Cytomegalovirus (CMV) is a member of the Herpesviridae family and is known to infect people of all ages. In most cases, CMV infection is asymptomatic, and the virus is cleared from the host without showing any significant symptoms. However, 1 out of 200 babies are born with congenital CMV infection, which affects multiple organs, including the brain, liver, spleen, lung, and inner ear. One long-term health problem in 1 out 5 babies born with congenital CMV infection is hearing loss. The progression of CMV-associated hearing loss in the first two years of life may lead to developmental delays in language, learning, and communication. Currently, for serological testing of CMV in patients older than 12 months, real-time polymerase chain reaction (rtPCR) is used. Although the rtPCR method quantitatively detects the CMV, this method is expensive and needs highly skilled technicians to perform the assay. Therefore, there is a need for a cost-effective, simple, and rapid diagnostic tool that can help detect CMV in newborn babies and prevent CMV-mediated pathology in pediatric as well as in future adult conditions. In recent scientific studies, gold nanoparticles (AuNP) of 100 nm, 40 nm, and 15 nm sizes have shown promising results in detecting various viruses. In our study, we developed an AuNP-based colorimetric assay for detecting CMV in pediatric-derived urine samples. For comparison purposes, the pediatric urine samples were screened through quantitative PCR (qPCR) for positive and negative CMV determination. In our assay, we explored the ability of different-sized nanoparticles to detect CMV in pediatric urine samples. Using purified CMV virions as a positive control, we have shown that AuNP can effectively detect the presence of CMV in pediatric urine samples. Therefore, this colorimetric assay may be a basis for a useful diagnostic tool for detecting CMV in newborn babies. It is a rapid and non-invasive high-throughput assay for screening for the presence of CMV at the point of care and may help provide better therapeutic outcomes for pediatric patients.

No / Lymphocyte responses from 208 individuals: 20 with melanoma, 34 with colon cancer, and 4 with lung cancer (58), 18 with suspected melanoma, 28 with polyposis, and 10 with COPD (56), and 94 healthy volunteers were examined. The natural logarithm of the Olive tail moment (OTM) was plotted for exposure to UVA through 5 different agar depths (100 cell measurements/depth) and analyzed using a repeated measures regression model. Responses of patients with cancer plateaued after treatment with different UVA intensities, but returned toward control values for healthy volunteers. For precancerous conditions and suspected cancers, intermediate responses occurred. ROC analysis of mean log OTMs, for cancers plus precancerous/suspect conditions vs. controls, cancer vs. precancerous/suspect conditions plus controls, and cancer vs. controls, gave areas under the curve of 0.87, 0.89, and 0.93, respectively (P<0.001). Optimization allowed test sensitivity or specificity to approach 100% with acceptable complementary measures. This modified comet assay could represent a stand-alone test or an adjunct to other investigative procedures for detecting cancer.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25172号 / 医博第5058号 / 新制||医||1071(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 竹内 理, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

O objetivo deste estudo é descrever o dano de DNA através das variáveis Tail Moment e Tail Intensity e investigar a associação entre o dano do DNA, padrão alimentar e concentrações plasmáticas de vitaminas em crianças e adolescentes em Ribeirão Preto (São Paulo, Brasil). Estudantes de 9 a 13 anos foram selecionados de três escolas de Ribeirão Preto (São Paulo, Brasil), totalizando uma amostra de 120 indivíduos. A coleta de dados incluiu antropometria, composição corporal, avaliação da ingestão utilizando um questionário de frequência alimentar (QFA) e o Índice de Qualidade da Dieta Revisado (IQD-R) e coleta de sangue para a dosagem de vitaminas e determinação do dano do DNA pelo método de eletroforese em gel de célula única - comet assay. Os indivíduos foram divididos em dois grupos opostos de dano do DNA usando duas técnicas distintas: análise de k-cluster e classificação por escala de dano. Padrões de ingestão de nutrientes também foram gerados, utilizando robust sparse k-means cluster, e associados com o dano de DNA. Para análise utilizaram-se os testes T de Student, Mann-Whitney e ANCOVA. Na primeira análise, o cluster 1 (n = 73), com menor dano de DNA, apresentou maior consumo de vegetais verdes escuros e alaranjados (p = 0,047), vegetais totais (p = 0,041) e carnes, ovos e leguminosas (p = 0,022), através do IQD-R, e um escore de IQD total maior (p = 0,030), indicando melhor qualidade de dieta em comparação ao cluster 2 (n = 47), de maior dano de DNA. O cluster 2 apresentou maior consumo de laticínios, em comparação ao cluster 1 (p = 0,008). Em relação a vitaminas plasmáticas, o cluster 1 apresentou maior concentração de riboflavina (p = 0,004). No segundo método de divisão, o grupo 1 (n = 108), com menor dano de DNA, apresentou maior concentração de retinol (p = 0,010), beta-caroteno (p = 0,017) e riboflavina (p = 0,046), após teste de ANCOVA ajustado para o índice de massa corporal (IMC) em comparação ao grupo 2 (n = 12). Na divisão por padrão alimentar, o cluster 2 (n = 58), com menor consumo de aminoácidos e micronutrientes, apresentou maior consumo de energia (p = 0,001) e uma tendência de maior dano do DNA (p = 0,063) em relação ao cluster 1 (n = 27), com maior ingestão de nutrientes. Estes achados corroboram a literatura, afirmando o papel protetor de vitaminas e antioxidantes contra o dano do DNA. / The aim of this study is to describe the DNA damage using values of Tail Moment and Tail Intensity and investigate the association between DNA damage, dietary pattern and vitamin status in children and adolescents in Ribeirão Preto (São Paulo, Brazil). 9 to 13 year old students were selected from three schools in the city of Ribeirão Preto (São Paulo, Brazil), totalizing a sample of 120 subjects. Data collection included anthropometry, body composition, assessment of food intake using a food frequency questionnaire (FFQ), quality of diet through Revised Health Eating Index (HEI-R), and blood sampling for vitamins dosage and determination of DNA damage with single cell gel electrophoresis - comet assay. The subjects were divided into two opposing groups according to DNA damage using two different methods: k-cluster analysis and classification by scale of damage. Nutrients intake patterns were also generated through robust sparse k-means cluster and associated with DNA damage. The statistical analysis were performed using Student\'s T test, Mann-Whitney test and ANCOVA. In the first analysis, cluster 1 (n = 73), with less DNA damage presented higher intake of dark green and orange vegetables (p = 0,047), total vegetables (p = 0,041), meat, eggs and legumes (p = 0,022), assessed through HEI, as well as a higher total HEI-R level (p = 0,030), indicating higher quality of diet compared to cluster 2 (n = 47), with increased DNA damage. Cluster 2 presented a higher intake of milk and dairy products, compared to cluster 1 (p = 0,008). In relation to vitamins plasma levels, cluster 1 presented higher levels of riboflavin (p = 0,004). In the second method of division, group 1 (n = 108), with less DNA damage, presented higher levels of retinol (p = 0,010), beta-carotene (p = 0,017) and riboflavin (p = 0,046), after ANCOVA teste adjusted for body mass index (BMI) compared to group 2 (n = 12). In the division by dietary pattern, cluster 2 (n = 58) with a lower consumption of amino acids and micronutrients had higher energy intake (p = 0,001) and a tendency of higher DNA damage (p = 0,063) compared to cluster 1(n = 27), with higher intake of nutrients. These findings corroborate the literature, asserting protective role of vitamins and antioxidants against DNA damage.

O objetivo deste estudo é descrever o dano de DNA através das variáveis Tail Moment e Tail Intensity e investigar a associação entre o dano do DNA, padrão alimentar e concentrações plasmáticas de vitaminas em crianças e adolescentes em Ribeirão Preto (São Paulo, Brasil). Estudantes de 9 a 13 anos foram selecionados de três escolas de Ribeirão Preto (São Paulo, Brasil), totalizando uma amostra de 120 indivíduos. A coleta de dados incluiu antropometria, composição corporal, avaliação da ingestão utilizando um questionário de frequência alimentar (QFA) e o Índice de Qualidade da Dieta Revisado (IQD-R) e coleta de sangue para a dosagem de vitaminas e determinação do dano do DNA pelo método de eletroforese em gel de célula única - comet assay. Os indivíduos foram divididos em dois grupos opostos de dano do DNA usando duas técnicas distintas: análise de k-cluster e classificação por escala de dano. Padrões de ingestão de nutrientes também foram gerados, utilizando robust sparse k-means cluster, e associados com o dano de DNA. Para análise utilizaram-se os testes T de Student, Mann-Whitney e ANCOVA. Na primeira análise, o cluster 1 (n = 73), com menor dano de DNA, apresentou maior consumo de vegetais verdes escuros e alaranjados (p = 0,047), vegetais totais (p = 0,041) e carnes, ovos e leguminosas (p = 0,022), através do IQD-R, e um escore de IQD total maior (p = 0,030), indicando melhor qualidade de dieta em comparação ao cluster 2 (n = 47), de maior dano de DNA. O cluster 2 apresentou maior consumo de laticínios, em comparação ao cluster 1 (p = 0,008). Em relação a vitaminas plasmáticas, o cluster 1 apresentou maior concentração de riboflavina (p = 0,004). No segundo método de divisão, o grupo 1 (n = 108), com menor dano de DNA, apresentou maior concentração de retinol (p = 0,010), beta-caroteno (p = 0,017) e riboflavina (p = 0,046), após teste de ANCOVA ajustado para o índice de massa corporal (IMC) em comparação ao grupo 2 (n = 12). Na divisão por padrão alimentar, o cluster 2 (n = 58), com menor consumo de aminoácidos e micronutrientes, apresentou maior consumo de energia (p = 0,001) e uma tendência de maior dano do DNA (p = 0,063) em relação ao cluster 1 (n = 27), com maior ingestão de nutrientes. Estes achados corroboram a literatura, afirmando o papel protetor de vitaminas e antioxidantes contra o dano do DNA. / The aim of this study is to describe the DNA damage using values of Tail Moment and Tail Intensity and investigate the association between DNA damage, dietary pattern and vitamin status in children and adolescents in Ribeirão Preto (São Paulo, Brazil). 9 to 13 year old students were selected from three schools in the city of Ribeirão Preto (São Paulo, Brazil), totalizing a sample of 120 subjects. Data collection included anthropometry, body composition, assessment of food intake using a food frequency questionnaire (FFQ), quality of diet through Revised Health Eating Index (HEI-R), and blood sampling for vitamins dosage and determination of DNA damage with single cell gel electrophoresis - comet assay. The subjects were divided into two opposing groups according to DNA damage using two different methods: k-cluster analysis and classification by scale of damage. Nutrients intake patterns were also generated through robust sparse k-means cluster and associated with DNA damage. The statistical analysis were performed using Student\'s T test, Mann-Whitney test and ANCOVA. In the first analysis, cluster 1 (n = 73), with less DNA damage presented higher intake of dark green and orange vegetables (p = 0,047), total vegetables (p = 0,041), meat, eggs and legumes (p = 0,022), assessed through HEI, as well as a higher total HEI-R level (p = 0,030), indicating higher quality of diet compared to cluster 2 (n = 47), with increased DNA damage. Cluster 2 presented a higher intake of milk and dairy products, compared to cluster 1 (p = 0,008). In relation to vitamins plasma levels, cluster 1 presented higher levels of riboflavin (p = 0,004). In the second method of division, group 1 (n = 108), with less DNA damage, presented higher levels of retinol (p = 0,010), beta-carotene (p = 0,017) and riboflavin (p = 0,046), after ANCOVA teste adjusted for body mass index (BMI) compared to group 2 (n = 12). In the division by dietary pattern, cluster 2 (n = 58) with a lower consumption of amino acids and micronutrients had higher energy intake (p = 0,001) and a tendency of higher DNA damage (p = 0,063) compared to cluster 1(n = 27), with higher intake of nutrients. These findings corroborate the literature, asserting protective role of vitamins and antioxidants against DNA damage.

Chronic inflammation is intimately linked with cancer development and progression and therefore reducing or eliminating inflammation represents a logical treatment and prevention strategy. Studies have shown that anti-inflammatory agents have anti-tumour effects in cancers, with reduced metastases and mortality. Current use of anti-inflammatory agents in the treatment and prevention of cancer is limited by their toxicity and side effects. The emerging field of nanotechnology allows the fundamental properties of a drug to be altered, creating a product with improved reactivity and bioavailability, leading to more targeted treatments and reduced dosage. In the present study, the genotoxic effects of three commonly used anti-inflammatory drugs; aspirin, ibuprofen and hydrocortisone, in their bulk and nano forms were evaluated on peripheral blood lymphocytes of healthy donors using the comet assay and the micronucleus assay. In order to determine any anti-cancer effects, these agents were also tested in peripheral blood lymphocytes in patients with haematological cancers. The glucocorticoid hydrocortisone was also evaluated for anti-oxidant capacity. Our results demonstrate that the nano versions of each drug produced a different response than the bulk counterpart, indicating that a reduction in particle size had an impact on the reactivity of the drug. Our results also indicate that the nano versions of each drug were less genotoxic than the bulk formulation, further emphasising the potential of nanoparticles as an improvement to current treatment options. We also found an anti-oxidant effect with hydrocortisone, with a more profound effect seen with the nano formulation.

No description available.

Die Induktion antioxidativer Enzyme gilt als eine Möglichkeit, die antioxidative Kapazität von Zellen zu steigern und dadurch mit oxidativem Stress assoziierten Erkrankungen (z. B. Herz-Kreislauf-Erkrankungen, Neurodegeneration, Atherosklerose) vorzubeugen. Ausgehend davon wurde in der vorliegenden Arbeit der Dual-Luciferase-Reportergen-(DLR)-Assay zum Nachweis der Induktion der antioxidativen Enzyme Katalase (CAT), zytosolische Glutathion-Peroxidase (GPX1) und Kupfer-Zink-Superoxid-Dismutase (SOD1) entwickelt. Im Zuge dessen wurden drei Säugetierzelllinien (CaCo2, IEC-18, V79) auf ihre Eignung zur Modellzelllinie untersucht. Aufgrund der Transfektionseffizienz wurde die Fibroblastenzelllinie V79 ausgewählt. Zur Gewährleistung eines hohen Substanzdurchsatzes des DLR-Assays wurden bei der Etablierung Parameter wie Kulturplattenformat, DNA-Menge, Luciferasen-Kinetik berücksichtigt. Nach erfolgreicher Etablierung des Versuchs im 96-Well-Format wurden L-Carnitin, Catechin, Epigallocatechingallat, Genistein, Wasserstoffperoxid (H2O2), Natrium-Ascorbat, Paraquat, Quercetin, 12-O-Tetradecanoylphorbol-13-Acetat (TPA) und Trolox in nicht-zytotoxischen Konzentrationen hinsichtlich der Aktivierung des Ratten-CAT-, des humanen GPX1- und des humanen SOD1-Promotors untersucht. Die Bestimmung der maximal tolerierbaren Behandlungskonzentration erfolgte im Vorfeld mittels Resazurintest. Von den zehn Verbindungen zeichneten sich drei Substanzen als potente Induktoren für die SOD1 und die GPX1 aus. Die 24-stündige Behandlung von mit Reportergenkonstrukten transient transfizierten V79-Zellen mit 100 µM Paraquat resultierte in einer Verdopplung der relativen SOD1-Promotor-Aktivität und einer Erhöhung der relativen GPX1-Promotor-Aktivität auf 1,6 bzw. 1,7. Die Stimulation mit 20 µM Genistein oder 10 µM Quercetin führte wiederum zu einer Verdopplung bis Verdreifachung der relativen SOD1- und GPX1-Promotor-Aktivität. Der Promotor der Rattenkatalase konnte demgegenüber nur durch 50 µM H2O2 aktiviert werden (1,5fach). Für diesen DLR-Assays bieten sich folglich Genistein, Quercetin wie auch H2O2 als Referenzsubstanzen an. Um aber eine qualitative Charakterisierung der einzelnen Verbindungen hinsichtlich ihres Induktionspotentials zu gewährleisten, sollten von allen getesteten Substanzen Dosis-Wirkungskurven aufgenommen werden. Zudem wird für den routinemäßigen Einsatz die Verwendung stabil transfizierter Zellen zur Vermeidung von mit der Transfektion verbundenen experimentellen Schwankungen empfohlen. / The induction of antioxidative enzymes might be an opportunity to elevate the cellular antioxidative capacity and, thus, to prevent oxidative stress associated diseases (e. g. cardio-vascular disease, neurodegenerative disease, atherosclerosis). Based on this idea the dual luciferase reporter gene (DLR) assay was developed to demonstrate the induction of three antioxidative enzymes: catalase (CAT), cytosolic glutathione peroxidase (GPX1), and copper-zinc superoxide dismutase (SOD1). In the course of the development three mammalian cell lines (CaCo2, IEC-18, V79) were tested for their ability to serve as a model cell line. The line V79 was chosen due to the transfection efficiency. To give consideration to a high-throughput several parameters were studied (e. g. format of the cultural plates, amount of DNA, kinetics of the luciferases) and the DLR assay was successfully established in 96 well plates. Subsequently, L-carnitine, catechin, epigallocatechin gallate, genistein, hydrogen peroxide (H2O2), sodium ascorbate, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate (TPA) and trolox were tested in non-cytotoxic concentrations for the activation of the rat CAT, human GPX1 and human SOD1 promoter. The maximally tolerable concentrations were determined by resazurin test in advance. Three out of these ten compounds were identified as potent inducers of GPX1 and SOD1. Stimulation of reporter gene construct transient transfected V79 cells for 24 hours with 100 µM paraquat caused a duplication of the relative GPX1 promoter activity and a 1.6-/1.7-fold increase of the relative SOD1 promoter activity. The incubation with 20 µM gen-istein or 10 µM quercetin resulted in duplication to triplication of both, the relative GPX1 and SOD1 promoter activity. In contrast, the rat CAT promoter was activated by 50 µM H2O2 (1.5-fold). Consequently, genistein, quercetin, and H2O2 are considered to be suitable reference substances for this DLR assay. To further characterize the inducing potential of the tested compounds all of them should be tested in different concentrations. Furthermore, for the routinely performed DLR assay it is recommended to use stably transfected cells to eliminate transfection caused variations.

Im Bereich der medizinischen Diagnostik spielen DNA-Chips eine immer wichtigere Rolle. Dabei werden Glas- oder Silikon-Oberflächen mit Tausenden von einzelsträngigen DNA-Fragmenten, sog. Sonden, bestückt, die mit den passenden DNA-Fragmenten in der zugefügten Patientenprobe verschmelzen. Die Auswertung solcher Messungen liefert die Diagnose für Krankheiten wie z.B. Krebs, Alzheimer oder für den Nachweis pathogener Erreger. Durch fortschreitende Miniaturisierung dieser Meßsysteme können bis zu 40.000 Genfragmente des Menschen in einer einzigen Messung analysiert werden. Neben den DNA-Fragmenten können Bio-Chips auch für andere biologische Komponenten wie Antikörper und Proteine eingesetzt werden, wobei bei letzteren neben der Bindung auch die Aktivität ein wichtiger Diagnoseparamter ist. <br /> <br /> Am Fraunhofer-Institut für medizinische Technik und am Lehrstuhl für Analytische Biochemie der Universität Potsdam wurden im Rahmen einer Doktorarbeit Methoden entwickelt, die es ermöglichen auf nukleinsäuremodifizierten Sensoroberflächen die Aktivität von Proteinen zu messen. Es wurden Nukleinsäuren auf Oberflächen optischer Sensoren verankert. Diese fungierten als Rezeptor für die Proteine sowie auch als Substrat für Restriktionsenzyme, die Nukleinsäuren schneiden und Polymerasen, die Nukleinsäuren synthetisieren und verlängern können.<br /> <br /> Seine Anwendung fand diese Messmethode in der Messung der Aktivität des Proteins Telomerase, das in 90% aller Tumore erhöhte Aktivität gegenüber gesunden Zellen aufweist. Die Vorteile dieses neuen Assays gegenüber älteren Methoden liegt im Verzicht auf radioaktiv-markierten Komponenten und einer deutlich verkürzten Analysezeit. Die Arbeit schliesst mit einem funktionsfähigen Nachweis der Telomeraseaktivität im Zellextrakt von gesunden und kranken Zellen. Der direkte Einfluß von Hemmstoffen auf die Aktivität konnte sichtbar gemacht werden, und steht daher bei der Entwicklung neuer Tumor-Diagnostika und Therapeutika zur Verfügung. / In the field of medical diagnostic the importance of DNA chips is growing continuously. On silica surfaces hundreds of single stranded DNA fragments are immobilised which finally detect the complementary sequences in samples of patients by hybridisation. These methods enable the detection of serious diseases as cancer, Alzheimer's disease or infection by pathogens. Biomolecules as nucleic acids, antibodies and proteins can be used as receptors on the solid surfaces whereas in case of proteins not only the binding but also the activity are of high interest for medical diagnosis.<br /> <br /> In this thesis a biosensoric approach has been developed to determine the activity of nucleic-acid modifying enzymes. Optical sensors as, e.g., the grating coupler, were used to monitor the association and dissociation of unlabeled compounds on the sensor surface in real time, by virtue of evanescent-field. Furthermore sensors based on total internal reflection fluorescence measured the activity of restriction enzymes and polymerases. The general approach included the immobilisation of oligonucleotides which acted as the receptor for the enzymes as well as the substrate for the enzymatic reaction. Enzymes as EcoRI and Klenow were used to establish a model system to measure the activity of DNA-modifying enzymes on optical surfaces. As most nucleic acid detection systems use amplification steps such as polymerase chain reaction (PCR) to increase the amount of the probe the new optical systems facilitated the analysis of the enzymatic activity by measuring the DNA-synthesis or restriction directly.<br /> <br /> These systems were finally used to detect the activity of the telomerase, an enzymatic marker for the cancerous development of cells. In 90% of cancer cells the activity of telomerase is higher than in normal cells. Additionally the increase of the telomerase activity in cells induced by carcinogenic substances was detected. Furthermore no purification steps of the samples were required as all measurements were performed with crude cell extract.<br /> <br /> Also the effect of inhibitors of the telomerase could be shown in real time measurements underlining the potential of this assay for further developments of new cancer therapeutics.

The goal of this project was to incorporate bacteriophage with Bioluminescent Assay in Real-Time Loop-mediated isothermal amplification (BART-LAMP) for the rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP). As the causative agent of Johne’s Disease, there are no rapid detection methods that are suitable in specificity and sensitivity. A screening assay for phage isolation was developed, and over 400 samples were screened for the isolation of a bacteriophage against MAP. One novel Mycobacterium phage was isolated and characterized using transmission electron miscroscopy, host range studies, restriction enzyme digestion, and pH and temperature stability. It was sequenced, annotated, and underwent an in silico protein analysis. No pathogenic or lysogenic genes were detected, and it was found to be related to Gordonia phage GTE2. BART-LAMP was applied to the detection of the isolated phage using purely extracted DNA and crude phage lysate, showing that phages could be detected successfully. / Beef Cattle Research Council; Agriculture and AgriFood Canada through Growing Forward initiative