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Cervical cancer is the second most common cancer in women worldwide. The principal cause of cervical cancer is infection with human papillomavirus (HPV). HPV-16 is a high-risk virus and it is responsible for a high portion of all HPV-caused cancers. The HPV-16 genome consists of early and late genes. The virus initially infects basal cells of the cervix epithelium and in these cells early genes are expressed, whilst late genes, L1 and L2, are only expressed in the upper cell layers of the epithelium. Proteins encoded by the late genes are highly immunogenic, thus it is speculated that expression of the late genes earlier in the virus life cycle could lead to clearance of the virus due to interference of the immune system.     The aim of this study was to treat reporter cell lines with three different small molecule drugs to see if they had the ability to induce HPV-16 late gene expression. The reporter cell lines used in this study had been previously created by transfecting HeLa-cells with plasmids representing the HPV-16 genome. In these plasmids, L1 is replaced with a CAT reporter gene that encodes the CAT protein, which can be easily quantified using a sandwich ELISA.     Upon treating the reporter cell lines with TPA, a significant induction of late gene expression was detected. Furthermore, treatment with valproic acid showed some induction of late gene expression. In conclusion, TPA and valproic acid was deemed to have potential to act as a candidate drugs for treatment of HPV infections.

Cervical cancer is almost exclusively caused by the HPV virus, whit HPV 16 and 18 involved in the majority of cases. The HPV virus can be divided into high risk and low risk types, where the high risk types are most associated with cancer. HPV is spread by sexual skin to skin contact, many people get infected without getting cervical cancer. HPV is also involved in the development of several other types of cancers such as oral and other genital cancers. The HPV virus infects epithelium stem cells and disrupts basic functions of the cells. A high expression of the late genes early in an infection may result in that an HPV 16 infection dies out. The late gene expression was analysed by using a CAT ELISA method, in the cell lines used one of the late genes had been replaced by a CAT reporter gene. Several small molecules where investigated, to study the regulation of the late gene expression. The results of the study was that a regulation of the late gene expression could be seen when pBELMCAT was treated with TPA, TA and RA where TPA gave the highest increase in the late gene expression. TA/RA combined with TPA increased the expression even more. As a conclusion it seems possible for small molecules to be used in treatments for cervical cancer that is caused by HPV 16, to upregulate the late gene expression and maybe be able to eliminate the infection before serious damage and disease can develop.

Onchocerca volvulus is a filarial parasite transmitted to humans by female Simulium spp. black flies. Infection with this parasite can cause blindness and severe skin disease among humans in Africa and the Americas. Enzyme-linked Immunosorbent Assay serological testing of OV-16 antigen is a diagnostic tool for determining effective elimination of the parasite. Programs typically rely on OV–16 ELISA to evaluate the progress towards interruption and/or elimination of disease by mass drug distribution of ivermectin and vector larvicidal control efforts. As elimination grows closer, monoclonal antibody positive controls for OV-16 ELISA become important to develop for Onchocerca testing due to the limited availability of pooled sera positive controls. Recent evaluation of laboratory designed OV-16 ELISA coating antigen by the Unnasch Lab (University of South Florida) showed that polymorphisms occurred which may alter the ability of the humanized monoclonal antibody to recognize the cognate antigen. With this development, it was important to evaluate these polymorphisms and isolate them for further testing against the standardized monoclonal antibody and positive sera to determine the effects antigenic polymorphisms could have on diagnostic testing. Upon evaluation, the polymorphisms did influence signaling when testing the monoclonal antibody. However, little effect on the recognition of the antigen was seen when different isoforms were evaluated against sera from O. volvulus infected individuals. Data suggest that the epitope recognized by the synthetically produced monoclonal antibody is not immuno-dominant in infected individuals.

Distinct cellular level of the Ca2+ binding chaperone calreticulin (CRT) is essential for cardiac development and postnatal function. However, CRT is also a potential autoantigen eliciting formation of antibodies (Ab), whose role is not yet clarified. Immunization with CRT leads to cardiac injury, and overexpression of CRT in cardiomyocytes induces dilated cardiomyopathy (DCM) in experimental animals. Hence, we analysed levels of anti-CRT Ab and calreticulin in the sera of patients with idiopatic DCM and hypertrophic cardiomyopathy (HCM). ELISA and immunoblot using human recombinant CRT and Pepscan with synthetic, overlapping decapeptides of CRT were used to detect anti-CRT Ab. Significantly increased levels of anti-CRT Ab of IgA (P<0.001) and IgG (P<0.05) isotypes were found in patients with both DCM (12/34 seropositive for IgA, 7/34 for IgG) and HCM (13/38 seropositive for IgA, 11/38 for IgG) when compared with controls (2/79 for IgA, 1/79 for IgG). Titration analysis in seropositive DCM and HCM patients documented anti-CRT Ab detected at 1/1600 dilution for IgG and 1/800 for IgA (and IgA1) and at least at 1/200 dilution for IgA2, IgG1, IgG2 and IgG3. Pepscan identified several immunogenic CRT epitopes: EVKIDNSQVESGSLED, IDDPTDSKPE, DKAPEHIPDPDA and RKEEEEAEDKEDDAEDKDEDEEDE recognised by IgA and...

Abstract Breast carcinoma is a heterogeneous disease with a prognosis that varies from excellent to very poor. Traditional tumour parameters and biological factors that are also predictive for treatment response are used in determining breast carcinoma prognosis and selecting appropriate treatment. Gelatinases MMP-2 and MMP-9 have been shown to associate with tumour progression. Their tissue inhibitors TIMP-1 and -2 are multifunctional molecules that have been suggested as prognostic markers in some previous reports. In the present work, the expression and prognostic value of gelatinases MMP-2 and MMP-9 and their tissue inhibitors TIMP-1 and -2 were assessed in primary breast carcinoma. The material consisted of a total of 416 patients. Tissue expression of TIMP-1 and -2 was analysed in a population of 203 patients using immunohistochemistry. Circulating gelatinases and their inhibitors were studied using ELISA in two different populations of 71 at preoperative state and 213 patients at pre- and postoperative state. High expression of TIMP-1 immunoreactive protein positively correlated with high histological grade of the tumour and associated with aggressive disease course in grade 2–3 subpopulation. High preoperative plasma TIMP-1 was prognostic for relapse in a modern patient series after a median follow-up time of 18 months. TIMP-1 as a continuous variable was prognostic in Cox regression univariate analysis, and was an independent prognostic variable superior to nodal status in multivariate analysis. High preoperative serum TIMP-1 was an independent prognostic variable for poor disease-specific survival, and TIMP-1 was found to maintain its prognostic value when assessed independently with different ELISA analyses, and was not very sensitive for preanalytical conditions. In addition, low circulating preoperative serum MMP-2 was observed to associate with high stage and positive nodal status in breast carcinoma. These results indicate that circulating TIMP-1 may be a potential new marker of worsened prognosis in breast carcinoma, although careful validation of assay platforms and identification of the sources of physiological variation are needed before it can be adopted into clinical decision-making.

Memoria para optar al Título Profesional de Médico Veterinario / En el presente estudio se describe el desarrollo y validación de un Ensayo inmunoenzimático indirecto (ELISA-I) para el diagnóstico serológico de la infección por Brucella ovis (B. ovis) en ovinos, utilizando proteínas citosólicas de Brucella abortus RB51 (B. abortus RB51) como antígeno. Se probaron dos tipos de placas de poliestireno, NUNC 69620 y NUNC Maxisorp, dos tampones de sensibilización de diferente pH con dos alternativas de conjugado, el anticuerpo monoclonal anti IgG-caprino/ovino (Sigma A9452) y la proteína G (Sigma P81170). Las concentraciones óptimas de antígeno citosólico, sueros y conjugados fueron determinadas mediante titulación del tipo checkerboard. Para la validación del ensayo se utilizaron 55 sueros de ovinos considerados como infectados/expuestos, positivos a examen clínico, a un ELISA-I comercial, a la fijación del complemento (FC) y a la prueba de reacción en cadena de la polimerasa (PCR) y 338 sueros de ovinos no infectados, provenientes de un rebaño ovino libre de infección, negativos a la prueba de FC. El punto de corte diagnóstico fue determinado a través del método de las características del tipo receptor-operador (ROC). El ELISA-I desarrollado con anticuerpo monoclonal como conjugado mostró mejores valores de sensibilidad (90,9%) y especificidad (95,9%). Estos resultados, a pesar de ser significativos, son deficientes para ser considerados de utilidad diagnóstica y lograr un adecuado control de la infección en rebaños ovinos. La baja sensibilidad obtenida al utilizar proteínas citosólicas como antígeno estaría relacionada con la escasa exposición al antígeno y la corta memoria inmunológica que generarían este tipo de proteínas en un animal infectado.

Los antibióticos son ampliamente utilizados en veterinaria, produciendo beneficios inmensos, pero también conllevan riesgos medioambientales debidos a la aparición de residuos "incontrolados" que pueden desencadenar resistencias bacterianas, reacciones alérgicas y otros inconvenientes importantes. En esta tesis se han desarrollado varios sistemas inmunoquímicos para la determinación de residuos de distintos tipos de antibióticos, tanto mediante el formato tradicional en placa ELISA, como mediante el uso de microarrays utilizando CDs como plataformas analíticas. En primer lugar, se han desarrollado inmunoensayos en placa ELISA para sulfonamidas y tetraciclinas. Se han puesto a punto dos inmunoensayos genéricos para la determinación de seis sulfonamidas, con elevada sensibilidad (IC50 entre 1,32 y 12,82 ng mL-1) e inmunoensayos específicos para la determinación de SSZ y SMX con valores de IC50 de 0,51 y 0,16 ng mL-1, respectivamente. Asimismo, se ha puesto a punto un inmunoensayo específico para la determinación de CTC, con un valor de IC50 de 36,40 ng mL-1. Estos ELISAs se han aplicado con éxito a la cuantificación de residuos de sulfonamidas en alimentos (miel), aguas de depuradora y plasma humano. En otro apartado se presentan los resultados de las investigaciones sobre el desarrollo de microinmunoensayos utilizando la tecnología de discos compactos. Este soporte presenta gran área analítica donde depositar miles de biomoléculas sonda y la posibilidad de integrar tanto información numérica como bioquímica (identificando cada punto de cada matriz y almacenando los resultados del ensayo en la misma plataforma física en la que se desarrolla el análisis); por último, su fabricación a gran escala permite obtener productos de alta calidad a muy bajo precio. Además, los resultados analíticos son leídos y cuantificados por un lector comercial de CDs integrado en un ordenador personal convencional. / Gallego Iglesias, E. (2012). Desarrollo de inmunoensayos para antibióticos en microplaca y en disco compacto aplicados a la determinación multirresiduo de contaminantes [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/14720 / Palancia

Austrofundulus limnaeus is a species of annual killifish which inhabits ephemeral ponds in South America. The species is able to survive seasonally desiccating ponds due to their ability to produce robust embryos. The embryos of this species are capable of entering a developmental arrest, termed diapause II, which precedes the onset of drought. While in this arrested state embryos exhibit the greatest tolerance to anoxia of any characterized vertebrate at 25ºC. Furthermore, when raised at 30ºC, embryos escape the entrance to diapause II and go on to develop directly. Currently, little is known about the molecular mechanisms which induce and maintain this developmentally arrested state. In this study I have developed methods to analyze changes in histone modifications in the context of diapause II. Histone modifications were chosen due to their extreme conservation and well characterized role as modulators of gene expression in other systems. Results utilizing adapted immunobased assays show significant changes in the global amount of H3S10P, H3K27me and H3K4me, as the embryos progress from early embryogenesis through the exit of diapause. Additionally, it is revealed that there exists a degree of phenotypic plasticity with regards to the entrance into diapause II which is modulated by the environment (severe hypoxia 0.1% O2). This work builds a foundation for future histone modification studies and contributes the development of several tools to the field. This study contributes to a greater molecular understanding of the cue(s) which influence the remarkable phenomenon of obligate developmental arrest in a vertebrate embryo.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.