dégradation des performances de communications. Pour apporter une solution au problème posé, nous avons scindé celui-ci en deux parties. La première partie à consister à modéliser les signaux RL puis à caractériser la notion de variation de puissance dans le contexte RL. Nous avons défini un paramètre, le Power Ratio (PR) qui traduit cette notion de variation de puissance. Ensuite nous avons analysé le PR du cas le plus simple (monoporteuse) au cas le plus complexe (multi-standards). Cette analyse nous a conduit à proposer une nouvelle vision du Power Ratio dans le cas multiporteuse OFDM. Une analyse fortement inspirée du cas multiporteuse est proposée dans le contexte Radio Logicielle. Dans la seconde partie de notre étude nous avons proposé le concept novateur de «méthode des porteuses fantômes C-SOCP » pour réduire le PR. Cette méthode s’applique à tout type de signal. De plus elle est à compatibilité descendante et ne dégrade point le taux d’erreur binaire. Nous avons alors modélisé le problème de réduction du PR sous forme d’un problème d’optimisation convexe plus précisément sous forme d’un Second Order Cone Programming (SOCP). Nous ajoutons à notre modélisation initiale, des contraintes soit pour contrôler la puissance additionnelle transmise, soit pour respecter le gabarit du spectre spécifié dans les standards.

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Suemasu, Cintia Natsumi. "Caracterização dos genótipos da talassemia alfa delecional por MLPA (Multiplex Ligation-Dependent Probe Amplification)." [s.n.], 2010. http://repositorio.unicamp.br/jspui/handle/REPOSIP/308947.

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Orientador: Maria de Fátima Sonati
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-16T20:41:19Z (GMT). No. of bitstreams: 1 Suemasu_CintiaNatsumi_M.pdf: 6481597 bytes, checksum: f9093ca98b4efb60bfe4f81a7e80efee (MD5) Previous issue date: 2010
Resumo: A talassemia alfa (a) constitui um grupo de doenças hereditárias causadas pela redução ou ausência da síntese das cadeias 'alfa' da hemoglobina. Os genes responsáveis pela codificação dessas cadeias são duplicados ('alfa' 2 e 'alfa'1) e estão localizados em um agrupamento gênico ou cluster, situado próximo ao telômero do cromossomo 16 (16p13.3). As deleções são as principais causas da doença, podendo afetar um ou ambos os genes 'alfa' do cromossomo (formas 'alfa' + e 'alfa' 0, respectivamente). Nos últimos anos, várias técnicas foram desenvolvidas para identificar as deleções envolvendo o cluster dos genes a no cromossomo 16. A gap- PCR, o Southern blot e o FISH são comumente aplicados com esta finalidade; entretanto, muitas deleções ainda não são detectadas utilizando-se essas estratégias metodológicas. Em 2005, uma técnica simples e adequada a análises quantitativas rápidas, o Multiplex Ligation-dependent Probe Amplification (MLPA), foi adaptada ao diagnóstico das hemoglobinopatias. Esta técnica é baseada na ligação e na amplificação, em reação de PCR, de dois oligonucleotídeos (sondas) que se hibridizam de forma adjacente ao DNA alvo. Cada oligonucleotídeo é projetado para gerar um produto de comprimento único e, por possuírem terminações comuns, todas as sondas podem ser amplificadas utilizando um único par de iniciadores. Por fim, o uso de fluorocromos permite a separação dos fragmentos, que foram gerados pela amplificação das sondas, em sistema de seqüenciamento por capilaridade. No presente estudo nós utilizamos o MLPA para investigar as bases moleculares da talassemia alfa em nove pacientes não relacionados, cinco deles com Doença da Hb H, cujos diagnósticos moleculares não puderam ser esclarecidos pelas técnicas usuais, incluindo o seqüenciamento de DNA. No total, foram utilizadas 44 sondas, distribuídas desde o telômero ao gene MSLN, cobrindo uma região de cerca de 800 kb, na região 16p13.3. Oito diferentes deleções foram detectadas. Enquanto quatro delas comprometem uma região de grande extensão que inclui todo o locus a e seu elemento regulatório, com tamanhos mínimos de 240 kb, 470 kb, 500 kb e 720 kb, respectivamente (posições 97000-334571, 97000-570532, 97000-602492 and 97000- 816477 do UCSC Genome Browser, Fevereiro, 2009, respectivamente), quatro outras encontram-se limitadas às regiões que contém o elemento regulatório, deixando os genes a intactos, porém sem expressão. Essas últimas apresentam tamanhos mínimos de 0.4 kb, em um dos casos, e de 95 kb a 100 kb nos demais (posições entre 163464-163904, 97000- 193847, 97000-202417 e 97000-202417 do UCSC Genome Browser, Fevereiro, 2009, respectivamente). Em um dos pacientes com fenótipo talassêmico heterozigótico foi demonstrada a presença de uma quadruplicação de cerca de 230 kb de DNA, incluindo todo o cluster a e seu elemento regulatório (97000 a 231370 do UCSC Genome Browser, Fevereiro, 2009). Este estudo representa o primeiro no Brasil a empregar o método de MLPA na caracterização das bases moleculares da talassemia a. A ampla diversidade de alterações identificadas enfatiza a necessidade de se investigar todos os casos cujos valores hematológicos revelem hipocromia e microcitose, na ausência de deficiência de ferro e níveis normais de HbA2
Abstract: Alpha (a) thalassemia is an inherited hemoglobin disorder characterized by reduction or absence of the a-globin chain synthesis. These chains are encoded by duplicated genes ('alpha' 2 and 'alpha' 1) that lie in a gene cluster near the telomeric end of the short arm of chromosome 16 (16p13.3). Deletions are the major molecular cause of the disease and may affect one or both 'alfa genes in the chromosome (the 'alpha' + and 'alpha' 0 forms, respectively). In recent years several techniques have been developed to identify deletions involving the 'alpha' -globin cluster in chromosome 16. The molecular tests commonly used to identify these deletions are gap- PCR, Southern Blot analysis and FISH. However, several thalassemia deletions remain uncharacterized by these methods. In 2005 a simple technique suitable for rapid quantitative analysis ¾ multiplex ligationdependent probe amplification (MLPA) ¾ was adapted for the diagnosis of thalassemias. The approach is based on the ligation and PCR amplification of two adjacently hybridizing oligonucleotides (probes). Each oligonucleotide pair is designed to give a product of unique length, and by using common ends all the probes can be amplified with one primer pair. Finally, the use of a fluorescent label allows the probe to be separated in a capillary sequencing system. In this study we used MLPA to investigate the molecular basis of thalassemia in nine unrelated patients (five with Hb H disease) in whom the molecular causes of the disease could not be detected by sequence analysis or other conventional techniques. In total, 44 probe pairs were used, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight different deletions were detected. While four of them affect a large region including the entire ? -globin gene locus and its upstream regulatory element, spanning genomic regions of at least 240 kb, 470 kb, 500 kb and 720 kb (positions 97000-334571, 97000-570532, 97000-602492 and 97000-816477 of the UCSC Genome Browser, February, 2009, respectively). The other four deletions were limited to a region that contains the upstream regulatory element; the ? -globin genes, although intact, are therefore not expressed. These deletions span a region of at least 0.4 kb in one case and from 95 kb to 100 kb in the other cases (positions 163464-163904, 97000- 193847, 97000-202417 and 97000-202417 of the UCSC Genome Browser, February, 2009, respectively). One carrier who was suspected of having heterozygous ? -thalassemia showed a segmental quadruplication spanning about 230 kb and including the a cluster and upstream regulatory element (97000-231370 do UCSC Genome Browser, February, 2009). This study is the first in Brazil to use the MLPA method to characterize thalassemias. The variety of rearrangements identified highlights the need to investigate all cases presenting with microcytosis and hypochromia without iron deficiency or elevated Hb A2 levels
Mestrado
Ciencias Biomedicas
Mestre em Ciências Médicas

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Ereku, Luck Tosan. "Design of microfluidic multiplex cartridge for point of care diagnostics." Thesis, Brunel University, 2017. http://bura.brunel.ac.uk/handle/2438/15331.

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A simple, but innovative microfluidic Lab-on-a-chip (LOC) device which is broadly applicable in point of care diagnostics of biological pathogens was designed, fabricated and assembled utilising explicit microfluidic techniques. The purpose of this design was to develop a cartridge with the capability to perform multiplex DNA amplification reactions on a single device. To achieve this outcome, conventional laboratory protocols for sample preparation; involving DNA extraction, purification and elution were miniaturized to suit this lab-on-a-chip device of 75mm X 50mm cross-sectional area. The extraction process was carried out in a uniquely designed microchamber embedded with chitosan membrane that binds DNA at pH 5.0 and elutes when a different solution at pH 9.0 flows through. Likewise, purification protocol that occurs in the designed waste reservoir is very significant in biomedical field because it is concerned with waste treatment and cartridge disposability, was performed with a super absorbent powder that converts liquid to a gel like substance. This powder is known as sodium polyacrylate, which is also they treated with anti-bacterial chemicals to prevent environmental contamination. Furthermore, this process also employed the use of a passive valve for a precise fluid handling operation involving flow regulation from extraction to waste reservoir. In order to achieve the intended multiplexing function a multiplexer was created to distribute flow simultaneously through a bifurcated network of channels connected to six similar amplification microchambers. Prior to fabrication, computational fluid dynamics (CFD) simulation was utilized at flowrates less than 10μL/s as the means to test the effectiveness of each design components and also to specifically deduct empirical values that can be analyzed to improve or understand the relationship between the fluid and geometrical constraints of the microfluidic modular elements. The device produced was a hybrid cartridge composed of PDMS and glass which is the most widely used materials microfluidics research due to their low cost and simplicity of fabrication by soft lithography technique. The choice of material also took into account the various physical and chemical properties advantages and disadvantages in their bio-medical applications. Such properties include but not limited to surface energy that determines the wetting fluid characteristics, biocompatibility, optical transparency. Subsequently, after a prototype cartridge was developed fluid flow experimentation using liquid coloured dye was used on the fully fabricated cartridge to test the efficacy of its microfluidic functionalities before expensive DNA amplification reagents were utilised at similar flowrates to the CFD simulations. This gave rise to comparison between similar and dissimilar flow Peculiarities in the microfluidic circuit of both experiments. The final experiment was performed with the aid of a recent molecular technique in DNA amplification known as of RPA kit (recombinase polymerase amplification reaction). It involved performing two main reaction experiments; first, was the positive experiment that bears the sample DNA and the latter, negative that served as the control without DNA. In the end, quantitative analysis of results was done using an agarose gel that showed 143 base pairs, for the positive samples, thus validating the amplification experiment.

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蘇昭燕 and Chiu-yin So. "Molecular characterization of large deletions in beta globin gene cluster using multiplex ligation-dependent probe amplification." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B40738164.

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So, Chiu-yin. "Molecular characterization of large deletions in beta globin gene cluster using multiplex ligation-dependent probe amplification." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B40738164.

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Cohn, Dianne Marie. "Utilizing multiplex ligation-dependent probe amplification to detect novel x-linked microduplications which cause intellectual disability." Connect to this title online, 2008. http://etd.lib.clemson.edu/documents/1239894829/.

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Huamán, Dianderas Francia Del Pilar. "Diagnóstico molecular de pacientes con retraso mental idiopático usando la técnica Multiplex Ligation Probe Amplification (MLPA)." Bachelor's thesis, Universidad Nacional Mayor de San Marcos, 2016. https://hdl.handle.net/20.500.12672/5468.

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Detecta las mutaciones de tipo deleción y/o duplicación causantes de RM idiopático síndrómico o no síndrómico en diferentes genes y regiones, por medio de la técnica MLPA, siguiendo los pasos propuestos por GIRMOGEN 2012. El lugar de ejecución fue el Centro de Genética y Biología Molecular (CGBM) del Instituto de Investigación de la Facultad de Medicina Humana de la Universidad San Martín de Porres. El material biológico consistió en una muestra de 3 a 4 mL sangre periférica, en tubos vacutainer con EDTA, de 50 pacientes diagnosticados clínicamente con retraso mental provenientes del Servicio de Genética - Departamento de Especialidades Médicas del Hospital Edgardo Rebagliati Martins – EsSalud.
Tesis

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Claesson, Cim. "Is Multiplex Ligation-dependent Probe Amplification a good method for screening formalin fixed paraffin embedded neuroblastoma tumors?" Thesis, Högskolan i Skövde, Institutionen för vård och natur, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-5442.

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Neuroblastoma is one of the most enigmatic solid tumors for scientists and pediatric oncologists. Neuroblastoma is primary a childhood form of cancer, consisting of neuroectodermal cells that originate from the neural crest and is destined for the adrenal medulla and sympathetic nervous system. The Neuroblastoma group at The University of Gothenburg received formalin-fixed paraffin-embedded tumor samples from Vietnam and this project was to examine if the quality of the DNA from, is good enough to run comparative genome hybridization array experiment on by using a cheaper technique Multiplex Ligation-dependent Probe Amplification technique. Multiplex Ligation-dependent Probe Amplification (MRC Holland) is a multiplex PCR method that can detect abnormalities such as deletions and amplifications. By using probes consisting of one short synthetic arm with a PCR primer sequence Y at the 3´end, and one long probe with a stuffer sequence, and a PCR primer sequence X at the 5´end that can hybridize and ligate. If these probes ligate it is possible to amplify them by PCR just using specific primers for the X and Y sequences. The resulting amplification products can then be analyzed bycapillary electrophoresis. These patient that the DNA was derived from had all stage 4 neuroblastoma, and that is why they all present many aberrations. Among the fascinating data from this experiment, there are many patients with both 11q deletions and has an extreme amplification of MYCN. In Sweden only a few cases has been discovered. In this material even though all patients are stage 4 patients, 16 have this combination.

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Sandberg, Julia. "Massively parallel analysis of cells and nucleic acids." Doctoral thesis, KTH, Genteknologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-45671.

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Recent proceedings in biotechnology have enabled completely new avenues in life science research to be explored. By allowing increased parallelization an ever-increasing complexity of cell samples or experiments can be investigated in shorter time and at a lower cost. This facilitates for example large-scale efforts to study cell heterogeneity at the single cell level, by analyzing cells in parallel that also can include global genomic analyses. The work presented in this thesis focuses on massively parallel analysis of cells or nucleic acid samples, demonstrating technology developments in the field as well as use of the technology in life sciences. In stem cell research issues such as cell morphology, cell differentiation and effects of reprogramming factors are frequently studied, and to obtain information on cell heterogeneity these experiments are preferably carried out on single cells. In paper I we used a high-density microwell device in silicon and glass for culturing and screening of stem cells. Maintained pluripotency in stem cells from human and mouse was demonstrated in a screening assay by antibody staining and the chip was furthermore used for studying neural differentiation. The chip format allows for low sample volumes and rapid high-throughput analysis of single cells, and is compatible with Fluorescence Activated Cell Sorting (FACS) for precise cell selection. Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences by constantly producing increasing amounts of data from one sequencing run. However, the reagent costs and labor requirements in current massively parallel sequencing protocols are still substantial. In paper II-IV we have focused on flow-sorting techniques for improved sample preparation in bead-based massive sequencing platforms, with the aim of increasing the amount of quality data output, as demonstrated on the Roche/454 platform. In paper II we demonstrate a rapid alternative to the existing shotgun sample titration protocol and also use flow-sorting to enrich for beads that carry amplified template DNA after emulsion PCR, thus obtaining pure samples and with no downstream sacrifice of DNA sequencing quality. This should be seen in comparison to the standard 454-enrichment protocol, which gives rise to varying degrees of sample purity, thus affecting the sequence data output of the sequencing run. Massively parallel sequencing is also useful for deep sequencing of specific PCR-amplified targets in parallel. However, unspecific product formation is a common problem in amplicon sequencing and since these shorter products may be difficult to fully remove by standard procedures such as gel purification, and their presence inevitably reduces the number of target sequence reads that can be obtained in each sequencing run. In paper III a gene-specific fluorescent probe was used for target-specific FACS enrichment to specifically enrich for beads with an amplified target gene on the surface. Through this procedure a nearly three-fold increase in fraction of informative sequences was obtained and with no sequence bias introduced. Barcode labeling of different DNA libraries prior to pooling and emulsion PCR is standard procedure to maximize the number of experiments that can be run in one sequencing lane, while also decreasing the impact of technical noise. However, variation between libraries in quality and GC content affects amplification efficiency, which may result in biased fractions of the different libraries in the sequencing data. In paper IV barcode specific labeling and flow-sorting for normalization of beads with different barcodes on the surface was used in order to weigh the proportion of data obtained from different samples, while also removing mixed beads, and beads with no or poorly amplified product on the surface, hence also resulting in an increased sequence quality. In paper V, cell heterogeneity within a human being is being investigated by low-coverage whole genome sequencing of single cell material. By focusing on the most variable portion of the human genome, polyguanine nucleotide repeat regions, variability between different cells is investigated and highly variable polyguanine repeat loci are identified. By selectively amplifying and sequencing polyguanine nucleotide repeats from single cells for which the phylogenetic relationship is known, we demonstrate that massively parallel sequencing can be used to study cell-cell variation in length of these repeats, based on which a phylogenetic tree can be drawn.
QC 20111031

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Werne, Solnestam Beata. "Interpreting the human transcriptome." Doctoral thesis, KTH, Genteknologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-158320.

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The human body is made of billions of cells and nearly all have the same genome. However, there is a high diversity of cells, resulted from what part of the genome the cells use, i.e. which RNA molecules are expressed. Rapid advances within the field of sequencing allow us to determine the RNA molecules expressed in a specific cell at a certain time. The use of the new technologies has expanded our view of the human transcriptome and increased our understanding of when, where, and how each RNA molecule is expressed. The work presented in this thesis focuses on analysis of the human transcriptome. In Paper I, we describe an automated approach for sample preparation. This protocol was compared with the standard manual protocol, and we demonstrated that the automated version outperformed the manual process in terms of sample throughput while maintaining high reproducibility. Paper II addresses the impact of nuclear transcripts on gene expression. We compared total RNA from whole cells and from cytoplasm, showing that transcripts with long, structured 3’- and 5’-untranslated regions and transcripts with long protein coding sequences tended to be retained in the nucleus. This resulted in increased complexity of the total RNA fraction and fewer reads per unique transcript. Papers III and IV describe dynamics of the human muscle transcriptome. For Paper III, we systematically investigated the transcriptome and found remarkably high tissue homogeneity, however a large number of genes and isoforms were differentially expressed between genders. Paper IV describes transcriptome differences in response to repeated training. No transcriptome-based memory was observed, however a large number of isoforms and genes were affected by training. Paper V describes a transcript profiling protocol based on the method Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification. We designed the method for a few selected transcripts whose expression patterns are important for detecting breast cancer cells, and optimized the method for single cell analysis. We successfully detected cells in human blood samples and applied the method to single cells, confirming the heterogeneity of a cell population.
Människokroppen är uppbyggd av miljarder celler och nästan alla innehåller samma arvsmassa. Trots detta finns det många olika celler med olika funktioner vilket är en följd av vilken del av arvsmassan som cellerna använder, dvs vilka RNA-molekyler som finns i varje cell. Den snabba utvecklingen av sekvenseringstekniker har gjort det möjligt att studera när, var och hur varje RNA-molekyl är uttryckt och att få en djupare förståelse för hur människans celler fungerar. Arbetet som presenteras i denna avhandling fokuserar på analys av RNA-molekyler i människans celler. I artikel I beskriver vi en automatiserad metod för att förbereda cellprov för RNA-sekvensering. Det automatiserade protokollet jämfördes med det manuella protokollet, och vi visade att det automatiserade protokollet överträffade det manuella när det gällde provkapacitet samtidigt som en höga reproducerbarheten behölls. I artikel II undersökte vi effekterna som RNA-molekyler från en del av cellen (cellkärnan) har på den totala mängden uttryckta RNA-molekyler. Vi jämförde RNA från hela cellen och från en del av cellen (cytoplasman) och visade att RNA-molekyler med långa och strukturerade 3'- och 5'-otranslaterade regioner och RNA-molekyler med långa proteinkodande sekvenser tenderade att hållas kvar i cellkärnan till en högre grad. Detta resulterade i en ökad komplexitet av RNA-molekylerna i hela cellen, medan vi i cytoplasma-fraktionen lättare kunde hitta de korta och svagt uttryckta RNA-molekyler. I Artikel III och IV studerar vi RNA-molekyler i människans skelettmuskler. I artikel III visar vi att andelen RNA-molekyler uttryckta i skelettmuskler är väldigt lika mellan muskler och mellan olika personer, men att ett stort antal RNA-molekyler var uttryckta i olika nivåer hos kvinnor och män. Artikel IV beskriver RNA-nivåer som svar på upprepade perioder av uthållighetsträning. Artikel V beskriver en metod för att studera ett fåtal utvalda RNA-molekyler. Vi valde RNA-molekyler vars uttryck är viktigt vid analys av bröstcancerceller, och optimerade metoden för analys av enskilda celler. Vi analyserade cancerceller från blodprov och använde metoden för att titta på RNA-nivåer i enskilda celler från en grupp av celler och visade på skillnader i RNA-nivåer inom gruppen.

QC 20150115

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Thompson, Lindsay Paige. "Degenerate Oligonucleotide Primed - Polymerase Chain Reaction Evaluation And Optimization To Improve Downstream Forensic STR Analysis Of Low Quality/Low Quantity DNA." VCU Scholars Compass, 2006. http://scholarscompass.vcu.edu/etd/1299.

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When forensic biological samples yield low quality/low quantity DNA, thecurrent STR analysis methods do not generate acceptable profiles. Whole genomeamplification can be used to pre-amplify the entire genome for downstream analyses. A commercially available kit for DOP-PCR, a form of WGA, is currently being used in the clinical for downstream single locus targets. Forensic analyses utilize a multiplex amplification. This study determined that the "home brew" created by our lab performs the same as the commercially available kit. Future optimization studies of DOP-PCR can utilize this "home brew". Additionally, this research determined that a 10 second increase in electrokinetic injection time onto the Capillary Electrophoresis (CE) in combination with a post-STR amplification purification and elution into formamide produces a slightly higher percent STR allele success over the standard protocol. After future optimization studies, this may be a useful method to obtain more accurate and complete STR profiles from low quality/low quantity biological samples.

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Saathoff, Katharina [Verfasser]. "Deletion Mapping and Phenotype-Genotype Analysis by Multiplex Ligation-dependent Probe Amplification in German Patients with Williams-Beuren-Syndrome / Katharina Saathoff." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2020. http://d-nb.info/1237415020/34.

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Santana, Ariane Rolins de. "Transferibilidade e desenvolvimento de sistema multiplex de genotipagem de marcadores microssatélites para Pterodon emarginatus Vogel (Fabaceae)." Universidade Federal de Goiás, 2014. http://repositorio.bc.ufg.br/tede/handle/tede/4501.

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
Microsatellite markers are widely used to assess the genetic variability in natural populations. These markers can be developed from derived Expressed sequence tag (ESTs) or genomic libraries. A feasible and economical alternative for obtaining of these markers for species that do not already have them is the transferability from primers designed for species evolutionarily close. The species Pterodon emarginatus Vogel (sucupira) belongs to the Fabaceae family and shows great potential for use timber and medicinal, which makes them a target of exploitation. Thus, studies that contribute to their use and conservation are needed, including the genetic population. Therefore, the aim of this study was to evaluate the potential for cross-amplification primers developed for the species Phaseolus vulgaris to P. emarginatus and to analyze the polymorphism of the loci transferred from multiplex genotyping in three populations of this species. For this, were evaluated 539 primers developed for Phaseolus vulgaris, with 345 derived from ESTs libraries and 194 genomics. Amplification tests were performed using three individuals, while the assessment initial of the polymorphism with eight. Finally, genetic diversity parameters were estimated using 88 plants from three natural populations of P. emarginatus. Primers that showed amplification products visualized on agarose gel were then evaluated in 6% acrylamide gel stained with silver nitrate. Polymorphic loci were synthesized with fluorescence for evaluation and genotyping by capillary electrophoresis in ABI3500 automatic DNA analyzer. The loci were transferred to P. emarginatus 23 (4 %) being 7 polymorphic. Polymorphic, 6 electropherograms profiles. These 6 loci were evaluated in populations along with three polymorphic genomic loci previously standardized sucupira, totaling nine loci suitable for genotyping. It was possible to develop two multiplex systems, a compound of 5primers and another for 4. The number of alleles per locus ranged from two to 13, with an average of five alleles per loci. The mean values of He and Ho for both populations were 0,563 and 0,463 respectively. The value of f was not significant for the total population and equal to 0,144. The values FIT and FST were significant and equal to 0,06 and 0,195 respectively. Microsatellite markers transferred and polymorphic for P. emarginatus showed polymorphism and can be used in studies genetic population more depth to the species.
Os marcadores microssatélites são amplamente utilizados para acessar a variabilidade genética em populações naturais. Estes marcadores podem ser desenvolvidos a partir de bibliotecas derivadas de Sequências Expressas Transcritas (ESTs) ou genômicas. Uma alternativa possível e econômica para a obtenção destes marcadores para espécies que ainda não os possuem, é a transferibilidade a partir de primers desenhados para espécies evolutivamente próximas. A espécie Pterodon emarginatus Vogel (sucupira) pertence à família Fabaceae e apresenta grande potencial de uso medicinal e madeireiro, o que faz dela alvo da exploração extrativista. Assim, estudos que contribuam com o seu melhor uso e conservação são necessários, dentre eles os genético-populacionais. Diante disso, o objetivo deste estudo foi avaliar o potencial de amplificação cruzada de primers desenvolvidos para a espécie Phaseolus vulgaris para P. emarginatus e analisar o polimorfismo dos locos transferidos a partir de sistema multiplex de genotipagem em três populações desta espécie. Para tanto, foram avaliados 539 primers desenvolvidos para Phaseolus vulgaris, sendo 345 derivados de bibliotecas de ESTs e 194 genômicos. Os testes de amplificação foram realizados utilizando três indivíduos, enquanto a avaliação inicial do polimorfismo com oito. Por fim, os parâmetros genéticos de diversidade foram estimados utilizando 88 plantas, provenientes de três populações naturais de P. emarginatus. Os primers que apresentaram produtos de amplificação visualizados em gel de agarose foram, posteriormente, avaliados em gel de acrilamida 6% corado com nitrato de prata. Os locos polimórficos foram sintetizados com fluorescência para a avaliação e genotipagem via eletroforese capilar, no analisador automático de DNA ABI3500. Os locos transferidos para P. emarginatus foram 23 (4%) sendo 7 polimórficos. Dos polimórficos, 6 apresentaram bons perfis de eletroferogramas. Estes 6 locos foram avaliados nas populações juntamente com três locos genômicos polimórficos previamente padronizados para sucupira, totalizando nove locos adequados para genotipagem. Foi possível o desenvolvimento de dois sistemas multiplex, um composto de 5 primers e outro por 4. O número de alelos por loco variou de dois a 13, com uma média de cinco alelos por loco. Os valores médios de He e Ho para o conjunto das populações foram de 0,563 e 0,463 respectivamente. O valor de f não foi significativo para o total das populações e igual a 0,144. Os valores de FST e FIT foram significativos e iguais a 0,06 e 0,195 respectivamente. Os marcadores microssatélites transferidos e polimórficos para P. emarginatus apresentaram bom polimorfismo e podem ser utilizados em estudos genético-populacionais mais aprofundados com a espécie.

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Honjo, Rachel Sayuri. "Detecção da microdeleção 7q11.23 por MLPA® e estudo clínico dos pacientes com síndrome de Williams-Beuren." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/5/5141/tde-13082012-100426/.

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INTRODUÇÃO: A síndrome de Williams-Beuren (SWB) é uma doença genética causada por uma microdeleção na região 7q11.23 e caracterizada por dismorfismos faciais típicos, deficiência intelectual, comportamento hipersociável, cardiopatia congênita, principalmente a estenose aórtica supravalvar (EASV), e outras malformações variáveis. MÉTODOS: Foram avaliados 65 pacientes (40 do sexo masculino, 25 do sexo feminino), com idades entre 2 e 59 anos (mediana = 14 anos), com características clínicas sugestivas de SWB. Todos os pacientes eram filhos de pais normais. A técnica de Multiplex Ligation-dependent Probe Amplification® (MLPA®) foi usada com kit específico com sondas da região da SWB (MRC Holland). As sondas foram hibridadas ao DNA e os fragmentos ligados foram amplificados por PCR e analisados com software específico. RESULTADOS: A deleção de todas as sondas da região 7q11.23 testadas foi detectada por MLPA® em 55/65 pacientes. Um caso de deleção atípica, ou seja, menor que 1,5 Mb, foi observada em um paciente com quadro clínico parcial da síndrome. Os nove pacientes sem deleção tinham um diagnóstico clínico duvidoso da SWB. Dois pacientes tiveram MLPA® positivo para SWB embora apresentassem resultados de FISH negativos. Os achados clínicos dos pacientes com deleção típica foram: fácies típica (98,2%), atraso do desenvolvimento neuropsicomotor (98,2%), comportamento hipersociável (94,5%), hiperacusia (94,5%) e cardiopatia (81,8%). Dentre os pacientes com cardiopatia, 42,2% apresentavam EASV (isolada ou associada a outras anomalias cardíacas), 26,7% apresentavam estenose pulmonar e 31,1% apresentavam outras cardiopatias isoladas ou em associação. Outros achados dos pacientes com deleção foram: anormalidades geniturinárias (85,4%), escoliose (56,4%), baixa estatura (43,6%), hérnias inguinais e/ou umbilicais (36,4%), hipertensão arterial (36,4%, com 20% destes apresentando estenose de artérias renais), estrabismo (34,5%), microcefalia (30,9%), sinostose radioulnar (10,9%), hipotireoidismo (14,5%) e hipotireoidismo subclínico (7,3%). Hipercalcemia foi detectada em um paciente apenas. Outros dois pacientes apresentaram nefrocalcinose e um paciente apresentou hipercalciúria, com níveis de cálcio sérico normais. Três pacientes adolescentes foram a óbito por causas cardiovasculares, incluindo um caso de óbito após transplante cardíaco. CONCLUSÕES: A técnica de MLPA® foi eficaz na detecção da microdeleção na região 7q11.23 possibilitando a confirmação diagnóstica da SWB em 84,6% dos pacientes estudados. Além disso, foi possível detectar uma deleção menor atípica em um paciente com fenótipo parcial e confirmar o diagnóstico em dois pacientes com quadro clínico típico de SWB e resultados de FISH negativos. Portanto, o MLPA® constitui-se um método promissor na investigação diagnóstica da SWB. Por ser uma doença multissistêmica, a SWB exige cuidados multidisciplinares e acompanhamento específico a fim de se prevenir complicações
INTRODUCTION: Williams-Beuren syndrome (WBS) is a genetic disorder caused by a microdeletion in 7q11.23 region. It is characterized by typical facial dysmorphisms, mental retardation, hipersociable behavior, congenital heart disease, mainly supravalvular aortic stenosis (SVAS), and other variable congenital malformations. METHODS: 65 patients (40 males, 25 females), aged 2-59 years old (median = 14 years old), with clinical characteristics suggesting WBS, were evaluated. All patients had normal parents. Multiplex Ligation-dependent Probe Amplification® (MLPA®) was performed with a kit with probes in WBS region (MRC Holland). The probes were hybridized to the DNA and the ligated fragments were amplified by PCR and analyzed with specific software. RESULTS: The deletion for all tested probes in the 7q11.23 region was detected by MLPA® in 55/65 patients. One case of atypical deletion, smaller than 1.5 Mb, was observed in one patient with partial clinical picture of the syndrome. The nine patients without the deletion did not have a definitive clinical diagnosis of WBS. Two patients had positive MLPA® results even though they had negative FISH for WBS. The clinical characteristics of the patients with the typical deletion were: typical facies (98.2%), neuropsicomotor delay (98.2%), hypersociable behavior (94.5%), hyperacusis (94.5%) and congenital heart disease (81.8%). Among the patients with cardiac abnormalities, 42.2% had SVAS (isolated or not), 26.7% had pulmonary valve stenosis and 31.1% had other cardiac anomalies (isolated or grouped). Other findings in patients with deletion comprised: genitourinary abnormalities (85.4%), scoliosis (56.4%), short stature (43.6%), inguinal and/or umbilical hernias (36.4%), arterial hypertension (36.4%, with 20% of these presenting renal arteries stenosis), strabismus (34.5%), microcephaly (30.9%), radioulnar synostosis (10.9%), hypothyroidism (14.5%), and subclinical hypothyroidism (7.3%). Hypercalcaemia was detected in only one patient. Two other patients had nephrocalcinosis and one patient had hypercalciuria, with normal serum calcium levels. Three adolescents died due to cardiovascular problems, including one case that died after a cardiac transplantation. CONCLUSIONS: MLPA® was effective to detect the microdeletion in 7q11.23 region confirming the diagnosis of WBS in 84.6% of the patients. It was also possible to detect a small atypical deletion in one patient with partial phenotype and confirm the diagnosis in two patients with typical clinical characteristics of WBS and negative FISH results. Thus, MLPA® is a promising method in the diagnostic investigation of WBS. WBS is a multisystemic disorder and therefore requires multidisciplinary care and specific follow-up in order to prevent complications

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O'Brien, Michelle. "Detection of large deletions in the factor VIII (F8) and von Willebrand factor (VWF) genes using multiplex ligation-dependent probe amplification (MLPA)." Thesis, Manchester Metropolitan University, 2013. http://e-space.mmu.ac.uk/314016/.

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Haemophilia A and von Willebrand Disease (VWD) are disorders which can cause mild to severe bleeding in an individual. In many cases, knowledge of the molecular basis of these disorders is required to enable appropriate genetic counselling within families, carrier diagnosis and, in some cases, prenatal diagnosis. The current ‘gold standard’ for diagnosing patients with Haemophilia A and VWD disorders is by direct sequencing of genomic DNA to identify mutations. However this method may not reveal the presence of larger heterozygous deletions or duplications of F8 and VWF due to masking by an unaffected allele. Therefore the genetic basis of disease in a proportion of cases may remain unresolved. Multiplex ligation-dependent probe amplification (MLPA) is a method based on gene dosage that can be applied to the detection of large gene deletions or duplications in Haemophilia and VWD, aiding effective genetic diagnosis. This project developed and applied MLPA to investigate the molecular basis of disease in a selected cohort of patients diagnosed with Haemophilia A, type 1 and type 3 VWD, where standard direct sequencing protocols of F8 and VWF had failed to reveal causative mutations, and to assess its application in the genetic diagnosis of these disorders. This extended previous work on the molecular basis of Haemophilia A and VWD carried out in the Molecular Diagnostics Centre (MDC) at Central Manchester NHS Foundation Trust. MLPA confirmed the presence of large deletions in seven Haemophilia A index patients, two type 1 VWD patients, seven type 3 VWD patients and one possible duplication in a female haemophiliac. The presence of a masked gene abnormality was also confirmed in two carrier females. This demonstrates that MLPA is a useful diagnostic tool for detecting or confirming larger scale mutations not suited to DNA sequence analysis. MLPA is an important addition to the range of molecular diagnostic tools for the investigation of these haemostatic disorders and may be utilised when conventional mutation screening has failed to identify a causative mutation.

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Bastos, Caroline Peixoto. "Multiplex PCR para identificação de S. aureus, S. intermedius e S. hyicus." Universidade Federal de Pelotas, 2008. http://repositorio.ufpel.edu.br/handle/ri/1310.

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Made available in DSpace on 2014-08-20T13:42:05Z (GMT). No. of bitstreams: 1 Dissertacao_ Caroline_Peixoto_ Bastos.pdf: 482424 bytes, checksum: 0f1a1fb787f50f0f7f89d2501df4b4db (MD5) Previous issue date: 2008-07-02
Staphylococcus aureus, Staphylococcus hyicus e Staphylococcus intermedius are species of genus Staphylococcus capable of producing enterotoxins, as well as the enzymes termonuclease and coagulase, and are called Staphylococcus Coagulase Positive (SCP). They show very similar biochemical and morphological characteristics, this fact, makes their differentiation and they identification in food by phenotypic techniques very difficult. In this way, the aim of this work was the development of a Multiplex PCR (mPCR), targetting the nuc gene (encoding for thermonuclease) as target, for the identification of S. aureus, S. intermedius, and S. hyicus. Initially, the mPCR was standartizeted using reference strains of S. aureus, S. warneri, S. lugdunensis and Listeria monocytogenes and isolates of S. hyicus and S. intermedius, affer words it was tested with 16 isolates of the three species of SCP. The primers annealing to each of the three species, the conditions for mPCR, the suitability of the Internal Amplification Control (IAC) for the genus Staphylococcus, and the sequences obtained through the amplification by PCR were evaluated. After this, the results were compared with those achieved by biochemical tests for differentiation of SCP. Primers NUC1-NUC2 (for sequences of the S. aureus nuc gene), NUC5-NUC6 (for sequences of the S. intermedius nuc gene), NUC7-NUC8 (for sequences of the S. hyicus nuc gene) and, as IAC, the primers 16S1 16S2 (for sequences of the 16S of rRNA gene) were used. The mPCR proposed enable the identification of S. aureus, S. hyicus e S. intermedius, and the IAC was suitable for the genus. The sequences obtained in PCR showed a high degree of similarity with the sequences of the thermonuclease gene deposited in GenBank. The mPCR showed greater discriminatory power that the biochemical tests for identification the vii S. aureus, S. hyicus e S. intermedius, as well as reduced the time required for the identification of ECP (reduction of 24h) or identification of specie (reduction of 72h).
Staphylococcus aureus, Staphylococcus hyicus e Staphylococcus intermedius são espécies do gênero Staphylococcus com capacidade de produzir enterotoxinas, bem como as enzimas termonuclease e coagulase, sendo denominadas de Estafilococos Coagulase Positiva (ECP). Apresentam características morfológicas e bioquímicas extremamente semelhantes, o que torna difícil sua diferenciação assim como sua identificação em alimentos através de técnicas fenotípicas de análise microbiológica. Dessa forma, este trabalho teve como objetivo o desenvolvimento de um Multiplex PCR (mPCR), tendo-se como alvo o gene nuc (codifica para a termonuclease), para a identificação de S. aureus, S. intermedius e S. hyicus. Inicialmente, o mPCR foi padronizado utilizando-se cepas padrão de S. aureus, S. warneri, S. lugdunensis e Listeria monocytogenes e isolados de S. hyicus e S. intermedius, e, posteriormente, foi testado em 16 isolados das três espécies de ECP. Foram avaliadas a sensibilidade dos primers para cada uma das três espécies, as condições da mPCR, a sensibilidade do Controle Interno de Amplificação (IAC) para o gênero Estafilococos, e as seqüências obtidas através da amplificação por PCR. Posteriormente, os resultados foram comparados aos obtidos com testes bioquímicos de diferenciação de ECP. Foram utilizados os primers NUC1-NUC2 (para seqüências do gene nuc de S. aureus), NUC5-NUC6 (para seqüências do gene nuc de S. intermedius), NUC7-NUC8 (para seqüências do gene nuc de S. hyicus) e, para amplificação do IAC, os primers 16S1 16S2 (para seqüências do gene 16S do rRNA). O mPCR proposto possibilitou a identificação das espécies S. aureus, S. hyicus e S. intermedius, bem como o IAC utilizado apresentou sensibilidade para o gênero. As seqüências obtidas na PCR apresentaram elevado v grau de similaridade com as seqüências do gene da termonuclease depositadas no GenBank. A mPCR apresentou maior poder discriminatório que os testes bioquímicos para identificação de S. aureus, S. hyicus e S. intermedius, bem como reduziu significativamente o tempo necessário para a identificação de ECP (redução de 24h) ou de identificação em nível de espécie (redução de 72h).

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Broberg, John. "Development of novel multiplexed systems for in situ PLA." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-161126.

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The in situ proximity ligation assay (in situ PLA) is an immunoassay that enables directvisualisation of single protein targets or protein interactions in cell or tissue samples. This project revolves around designing and introducing several novel multiplexable components tobe used in conjunction with Olink Bioscience's Duolink product line. In this report, a novel in silico approach to DNA oligomer interaction design is presented. Using this in silico method, a multiplexed system of DNA oligomers has been designed andevaluated using in situ PLA and fluorescence microscopy.

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Dutra, Roberta Lelis. "Investigação da variação no número de cópias genômicas (CNVs) em pacientes com anomalias congênitas e atraso de desenvolvimento neuropsicomotor (ADNPM) pela técnica de MLPA (Multiplex Ligation-dependent Probe Amplification)." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5141/tde-25112014-120744/.

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INTRODUÇÃO: Os desequilíbrios genômicos constituem causa frequente de abortamento, anomalias congênitas (AC) e atraso de desenvolvimento neuropsicomotor (ADNPM). O aprimoramento de novas técnicas de diagnóstico citogenômico, como por exemplo, a MLPA (Multiplex Ligation-dependent Probe Amplification) e a triagem ampla do DNA utilizando arrays, mostraram que a alteração no número normal de cópias genômicas (CNVs) influencia na patogenicidade dos fenótipos em diversas síndromes. OBJETIVOS: Com isso, os objetivos do presente estudo foram identificar CNVs em pacientes com MC e ADNPM utilizando a técnica de MLPA e, a partir dos resultados alterados, aplicar da técnica de array para a identificação de possíveis rearranjos complexos, além de associar as alterações moleculares encontradas com o fenótipo dos pacientes. MÉTODOS: Participaram do estudo 416 pacientes com MC e ADNPM. As amostras de DNA foram analisadas utilizando a técnica de MLPA com kits comerciais para as principais síndromes de microdeleções (P064) e regiões subteloméricas (P036 e P070). Dois kits de MLPA específicos para as regiões 7q11.23 (P029) e 22q11.2 (P250) também foram utilizados para complementar a identificação de CNVs atípicas. Entre os casos que apresentavam alterações pela técnica de MLPA, 15 pacientes foram submetidos à técnica de array, utilizando três diferentes plataformas: Agilent SurePrint G3 Genoma Humano microarray 180 K, HumanCytoSNP-12 BeadChip, CytoScan(TM) HD array 6.0 Affymetrix®. RESULTADOS: A análise molecular pela técnica de MLPA possibilitou a detecção de microdeleções e/ou microduplicações em 97 pacientes sendo que: em 46 pacientes foi possível encontrar alterações utilizando apenas o kit P064 (microdeleções), em 34 pacientes utilizando apenas os kits P036 e P070 (regiões subteloméricas) e em quatro pacientes só foi possível identificar a alteração utilizando outro kit de MLPA (P250), específico para alterações genômicas em 22q11.2. Rearranjos complexos, envolvendo mais de três cromossomos, foram observados em 10 pacientes. DISCUSSÃO: A MLPA permitiu detectar CNVs em 97/416 pacientes (23,3%), sendo uma técnica ideal para ser aplicada em pacientes com sinais fenotípicos inespecíficos. Algumas alterações genômicas encontradas estão relacionadas também com alterações específicas, como a presença de malformação cardíaca ou convulsões. E em outros casos a alta variabilidade fenotípica pode ser associada a um conjunto de CNVs consideradas patogênicas. Além disso, a inclusão de outra técnica de triagem, com maior cobertura do genoma permitiu detectar rearranjos complexos antes não observados mesmo em síndromes bem descritas como as síndromes de midrodeleções 7q11.23 e 22q11.2. CONCLUSÃO: A MLPA com kits combinados, por possuir maior abrangência de regiões detectadas e menor custo, é uma ferramenta valiosa para ser utilizada como um teste de triagem diagnóstica
INTRODUCTION: Genomic imbalances are the most common cause of miscarriage, congenital anomalies (CA) and mental retardation (MR). With the improvement of new cytogenomics diagnostic techniques, such as the MLPA (Multiplex Ligation-dependent Probe Amplification) and the array techniques, it have been shown that changes in the normal gene copy number influence the pathogenic variability of phenotypes in different syndromes. AIMS: The aims of the present study were to identify CNVs in patients with CM and RM using the MLPA technique and, from the abnormalities results, to apply the array methodology for the identification of complex rearrangements. Furthermore, the study aimed to associate the alterations found by molecular techniques with the phenotype of patients. METHODS: 416 patients with CM and RM participated in the study. The samples were analysed by MLPA technique with commercial kits for the main microdeletion syndromes (P064) and subtelomeric regions (P036 and P070). Two more MLPA kits for specific regions 7q11.23 (P029) and 22q11.2 (P250) were used to confirm the altered results and to complement some results with the identification of atypical abnormalities. From the patients who presented abnormalities by MLPA technique, 15 underwent by microarray-based comparative genomic hybridization (CGH-array) technique, using three different platform: Agilent SurePrint G3 Human Genome microarray 180 kb, HumanCytoSNP -12 BeadChip, CytoScan(TM) HD ® and Affymetrix 6.0. RESULTS: The molecular analysis by MLPA technique allowed the detection of microdeletions and/or microduplications in 97 patients. In 46 patients it was possible to find genomic alteration using only MLPA kit P064 and in 34 patients using only the subtelomeric kits P036 and P070. For four patients it was only possible to identify the genomic abnormalities using another specific MLPA kit (P250), involving the 22q11.2 region. Complex rearrangements involving more than three chromosomes were detected in 10 patients. DISCUSSION: The MLPA technique was capable of detecting CNVs in 97/416 (23,3%) of patients, being an ideal technique to be applied in patients with non-specific signs phenotypic. Some genomic alterations found are, also related to specific changes, such as the presence of cardiac malformation or convulsions. In other cases, the high phenotypic variability may be associated to certain group of pathogenic CNVs. Moreover, the inclusion of additional screening method, with greater coverage, allowed the detection of complex rearrangements not seen before even in syndromes as well described microdeletions syndromes on 7q11.23 and 22q11.2 regions. CONCLUSION: The MLPA technique can be a valuable tool used as a molecular screening test, because it has greater coverage and lower cost of detected regions

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Haoud, Khadidja. "Etude de la prévalence des aneuploïdies dans les produits d'avortements spontanés : intéret des techniques FISH et MLFA pour la détection des remaniements chromosomiques." Thesis, Clermont-Ferrand 1, 2014. http://www.theses.fr/2014CLF1MM29/document.

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L’avortement spontané (AS) désigne la perte du produit de conception avant sa viabilité, c'est-à-dire avant la 22e semaine d’aménorrhée, ou un poids fœtal inférieur à 500 g. La cause génétique est à l’origine de plus des deux tiers des AS, les aneuploïdies autosomiques, représentant à elles seules jusqu’à 70% des pertes fœtales du 1er trimestre. Le caryotype présente une très bonne sensibilité en ce qui concerne le dépistage des trisomies autosomiques (13, 18 et 21) et des aneuploïdies affectant les chromosomes sexuels, mais il montre d’importantes limites, d’une part en raison des échecs de culture cellulaire et d’autre part en raison de l’existence de remaniements non détectables au caryotype standard. Actuellement plusieurs techniques moléculaires de dépistage rapides des aneuploïdies liées aux échecs de grossesses ont été vérifiées : 1°) la fluorescence in situ par hybridation (FISH) 2°) l’amplification multiplex de sondes nucléiques dépendant des ligatures (MLPA). Ces deux méthodes présentent l’avantage d’être réalisables, sans culture préalable, sur noyaux en interphase ou sur ADN extrait et de permettre la détection d’anomalies cryptiques. Notre étude repose sur l’étude cytogénétique des produits d’AS pour mettre en évidence les anomalies chromosomiques les plus fréquentes à l’origine de ces pertes fœtales et d’en mieux appréhender les mécanismes de survenue. Elle a été réalisée sur 220 patientes âgées de 19 à 45 ans, et était fondée sur l’analyse directe par FISH sur noyaux interphasiques (AneuVysionTM) de prélèvements de villosités choriales et sur l’analyse de l’ADN extrait de tissus fœtaux par MLPA afin de révéler d’éventuelles aneuploïdies et micro-remaniements. L’âge gestationnel au moment des prélèvements était compris entre 7 et 38 semaines d’aménorrhée. Sur un total de 151 échantillons analysés par AneuVysionTM, 10 anomalies chromosomiques ont été observées: 3 trisomies 21, 1 trisomie 18, 1 trisomie 13, 1 mosaïque 46,XX/47,XX+21, 3 triploïdies et 1 monosomie X (Turner). Par ailleurs, sur les 69 autres échantillons analysés par MLPA, 6 étaient ininterprétables. Les anomalies trouvées par cette technique étaient: 2 monosomies X. Pour les échantillons restants, la MLPA a été négative. Nous avons en parallèle réalisé une étude rétrospective fondée sur l’analyse comparative d’un échantillon recruté à Sidi Bel Abbès, de femmes ayant subi un AS et admises à la maternité del’hôpital Hassani Abdelkader de Sidi Bel Abbès et d’un échantillon recruté à Clermont-Ferrand de femmes ayant subi un AS et pour lesquelles un prélèvement pour établir le caryotype du produit de fausse-couche avait été adressé dans le service de cytogénétique du CHU Estaing de Clermont-Ferrand. Cette étude a couvert une période de six années, allant de janvier 2005 à décembre 2010. Les techniques de FISH et de MLPA représentent des outils simples, rapides et sensibles pour la détection des remaniements chromosomiques. Elles représentent une alternative très intéressante à la culture cellulaire, et permettent le diagnostic de désordres génomiques indécelables par les techniques conventionnelles
Spontaneous abortion (SA) is the loss of the product of fertilization before its viability, that is, before22 weeks of gestation or fetal weight less than 500 g. Genetic causes account for more than two thirds of SA, autosomal aneuploidies alone accounting for up to 70% fetal loss. Chromosomal cytogenetic techniques show significant limitations on the one hand because of the failures of cell culture, and secondly because of the existence of undetectable alterations to the standard karyotype. It was therefore planned to use molecular techniques :- Fluorescent in situ hybridization (FISH)- Multiplex ligation-dependent probe amplification (MLPA). Both techniques have the advantage of being achievable without prior culture of cores interphase or DNA extracted and to enable detection of cryptic abnormalities. The project is based on cytogenetic study of AS products to highlight the most frequent chromosomal abnormalities causing fetal losses, and to better understand their occurrence. Our study was performed on 220 patients from 19 to 45 years, and was based on the direct analysis by FISH on interphase nuclei (AneuVysionTM) of chorionic villus sampling and analysis of DNA extracted fetal tissue by MLPA to reveal any aneuploidy and rearrangements. The gestational age of the samples ranged from the 7th to the 38th week of gestation. In a total of 151 samples analyzed by AneuVysionTM, 10 chromosomal abnormalities were observed: three trisomies 21, one trisomy 18, one trisomy 13, one mosaic 46,XX/47,XX+21, 3 triploidies and one monosomy X (Turner). In addition, among the other 69 samples analyzed by MLPA, 6 were uninterpretable. The abnormalities found by this technique were 2 monosomies X. For the remaining samples, the MLPA was negative. We conducted a retrospective parallel study based on the analysis of a sample recruited in Sidi Bel Abbes, women who have had an AS and were admitted to the maternity hospital Abdelkader Hassani, Sidi Bel Abbes ; and a sample recruited in Clermont-Ferrand : women who underwent AS for which a levy to establish the karyotype product miscarriage had been addressed in the Department of Cytogenetics of CHU Estaing, Clermont-Ferrand. This study covered a period of six years, from January 2005 to December 2010. The techniques of FISH and MLPA are simple, rapid and sensitive tools for the detection of chromosomal rearrangements. They represent a very interesting alternative to cell culture and allow diagnosis for genomic disorders undetectable by conventional techniques

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20

Rosolem, Debora Cristina Batista. "Avaliação citogenética molecular de células do líquido pleural de pacientes com derrame pleural maligno." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5150/tde-09122014-115852/.

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Introdução O diagnóstico de derrame pleural maligno (DPM) se baseia no achado de células tumorais no líquido ou no tecido pleural. Resultados falsos positivos ou falsos negativos influenciam na escolha da melhor conduta terapêutica a ser tomada, além de alterar substancialmente o prognóstico desses pacientes. A sensibilidade do exame citológico é geralmente inferior a 70%, motivo pelo qual, métodos complementares são frequentemente associados. Fatores como tipo histológico, sítio primário e grau de invasibilidade do tumor são os principais responsáveis por esta variação. Dentre os exames complementares propostos, destacam-se a dosagem de marcadores tumorais no líquido pleural (LP), as técnicas citoquímicas, imunocitoquímicas e de marcadores de proliferação celular em células do LP, a análise da ploidia de DNA por citometria de fluxo (CF) ou estática (CE) e, mais recentemente, as técnicas de citogenética e de biologia molecular, como a técnica de hibridação in situ por fluorescência (FISH) e a técnica de amplificação multiplex por sondas ligação - dependentes (MLPA) estas, capazes de detectar alterações em regiões gênicas consideradas \"alvo\" para o desfecho neoplásico. Objetivos 1) Padronizar as técnicas de DNA ploidia, FISH e MLPA em amostras frescas de líquido pleural; 2) Testar a eficiência diagnóstica dos métodos da DNA ploidia e da FISH no diagnóstico de derrame pleural maligno e 3) Avaliar alterações no número de cópias no gene EGFR em metástases pleurais utilizando a técnica de MLPA. Métodos Foram incluídos 200 pacientes adultos portadores de derrame pleural (DP) com indicação de toracocentese. O diagnóstico histológico foi o padrão ouro para malignidade. Características clínicas, radiológicas, histológicas e de seguimento clínico foram considerados para a exclusão de malignidade, de maneira que 130 casos foram classificados como malignos e 70 como benignos. As 200 amostras de LP foram submetidas ao exame citológico e à FISH utilizando sondas centroméricas para os cromossomos 11 e 17. A análise da ploidia de DNA por CF foi realizada em 45 casos de DP e a MLPA com o kit do gene do receptor do fator de crescimento epidérmico (EGFR) em 50 casos. Resultados A análise da ploidia de DNA por CF apresentou sensibilidade inferior ao exame citológico, com especificidade próxima (57,0%vs 96,2%; 70,0% vs 66,7%, respectivamente). A FISH isoladamente apresentou sensibilidade de 98,5% e especificidade de 98,6% e de 98,0% e 99,% quando associada ao exame citológico, com apenas um caso falso positivo e dois casos falsos negativos. A técnica de MLPA, padronizada para LP, demonstrou alterações na sequência do gene do EGFR em 28,2% dos casos malignos. Nenhuma amostra de líquido pleural dos casos benignos (controle) apresentou alteração no número de cópias e/ou rearranjos estruturais. Conclusão A análise citogenética de amostras frescas de líquido pleural por FISH é um valioso complemento ao exame citológico no diagnóstico de derrame pleural maligno, particularmente nos casos em que o resultado da citologia oncótica é inconclusiva
Introduction The diagnosis of malignant pleural effusion (MPE) is based on the finding of tumor cells in the pleural fluid or tissue. False positive or false negative results influence the choice of the best therapeutic approach to be used with these patients and substantially change their prognosis. The sensitivity of the cytology is generally lesser than 70%, for which complementary methods are often associated. Factors such as tumor histological type, staging, primary site and potential of invasiveness are responsible for this variation. Among the proposed ancillary tests, we highlight the dosage of tumor markers in pleural fluid (PF), the cytochemical and immunocytochemical techniques, including markers of cell proliferation, DNA ploidy analysis by flow cytometry (FC) or static cytometry (EC) and more recently, the cytogenetics and molecular techniques, as the fluorescence in situ hybridization (FISH) and the multiplex ligation - dependent probe amplification (MLPA), capable of detecting changes in gene regions considered \"target\" for the neoplastic outcome. Objectives 1) To standardize the techniques of DNA ploidy, FISH and MLPA in fresh samples of pleural fluid; 2) To test the diagnosis efficiency of DNA ploidy and FISH in the diagnosis of malignant pleural effusion and 3) To evaluate changes in the copy number of the EGFR gene by using the MLPA technique in cases of pleural metastases. Methods We included 200 adult patients with pleural effusion and clinical indication for thoracentesis. The histological diagnosis was considered the gold standard for malignancy. Clinical follow-up, radiological and histological characteristics were considered for exclusion of malignancy, which ranked de cases as 130 malignant effusions and 70 as benign ones. All cases were submitted to cytology and FISH using centromeric probes for the chromosomes 11 and 17. Analysis of DNA ploidy by FC was performed in 45 cases and the MLPA for epidermal growth factor receptor (EGFR) gene in 50 cases. Results DNA ploidy analysis showed less sensitivity than PF cytology, with similar specificity (57.0% vs 96.2% and 70.0% vs 66.7%, respectively). FISH alone had a sensitivity of 98.5% and specificity of 98.6%, and of 98.0% and 99% when associated with cytology. Only one false positive and two false negative cases were observed. The MLPA technique, standardized for PF, showed changes in the EGFR gene in 28.2% of the malignant cases. No samples of pleural fluid from benign cases (control) showed changes in copy number and/or structural rearrangements. Conclusion The cytogenetic analysis of fresh pleural fluid samples by FISH seems to be a valuable method to be associated to cytology in the diagnosis of malignant pleural effusion, particularly in cases of inconclusive cytological results

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Oliveira, Maythsulene Inácio de Sousa. "Desenvolvimento de métodos moleculares para detecção simultânea de fusarium oxysporum f. sp. phaseoli, fusarium solani e curtobacterium flaccumfaciens pv. flaccumfaciens." Universidade Federal de Goiás, 2015. http://repositorio.bc.ufg.br/tede/handle/tede/8762.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Common bean (Phaseolus vulgaris L.) is grown in Brazil in three different cropping seasons, and in diverse agroecosystems. In such different environments, the crop is exposed to several constraints responsible for yield losses, such as pathogenic organisms. Among common bean relevant diseases, fusarium wilt (Fusarium oxysporum f. sp. phaseoli), dry root-rot (Fusarium solani) and Curtobacterium wilt (Curtobacterium flaccumfaciens pv. flaccumfaciens) have similar symptoms, hindering diagnosis in the field, and whose identification in seed health testing is also limited. In both cases, identification at species level is an important step to manage this root pathogen complex, whose detection can be improved by molecular biology tools. Therefore, this study aimed to: 1) to develop and validate a multiplex PCR (m-PCR) method for simultaneous identification of three common bean pathogens, F. oxysporum f. sp. phaseoli, F. solani and C. flaccumfaciens pv. flaccumfaciens; and 2) develop an isothermal amplification of DNA (LAMP) method to detect of F. oxysporum f. sp. phaseoli on seeds. M-PCR method was developed for identification of isolated colonies, as well as infected seeds. In seeds, total DNA was obtained by alkaline lysis method, which inactivates nucleases during the extraction process. M-PCR allowed the identification of all pathogens, with detection of C. flaccumfaciens pv. flaccumfaciens, F. oxysporum f. sp. phaseoli and F. solani amplicons in agarose gel with respectively 306, 609 and 143 base pairs. Furthermore, m-PCR also reduced costs and time to detect Fusarium oxysporum f. sp. phaseoli from 10 days to three hours. It was not possible to develop an optimized protocol for detection of F. oxysporum f. sp. phaseoli by the LAMP method, using only the tf1 gene for design of primers, since such primers were functional only for amplifying large amounts of target DNA. Based on the negative results with LAMP, it is suggested that further studies should be performed using other DNA sequences available in GenBank database.
O feijoeiro-comum (Phaseolus vulgaris L.) é cultivado durante todo o ano no território brasileiro, em três épocas distintas e em vários agroecosistemas. Nestes ambientes distintos, a cultura está exposta a diversos fatores que causam perdas de rendimento, como o ataque de patógenos. Dentre as doenças do feijoeiro-comum encontram-se a murcha-de-fusarium (Fusarium oxysporum f. sp. phaseoli), a podridão-radicular-seca (Fusarium solani) e a murcha-de-curtobacterium (Curtobacterium flaccumfaciens pv. flaccumfaciens) que apresentam sintomas semelhantes, dificultando seu diagnóstico no campo, e cuja identificação em testes de sanidade de sementes também é limitada. Em ambos os casos, a identificação em nível de espécie é uma importante etapa do manejo deste complexo de patógenos, cuja detecção pode ser aperfeiçoada com a adoção de ferramentas de biologia molecular. Portanto, este estudo teve como objetivos: 1) Desenvolver e validar um método de multiplex PCR (m-PCR) para identificação simultânea de três espécies de patógenos do feijoeiro-comum, F. oxysporum f. sp. phaseoli, F. solani e C. flaccumfaciens pv. flaccumfaciens; e 2) desenvolver a técnica de amplificação isotérmica de DNA (LAMP) para detecção de F. oxysporum f. sp. phaseoli em sementes. O método de m-PCR foi desenvolvido para identificação de colônias isoladas bem como sementes infectadas. Nas sementes, o DNA total foi obtido pela lise alcalina, método que inativa nucleases durante o processo de extração. A m-PCR possibilitou a identificação de todos os patógenos, com detecção de C. flaccumfaciens pv. flaccumfaciens, F. oxysporum f. sp. phaseoli e F. solani em bandas formadas em gel de agarose respectivamente com 306, 609 e 143 pares de base. Além disso, a extração do DNA total das sementes pela lise alcalina em combinação com a m-PCR também possibilitou redução de custos e tempo de realização do diagnóstico de Fusarium oxysporum f. sp. phaseoli, de 10 dias para três horas. Não foi possível estabelecer um protocolo otimizado para detecção de F. oxysporum f. sp. phaseoli pelo método LAMP, utilizando somente o gene tf1 para desenho dos iniciadores, uma vez que, os iniciadores revelaram-se funcionais apenas para a amplificação com grandes quantidades de DNA alvo. Diante dos resultados obtidos com LAMP, sugere-se que estudos posteriores sejam realizados empregando outras sequências de DNA disponíveis no banco de dados GenBank.

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How, Siew-Eng. "Multiple valency and assay amplification." Thesis, University of Southampton, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.415237.

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Gunnar, Erika. "Characterization of the genetic basis in two cases of abetalipoproteinemia reveals two novel mutations." Thesis, Linköping University, Department of Physics, Chemistry and Biology, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-58620.

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BACKGROUND: Abetalipoproteinemia (ABL) is a rare autosomal recessive disorder caused by mutations in the gene coding for microsomal triglyceride transfer protein (MTTP).

AIM: To characterize the genetic basis of ABL in two unrelated patients.

RESULTS: In the first patient, the substitution c.1911C>T in exon 12 of the MTTP gene, resulting in the protein substitution p.P552L, was discovered using mutation screening. The parents are heterozygous and the proband is a homozygous carrier of this substitution. Using restriction fragment length polymorphism (RFLP), 100 control subjects were analyzed and none carried the substitution indicating that it is a novel MTTP mutation. Sequencing of the other ABL patient showed that the proband carried a homozygous single base insertion, at position c.2342IVS16+2-3insT, located at the donor splice-site of intron 16 resulting in skipping of exon 16 and truncation of the protein. The proband's mother is heterozygous for the insertion while the father does not carry the insertion. Multiplex ligation-dependent probe amplification (MLPA) did not identify any deletion encompassing exon 16 in the proband, father or mother. Nonpaternity was excluded using polymorphic markers from several chromosomes. Haplotype analysis using markers spanning chromosome 4 revealed heterodisomy (two homologous chromosomes) of 4p and the distal part of 4q, and isodisomy (duplication of one chromosome) of 4q12-4q26.

CONCLUSION: These data show that the cause of ABL in one of the patients is a missense mutation, p.P552L, while the cause of ABL in the other patient is due to uniparental disomy, probably resulting from non-disjunstion in meiosis I.

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Astúcia, Vítor Hugo Soares. "Linear amplification with multiple nonlinear devices." Master's thesis, Faculdade de Ciências e Tecnologia, 2012. http://hdl.handle.net/10362/8229.

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Dissertação para obtenção do Grau de Mestre em Engenharia Electrotécnica e Computadores
In mobile wireless systems, where there are strict power and bandwidth constrains it is desirable to adopt energy efficient constellations combined with powerful equalizer. However, this increased spectral efficiency of multilevel modulations comes at the expense of reduced power efficiency, which is undesirable in systems where power consumption is a constraint. Hence, minimization of the transmitted energy would enable a significant reduction in the total energy consumption of the wireless mobile devices. A simple and practical constellation optimization design would optimize the transmitted energy with a minimum increase in system complexity. The constellation decomposition in terms of a sum of BPSK (Bi-Phase Shift Keying) sub-constellations, relies on an analytical characterization of the mapping rule were the constellation symbols are written as a linear function of the transmitted bits. Moreover, large constellations in general and non-uniform constellations in particular are very sensitive to interference, namely the residual ISI (Inter-Symbol Interference) at the output of a practical equalizer that does not invert completely the channel effects. IB-DFE(Iterative Block DFE) is a promising iterative frequency domain equalization technique for SC-FDE schemes (Single-Carrier with Frequency Domain Equalization) that allows excellent performance. Therefore it is possible to use the decomposition of constellations on BPSK components to define a pragmatic method for designing IB-DFE receivers that can be employed with any constellation. In this thesis we consider SC-DFE schemes based on high orderM-ary energy optimized constellations with IB-DFE receivers. It is proposed a method for designing the receiver that does not require a significant increase in system complexity and can be used for the computation of the receiver parameters for any constellation. This method is then employed to design iterative receivers, implemented in the frequency-domain, which can cope with higher sensitivity to ISI effects of the constellations resulting from the energy optimization process.
Fundação para a Ciência e Tecnologia - MPSat (PTDC/EEA-TEL/099074/2008) project

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Zajac, Pawel. "Parallel target selection by trinucleotide threading." Doctoral thesis, KTH, Genteknologi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-11284.

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DNA is the code for all life. Via intermediary RNA the information encoded by the genome is relayed to proteins executing the various functions in a cell. Together, this repertoire of inherently linked biological macromolecules determines all characteristics and features of a cell. Technological advancements during the last decades have enabled the pursuit of novel types of studies and the investigation of the cell and its constituents at a progressively higher level of detail. This has shed light on numerous cellular processes and on the underpinnings of several diseases. For the majority of studies focusing on nucleic acids, an amplification step has to be implemented before an analysis, scoring or interrogation method translates the amplified material into relevant biological information. This information can, for instance, be the genotype of particular SNPs or STRs, or the abundance level of a set of interesting transcripts. As such, amplification plays a significant role in nucleic acid assays. Over the years, a number of techniques – most notably PCR – has been devised to meet this amplification need, specifically or randomly multiplying desired regions. However, many of the approaches do not scale up easily rendering comprehensive studies cumbersome, time-consuming and necessitating large quantities of material.Trinucleotide threading (TnT) – forming the red thread throughout this thesis – is a multiplex amplification method, enabling simultaneous targeted amplification of several nucleic acid regions in a specific manner. TnT begins with a controlled linear DNA thread formation, each type of thread corresponding to a segment of interest, by a gap-fill reaction using a restricted trinucleotide set. The whole collection of created threads is subsequently subjected to an exponential PCR amplification employing a single primer pair. The generated material can thereafter be analyzed with a multitude of readout and detection platforms depending on the issue or characteristic under consideration.TnT offers a high level of specificity by harnessing the inherent specificities of a polymerase and a ligase acting on a nucleotide set encompassing three out of the four nucleotide types. Accordingly, several erroneous events have to occur in order to produce artifacts. This necessitates override of a number of control points.The studies constituting this thesis demonstrate integration of the TnT amplification strategy in assays for analysis of various aspects of DNA and RNA. TnT was adapted for expression profiling of intermediately-sized gene sets using both conventional DNA microarrays and massively parallel second generation 454 sequencing for readout. TnT, in conjunction with 454 sequencing, was also employed for allelotyping, defined as determination of allele frequencies in a cohort. In this study, 147 SNPs were simultaneously assayed in a pool comprising genomic DNA of 462 individuals. Finally, TnT was recruited for parallel amplification of STR loci with detection relying on capillary gel electrophoresis. In all investigations, the material generated with TnT was of sufficient quality and quantity to produce reliable and accurate biological information.Taken together, TnT represents a viable multiplex amplification technique permitting parallel amplification of genomic segments, for instance harboring polymorphisms, or of expressed genes. In addition to these, this versatile amplification module can be implemented in assays targeting a range of other features of genomes and transcriptomes.
QC 20100819

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Nyström, Anna-Maja. "RAS-MAPK syndromes - a Clinical and Molecular Investigation." Doctoral thesis, Uppsala universitet, Institutionen för genetik och patologi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-100804.

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The RAS-MAPK syndromes are a group of clinically and genetically related disorders, characterized by cardiac defects, facial dysmorphism, cutaneous abnormalities and neurocognitive impairment. The pathogenesis is dysregulation of the RAS-MAPK pathway, and several genes within the pathway are involved. The present thesis aimed at identifying genetic causes in three of the RAS-MAPK syndromes - Noonan syndrome (NS), cardio-facio-cutaneous syndrome (CFC) and Neurofibromatosis-Noonan syndrome (NFNS) - and at correlating genotype with phenotype. A mutation analysis of six genes associated with the RAS-MAPK syndromes in NS and CFC patients revealed mutations in 10/31 patients. The results suggested more complex genetic overlap and genetic heterogeneity among these syndromes than previously believed. Subsequently, gene dosage imbalances of seven RAS-MAPK-syndrome-related genes were investigated in mutation-negative patients. A multiplex ligation-dependent probe amplification strategy was developed that excluded copy number changes of these genes as a common mechanism in NS. Genetic causes of clinical variability in NS were investigated where an atypical and severe NS patient was described. In addition, multiple café-au-lait (CAL) spots affected the patient and four otherwise healthy family members. Molecular analysis of four candidate genes revealed a previously described de novo PTPN11 mutation and an inherited NF1 variant in the patient. Neither of them explained the CAL spots trait, which consequently represented a distinct entity. The results suggested that the atypical and severe phenotype in the patient could be a consequence of an additive effect. Finally, a family displaying NFNS was investigated clinically and molecularly revealing a novel mutation in the GAP-domain of NF1. Furthermore, the results suggested that other RAS-MAPK-syndrome-related genes are not involved in NFNS. A proposal of prioritizing the GAP-domain of NF1 in NFNS was presented. Conclusively, these studies contribute to further understanding of the RAS-MAPK syndromes and facilitate the diagnostic process and future prognosis prediction.

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Sabbag, Adriana Rosolia Costa. "Pesquisa de síndromes de microdeleção em pacientes com deficiência intelectual por meio da técnica de MLPA - Amplificação de Múltiplas Sondas Dependentes de Ligação." Universidade Federal de São Carlos, 2012. https://repositorio.ufscar.br/handle/ufscar/7009.

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Universidade Federal de Sao Carlos
Intellectual disability (ID) is manifest sign of more than 2,000 different clinical conditions and is present in 5% of the population. Because it is a heterogeneous group of clinical conditions, with different causal factors and simultaneously involved, about 50% of patients with ID have no defined etiology. Chromosomal microdeletions, situations in which there is loss of a fragment of up to 5Mb of the genome resulting in haploinsufficiency of one or multiple genes, are one of the possible causes of ID. The aim of this study was to standardize genetic testing with the technique of MLPA (Multiplex Ligation Probe Amplification) to investigate the presence of microdeletion syndromes in a sample of patients with idiopathic DI or with diagnosis not yet confirmed by molecular genetic testing. The MLPA kit used (SALSA® MLPA® P064-B2 MR1) detected, at the same time, 11 microdeletion syndromes: 1p36 deletion, Sotos, Miller-Dieker, 22q11.2 deletion, Saethre-Chotzen, Prader-Willi, Angelman, Williams, Alagille, Smith-Magenis and Canavan. We selected 57 patients with idiopathic ID and facial dysmorphias, with normal conventional karyotyping and normal brain imaging studies. The presence of microdeletion was identified in 4 patients (7%), 3 patients had Williams syndrome and 1 had 22q11.2 deletion. The results reinforce the usefulness of MLPA in the etiologic research of ID, helping in the clinical management and familial genetic counseling.
Deficiência intelectual (DI) é sinal manifesto de mais de 2.000 condições clínicas diferentes e está presente em 5% da população. Por se tratar de um grupo heterogêneo de condições clínicas, com fatores causais distintos e simultaneamente envolvidos, cerca de 50% dos pacientes com DI não têm sua etiologia definida. Entre as possíveis causas de DI estão as microdeleções cromossômicas, situações nas quais há perda de um fragmento de até 5Mb do genoma, implicando na haploinsuficiência de um ou múltiplos genes. O objetivo desse trabalho foi padronizar testes genéticos fundamentados na técnica de MLPA (Amplificação de Múltiplas Sondas dependentes de Ligação) para investigar a presença de síndromes de microdeleção em uma amostra de pacientes com DI idiopática ou com hipótese diagnóstica ainda não confirmada por teste genético molecular. O kit de MLPA utilizado (SALSA® MLPA® P064-B2 MR1) detectava, simultaneamente, 11 síndromes de microdeleção: deleção 1p36, Sotos, Miller-Dieker, deleção 22q11.2, Saethre- Chotzen, Prader-Willi, Angelman, Williams, Alagille, Smith-Magenis e Canavan. Foram selecionados clinicamente 57 pacientes com DI e dismorfias faciais, com cariótipo convencional e exame de imagem de encéfalo normais. A presença de microdeleção foi identificada em 4 pacientes (7%), tendo sido detectados 3 pacientes com síndrome de Williams e 1 com deleção 22q11.2. Os resultados reforçam a utilidade da técnica de MLPA na investigação etiológica da DI, ajudando no manejo clínico dos pacientes e no aconselhamento genético familiar.

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Pettersson, Erik. "Interrogation of Nucleic Acids by Parallel Threading." Doctoral thesis, Stockholm : Bioteknologi, Alba Nova, Kungliga Tekniska högskolan, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4546.

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Konstantinidis, Michalis. "Preimplantation genetic diagnosis : new methods for the detection of genetic abnormalities in human preimplantation embryos." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:28611f65-7729-4293-9c3f-4fc3f0cc39d7.

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Preimplantation genetic diagnosis (PGD) refers to the testing of embryos produced through in vitro fertilization (IVF) in order to identify those unaffected by a specific genetic disorder or chromosomal abnormality. In this study, different methodologies were examined and developed for performance of PGD. Investigation of various whole genome amplification (WGA) methods identified multiple displacement amplification as a reliable method for genotyping single cells. Furthermore, this technology was shown to be compatible with subsequent analysis using single nucleotide polymorphism (SNP) microarrays. Compared to conventional methods used in this study to perform single cell diagnosis (e.g. multiplex PCR), WGA techniques were found to be advantageous since they streamline the development of PGD protocols for couples at high risk of transmitting an inherited disorder and simultaneously offer the possibility of comprehensive chromosome screening (CCS). This study also aimed to develop a widely applicable protocol for accurate typing of the human leukocyte antigen (HLA) region with the purpose of identifying embryos that will be HLA-identical to an existing sibling affected by a disorder that requires haematopoietic stem cell transplantation. Additionally, a novel microarray platform was developed that, apart from accurate CCS, was capable of reliably determining the relative quantity of mitochondrial DNA in polar bodies removed from oocytes and single cells biopsied from embryos. Mitochondria are known to play an important role in oogenesis and preimplantation embryogenesis and their measurement may therefore be of clinical relevance. Moreover, real-time PCR was used for development of protocols for CCS, DNA fingerprinting of sperm samples and embryos and the relative quantitation of telomere length in embryos (since shortened telomeres might be associated with reduced viability). As well as considering the role of genetics in terms of oocyte and embryo viability assessment and the diagnosis of inherited genetic disorders, attention was given to a specific gene (Phospholipase C zeta) of relevance to male infertility. A novel mutation affecting the function of the resulting protein was discovered highlighting the growing importance of DNA sequence variants in the diagnosis and treatment of infertility.

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Pak, Nikita. "Simultaneous amplification of multiple dna targets with optimized annealing temperatures." Thesis, Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/44901.

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The polymerase chain reaction (PCR) is an extremely powerful tool for viral detection and screening because it can detect specific infectious agents with great sensitivity and specificity. It works by exponentially amplifying a target viral DNA sequence to high enough concentrations through the use of specific reagents and thermal cycling. It has surpassed culture based methods as the gold standard for viral detection because of the increased speed and sensitivity. Microfluidic approaches to PCR have focused on decreasing the time to thermally cycle, the volumes used for reactions, and they have also added upstream and downstream processes that are of benefit for on-chip viral detection. While these improvements have made great strides over commercially available products in terms of speed, cost, and integration, a major limitation that has yet to be explored is the throughput associated with running PCR. Since each PCR reaction relies on primers with a unique annealing temperature to detect specific viral DNA, only a single virus can be screened for at a time. The device presented here uses two infrared laser diodes that are driven identically by the same laser driver to independently thermally cycle two chambers on the same microfluidic chip. Different temperatures are achieved in the two chambers by modulating the radiation reaching one of those chambers with an optical shutter. Closed loop temperature feedback in both chambers is done with a Labview program and thermocouples embedded in the polymer chip. This allows for accurate temperature measurement without inhibiting the reaction. To demonstrate the capabilities of this device, two different reactions were simultaneously amplified successfully on the same device that have annealing temperatures that differ by 15°C.

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Clausson, Carl-Magnus. "Making Visible the Proximity Between Proteins." Doctoral thesis, Uppsala universitet, Science for Life Laboratory, SciLifeLab, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-217772.

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Genomic DNA is the template of life - the entity which is characterized by a self-sustaining anatomical development, regulated signaling processes, the ability to reproduce and to respond to stimuli. Through what is classically known as the central dogma, the genome is transcribed into mRNA, which in turn is translated into proteins. The proteins take part in most, if not all, cellular processes, and it is by unraveling these processes that we can begin to understand life and disease-causing mechanisms. In vitro and in vivo assays are two levels at which protein communication may be studied, and which permit manipulation and control over the proteins under investigation. But in order to retrieve a representation of the processes as close to reality as possible, in situ analysis may instead be applied as a complement to the other two levels of study. In situ PLA offers the ability to survey protein activity in tissue samples and primary cell lines, at a single cell level, detecting single targets in their natural unperturbed environment. In this thesis new developments of the in situ PLA are described, along with a new technique offering in situ enzyme-free detection of proximity between biomolecules. The dynamic range of in situ PLA has now been increased by several orders of magnitude to cover analogous ranges of protein expression; the output signals have been modified to offer a greater signal-to-noise ratio and to limit false-positive-rates while also extending the dynamic range further; simultaneous detection of multiple protein complexes is now possible; proximity-HCR is presented as a robust and inexpensive enzyme-free assay for protein complex detection. The thesis also covers descriptions on how the techniques may be simultaneously applied, also together with other techniques, for the multiple data-point acquisition required by the emerging realm of systems biology. A future perspective is presented for how much more information may be simultaneously acquired from tissue samples to describe biomolecular interactions in a new manner. This will allow new types of biomarkers and drugs to be discovered, and a new holistic understanding of life.

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Anscombe, C. J. "Multiple displacement amplification and whole genome sequencing for the diagnosis of infectious diseases." Thesis, Queen Mary, University of London, 2016. http://qmro.qmul.ac.uk/xmlui/handle/123456789/18409.

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Next-generation sequencing technologies are revolutionising our ability to characterise and investigate infectious diseases. Utilising the power of high throughput sequencing, this study reports, the development of a sensitive, non-PCR based, unbiased amplification method, which allows the rapid and accurate sequencing of multiple microbial pathogens directly from clinical samples. The method employs Φ29 DNA polymerase, a highly efficient enzyme able to produce strand displacement during the polymerisation process with high fidelity. Problems with DNA secondary structure were overcome and the method optimised to produce sufficient DNA to sequence from a single bacterial cell in two hours. Evidence was also found that the enzyme requires at least six bases of single stranded DNA to initiate replication, and is not capable of amplification from nicks. Φ29 multiple displacement amplification was shown to be suitable for a range of GC contents and bacterial cell wall types as well as for viral pathogens. The method was shown to be able to provide relative quantification of mixed cells, and a method for quantification of viruses using a known standard was developed. To complement the novel molecular biology workflow, a data analysis pipeline was developed to allow pathogen identification and characterisation without prior knowledge of input. The use of de novo assemblies for annotation was shown to be equivalent to the use of polished reference genomes. Single cell Φ29 MDA samples had better assembly and annotation than non-amplification controls, a novel finding which, when combined with the very long DNA fragments produced, has interesting implications for a variety of analytical procedures. A sampling process was developed to allow isolation and amplification of pathogens directly from clinical samples, with good concordance shown between this method and traditional testing. The process was tested on a variety of modelled and real clinical samples showing good application to sterile site infections, particularly bacteraemia models. Within these samples multiple bacterial, viral and parasitic pathogens were identified, showing good application across multiple infection types. Emerging pathogens were identified including Onchocerca volvulus within a CSF sample, and Sneathia sanguinegens within an STI sample. Use of Φ29 MDA allows rapid and accurate amplification of whole pathogen genomes. When this is coupled with the sample processing developed here it is possible to detect the presence of pathogens in sterile sites with a sensitivity of a single genome copy.

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Bonnefond, Sylvain. "Amplification de la fluorescence par diffusion multiple : une étude exploratoire vers des conditions biocompatibles." Thesis, Université Côte d'Azur (ComUE), 2019. http://www.theses.fr/2019AZUR4080.

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L’objet de cette thèse a consisté à mettre au point une méthode d’amplification de la fluorescence en utilisant la diffusion multiple (introduction de nanoparticules diélectriques, élastiques et passives) qui sera applicable sur des échantillons biologiques. Cela a nécessité de faire le lien entre le concept de laser biologique et de laser aléatoire pour une utilisation dans un régime amplifié, qui précède le régime laser, applicable aux marquages cellulaires et tissulaires. La génération d’une amplification passe par l’utilisation de diffuseurs (nanoparticules de dioxyde de titane dans ce travail) répartis de façon homogène. Il a fallu mettre au point des conditions de stabilisation colloïdale par adsorption de protéine (BSA) pour éviter l’agrégation des nanoparticules en tampons et milieux biologiques. Un montage optique a été mis au point pour exciter les fluorophores de façon reproductible, en évaluant précisément l’énergie de pompe et la réponse impulsionnelle de fluorescence, tout en optimisant la fenêtre d’observation pour limiter le photoblanchiment des fluorophores et la phototoxicité des cellules. Une première amplification stimulée a été montrée et validée avec une dispersion colloïdale dans une solution aqueuse homogène de FITC à une concentration de 200 µM. Cette expérience a prouvé qu’il est possible d’atteindre un gain de fluorescence jusqu’à 40 associé à une diminution de la largeur spectrale jusqu’à 5 nm, et une réduction globale de la durée de vie. La présence du processus d’émission stimulée dans l’amplification est confirmée par la corrélation entre la fluorescence et la concentration des nanoparticules ou l’énergie de pompe. Cette première démarche a été étendue à des concentrations de 2 et 20 µM pour lesquelles une saturation rapide du gain (respectivement ~ 10 et 20) a été constatée. Enfin une amplification a été montrée et validée sur des cellules en suspension marquées par la CFSE ou exprimant la GFP dans leur milieu de culture. Dans ces conditions une diminution du gain par rapport aux conditions précédentes a été constatée (jusqu’à 6 – 10 fois). L’explication de ce gain moindre a été explorée en testant les modifications des milieux sur l’amplification, et la présence d’une couche de BSA entourant les nanoparticules semble en être la cause car elle diminue probablement l’indice de réfraction effectif de la nanoparticule conduisant à une faible diffusion
The purpose of this PhD has been to develop a method of amplification of the fluorescence by using the multiple scattering (introduction of dielectric, elastic and passive nanoparticles) that will be applicable to biological samples. This required doing the link between the biological laser and random laser concept for a use in an amplified regime, which precedes the laser regime, applicable to cell and tissue labelling. The generation of an amplification passes by the use of scatterers (nanoparticles of titanium dioxide in this work) homogeneously distributed. It required developing conditions of the colloidal stabilization to avoid the aggregation of nanoparticles into biological media and buffers by the adsorption of proteins (BSA). An optical setup has been developed to excite the fluorescence, by accurately evaluating the pump energy and the pulse response of fluorescence, while optimizing the observation window to limit photobleaching of fluorophores and cell toxicity. A first stimulated amplification was shown and validated with a colloidal suspension in a homogeneous aqueous solution of FITC at a concentration of 200 µM. This experiment has shown that it is possible to achieve a fluorescence gain up to 40 associated with a decrease in spectral width up to 5 nm, and an overall reduction in lifetime. The presence of the stimulated emission process in amplification is confirmed by the correlation between fluorescence and nanoparticle concentration or pump energy. This first step was extended to concentrations of 2 and 20 µM for which a rapid saturation of the gain (respectively ~ 10 and 20) was observed. Finally, amplification has been shown and validated on CFSE-labeled or GFP-expressing suspension cells in their culture medium. In these conditions, a decreasing of the gain compared with the previous conditions has been observed (up to 6 – 10 times). The explanation of this lower gain has been explored by testing the changes of media on amplification, and the presence of a layer of BSA surrounding the nanoparticles seems to be the cause because the protein decreases probably the refractive index of the nanoparticle leading a weak scattering

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Muharam, Firman Alamsyah. "Overcoming problems with limiting DNA samples in forensics and clinical diagnostics using multiple displacement amplification." Queensland University of Technology, 2006. http://eprints.qut.edu.au/16207/.

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The availability of DNA samples that are of adequate quality and quantity is essential for any genetic analysis. The fields of forensic biology and clinical diagnostic pathology testing often suffer from limited samples that yield insufficient DNA material to allow extensive analysis. This study examined the utility of a recently introduced whole genome amplification method termed Multiple Displacement Amplification (MDA) for amplifying a variety of limited sample types that are commonly encountered in the fields of forensic biology and clinical diagnostics. The MDA reaction, which employs the highly processive bacteriophage φ29 DNA polymerase, was found to generate high molecular weight template DNA suitable for a variety of downstream applications from low copy number DNA samples down to the single genome level. MDA of single cells yielded sufficient DNA for up to 20,000,000 PCR assays, allowing further confirmatory testing on samples of limited quantities or the archiving of precious DNA material for future work. The amplification of degraded DNA material using MDA identified a requirement for samples of sufficient quality to allow successful synthesis of product DNA templates. Furthermore, the utility of MDA products in comparative genomic hybridisation (CGH) assays identified the presence of amplification bias. However, this bias was overcome by introducing a novel modification to the MDA protocol. Future directions for this work include investigations into the utility of MDA products in short tandem repeat (STR) assays for human identifications and application of the modified MDA protocol for testing of single cell samples for genetic abnormalities.

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Eulálio, Inês Mariana Cardoso. "Developmente of a CNVs detection method through qPCR." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22507.

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Mestrado em Biotecnologia
Os copy number variations (CNVs) consistem em segmentos de DNA de uma kilobase ou mais, que se encontram num número variável de cópias, em comparação com um genoma de referência. A deteção de CNVs é convencionalmente realizada através de técnicas de citogenética, como fluorescence in situ hybridization e array comparative genomic hybridization, ou com base em PCR, como multiplex ligation-dependent probe amplification, SNP arrays ou deep sequencing. Porém, a evolução da técnica de PCR quantitativo em tempo real (qPCR) permitiu que fosse, actualmente, considerada o método gold standard para a detecção de CNVs devido, sobretudo, ao elevado rendimento, sensibilidade, precisão e versatilidade. O presente trabalho descreve o desenvolvimento e validação de um método de deteção de CNVs através da técnica de qPCR. A metodologia adotada provou ser um método preciso e sensível para a detecção de CNVs em regiões específicas.
Copy number variations (CNVs) consist in DNA segments of one kilobase or larger, that are present in variable copy number, in comparison to a reference genome. CNVs detection is conventionally performed through cytogenetic, such as fluorescence in situ hybridization and array comparative genomic hybridization, or PCR-based techniques, like multiplex ligation-dependent probe amplification, SNP arrays or deep sequencing. However, the evolution of quantitative PCR (qPCR) allows it to be considered the gold standard for CNVs detection, mainly due to its high throughput, precision and versatility. The present work describes the development and validation of a qPCR method for CNVs detection. This method proved to be an accurate and sensitive method for CNVs detection in targeted regions.

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Dubois, Jérôme. "Homogénéisation dynamique de milieux aléatoires en vue du dimensionnement de métamatériaux acoustiques." Thesis, Bordeaux 1, 2012. http://www.theses.fr/2012BOR14512/document.

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Les métamatériaux sont des milieux prometteurs pour l'imagerie acoustique. Grâce à ces milieux, il est possible de concevoir des lentilles à faces parallèles pouvant dépasser la limite conventionnelle de résolution d'une lentille et par conséquent améliorer les systèmes d'imagerie. Malgré l'intérêt grandissant des chercheurs pour les métamatériaux, le comportement des ondes acoustiques dans ces milieux n'est pas totalement connu. Nous proposons de développer la problématique de la propagation des ondes acoustiques dans un milieu de type métamatériau en détail dans ce manuscrit. Cette étude a permis d'extraire un critère discriminant un métamatériau d'un matériau classique et d'apporter un regard nouveau sur l'amplification des ondes évanescentes dans les métamatériaux.Nous explorons une piste peu empruntée en vue du dimensionnement de métamatériaux : les milieux aléatoires. Nous nous focalisons sur les milieux à deux dimensions dont les phases sont fluides. Dans cette optique, une phase de validation de techniques d'homogénéisation dynamique existantes est réalisée \textit{via} la comparaison des réponses acoustiques d'un écran de diffuseurs répartis aléatoirement obtenues par des simulations numériques FDTD avec celles prédites par des modèles analytiques. L'étude de ces modèles, utiles au dimensionnement de structures aléatoires présentant des réponses acoustiques ciblées, nous a amené à examiner avec attention leur comportement quasi-statique. Une technique d'homogénéisation permettant de prendre en compte explicitement les interactions entre diffuseurs est proposée dans ce contexte. Développée dans le cadre de la diffusion simple et multiple, elle relie les propriétés mécaniques effectives aux moyennes des champs acoustiques dans un volume représentatif.Finalement, l'analyse du comportement d'un milieu aléatoire \og réaliste \fg~possédant théoriquement des bandes fréquentielles à réfraction négative, grâce à des diffuseurs résonants à basses fréquences, a été menée. Différents régimes de fonctionnement atypiques sont identifiés à l'aide de simulations numériques. La confrontation des réponses de ce milieu avec celles d'un cristal phononique est ensuite présentée et révèle une étonnante similitude entre les deux arrangements
Metamaterials are promising media for acoustic imaging. For example, such media give the possibility to build flat lenses exhibiting sub-diffraction-limit resolution, thereby improving imaging setup. Despite the growing interest of the researcher for metamaterials, acoustic wave propagation is still not widely known. This work addresses the topic of wave propagation in metamaterials. In this work, we have defined a criterion which differentiate metamaterial from classical material and provide a new insight in the amplification of evanescent waves.We explore how to design metamaterials with random media. We focus on two dimensional media with fluid components. A validation process of existing dynamic homogenization techniques is done via the comparison between the responses of a screen of scatterers obtained by numerical simulations from FDTD with those predict by the analytical models. The study of those models, useful for designing random media with atypical responses, lead us to consider their quasi-static limit. In this context, we propose a homogenization technique which includes explicitly the interactions between scatterers. It is developed for multiple and simple scattering and link the effective properties to the averages of the acoustic fields in a representative volume.Finally, the analysis of the acoustic responses of a realistic random medium having theoretical negative refraction frequency bandwidth, thanks to low frequency resonant scatterers is done. Different atypical responses are identified from the numerical simulations. The comparison between the responses of this medium and those of phononic crystals is presented and shows a surprising similarity of the two arrangements

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Krüger, Petrus Paulus. "A feasibility study of broadband low–noise amplifiers with multiple amplification paths for radio astronomy / P.P. Krüger." Thesis, North-West University, 2010. http://hdl.handle.net/10394/4507.

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Multipath amplifier theory: In this thesis it is proven that the theoretical minimum noise measure of a multipath amplifier (an amplifier which has multiple parallel amplifiers) is achieved by using the optimum source impedance for the amplifier and the optimum gain for each amplification path. This optimum source impedance and gain can be calculated by using the optimum–loaded input network, i.e. by replacing each amplifier with its optimum source impedance. The resulting noise measure is the same as the minimum noise measure of the amplifiers used in the amplification paths. Whereas single–path amplifiers can achieve this minimum noise measure over narrow bandwidths, multipath amplifiers are theoretically able to achieve the minimum noise measure over very broad bandwidths. The theory is demonstrated by applying it to distributed amplifiers. In an ideal distributed amplifier, the magnitude of the optimum gain of the amplification paths decreases and the phase delay increases the farther the stage is from the input, with the decrease in gain being faster for higher frequencies. The challenge in designing broadband, low–noise, distributed amplifiers is to achieve optimum gain matching over broad bandwidths. Multipath amplifier design procedure: Based on the theory, a three step design and optimisation procedure is introduced. Firstly, unconditionally stable amplification paths are designed to have small minimum noise measures, then an input network is designed for optimum source impedance matching and lastly an output network is designed for gain matching. Multipath amplifier prototype: The theory and design procedure is demonstrated by optimising a 0.5–2 GHz distributed amplifier. An average noise measure of 0.3 dB is achieved, which is only 0.1 dB higher than the minimum noise measure of the amplification stages used. This increase is mainly due to transmission line loss and gain mismatch. Radio telescope feasibility: Multipath amplifiers break the trade–off between noise temperature, bandwidth and source termination that a single–path amplifier has, because they have much more design freedom when designing the input network. In general, the more paths, the larger the low–noise bandwidth, but the larger and more complex the amplifier. Roughly two to three amplification paths are required per octave of bandwidth for LNAs around 1 GHz. When the bandwidth is very narrow, a single path is sufficient. Multipath amplifiers have similar trade–offs between linearity and power consumption, between noise temperature and noise resistance, and between noise temperature and size to a single–path amplifier. Multipath amplifiers are therefore a feasible alternative for use in radio telescopes.
Thesis (Ph.D. (Space Physics))--North-West University, Potchefstroom Campus, 2011.

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Mehdi, Hassan. "Modélisation Bilatérale de systèmes MIMO pour la simulation de niveau circuit et sous système, application à la fonction amplification de puissance." Limoges, 2010. https://aurore.unilim.fr/theses/nxfile/default/f26f7d4a-e4f9-46b9-89cb-6db11e6c6ed9/blobholder:0/2010LIMO4032.pdf.

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Ce travail de thèse concerne la modélisation comportementale des systèmes RF MIMO. L'objectif est la prédiction des performances systèmes d'un amplificateur de puissance qui intègre des sous circuits passifs représentés par des paramètres S. L'approche présenté se base sur les techniques de Vector Fitting qui permettant l'approximation rationnelle efficace de donnés fréquentielles. La première application concerne la synthèse équivalente d'un sous circuit passif décrits par des [S] à partir d'éléments simples R, L et C. Cette approche permet dans un simulateur de niveau circuit, d'apporte une alternative aux problèmes de convergence en transitoire d'enveloppe et d'accéder aux performances du circuit face à un stimulus réel. La deuxième application concerne la modélisation et l'implémentation de ces modèles comportementaux dans un environnement système Scilab/Scicos qui permet la résolution de systèmes implicites. La méthode permet d'intégrer des modèles en enveloppe quelque soit le nombre d'accès considérés. Ces modèles permettant d'envisager l'implémentation du modèle topologique d'amplificateur de puissance dans un environnement systèmes
This work concerns the behavioural modelisation of MIMO RF systems. The objective is to predict the power amplifier system performance that integrates into sub-"passive circuits" in which they are presented in S-parameters. This approach is based on Vector Fitting technique permits the effective rational approximation of frequency data. The first application concerns the equivalent synthesis of a sub passive circuit described from [S] parameters and simulated using simple R, L, C elements. This approach allows us to have an alternative solution of the transient envelop convergence problems and to have an access to circuit performances in the presence of real stimulus. The second application concerns the modelisation and implementation of behavioural models in a "Scilab/Scicos" environment, in which, solving an implicit systems becomes a doable task, also, it takes into account the integration of the model nomatter what the number of accesses is. These models permit to implement a power amplifier's topological model in a system environment (Scicos)

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Rönn, Ann-Charlotte. "Analysis of Nucleotide Variations in Non-human Primates." Doctoral thesis, Uppsala University, Molecular Medicine, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7904.

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Many of our closest relatives, the primates, are endangered and could be extinct in a near future. To increase the knowledge of non-human primate genomes, and at the same time acquire information on our own genomic evolution, studies using high-throughput technologies are applied, which raises the demand for large amounts of high quality DNA.

In study I and II, we evaluated the multiple displacement amplification (MDA) technique, a whole genome amplification method, on a wide range of DNA sources, such as blood, hair and semen, by comparing MDA products to genomic DNA as templates for several commonly used genotyping methods. In general, the genotyping success rate from the MDA products was in concordance with the genomic DNA. The quality of sequences of the mitochondrial control region obtained from MDA products from blood and non-invasively collected semen samples was maintained. However, the readable sequence length was shorter for MDA products.

Few studies have focused on the genetic variation in the nuclear genes of non-human primates. In study III, we discovered 23 new single nucleotide polymorphisms (SNPs) in the Y-chromosome of the chimpanzee. We designed a tag-microarray minisequencing assay for genotyping the SNPs together with 19 SNPs from the literature and 45 SNPs in the mitochondrial DNA. Using the microarray, we were able to analyze the population structure of wild-living chimpanzees.

In study IV, we established 111 diagnostic nucleotide positions for primate genera determination. We used sequence alignments of the nuclear epsilon globin gene and apolipoprotein B gene to identify positions for determination on the infraorder and Catarrhini subfamily level, respectively, and sequence alignments of the mitochondrial 12S rRNA (MT-RNR1) to identify positions to distinguish between genera. We designed a microarray assay for immobilized minisequencing primers for genotyping these positions to aid in the forensic determination of an unknown sample.

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Halilovic, Amina. "SÄKERSTÄLLNING AV SÄLLSYNTA DNA-KONTROLLER MED HELGENOMAMPLIFIERING I KLINISKT SYFTE." Thesis, Malmö högskola, Fakulteten för hälsa och samhälle (HS), 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-24387.

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Vid klinisk enbaspolymorfi (SNP) analys inkluderas DNA-kontroller med kända genotyper i varje analysomgång för att säkerställa riktigheten vad gäller analysresultatet. DNA-kontrollerna har en central roll för resultatens trovärdighet vid genotypningen. Vissa kontrollprover som används är av sällsynt genotyp och kan vara mycket svåra att få tag på. Detta arbete har utförts för att undersöka om det går att erhålla DNA-material från sällsynta genotyper med hjälp av helgenomamplifiering och på så sätt säkerställa en tillgång till dessa. I arbetet testades helgenomamplifiering med hjälp av två olika kit. De helgenomamplifierade produkternas kvantitet och kvalitet analyserades och jämfördes med det ursprungliga DNA:t, med avsikt att redogöra för det mest fördelaktiga kitet för SNP-analys i kliniskt syfte. Båda helgenomamplifierings-kiten påvisade god förmåga att amplifiera genomiskt DNA med hög kvalité. Helgenomamplifierat DNA från det bästa kitet sekvenserades och här var skillnader mellan ursprungligt och helgenomamplifierat DNA marginella. Vid sekvensanalys av ett 464 baspar långt fragment av faktor II genen och 585 baspar långt fragment av ApoE genen på fem helgenomamplifierade DNA-prover påvisades endast en eventuell diskrepans.
Clinical single nucleotide polymorphisms (SNP) analysis includes DNA controls with known genotypes in each run to ensure the accuracy of the analysis results. DNA controls have a central role for the credibility of the results in the genotyping process. Some of the used control samples are rare and can be very difficult to obtain. This work was carried out to investigate whether it is possible to obtain DNA from samples with a rare genotype using whole genome amplification and as a result ensure access to these samples. In this work the whole genome amplification method was tested by two different kits. The quantity and quality of the whole genome amplification products were analyzed and compared with the original DNA, with the intention to describe the most advantageous kit for clinical SNP analysis. Both tested kits demonstrated a good ability to amplify genomic DNA with high quality. Whole genome amplified DNA from the best kit was sequenced and the difference between the original DNA and whole genome amplified DNA was negligible. Sequence analysis of 464 base pairs of the factor II gene and 585 base pairs of the ApoE gene in five whole genome amplified DNA samples indicated only one possible discrepancy.

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Akinbami, Omolola Adetola. "Use of multiple displacement amplification based approaches for detection and analysis of environmentally significant and contaminating bacteria in fresh water." Thesis, Queen's University Belfast, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.602407.

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One of the great challenges of microbial analysis in natural environments derives from the fact that a large proportion of microorganisms present is not culturable in standard conditions. To overcome this, various molecular approaches are widely used. Multiple Displacement Amplification (MDA) is especially useful as it can be applied in conjunction with other techniques to identify genes derived from individual microbial cells. The aim of this project was to study freshwater samples obtained from various environments in Ireland in order to identify dominant bacterial species and key genes associated with them that were likely to be involved in biodegradation of contaminating compounds. In order to do this, various molecular approaches were applied, with the most important being MDA assisted PCR. The tlree fresh water environments studied were: commercial bottled water, agriculturally contaminated ground water obtained from Co. Kilkenny, Ireland and ground water samples obtained from a diesel-contaminated site in Northern Ireland. In these environments, dominant bacterial strains were identified using MDA assisted PCR. Strains related to Rhodoferax ferrireducens were identified in commercial bottled water; Pseudomonas fluorescens and Polaromonas sp. in agriculturally-contaminated ground water samples; Dechloromonas aromatica and Pseudomonas putida in diesel-contaminated ground water samples. Functional genes (nitrate reductase and naphthalene dioxygenase) which are known to be involved in biodegradation were shown to be present in some of the strains. It was shown that the narG gene (nitrate reductase) was associated with strains related to Pseudomonas fluorescens, Alicycliphilus denitrificans and Polaromonas naphthalenivorans detected in agricultural-contaminated ground water. Naphthalene dioxygenase gene (nahAc) was associated with strains related to Pseudomonas fluorescens, Pseudomonas putida and nagAc (another naphthalene dioxygenase gene) with Ralstonia pickettii detected III ground water samples obtained from a diesel-contaminated site in Northern Ireland. To achieve a more comprehensive characterization of the studied environments corresponding 16S rRNA gene libraries were obtained and analysed. Sequences found in these libraries were affiliated with Proteobacteria, Bacteroidetes, Firmicutes, Chloroflexi, Actinobacteria and Cyanobacteria

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Brito, Luciana Carla Neves. "Análise microbiológica de infecções endodônticas utilizando a associação das técnicas do Multiple Displacement Amplification (MDA) e da Hibridização DNA-DNA (Checkerboard)." Universidade Federal de Minas Gerais, 2007. http://hdl.handle.net/1843/ZMRO-77LM2N.

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Multiple Displacement Amplification (MDA) has been used to uniformly amplify bacterial genomes present in small samples, providing abundant targets for molecular analysis. The purpose of this investigation was to combine MDA and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections. 66 samples were collected from teeth with endodontic infections. Non-amplified and MDA amplified samples were analyzed by checkerboard DNA-DNA hybridization for levels and proportions of 77 bacterial taxa. Counts, % DNA probe counts and % of teeth colonized for each species in amplified and non-amplified samples were computed. Significance of differences for each species between amplified and non- amplified samples was determined using the Wilcoxon signed ranks test and adjusted for multiple comparisons. The average amount of DNA present in clinical samples ranged from 6.80 (± 5.2) ng before to 6.26 (± 1.73) ìg after MDA. 70 of the 77 DNA probes hybridized with one or more of the non-amplified samples, while all probes hybridized with at least one sample after amplification. Most commonly detected species at levels > 104 in both amplified and non-amplified samples were Prevotella tannerae and Acinetobacter baumannii at frequencies ranging from 89-100% of samples. The mean number (± SEM) of species at counts >104 in amplified samples was 51.2 ± 2.2 and in non-amplified samples was 14.5 ± 1.7. The combination of MDA and checkerboard DNA-DNA hybridization demonstrated the presence of wide range of bacterial species in endodontic samples and could facilitate studies evaluating the microbiota of endodontic infections.
A técnica do Multiple Displacement Amplification (MDA) vem sendo utilizado para amplificar uniformemente o genoma bacteriano presente em pequenas amostras, fornecendo grandes melhorias nas análises moleculares. O propósito desta pesquisa foi associar o MDA e a hibridização DNA-DNA (checkerboard) para examinar a microbiota de infecções endodônticas. Sessenta e seis amostras foram coletadas de infecções endodônticas. As amostras não amplificadas e aquelas amplificadas pelo MDA foram analisadas pelo checkerboard para a determinação dos níveis e proporções de 77 taxas bacterianas. Computaram-se a contagem, percentagem do total de DNA e percentagem de dentes colonizados para cada espécie em amostras amplificadas e não amplificadas. As diferenças significantes para cada espécie entre as amostras amplificadas e não amplificadas foram determinadas utilizando-se o teste de Wilcoxon e ajustado para comparações múltiplas. A quantidade média de DNA presente nas amostras clínicas variou de 6,80 (± 5,2) ng sem amplificação a 6,26 (± 1,73) ìg após a utilização do MDA. Setenta das 77 sondas de DNA hibridizaram com uma ou mais das amostras não amplificadas, enquanto todas as sondas hibridizaram com no mínimo uma amostra após a amplificação. As espécies mais comumente detectadas no nível > 104 células bacterianas, nas amostras amplificadas e não amplificadas, foram Prevotella tannerae e Acinetobacter baumannii numa freqüência que variou de 89-100% das amostras. O número médio (± SEM) de espécies nas contagens >104 células bacterianas, nas amostras amplificadas, foi de 51,2 ± 2,2 e, nas não amplificadas, foi de 14,5 ± 1,7. A combinação do MDA e da hibridização DNA-DNA (checkerboard) demonstrou a presença de uma grande variedade de espécies bacterianas nas amostras endodônticas demonstrando sua utilidade naqueles estudos que avaliam a microbiota presente nas infecções endodônticas.

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Tavares, Warley Luciano Fonseca. "Estudo das comunidades microbianas associadas às infecções endodônticas de dentes decíduos sintomáticos e assintomáticos pelas técnicas do Multiple-Displacement Amplification e Checkerboard DNA-DNA Hybridization." Universidade Federal de Minas Gerais, 2008. http://hdl.handle.net/1843/BUOS-9K2GUG.

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Multiple Displacement Amplification (MDA) has been used to uniformly amplify the genome from bacterial species in different types of oral samples. MDA is particularly useful in small samples, since it generates abundant target for microbial analysis. The aim of the present study was to combine MDA and Checkerboard DNA-DNA hybridization to evaluate the microbiota of endodontic infections in deciduous teeth A total of 35 children, 4 to 10 years old, having teeth with intact roots or less than 2/3 of physiological root resorption were involved in this study. Forty root canal samples were collected.and amplified. Amplified samples were analyzed by checkerboard DNA-DNA hybridization for levels of 83 bacterial taxa. . Percentages of teeth colonized by each species at different thresholds in amplified samples were computed. Levels of bacterial species present in different clinical conditions were analyzed. Significance of differences between mean proportions of each species were determined for root canal samples taken from teeth with (open tooth) or without (closed tooth) pulp chamber exposure to oral cavity, sinus tract, swelling, and pain. Significance of differences for each species in these clinical scenarios was sought with Kruskall-Wallis test. The mean amount of DNA (± SD) in the samples prior to amplification was 5.2 (± 4.7) ng. After MDA, samples contained, on average, 6.05 (± 2.3) ìg of DNA. Eighty of 83 DNA probes hybridized with one or more samples. Most prevalent bacterial species at levels > 104 bacterial cells were Actinomyces naeslundii 1 and Prevotella intermedia, both present in 93.8%.of sampled teeth. The mean number of species (± SEM) detected per tooth at the > 104 level was 20.19 (± 3.27).The most commonly detected species at this level were Actinomyces naeslundii 1 and Prevotella intermedia When mean DNA probe counts x 105 (± SEM) were analyzed, the most abundant species were A. naeslundii 1 (17.07±3.17), Prevotella nigrescens (1.12 ± 0.55) and P. intermedia (1.01 ± 0.30). Eikenella corrodens, Haemophilus aphrophilus, and Helicobacter pylori were not detected in any of the samples. Upon the analysis of the microbiota associated with the different clinical signs and symptoms investigated, statistically significant differences could be detected in a few of them. Twenty seven species were statistically significantly increased in the open tooth group. A. naeslundii 1, Veillonella parvula, Gemella morbillorum. Streptococcus oralis, Aggregatibacter actinomycetemcomitans and Neisseria mucosa were statistically significant increased in teeth with pulp chamber exposure to oral cavity. P. intermedia, Neisseria. mucosa, Streptococcus anginosus, Selenomonas noxia and Streptococcus sanguinis were detected in higher mean counts in teeth without sinus tract. There were no statistically significant differences in the microbiota associated with presence or absence of swelling. Painful teeth presented increased counts of Prevotella nigrescens and Prevotella oris. The microbiota associated with root canals from deciduous teeth seems to be more complex than previously anticipated In conclusion, results suggest that selected species are associated with the clinical signs and symptoms detected in primary root canal infections.
O Mulltiple Displacement Amplification (MDA) tem sido utilizado para amplificação uniforme do genoma de espécies bacterianas em diferentes amostras da cavidade oral. O MDA é particularmente útil em pequenas amostras, visto que o mesmo gera uma quantidade de amostra de DNA abundante para a análise microbiana. O objetivo do presente estudo foi avaliar a microbiota de infecções endodônticas de dentes decíduos. Um total de 35 crianças, de 4 a 10 anos de idade, apresentando dentes com raízes intactas ou menos que 2/3 de rizólise foram envolvidas no estudo. Quarenta amostras foram coletadas e amplificadas pela técnica do MDA. As amostras amplificadas foram analisadas pela hibridização DNA-DNA (Checkerboard) para taxas de 83 espécies bacterianas. Foram computadas as porcentagens de dentes colonizados por cada uma das espécies em diferentes limiares nas amostras amplificadas. Os níveis das espécies bacterianas encontradas em diferentes condições clínicas foram analisados. A significância das diferenças entre as proporções de cada espécie foram determinadas para amostras de canais radiculares de dentes com ou sem câmara pulpar exposta à cavidade oral, fístula, edema, e dor. A significância das diferenças para cada espécie nos diferentes cenários clínicos foi analisada pelo teste Kruskall-Wallis. A quantidade de DNA (± DP) nas amostras antes da amplificação era 5.2 (± 4.7) ng. Após o MDA, as amostras continham, em média, 6.05 (± 2.3) ìg de DNA. Oitenta das 83 sondas de DNA hibridizaram com uma ou mais amostras. As espécies bacterianas mais prevalentes em níveis > 104 células bacterianas foram Actinomyces naeslundii 1 e Prevotella intermedia, ambas presentes em 93.8% dos dentes analisados. O número médio de espécies (± DPM) detectadas por dente no nível de > 104 foi 20.19 (± 3.27). As espécies mais comumente encontradas neste nível foram Actinomyces naeslundii 1 e Prevotella intermédia. Quando a média de sondas de DNA x 105 (± DPM) foi analisada, as espécies mais abundantes foram A. naeslundii 1 (17.07±3.17), Prevotella nigrescens (1.12 ± 0.55) e P. intermedia (1.01 ± 0.30). Eikenella corrodens, Haemophilus aphrophilus, e Helicobacter pylori não foram detectados em nenhuma das amostras. Em relação à análise da microbiota associada a diferentes sinais e sintomas clínicos, diferenças estatisticamente significantes foram detectadas em algumas situações. Vinte e sete amostras foram estatisticamente significantes ao serem encontradas em maiores contagens em dentes abertos. A. naeslundii 1, Veillonella parvula, Gemella morbillorum. Streptococcus oralis, Aggregatibacter actinomycetemcomitans e Neisseria mucosa foram estatisticamente significantemente encontradas em maior número em dentes com exposição da câmara pulpar à cavidade oral. P. intermedia, Neisseria mucosa, Streptococcus anginosus, Selenomonas noxia e Streptococcus sanguinis foram detectados em contagens médias mais altas em dentes sem fístula. Não houve diferenças estatisticamente significantes na microbiota associada à presença ou ausência de edema. Dentes com dor apresentaram contagens elevadas de Prevotella nigrescens e Prevotella oris. A microbiota associada a canais radiculares de dentes decíduos demonstra ser mais complexa do que antes imaginado. Em conclusão, os resultados sugerem que espécies selecionadas estão associadas com os sinais e sintomas clínicos detectados em infecções endodônticas de dentes decíduos.

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Rodriguez, Carmen. "Etude moléculaire de mécanismes de la progression tumorale : IL-6R et GP130, émergence de la tumeur. CD44, métastases. MDR1 et GSTmu, chimiorésistance." Montpellier 2, 1994. http://www.theses.fr/1994MON20250.

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Le cancer est la deuxieme cause de mortalite, il touche en moyenne une personne sur trois. Les principales causes d'aggravation conduisant a l'issue fatale sont la dissemination metastatique et la resistance aux agents anticancereux. La transformation maligne, l'emergence d'un clone tumoral et son evolution vers des stades plus agressifs resultent de l'alteration de certaines proteines ou de leurs taux d'expression. L'identification des proteines impliquees dans chacune des etapes de la progression tumorale et l'evaluation de leurs impacts respectifs sont essentiels pour une meilleure comprehension. Au cours de cette etude, nous nous sommes interesses a trois groupes de proteines potentiellement impliquees dans ces processus de transformation et d'aggravation. Nous nous sommes interroges sur l'existence de mutations dans les genes codant pour les chaines receptrice (il-6r) et transductrice (gp130) du recepteur de l'il-6, qui pourraient induire l'emergence du clone tumoral dans le myelome multiple (neoplasie b dont le facteur central de proliferation est l'il-6). Cette etude a revele la presence de mutations ponctuelles dans le domaine intracytoplasmique de la gp130 pour 2 des 28 echantillons de myelome analyses. L'importance des sequences adjacentes non analysees est discutee. Nous avons identifie les isoformes du cd44 exprimees dans les cancers du sein et du colon, et examine leurs influences respectives sur le potentiel metastatique des tumeurs. Une grande diversite d'expression est observee, avec des profils parfois tres complexes. L'expression de variants contenant l'exon 10 est correlee avec la presence de metastases distales dans les cancers du colon. Nous nous sommes interesses a l'impact des mecanismes de resistance impliquant la p-gp et la gst- dans les lymphomes malins non hodgkiniens. Bien que la consideration simultanee de ces deux mecanismes semble d'une meilleure valeur pronostique que leur consideration individuelle, ils ne suffisent pas a expliquer la chimioresistance observee. L'identification recente d'autres mecanismes de resistance est discutee

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Chaillat, Stéphanie. "Méthode multipôle rapide pour les équations intégrales de frontière en élastodynamique 3D. Application à la propagation d'ondes sismiques." Phd thesis, Ecole des Ponts ParisTech, 2008. http://tel.archives-ouvertes.fr/tel-00359461.

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La simulation de la propagation d'ondes pour des configurations 3D est un domaine de recherche très actif. Le principal avantage de la BEM est de ne discrétiser que les frontières du domaine. Elle est ainsi bien adaptée aux domaines infinis. Cependant, la BEM classique conduit à des matrices pleines et donc à des coûts de calcul et mémoire importants.
La FMM a permis d'augmenter de manière significative les capacités de la BEM dans beaucoup de domaines d'application.
Dans ce travail, la FMM est étendue aux équations de l'élastodynamique 3D dans le domaine fréquentiel, pour des domaines homogènes puis, grâce à une stratégie de couplage BE-BE, aux problèmes multi-domaines. D'autres améliorations de la méthode sont aussi présentées: préconditionnement, réduction du nombre de moments, développement multipôle pour les fonctions de Green du demi-espace. Des applications en sismologie sont présentées pour des modèles canoniques ainsi qu'au modèle de la vallée de Grenoble.

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Cabrera, Salas Dwight José. "Low phase noise Mm-wave frequency generation for backhauling applications on BiCMOS technology." Thesis, Bordeaux, 2015. http://www.theses.fr/2015BORD0432/document.

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Cette thèse porte sur l’analyse et la conception de générateurs de fréquence millimétrique à faible bruit de phase destinés à des applications de communication sans fil de très haut débit sur une technologie BiCMOS 0.25m. Spécifiquement, des applications backhauling sont visées sur le protocole de communication P2P (point-to-point), pour un système de radio hétérodyne (à faible fréquence intermédiaire) approprié pour les bandes entre 30–38GHz et de faible profondeur de modulation (2-3 bits /symbole). Une étude rigoureuse du comportement du bruit de phase en 1/f2 d’un oscillateur contrôlé en tension (du type paire différentielle croisée) en fonction de la fréquence d’oscillation est développée. Des facteurs essentiels pour la conception de ces oscillateurs tels que la plage de fréquence et la charge de la paire croisée sur le résonateur sont pris en compte dans l’analyse. L’étude révèle que lorsque la fréquence augmente, l’oscillateur passe à travers deux régimes d’opération différents, ici appelés région QL-limited et région QC- limited, qui résultent de la dépendance du facteur de qualité du résonateur à sa partie inductive (pour les basses fréquences d’oscillation) et sa partie capacitive (pour les hautes fréquences d’oscillation). De plus, l’impact de la plage de fréquence sur l’évolution du bruit de phase en 1/f2 a été considéré en utilisant un circuit classique à base d’un varactor et d’un condensateur du type MiM. Des équations simples et précises ont été calculées pour les paramètres du circuit afin d’obtenir une fréquence centrale souhaitée avec la variation de la capacité requise. Pour ce circuit, il a été démontré (et vérifié à travers des simulations du circuit) que le pire scénario du facteur de qualité peut être associé à la constante de temps d’un condensateur. Ce dernier a permis d’estimer aisément le facteur de qualité minimal de la partie capacitive du résonateur LC de l’oscillateur, pour une plage de fréquence donnée, en fonction de la fréquence d’oscillation. D’une manière similaire, et basée sur une analyse à petit signal, la constante de temps de la capacité de sortie de la paire croisé a été déterminée. Notamment cette constante de temps présente un comportement constant sur une large gamme de fréquences, ce qui permet d’évaluer facilement son facteur de qualité. Cette étude fournit les bases théoriques qui permettent l’évaluation du bruit de phase en 1/f2 d’une source de signal basée sur un oscillateur en mode fondamental, super-harmonique ou sous-harmonique. En effet, la supériorité des oscillateurs sous-harmoniques est démontrée et des équations simples sont proposées pour déterminer la performance maximale et les conditions dans lesquelles elles peuvent être atteintes. Enfin, un système de génération de signal est ainsi conçu et vérifié par des mesures sur un prototype. Le système est composé d’un VCO sous-harmonique suivi d’un tripleur de fréquence (ILFT) –verrouillé par injection. Le circuit est implémenté sur une technologie SiGe:C BiCMOS 0.25 m. Le tripleur implémente une configuration à émetteur commun, polarisé en courant, qui exploite la seconde harmonique du VCO afin d’améliorer l’efficacité de la génération du signal responsable de verrouiller le ILFT. A 30.8 GHz, le système atteint un bruit de phase de -112 dBc/Hz à 1MHz d’offset. La consommation totale de courant est de 38mA pour une tension d’alimentation de 2.5V. Un deuxième prototype a été réalisé pour un système de génération multibande, offrant ainsi trois sorties RF à 18 GHz, 34GHz et 68 GHz. Avec une plage de fréquence de 10% (mesurée par rapport à la fréquence centrale) pour chaque sortie RF. Le bruit de phase mesuré à 1MHz d’offset est respectivement de -113dBc/Hz, -107dBc/Hz et -100dBc/Hz
This thesis deals with the analysis and design of Low phase Noise Local-Oscillator(LO) sources suitable for backhauling applications on the frequencies 30-38GHz. The LO is intended to be used in a low-IF architecture for low order modulations (2-3 bits/symbol). This work was developed in collaboration with NXP Semiconductorsat CAEN, France, within the project RF2THz of the European program CATRENE.The original contributions in this work include a rigorous study of the 1/f2 phasenoise characteristics of the VCO (bipolar cross-coupled pair Voltage-Controlled-Oscillator) with the oscillating frequency. Key factors in the design of VCOs such as tuning range and the tank load given by the cross-coupled pair are considered in the analysis. The study reveals that as the frequency scales, the VCO passes through two different zones, named the QL-limited and the QC-limited region, that results from the dependence of the resonator quality factor on its inductive part (for low oscillating frequencies) and its capacitive part (for high oscillating frequencies). Moreover, the impact of the tuning range on the 1/f2 phase noise evolution was captured by using a classical circuit based on an AC-coupled varactor and a MiM capacitor. Simple and accurate equations were derived for the circuit parameters in order to achieve a desired central frequency with the required capacitance variation. For this circuit, it is demonstrated (and verified through circuit simulations) that the lowest quality factor scenario can be associated to the time-constant of a lossy capacitor. The latter allows to estimate easily the minimum quality factor of the capacitive part of the VCO LC tank, for a given tuning range, as a function of the oscillating frequency. In a similar way, and based on a small signal analysis, the time-constant of the output capacitance of the bipolar cross-coupled pair was derived. Interesting, this time constant shows a constant behavior over a wide frequency range, thereby allowing to estimate easily its quality factor. This study set the bases for an analytical framework that enables the evaluation of the 1/f2 phase noise performances of local oscillator sources working either on fundamental,super-harmonic or sub-harmonic mode. The superiority in terms of 1/f2 phase noise of local oscillators based on sub-harmonic oscillators is thus demonstrated and simple equations are derived to determine the maximum performance and the conditions on which this can be achieved. Finally, a signal generation system intended for a low-IF point-to-point fixed radio system in the Ka-Band band is thus designed and verified through prototype measurements.The system is composed by a sub-harmonic VCO followed by an injectionlocked frequency tripler (ILFT) and it is designed in a 0.25m BiCMOS SiGe:C technology. The ILFT implements a cascode current-biased common emitter configuration that exploits the second harmonic of the VCO to enhance the efficiency in the generation of the injecting signal responsible for the ILFT locking. At 30.8GHz, the system achieves a phase noise of -112dBc/Hz at 1MHz offset. The total current consumption is 38mA for a supply voltage of 2.5V. A second prototype is designed for a multiband LO generation, providing thus three RF outputs at 18GHz, 34GHz and 68GHz. With a measured tuning range of 10% for each RF output, the measured phase noise at 1MHz is -113dBc/Hz, -107dBc/Hz and -100dBc/Hz respectively

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NOBILET, Stéphane. "Etude et optimisation des techniques MC-CDMA pour les futures générations de systèmes de communications hertziennes." Phd thesis, INSA de Rennes, 2003. http://tel.archives-ouvertes.fr/tel-00004081.

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Les futurs systèmes de radiocommunications devront proposer des services nécessitant des transferts de données à haut débit, tout en garantissant une grande mobilité aux utilisateurs. Pour y parvenir, de nouvelles techniques de traitement de signal doivent être mises en oeuvre. Une de ces approches constituant actuellement un axe important de la recherche dans ce domaine est la technique MC-CDMA. Cette dernière repose sur la combinaison de deux techniques : les modulations à porteuses multiples et l'étalement de spectre. Les travaux de recherche présentés dans cette thèse ont pour buts l'étude et l'optimisation des systèmes de communications mettant en oeuvre cette technique MC-CDMA.

Après une présentation générale des différentes façons de combiner les techniques de modulations à porteuses multiples et d'étalement de spectre, les performances des systèmes MC-CDMA sont présentées sur des canaux de Rayleigh et BRAN dans le cas de détection mono-utilisateur et multi-utilisateur.

Puis, l'influence des codes d'étalement sur la variation de la dynamique de l'enveloppe du signal émis, et sur l'interférence produite par la cohabitation des données de plusieurs utilisateurs sur les mêmes ressources fréquentielles et temporelles est étudiée. Pour cela, nous nous sommes intéressés aux variations de l'enveloppe des sisgnaux MC-CDMA à travers l'étude du facteur de crête et du facteur de crête global. Ces quantités permettent d'estimer les fluctuations du signal transmis respectivement en voie montante et descendante. En ce qui concerne la minimisation de l'interférence d'accès multiple, une technique reposant sur l'allocation des séquences d'étalement est décrite et plusieurs critères de sélection des codes d'étalement sont proposés.

Enfin, une attention particulière est portée à l'optimisation de la voie montante des systèmes MC-CDMA. Afin d'éviter l'insertion de Nu jeux de sous-porteuses pilotes servant à estimer les Nu canaux de la voie montante, une solution reposant sur le principe de la réciprocité du canal radiomobile est ici proposée. Cette alternative consiste à effectuer une prédistorsion du signal utile dans le terminal avant son émission. Deux variantes sont envisagées, la première repose sur un multiplexage temporel des voies montante et descendante alors que la seconde repose sur un multiplexage fréquentiel de ces deux voies.

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Oukacine, Farid. "Nouvelle méthodologie analytique pour l'étude de l'activité antibactérienne des dendrimères greffés de la L-lysine par électrophorèse capillaire." Thesis, Montpellier 2, 2011. http://www.theses.fr/2011MON20089/document.

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Ces travaux de thèse ont permis de mettre en œuvre une méthode de criblage de l'activité antibactérienne des dendrimères greffés de la L-lysine (DGL) par électrophorèse capillaire (EC). Le principe de la méthode est basé sur le suivi du profil électrophorétique des bactéries avant et après leur rencontre avec une bande de composé cationique dont on souhaite cribler l'activité. La mise en œuvre de cette méthode a nécessité plusieurs étapes. Dans la première étape, une nouvelle méthodologie permettant la focalisation, la mobilisation et la quantification des bactéries par EC a été développée. Cette méthode a été appliquée pour la quantification de la flore totale dans des eaux naturelles. Dans la seconde étape, plusieurs revêtements neutres de capillaire ont été comparés pour l'analyse simultanée de composés polycationiques et polyanioniques. Dans la dernière partie, la méthodologie de criblage a été mise en œuvre pour l'étude de l'activité des DGL
In this work, a new analytical methodology has been implemented for the screening of antibacterial activity of dendrigraft poly-L-lysines (DGL) by capillary electrophoresis (CE). The principle of this methodology is based on the monitoring of the electrophoretic profile of bacteria before and after the meeting with a zone containing the cationic compound to be screened. The implementation of this methodology has required several steps. In a first experimental part, a new methodology has been developed for the focalization, mobilization and quantification of bacteria. This focusing mode has been applied for the quantification of bacteria in natural waters. In a second experimental part, several neutral capillary coatings were compared for the simultaneous CE analysis of polyanionic and polycationic compounds. In the last experimental part, the screening of antibacterial activity has been implemented on DGL

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Chou, Yu-Pao, and 周育葆. "Primer Design for Multiplex Polymerase Chain Reaction and Multiplex Isothermal Recombinase Polymerase Amplification." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/65p49j.

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碩士
國立交通大學
生物資訊及系統生物研究所
106
At present, most of the deoxyribonucleic acid (DNA) amplification techniques such as polymerase chain reaction (PCR). PCR relies on the thermal cycle machine, through denaturation, annealing, extension, the process requires precise control the temperature. Recombinase polymerase amplification (RPA) technology is developed by TwistDx in 2006. First, it makes the primer sequence and protein form a complex, the complex will find the location of the homologous sequence on the DNA template and open double-stranded DNA helix structure. Next, amplification was performed by recombinase polymerase. The temperature of the whole process is maintained at about 37 to 42°C, which will allow the DNA amplification technology get a new breakthrough. DNA amplification technology at constant temperature will no longer need to rely on the thermal cycle machine, enhances this DNA amplification technology’s portability and convenience. However, the most important part of the technology is how to design primers to make the RPA correctly amplify the target gene or sequence. In addition, design primers for multiplex PCR or RPA, it needs to avoid the two primers because the sequence with excessive similarity leads to form primer dimers so that reduce the amplification efficiency. So far, it is still not found that someone provides a primer design for multiplex RPA platform. In this study, we collect RPA primers from literature, and statistics out RPA primer features and integrate the recommendations of primer design from literature. Next, according to as above, use a series of bioinformatics methods like we use Primer3 to generate candidate primer groups, and then we use Bowtie to confirm the specificity of each primer pairs. Finally, the genetic algorithm was used to find out optimized primer group that the temperature between the two primers will not be too high to form primer dimers. In this study, we respectively designed primer sets for multiplex PCR and multiplex RPA to provide future experimental verification, such as gel electrophoresis, next-generation sequencing or Nanopore MinION sequencing platform. In summary, this study develops a web platform and a standalone tool allows users to design multiplex PCR or RPA primer sets that meet their own experimental needs.

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官元揚. "The study of pigeon relative identification by STR multiplex amplification on forensic applicatons." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/nnbbyz.

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