• Refine Query
  • Source
  • Publication year
  • to
  • Language
  • 3
  • 2
  • 1
  • Tagged with
  • 8
  • 8
  • 3
  • 2
  • 2
  • 2
  • 2
  • 2
  • 2
  • 2
  • 2
  • 2
  • 2
  • 2
  • 2
  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Enabling Technologies for Synthetic Biology: Gene Synthesis and Error-Correction from a Microarray-Microfluidic Integrated Device

Saaem, Ishtiaq January 2011 (has links)
<p>Promising applications in the design of various biological systems hold critical implications as heralded in the rising field of synthetic biology. But, to achieve these goals, the ability to synthesize in situ DNA constructs of any size or sequence rapidly, accurately and economically is crucial. Today, the process of DNA oligonucleotide synthesis has been automated but the overall development of gene and genome synthesis technology has far lagged behind that of gene and genome sequencing. This has meant that numerous ideas go unfulfilled due to scale, cost and impediments in the quality of DNA due to synthesis errors. </p><p>This thesis presents the development of a multi-tool ensemble platform targeted at gene synthesis. An inkjet oligonucleotide synthesizer is constructed to synthesize DNA microarrays onto silica functionalized cylic olefin copolymer substrates. The arrays are married to microfluidic wells that provide a chamber to for enzymatic amplification and assembly of the DNA from the microarrays into a larger construct. Harvested product is then amplified off-chip and error corrected using a mismatch endonuclease-based reaction. This platform has the potential to be particularly low-cost since it employs standard phosphoramidite reagents and parts that are cheaper than optical and electrochemical systems. Genes sized 160 bp to 993 bp were successfully harvested and, after error correction, achieved up to 94% of intended functionality.</p> / Dissertation
2

Development Of A Sandwich-type Dna Array Platform For The Detection Of Label-free Oligonucleotide Targets

Cansiz, Sena 01 October 2010 (has links) (PDF)
DNA arrays have become a major bioanalytical method as they enable high-throughput screening and they can be manufactured on different surfaces depending on the nature of diagnostic purpose. However, current technologies to produce and detect arrays of DNA probes are expensive due to the requirement of specialized instrumentation. In this study we have established an array platform in sandwich hybridization format for the detection of label-free nucleic acid targets. Unlike direct hybridization, which is the main microarray hybridization principle, sandwich assay enables unlabeled target detection, lowering the cost and assay time. To this end, sequence specific signal development was achieved by a sandwich complex which is composed of a surface immobilized capture DNA probe (Probe1) and a fluorescein-tagged signal DNA probe (Probe 2), which are partially complementary to the sequence to be analyzed (target oligonucleotide). As the solid support of the array platform both 3-aminopropyl-3-methoxysilane (APTMS) activated and commercially purchased poly-L lysine coated glass slides were used and due to the less background noise property the latter one was preferred. Similarly, for the immobilization of the capture Probe (P1) onto the solid support two different methods were tried / heat immobilization and immobilization via a heterobifunctional cross-linker (HBCL). In regard to the experiments, it is observed that using a cross-linker instead of heat immobilization reduces the ratio of false negative control results in a significant manner. Following the solid support and immobilization method choice comparative optimization studies which include cross-linker type, probe concentration, sensitivity of the platform and hybridization conditions (sequence, temperature and duration) were conducted. Optimum hybridization signal was obtained with a 32.5
3

Authentication and investigation of potential hepatotoxicity of Black Cohosh

Williams, Sarah January 2017 (has links)
Black Cohosh (Actaea racemosa) is one of the highest selling medicinal plants, ranking as the sixth best seller in the US in 2015 (Smith et al., 2016). However, this popularity has been tarnished by claims of hepatotoxicity. The investigation of these reports has determined that implicated products did not contain Black Cohosh plant material. Other reports were shown to be incomplete or had other factors contributing. This has led to the suspicion that cases of adverse reactions may in fact be linked to cases of substitution or adulterations with Asian species of Actaea, rather than to A. racemosa. (Jordan et al., 2010). This shows the need for authentication of Black Cohosh products. In this study various DNA based authentication methods were developed. The first, PlantID is capable of discriminating between Actaea racemosa and four potential adulterant species; Actaea cimicifuga, Actaea cordifolia, Actaea podocarpa and Caulophyllum thalictroides, in a single PCR reaction. The resulting fragments are scrutinized using gel electrophoresis. Other platforms of analysis were trialled with little success. The second was a qPCR based method. These assays are competent in detecting A. racemosa, A. cimicifuga and A. dahurica species and are compared to a generic primer capable of amplification of ten Actaea species. This enables the user to detect specific species in comparison to how much Actaea species are present as a whole. This assay was extensively tested on many materials and products available in the UK and the USA. Out of 34 products assessed it was possible to extract DNA from 32. From the UK market it was found that five products contained undeclared species. From the US market it was found that six products contained undeclared species. All of the THR registered products were found to contain only the authentic species Actaea racemosa. This was a reassuring result from the analysis and adds further value to the scheme of THR. Sequence data from GenBank was used to assist in assigning species to sequenced DNA samples. The data contained on GenBank was scrutinised using various bioinformatics tools. Sequences were organised into molecular taxonomic units using tree diagram software. This showed efficiently and iii visually which sequence entries were reliable to use based upon grouping. This analysis showed that the nuclear internal transcribed spacer (nrITS) was an ideal barcoding region and that maturase K (MatK) was a poor choice for Actaea species. To address the issue of hepatotoxicity claims, cultured human hepatocyte derived cells were treated with 60% ethanol extracts of Actaea racemosa and Asian Actaea. A qPCR array was utilised to assess 84 genes associated with hepatotoxicity across various concentrations of extract. The collective array output gave a plethora of data which was analysed using bespoke online software from the manufacturer. Stringent quality controls were included on the arrays which gave confidence of results. There were small changes noted for Actaea racemosa and some activity for the Asian Actaea treated cells was also seen. An LDH and MTT assay were used to assess cell viability and toxicity in two human hepatocyte derived cell lines. Actaea racemosa showed no significant effects whereas the Asian Actaea extract showed a notable decrease in cell viability and significant release of LDH indicating toxicity. The Asian Actaea material used to manufacture extracts was of questionable species origin but determined to be either A. dahurica or A. cimicifuga. The results from these experiments were unfortunately not as conclusive as hoped, but did show some evidence of a more likely culprit of toxicity originating from Asian Actaea species.
4

Effects of Microparticulate Drug Delivery Systems : Tissue Responses and Transcellular Transport

Ragnarsson, Eva January 2005 (has links)
<p>Over the past decade, the development of macromolecular drugs based on peptides, proteins and nucleic acids has increased the interest in microparticulate drug delivery, i.e., the delivery of drug systems in the nanometer and micrometer ranges. However, little is known so far about the effect that microparticulate systems have on various tissues after administration. Additionally, the knowledge of mechanisms responsible for the uptake and transport of microparticles across the human intestine is incomplete and requires further investigation to improve both the safety profiles and the efficiency of these drug delivery systems.</p><p>This thesis is comprised of two parts. The first one investigates gene expression responses obtained from DNA arrays in local and distal tissues after microparticulate drug delivery. The second part focuses on the mechanisms responsible for the transport of microparticles across epithelial cells lining the intestine.</p><p>The results presented in the first part demonstrated that gene expression analysis offers a detailed picture of the tissue responses after intramuscular or pulmonary administration of microparticulate drug delivery systems compared to the traditional techniques used for such evaluations. In addition, DNA arrays provided a useful and sensitive tool for the initial characterization and evaluation of both local and distal tissue responses, making it possible to distinguish between gene expression patterns related to each studied delivery system.</p><p>The results presented in the second part demonstrated that the surface properties of the microparticle were important for the extent of transport across an <i>in vitro</i> model of the follicle-associated epithelium (FAE), comprised of intestinal epithelial cells specialized in particle transport (M cells). Another important finding was that the enteropathogen bacterium, <i>Yersinia pseudotuberculosis</i>, induced microparticle transport across the normal intestinal epithelium, represented by Caco-2 cells and excised human ileal tissue. This transport was most probably mediated by an increased capacity for macropinocytosis in the epithelial cells.</p>
5

Effects of Microparticulate Drug Delivery Systems : Tissue Responses and Transcellular Transport

Ragnarsson, Eva January 2005 (has links)
Over the past decade, the development of macromolecular drugs based on peptides, proteins and nucleic acids has increased the interest in microparticulate drug delivery, i.e., the delivery of drug systems in the nanometer and micrometer ranges. However, little is known so far about the effect that microparticulate systems have on various tissues after administration. Additionally, the knowledge of mechanisms responsible for the uptake and transport of microparticles across the human intestine is incomplete and requires further investigation to improve both the safety profiles and the efficiency of these drug delivery systems. This thesis is comprised of two parts. The first one investigates gene expression responses obtained from DNA arrays in local and distal tissues after microparticulate drug delivery. The second part focuses on the mechanisms responsible for the transport of microparticles across epithelial cells lining the intestine. The results presented in the first part demonstrated that gene expression analysis offers a detailed picture of the tissue responses after intramuscular or pulmonary administration of microparticulate drug delivery systems compared to the traditional techniques used for such evaluations. In addition, DNA arrays provided a useful and sensitive tool for the initial characterization and evaluation of both local and distal tissue responses, making it possible to distinguish between gene expression patterns related to each studied delivery system. The results presented in the second part demonstrated that the surface properties of the microparticle were important for the extent of transport across an in vitro model of the follicle-associated epithelium (FAE), comprised of intestinal epithelial cells specialized in particle transport (M cells). Another important finding was that the enteropathogen bacterium, Yersinia pseudotuberculosis, induced microparticle transport across the normal intestinal epithelium, represented by Caco-2 cells and excised human ileal tissue. This transport was most probably mediated by an increased capacity for macropinocytosis in the epithelial cells.
6

Detecção de instabilidade genômica por hibridização genômica comparativa baseada em microarranjos (array CGH) em fetos dismórficos / Detection of genomic instability by microarray-based comparative genomic hybridization (array CGH) in dysmorphic fetuses

Machado, Isabela Nelly 16 August 2018 (has links)
Orientador: Ricardo Barini / Tese (doutorado) - Universidade Estadual de Campinas. Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-16T03:43:19Z (GMT). No. of bitstreams: 1 Machado_IsabelaNelly_D.pdf: 3476920 bytes, checksum: d5a5716fb5a528c323d17a22ef6c28d0 (MD5) Previous issue date: 2010 / Resumo: Introdução: Para uma parcela significativa de fetos com defeitos congênitos o diagnóstico sindrômico permanece indefinido, dificultando a abordagem perinatal, o estabelecimento de prognóstico e o aconselhamento genético. A incapacidade de detecção de pequenas instabilidades genômicas, atualmente apontadas como provável fator causal nestas condições dismórficas, é a principal limitação do estudo cromossômico microscópico pelo bandamento G (cariótipo convencional). A hibridização genômica comparativa (comparative genomic hybridization-CGH) é capaz de identificar perdas e ganhos de material genômico com alta resolução, sem envolver o cultivo celular e o conhecimento prévio da região genômica envolvida. Objetivo: Avaliar a aplicabilidade da técnica de array CGH em sangue fetal para o diagnóstico de perdas e ganhos genômicos em um grupo de fetos dismórficos. Sujeitos/Método: Foi realizado um estudo prospectivo descritivo a partir de amostras sanguíneas de fetos dismórficos e com cromossomos numericamente normais ao bandamento G, admitidos no Setor de Medicina Fetal do Centro de Atenção Integral à Saúde da Mulher (CAISM) da Universidade Estadual de Campinas (Unicamp). Foi realizada a caracterização da amostra estudada e uma análise descritiva dos achados moleculares através da técnica de array CGH. Resultados: Foram incluídos no estudo 50 fetos, dos quais 49 preencheram os critérios de qualidade da técnica. A taxa de detecção de alterações cromossômicas pela técnica de array CGH não detectadas pelo cariótipo convencional foi de 93,7% (45 fetos), e 27% foram consideradas significativas dos pontos de vista citogenético e clínico. Entre os fetos com alterações do número de cópias, 87% apresentaram pelo menos um clone para o qual já estão descritas variações do número de cópias (CNV) em indivíduos fenotipicamente normais. Adicionalmente, a técnica mostrou-se eficaz para o esclarecimento diagnóstico da origem, exata localização e dimensionamento do material adicional encontrado em um feto com anomalia cromossômica estrutural. Conclusões: A caracterização do perfil genômico por array CGH de fetos com defeitos congênitos permitiu complementar o diagnóstico citogenético convencional, aumentando a definição diagnóstica e a identificação de regiões cromossômicas associadas a algumas anomalias congênitas / Abstract: Introduction: A great number of fetuses with congenital defects remain without definitive diagnosis, making difficult the perinatal management, the prognosis establishment and the genetic counseling. The incapacity of detection of short sequence copy number changes, pointed as a probable etiology factor for some congenital defects, is the main limitation of routine G-banding. The Comparative Genomic Hybridization (CGH) overcome this limitation, and also does not require cellular culture or prior knowledge of the involved genomic region. Objective: To evaluate the applicability of the CGH method on fetal material for genomic gains and losses in a group of malformed fetuses. Methods: On a prospective descriptive study, fetal blood samples were collected from malformed fetuses with numerically normal chromosomes at G-banded karyotype, at the Fetal Medicine Unit of the Centro de Atenção Integral à Saúde da Mulher (CAISM) of the Universidade Estadual de Campinas (UNICAMP). Sample characterization and a descriptive analysis of the CGH-based technique results were accomplished. Results: Fifty fetuses were included in this study and 49 were considered optimal according to adopted quality control criteria. The detection rate of fetuses with copy number imbalances not detected by the G-banded karyotype was 93.7% (45 fetuses), with 27% of cytogenetic and clinical significance. Among fetuses with copy number imbalances, 87% presented at least one abnormal clone encompassing CNVs described for phenotipically normal individuals. Additionally, the array CGH showed to be effective for the identification of the additional genetic material origin, with its precise location and size, presented in one fetus with structural chromosomal anomaly. Conclusions: The genomic profile characterization of malformed fetuses through array CGH allowed complementing the conventional cytogenetic diagnosis, obtaining a higher precise diagnosis and the identification of chromosomal regions associated with some congenital anomalies / Doutorado / Tocoginecologia / Doutor em Tocoginecologia
7

Využití nových genomických technik ve studiu patogeneze vybraných vzácných dědičných onemocnění. / Application of novel genomic techniques in studies of pathogenesis of selected rare inherited disorders

Nosková, Lenka January 2013 (has links)
Rare diseases are a heterogeneous group of disorders. Knowledge of their molecular basis is poor and till recently there were no appropriate methodical approaches due to a limited number of patients. Novel genomic techniques, especially the DNA array technology and the next generation sequencing emerging in last few years, enabled studies of these diseases even in small families and sporadic cases. This PhD thesis focuses on application of novel genomic techniques in studies of rare inherited diseases. It describes a use of DNA array technology in linkage analysis, analysis of differential gene expression, analysis of copy number variations and homozygous mapping, and a use of next generation sequencing technology. Combination of these methods was used for identification of molecular basis of adult neuronal ceroid lipofuscinosis, Rotor syndrome, isolated defect of ATP synthase and mucopolysaccharidosis type IIIC.
8

Studium molekulární podstaty vybraných dědičně podmíněných onemocnění / Molecular basis of selected inherited rare diseases

Hartmannová, Hana January 2013 (has links)
Rare diseases represent a clinically and genetically heterogeneous group of diseases affecting various organs and presenting at different ages. Identification and functional characterization of genetic defects causing individual rare diseases represent unique opportunity to understand biological functions of human genes and gene products as well as to basic pathogenetic mechanisms of individual diseases. This knowledge is prerequisite for their effective diagnosis, specific treatment and prevention and it also opens up an avenue for better understanding of complex diseases. My thesis documents basic conceptual and methodological developments of biochemical genetics, functional cloning, genetic mapping, positional cloning, DNA microarrays and genomic sequencing, which have provided a universal framework for effective characterization of the genetic architecture of almost all human diseases. This conceptual and technological developments are demonstrated on several cases of rare genetic diseases - adenylosuccinate lyase deficiency, mucopolysacharidosis type IIIC, Rotor syndrome, deficiency of ATP synthase, neuronal ceroid lipofuscinosis, GAPO syndrome and X -linked restrictive cardiomyopathy, which genetic and molecular basis I have helped to elucidate.

Page generated in 0.1046 seconds