Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 4 uppsatser.

Tübingen, Univ., Diss., 2001.

Thesis (M. Sc.)--Carleton University, 1997. / Also available in electronic format on the Internet.

Universiẗat, Diss., 1999--Tübingen.

Protein biomarkers are important diagnostic tools for detection of non-communicable diseases, such as cancer and cardiovascular conditions. In order to be used as diagnostic tools they need to be detected at very low concentrations in biological samples (e.g. whole blood, serum or urine). This has been currently performed in central laboratories using expensive, bulky equipment and time consuming assays.

Le diagnostic précoce de l'hypothyroïdie permet d'initier le traitement au plus tôt et ainsi de préserver la santé du patient. Le bénéfice du traitement de l'hypothyroïdie franche a été depuis longtemps établi, mais les critères de prise en charge des patients en hypothyroïdie fruste sont encore difficiles à définir. En effet, les symptômes ne sont pas toujours présents et leur appréciation est subjective. Afin d'établir le diagnostic et la prise en charge, le médecin s'appuie sur le dosage de la thyréostimuline (TSH) dans le sang, qui peut éventuellement être complété par le dosage des hormones thyroïdiennes. Le dosage de la TSH, très sensible, peut présenter sur un même échantillon sanguin d'importantes variations qui rendent d'autant plus difficiles la décision du médecin et le suivi du patient. Le polymorphisme naturel de la TSH peut expliquer en partie ces variations. La TSH appartient en effet à la famille des hormones glycoprotéiques et sa glycosylation peut constituer jusqu'à 30% de son poids. Dans le cas de l'hypothyroïdie en particulier, ces glycanes sont modifiés et présentent une plus grande quantité d'acides sialiques terminaux. Ainsi, certaines variations entre les dosages de la TSH, qui freinent actuellement leur harmonisation, peuvent être dues à des différences de reconnaissance de glycoformes par les anticorps utilisés dans les dosages. Dans ce contexte, l'objectif de de ces travaux était de contribuer à la construction de dosages plus performants que ceux actuellement utilisés dans le diagnostic de l'hypothyroïdie. Un nouveau calibrateur recombinant sialylé plus proche de la TSH circulante dans l'hypothyroïdie a alors été produit. De nouvelles associations d'anticorps monoclonaux ont été utilisées pour construire des dosages. Les nouveaux dosages sélectionnés ont ensuite été calibrés avec la TSH sialylée produite et le calibrateur de référence international. Ils ont alors servi à doser plusieurs séries de sérums de patients. Ces travaux ont donc validé l'utilisation d'un nouveau calibrateur d'origine recombinante pour les dosages de la TSH, ce qui devrait à l'harmonisation des dosages existants. / If iodine deficiency is the first cause of low thyroid hormone levels in the world, there are also other etiologies to thyroid disorders. Diagnosis of those allow an early treatment to preserve patient's health. Although there is a general agreement concerning treatment of overt hypothyroidism, treatment of subclinical hypothyroidism is still under debate. In these cases, symptoms are, by definition, not always present. In order to establish diagnosis, the clinicians rely on the measurement of circulating thyroid stimulating hormone (TSH, potentially completed with thyroid hormones measurement). TSH assays are now very sensitive, but can present important between assays variations. The diagnosis and follow up of the patient are consequently complicated. Natural polymorphism of TSH can explain a part of this variability. TSH belongs to the glycoprotein hormones family and its glycans can count for more than 30% of its weight. In hypothyroidism, these glycans are subject of modulation and present higher levels of terminal sialylation. Variation in immuno-assays can be explained by these modifications of sialylation if recognition by antibodies used in immuno-assays is glycosylation dependent. In this context, the aim of this work was to contribute to the construction of new immuno-assays, more reliable in the early diagnosis of subclinical hypothyroidism. During this thesis a new recombinant standard closer to circulating TSH was produced. The total level of sialylation was higher and better mimic the circulating forms in hypothyroidism. In order to select the best antibodies associations in immuno-assays, new antibodies were obtained and associated with commercially available antibodies. New immuno assays improvement is based on the following two approaches: the first one is the use of a new standard which presents glycoformes closer to the circulating TSH and the second one consists in an appropriate selection of antibodies involved in the assays. The new assays were used to measure TSH concentration in blood samples. These studies associated with validation steps allow us to select four assays and constitute a proof of concept for the use of a new sialylated recombinant standard for TSH assays. This can contribute to the needed harmonization of TSH assays.

Being able to detect if drugs were used in the commission of a crime, and if so what drugs, is of great importance. For many cases these tests can be carried out on an intimate blood or urine sample (one recovered directly from the subject in question), however this may not always be the case. In cases where a dried bloodstain is the only source of biological material, identifying the presence of drugs affecting an individual at the time of stain deposition has not been well studied. Towards this goal, two methods of detection of drugs of abuse in dried bloodstains were evaluated: lateral flow immunoassay test cards and gas chromatography-mass spectrometry. Stains were created using certified drug-free blood spiked with analytes of interest, and were then extracted and introduced into each testing method. Both methods proved effective for the detection of one or two of the five chosen analytes (amphetamine, cocaine, morphine 3-ß-D-glucuronide, phencyclidine and 11-nor-Δ9-tetrahydracannabinol), even after 24 hours of drying at room temperature. With further method optimization and more thorough method development, these methods may, in the future, be effectively used for drug detection in forensic stains. However, neither method evaluated in this study was able to detect all of the drugs tested.

Objectives: There are three aims for this thesis: the first is to develop a field and laboratory protocol for the storage and analysis of transforming growth factor–beta 2 (TGF-β2) in human breastmilk; second, to validate this protocol and the immunoassay used to assess this new method; and lastly, to explore the ramifications of biosimilar TGF-β2 across multiple milks on human health, growth, and immunity through the review of laboratory findings and previous literature. Rational: Little anthropological research has been done on TGF-β2 in human milk. Anthropology as a discipline is well positioned to provide insight into TGF-β2, combining biocultural, evolutionary, and ecological approaches to holistically illustrate the effects this cytokine has on human immunity. This thesis provides an applied anthropological perspective and methodology on TGF-β2 in human milk. Methods: A protocol was developed for a new method of drying breastmilk on polystyrene microplates. Samples were then reconstituted using reagent diluent with 1% BSA and assayed using a Human TGF-beta 2 DuoSet enzyme-linked immunosorbent assay (ELIZA) assay kit from R&D Systems. Other mammalian milks and infant formula samples were also dried and tested for TGF-β2 concentrations. Validity of the assay and TGF-β2 concentrations were then statistically measured using linear regression analysis and Bland-Altman plots. Results: The results of the first objective in the development of a laboratory and field protocol for drying breastmilk on polystyrene plates for the extraction of TGF-β2 showed this method to hold promise for future application, but lacked statistical power in this study to confirm if this method is viable. The second objective of assay validation was unsuccessful, with the percent coefficient of variation for the intra-assay variation and inter-assay validation 38.28% and 17.70%, respectively indicating that this assay struggled to produce consistent and reliable results from the reconstituted samples. Results from the third objective suggest that biosimilar TGF-β2 in non-human milk can influence human growth and development, the extent of which, however, needs further study. Conclusions: Given these findings, more work with TGF-β2 in milk is required. TGF-β2 is a cytokine which could reveal a great deal about the developmental origins of human immunity and how it is maintained and altered across our life course and therefore an area of biology worth further research.

Submitted by MARCOS ANTONIO MAIOLI null (maioli_marcos@hotmail.com) on 2016-02-17T13:03:36Z No. of bitstreams: 1 Tese Maioli, M.A..pdf: 2279252 bytes, checksum: 484c7c928f7a1a221c494bcff9e436b0 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-02-17T16:54:14Z (GMT) No. of bitstreams: 1 maioli_ma_dr_araca.pdf: 2279252 bytes, checksum: 484c7c928f7a1a221c494bcff9e436b0 (MD5) / Made available in DSpace on 2016-02-17T16:54:14Z (GMT). No. of bitstreams: 1 maioli_ma_dr_araca.pdf: 2279252 bytes, checksum: 484c7c928f7a1a221c494bcff9e436b0 (MD5) Previous issue date: 2016-02-01 / Outra / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Esse estudo teve como objetivo a padronização de um ensaio imunoenzimático (ELISA) para a determinação das concentrações plasmáticas de IGF-I total, utilizando o sistema de amplificação biotina-estreptavidina peroxidase em um ensaio competitivo. O IGF-I foi extraído da IGFBP, utilizando o tampão glicina acidificado seguido de neutralização do pH com hidróxido de sódio. As microplacas foram sensibilizadas com anti IgG de coelho, e as dosagens realizadas utilizando duas abordagens, um método sem competição (incubação prévia das amostras com o anticorpo anti-h-IGF-I) e outro com competição (adição simultânea de IGF-I biotilinado e amostra). Os melhores resultados foram obtidos utilizando o método competitivo, com a sensibilização da placa com 0,25 μg/poço de IgG anti-coelho, o anticorpo específico na diluição 1:250.000 e 0,06 ng/poço de IGF-I biotinilado. O ensaio in house apresentou, um limite inferior de detecção de 50 ng/mL, uma correlação de 0.945 entre doses quando comparado à uma metodologia comercial. Além disso, após 33 ensaios (1114 amostras) a metodologia apresentou uma boa precisão, com coeficientes de variação inter-ensaio de 12,94% (345,8 ng/mL) para os controles alto e 20,71% (131,6 ng/mL) para o baixo. Dessa forma, conclui-se que a metodologia imunoenzimática para quantificação de IGF-I total utilizando o sistema de amplificação biotina-estreptavidina peroxidase em um ensaio competitivo está estabelecida e apresenta-se como uma ferramenta útil para estudos que visem o monitoramento das concentrações de IGF-I. / This study aimed to standardize an enzyme-linked immunosorbent assay (ELISA) to determine plasma concentrations of total IGF-I using the amplification biotin-streptavidin peroxidase system in a competitive assay. The IGF-I was extracted from IGFBPs using the acidified glycine buffer followed by the pH neutralization with sodium hydroxide. The microplates were coated with anti-rabbit IgG, thereafter the measurements were carried out using two approaches, one without competition (prior incubation of samples with the anti-hIGF-I antibody) and another with competition (simultaneous addition of IGF-I and biotinylated sample). The best results were obtained using the competitive method, with the following combination of reagents: microplates were coated with 0.25 µg/well of anti-rabbit IgG, the specific antibody at a dilution of 1:250.000 and 0.06 ng/well of biotinylated IGF-I. The in house methodology showed sensitivity of detection limit of 50 ng/ml, a correlation between doses of 0.945 when compared to a commercial method. In addition, after 33 assays (quantification of 1114 samples) the proposed methodology presented a good precision, with interassay variation coefficients of 12.94% and 20.71% for the high and low controls, respectively. Finally, we concluded that ELISA method for the quantification of total IGF-I using the system biotin-streptavidin-peroxidase amplification in a competitive assay is established and is presented as a useful tool for studies aimed at monitoring the IGF-I concentrations. / FAPESP: 2014/09135-9 / BEPE: 2015/08021-2

Made available in DSpace on 2015-04-09T12:28:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-05-12Bitstream added on 2015-04-09T12:47:39Z : No. of bitstreams: 1 000815970.pdf: 611466 bytes, checksum: 271b827fb3d2f7f3486a8357d3947ec5 (MD5) / Macacos-da-noite são tidos como excelentes modelos experimentais, que podem contribuir com o desenvolvimento de biotécnicas da reprodução em primatas. O monitoramento do ciclo é um procedimento básico, porém complexo em animais selvagens. Tem-se desenvolvido métodos não invasivos para avaliar o perfil hormonal reprodutivo desses animais expandindo o conhecimento sobre a fisiologia reprodutiva de primatas em cativeiro e vida livre. O presente estudo visa monitorar a atividade folicular e as concentrações fecais de metabólitos de progesterona para ampliar os conhecimentos sobre a fisiologia reprodutiva dessa espécie. Foram utilizadas 12 fêmeas adultas, pertencentes à colônia de reprodução de macacos-da-noite do CENP. O estudo foi realizado a partir do exame ultrassonográfico do ovário e colheita de fezes para monitoramento dos níveis de metabólitos de progesterona dos animais por enzimaimunoensaio. Por meio da dosagem de metabólitos de progesterona foi possível apenas sgerir o ciclo da espécie para o período estudado. Porém, os níveis basais (2,1 ± 0,8 ng/g de fezes secas), a médio do pico (36,6 ± 8,6 ng/g fezes secas), os níveis médios (4,7 ± 1,8 ng/g de fezes secas) e os valores mínimo (0,4 ng/g de fezes secas) e máximo (49,9 ng/g de fezes secas) de metabólitos de progesterona foram determinados para espécie em cativeiro sob estas condições. A determinação do pico e concentrações basais de metabólitos de progesterona, conjuntamente a avaliação ultrassonográfica são ferramentas não invasivas e factíveis na avaliação do ciclo estral de macacos-da-noite / Owl Monkeys are considered excellent experimental models and can contribute to the development of biotechnologies of reproduction in primates. Monitoring the reproductive cycle is a basic procedure, however complex in wild animals. Noninvasive methods has been developed to assess the reproductive hormonal profile of these animals expanding knowledge on reproductive physiology of primates in captivity and the wild. This study aims to monitor follicular activity and fecal progesterone levels to increase knowledge about reproductive physiology of this species. 12 adult females belonging to the breeding colony of owl monkeys of CENP were used. The study was performed using the ultrasound examination of the ovaries and feces collection for monitoring the levels of metabolites of progesterone by enzymeimuneassay. By progesterone assay we may suggest the cycle of the species for the period studied. However, the baseline levels (2.1 ± 0.8 ng/g), mean peak (36.6 ± 8.6 ng/g), mean levels (4.7 ± 1.8 ng/g) and the minimum (0.4 ng/g) and maximum (49.9 ng/g) metabolites of progesterone were determined for the specie in captivity. The determination of the peak and basal levels of progesterone together to the sonographic evaluation are noninvasive and feasible in the evaluation of the estrous cycle in owl monkeys