Chez l'humain, il y a présence de quatre petits ARN Y; soit les ARN hYl, hY3, hY4 et hY5. La longueur de ces ARN varie entre 84 (hY5) à 112 (hY1) nucléotides. Ces ARN forment une longue structure tige boucle pouvant être liée par une protéine nommée Ro60. Les ribonucléoprotéines Ro (Ro RNP) résultent de la liaison de Ro60 ainsi que celle de la protéine La aux ARN hY. Ces Ro RNP sont la cible d'auto-anticorps chez des patients atteints de pathologies du tissu conjonctif, comme le lupus érythémateux systémique. Les connaissances sur le rôle de ces petits ARN non-codants sont limitées. Cela rend leur étude d'autant plus importante. Par contre, quelques évidences suggèrent leur implication dans la réplication chromosomale. Récemment, environ mille pseudogènes Y ont été découverts dans le génome humain; leur présence suggère un mécanisme de rétrotransposition. L'étude des séquences adjacentes aux pseudogènes a permis d'identifier une signature de la machinerie des Long Interspersed Nuclear Elements 1 (LINE-1), suggérant un nouvel élément mobile similaire à Alu. De plus, la majorité des pseudogènes Y présentaient des mutations précises aux sites de liaison de plusieurs protéines, dont Ro60. Cela nous a amenés à explorer les différents aspects du mécanisme de rétroposition présumé des ARN hY. Pour ce faire, nous avons adapté un essai de rétrotransposition Ll dans les cellules HeLa. Notre principale hypothèse était de vérifier si l'absence de protéines liées à l'ARN Y pouvait influencer la rétrotransposition de l'ARN en question. La méthode de détection par dénombrement de colonies s'est avérée peu profitable puisqu'elle manquait de sensibilité. Les niveaux très faibles et le taux élevé de variation entre les différents essais ne permettaient pas d'émettre des conclusions claires et précises. Par la suite, nous avons développé une seconde méthode de détection basée sur la PCR quantitative en temps réel. Il s'agit d'une nouvelle application pour l'étude des événements de rétrotransposition. Celle-ci nous a permis de démentir notre hypothèse : la perte de liaison de certaines protéines ne favoriserait pas la prise en charge de l'ARN hY par la machinerie Ll .

The Proximity Ligation Assay (PLA) is a relatively new method which utilizes the strength of both immunoassays and DNA detection. PLA has the capacity of high multiplexing due to the high specificity achieved with both dual protein-binding and dual primer binding during detection with Real-Time PCR. We developed a multiplexed PLA protocol that can measure 28 biomarkers in human EDTA plasma. The method was tested on 46 individuals diagnosed with colorectal cancer and 48 age matched healthy controls. The results are very promising as we re-discover the most well-known biomarkers for colorectal cancer and also find some potential new markers (significance tested with students T-test with p<0.05). Further improvements of the protocol are needed to decrease the variation.

Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2016-08-31T19:39:59Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2015GeorgiaMuccilloDexheimer.pdf: 1692655 bytes, checksum: 44476f343c262e53d202386e916adf5c (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2016-09-05T19:27:37Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2015GeorgiaMuccilloDexheimer.pdf: 1692655 bytes, checksum: 44476f343c262e53d202386e916adf5c (MD5) / Made available in DSpace on 2016-09-05T19:27:37Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2015GeorgiaMuccilloDexheimer.pdf: 1692655 bytes, checksum: 44476f343c262e53d202386e916adf5c (MD5) Previous issue date: 2016-08 / Terapias antitumorais baseadas em efeitos anti-inflamatórios vêm sendo consideradas no tratamento do câncer. A sobrevivência, proliferação e, eventualmente, invasão e metástase das células tumorais são reguladas por mediadores inflamatórios locais. Citocinas inflamatórias primárias, como o Fator de Necrose Tumoral (TNF), podem ser alvos para a terapia anticâncer. Diversos anti-inflamatórios isolados de produtos naturais vêm ganhando importância como quimiopreventivos e agentes terapêuticos para o câncer. O objetivo deste trabalho foi investigar a expressão dos genes TNF-α, NF-κΒ e p38α, associados à proliferação e inflamação em células de linhagem Caco-2 tratadas com extratos etanólico e hexânico obtidos de Calyptranthes grandifolia O.Berg (Myrtaceae). Células Caco-2 foram cultivadas e tratadas com o extrato vegetal em diferentes concentrações (200, 100, 50 e 25 μg/mL), e estimuladas com LPS. Para análise da expressão gênica foi realizada a extração de RNA total pelo método de Trizol®, seguido da síntese de DNAc (DNA complementar) e análise por qPCR (Reação em Cadeia da Polimerase em Tempo Real). Dentre os genes avaliados, observou-se uma diminuição da expressão de TNF-α nas concentrações de 100 e 200 μg/mL apenas no extrato etanólico (p<0,025, ANOVA e Teste de Tukey). O gene p38α apresentou uma tendência a aumento da expressão apenas no tratamento com extrato etanólico e o gene NF-κΒ não apresentou variações significativas em ambos os extratos analisados. Pode-se concluir que o extrato etanólico pode apresentar atividade anti-inflamatória nas concentrações testadas através da diminuição de TNF-α. Contudo, estudos complementares como análises de genotoxicidade, proteína, dentre outras, devem ser realizadas a fim de confirmar o seu potencial efeito anti-inflamatório. / Anti-tumor therapies based on anti-inflammatory effects have been considered in cancer treatment. Survival, proliferation and, eventually, invasion and metastasis of tumor cells are regulated by local inflammatory mediators. Primary inflammatory cytokines, such as Tumor Necrosis Factor (TNF), can be targets for anticancer therapy. Several anti-inflammatory isolated from natural products are acquiring importance as chemopreventive and therapeutic agents for cancer. This study aimed to investigate the expression of TNF-α, NF-κΒ and p38α genes, associated with proliferation and inflammation in Caco-2 cell line treated with ethanolic and hexanic extracts of Calyptranthes grandifolia O.Berg (Myrtaceae). Caco-2 cells were cultured and treated with plant extract at different concentrations (200, 100, 50 and 25 ug/ml) and stimulated with LPS. For gene expression analysis was performed a total RNA extraction by Trizol® method, followed by the synthesis of cDNA (complementary DNA) and analysis by qPCR (Polymerase Chain Reaction in Real Time). Among the evaluated genes, there was a decrease in TNF-α expression in 100 and 200 ug/ml concentrations only on ethanolic extract (p <0.025, ANOVA and Tukey test). The p38α gene showed a tendency to increase expression only in treatment with ethanolic extract and NF-κΒ gene did not present significant variations in both analyzed extracts. It can be concluded that ethanolic extract may present an anti-inflammatory activity on tested concentrations by decreasing TNF-α. However, further studies as genotoxicity analysis, protein, among others, should be performed to confirm its potential anti-inflammatory effect.

The poultry industry has successfully applied breeding and production programmes to meet growing consumer demands for chicken meat and eggs. Over the last four decades, poultry breeders have selected birds not only for productivity, but also for improved health, welfare, fitness and environmental robustness. Intensive production settings contribute to faster spread of diseases and greater losses in production due to increased morbidity and mortality of the flock. Traditional methods of disease treatment and prevention have played a critical role in control of disease. However, growing resistance of pathogens to therapeutic measures and consumer concerns led to the withdrawal of antibiotics as growth promoting additives in chicken feed. In addition, some vaccines have been overcome by increasing variation and virulence of pathogens and are no longer successful in disease prevention. The emergence of virulent and drug resistant pathogens have emphasised the need to focus on other solutions to disease, particularly natural genetic resistance. Genetic loci or gene expression patterns associated with the differential resistance of lines to specific pathogens have been identified, providing valuable markers for selective breeding. However, to date relatively few of these have been successfully incorporated into commercial lines. An ability to suppress or resist multiple pathogens, by selection for improved innate immune robustness has also been studied but it has not been introduced in commercial production, partly as the phenotype is ill-defined. Previous studies that focused on pro-inflammatory cytokines and their mRNA levels expressed by innate immune effector cells (heterophils and macrophages) identified differences between resistant and susceptible chicken lines, with the former producing stronger responses, supporting efforts to select poultry with an efficient early innate response. Here, small-scale qPCR screening and cellular techniques were evaluated with the conclusion that a more rapid, cheaper and reproducible method needs to be applied. The main objective of this project was therefore to design and validate a diagnostic tool that could be used to phenotype the immune responses of chickens at the level of innate immunity. For this purpose, a panel of 89 genes was selected based on previously published infection studies and on RNA-seq results obtained from stimulation of heterophils, macrophages and dendritic cells with lipopolysaccharide (LPS). Target genes were cloned and sequenced to optimise the design of qPCR reactions and primers. A multiplex qPCR platform, the Fluidigm 96.96 Dynamic Array, was selected as the tool of choice with the capacity to measure transcription of 96 genes of interest in 96 samples simultaneously. The preamplification reaction was optimised and the platform validated using a commercial line of chickens housed in clean or pathogen-challenged environments. Lymphoid tissues, including bursa of Fabricius, spleen, ileum with Peyer’s patches, caecal tonsils, and blood leukocytes were isolated and transcript levels for immune-related genes defined between organs, birds and farms. For qPCR analysis, a panel of reference genes was normalised and TBP, ACTB and GAPDH genes were selected and validated as the most stable. The high-throughput qPCR analysis identified peripheral blood leukocytes as a potentially reliable indicator of immune responses among all the tissues tested with the highest number of genes significantly differentially expressed between birds housed in varying hygienic environments. The research described here could potentially aid the selection of poultry for improved immune robustness. The technical optimisation and validation of a new tool to simultaneously quantify expression of tens of relevant immune-related genes will prime research in many areas of avian biology, especially to define baseline immune gene expression for selection, the basis of differential resistance, and host responses to infection, vaccination or immuno-modulatory substances.

Cancer remains one of the most common causes for death in the world today. Researchers are continuously trying to improve old, and develop new, methods in order to strife this global problem. Much research is being made trying to find new specific biomarkers that can be used to detect and diagnose cancer in an early stage. One candidate protein for possible future use as a biomarker is tripeptidyl-peptidase II (TPPII) which has previously been shown to be up-regulated in Burkitt´s lymphoma. This paper focuses on the expression of TPPII on an mRNA-level to see if there is any difference between expression in human leucocytes from patients with a leukemia diagnosis and a healthy volunteer, in order to evaluate if the expression of TPPII have any future use as a biomarker. Patient samples were analyzed using real time qPCR, to study the expression of mRNA, and Western blot, in order to correlate the mRNA findings with protein expression. Three different cell lines with different characteristics regarding expression and function of TPPII were also used to validate the methods used and for comparison with the patient samples analyzed. A difference in expression of mRNA were seen between the different patient samples, both individually and between larger groups of samples with the same diagnosis, indicating a large individual variation, thus making future use in a clinical setting difficult. However, seeing as only a few samples were analyzed in this study, more research must be done in order to draw any final conclusions.

The thesis aimed to develop and characterize a novel detection approach, which we termed aptaPCR exploiting nucleic acid aptamers as combined recognition and reporter biocomponents for the ultrasensitive detection of analytes. Nucleic acid aptamers are synthetic ligands selected from vast combinatorial libraries through a process referred to as SELEX – Systematic Evolution of Ligand By Exponential Enrichment. As compared to other natural and synthetic receptor, aptamers possess unique chemical and biochemical characteristics, such as: a well known chemistry, remarkable stability, an ability to be selected against virtually any target even in non-physiological conditions

Tissue factor (TF), a 47 kDa glycoprotein, is the initiator of the extrinsic pathway of blood coagulation and consequently of the upmost importance when damage to blood vessel occurs. The source of TF in circulation has been investigated. However, the source of TF is still not clear. One theory is that platelets express and increases the expression of TF after stimulation and the aim of our report was to investigate whether platelets really are a source for TF in circulation. Using specific primers for TF mRNA, platelets in plasma from healthy volunteers and from patients suffering from cardiac infarction were analyzed by using real-time polymerase chain reaction (PCR). Gel electrophoresis was performed after amplification of TF mRNA to verify the results. The samples were negative for TF when using real-time PCR and the few positive all had cycle threshold (Ct) values above 35. The contamination by monocytes was analyzed by using real-time PCR, with primers for CD14 and showed low amounts. After analysis, our conclusion was that platelets do not express TF. Although some samples had positive real-time PCR, the Ct values were all above 35, meaning they had very few transcripts in the initial samples and that the biological importance is uncertain. Since contamination of CD14 positive cells were found in most samples it can’t be ruled out that the origin of the positive TF mRNA is from monocytes.

Clostridium perfringens is the aetiological agent of Necrotic enteritis (NE); a disease that impacts on the health and welfare of broilers. This disease is a large cost to the industry and presents as lesions in the small intestine hindering productivity. Antibiotics are commonly used to treat NE but as pressure increases to limit their use further information about disease onset and broiler responses to the bacteria and it’s virulence factors during infection is required to implement new preventative measures and treatments. NetB is a secreted toxin from C. perfringens which has an important role in NE onset. Using an in situ intestinal loop model we have been able to characterise: I) temporal broiler responses to NetB positive bacterial culture supernatant (Chapter 2), ii) early host responses to different isolates possessing NetB (virulent) or not (avirulent) in the presence or absence of bacterial cells (Chapter 3) and iii) the responses of two commercial broiler breeds (Chapter 4) four hours post exposure. Samples collected from these experiments have been used for histology, mRNA expression and immunohistology. We have shown differences in mRNA expression in the duodenum of broilers after exposure to C. perfringens cells as well as the culture supernatant from the isolates used after four hours. The presence of bacteria cells resulted in up-regulation of pro-inflammatory cytokine, IFN-γ, mRNA, whereas it resulted in down-regulation of B-LA, mRNA a gene involved in presentation of pathogens to immune cells. IL-6 mRNA expression was also reduced in the presence of virulent isolates. This could indicate a possible evasion strategy for C. perfringens in broilers. Immunohistochemical analysis indicated that slower growing broilers have increased numbers of immune cells (macrophages and γδ T cells) in their duodenum compared with faster growing broilers, although this did not appear to have an effect on mRNA expression levels of pro-inflammatory cytokines, 4h post antigen infusion. Overall we detect greater changes when bacteria are included with culture supernatant and have highlighted possible mechanisms for C. perfringens to avoid the broiler immune system. Induction of NE in the literature requires pre-disposing factors, including co-infection with other intestinal pathogens and dietary manipulation of the host. The final experiment trialled protocols administering a virulent isolate of C. perfringens in-feed and via gavage along with an increased protein source to induce NE (Chapter 5). These models were not considered to be consistent for further investigation of NE in the future.

BACKGROUND: In recent years, studies have increasingly pointed to a number of different mechanisms that potentially form the foundation for the neurodegenerative pathology seen in Post-Traumatic Stress Disorder (PTSD). One such mechanism is neural damage due to oxidative stress. This study attempted to identify significantly altered expression levels of particular genes of interest between PTSD and control groups, as well as between PTSD samples and samples exhibiting commonly seen PTSD comorbidities, major depressive disorder (MDD), and depressive disorder not otherwise specified (DepNOS). The genes of interest being pursued in the study encompass the production of reactive oxygen species, such as inflammatory response mechanisms, the processes that control the removal of reactive oxygen species (ROS), and genes that are activated in response to oxidative stress. Other genes of interest involve factors important in the structural integrity of the prefrontal cortex, such as junction proteins, the blood-brain barrier, such as aquaporins, and neuronal integrity. These genes were included due to the evidence of structural degeneration in PTSD patients. A total of 54 genes were tested in all four groups. OBJECTIVES: To identify and determine genetic differences amongst individuals with PTSD in comparison to non-PTSD sufferers, and sufferers of common PTSD comorbidities. METHODS: The expression levels of the genes of interest were measured using quantitative polymerase chain reaction (qPCR) techniques. RNA is extracted and collected from tissues samples of deceased military PTSD patients from the prefrontal cortex. The prefrontal cortex derived RNA is used as the experimental samples, due to the prevalence of the pathology of PTSD in this region. Complimentary DNA (cDNA) is reverse transcribed from the collected RNA, and then products of genes of interest are amplified during the qPCR reaction using specifically designed primers. The expression level of the genes of interest were then compared to the ubiquitously expressed gene 18s for normalization calculations. Genetic expression levels in the PTSD, MDD, and DepNOS cohorts were then normalized to the expression of non-PTSD controls. RESULTS: Of the 54 genes of interest analyzed, two genes, NQO1 and IL-6, exhibited significantly decreased and increased levels of expression in comparison to the control group, respectively (p < 0.05). Alongside this, NQO1 showed significantly decreased expression in comparison to the DepNOS cohort, while IL-6 exhibited significantly increased expression in comparison to the MDD cohort (p < 0.05). CONCLUSION: Two genes involved with the production and elimination of reactive oxygen species were identified with having significantly altered levels of expression. NQO1, which facilitates the removal of ROS was shown to have significantly lower expression when compared to the control group, indicating an inhibited ability to remove ROS readily. Furthermore, IL-6, a proinflammatory cytokine and a promotor of ROS production, exhibited significantly increased expression, indicating a potential increase in ROS formation. Together these results indicate a potential mechanism for the production and accumulation of ROS in patients with PTSD, leading to the observed neurodegeneration. / 2018-07-11T00:00:00Z

Submitted by Marcelo Andrade Silva (marcelo.andradesilva@ufpe.br) on 2015-03-09T13:42:42Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DissertacaoThays Almeida.pdf: 1479816 bytes, checksum: 7f1cecad4d86341af2b9e3222a0f79be (MD5) / Made available in DSpace on 2015-03-09T13:42:42Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DissertacaoThays Almeida.pdf: 1479816 bytes, checksum: 7f1cecad4d86341af2b9e3222a0f79be (MD5) Previous issue date: 2013 / CAPES e CNPq / As leishmanioses são doenças infecto-parasitárias e, no Brasil, a Leishmania (V.) braziliensis é a espécie de maior incidência para Leishmaniose Tegumentar Americana (LTA). Em todas as suas formas clínicas, a resposta imune é dependente de células T, por isso, torna-se importante compreender os mecanismos imunológicos responsáveis pela formação, progressão e cura da doença. O presente estudo teve como objetivo identificar, através da técnica PCR quantitativa em tempo real (qPCR), o perfil de expressão de mRNA para IFN-γ, TNF-α, TGF-β, IL-10 e IL-4 e da enzima iNOS em células mononucleares do sangue periférico (PBMC) de pacientes com LTA após estimulação com os antígenos solúvel e insolúvel de L.(V.) braziliensis. Associou-se o perfil de expressão desses mediadores ao tamanho, tempo e número de lesões, além da resposta a intradermorreação de Montenegro (IDRM). Células de 27 pacientes e 6 controles foram incubadas por 24 horas na presença do mitógeno fitohemaglutinina (PHA) ou dos antígenos solúvel (AgSol) e insolúvel (AgIns) de L. (V.) braziliensis. A expressão de mRNA para as citocinas e para a iNOS foi avaliada por qPCR e as análises dos resultados foram feitas através do método Ct comparativo e pelos testes não-paramétricos Wilcoxon e Mann-Whitney. Em relação ao AgSol, foi verificada diferença significava da expressão de IFN-γ e TNF-α em pacientes quando comparados aos controles. Quanto ao AgIns, houve expressão significativa das citocinas IFN-γ, TNF-α e IL-4 em pacientes quando comparados aos controles. As citocinas IL-10 e TGF- β foram expressas frente a todos os estímulos utilizados, não apresentando diferença significativa entre e intra-grupos. Não houve expressão de iNOS com nenhum dos estímulos utilizados. Comparando os antígenos de L.(V.) braziliensis utilizados, foi observado maior expressão de IFN-γ e TNF-α frente ao antígeno insolúvel do parasita. A partir desses dados, podemos sugerir que ambas frações antigênicas do parasita são capazes de induzir uma resposta imune específica em pacientes com LTA ativa. Além disso, os dados obtidos nesse estudo sugerem a existência de um perfil de resposta imune Th1 para o antígeno solúvel e um perfil de resposta imune Th1 e Th2 considerando o antígeno insolúvel do parasita. A expressão gênica significativa para as citocinas TNF-α e IFN-γ, sugere que os pacientes tenham montado uma eficiente resposta imune contra o parasita e a presença da citocina IL-10 que eles estariam promovendo um mecanismo imunorregulatório sobre a forte resposta imune inflamatória exercida pelas citocinas Th1. Dentre os antígenos utilizados, pode-se sugerir que, devido a sua composição, o antígeno insolúvel seja mais imunogênico que o antígeno solúvel e que, por isso, essa molécula antigênica seja uma possível candidata a vacina e alvos para a imunoterapia. A ausência de expressão da iNOS pode ter sido devido a presença de TGF-β, que estaria regulando negativamente a produção de NO e consequentemente de iNOS. Além disso, pode estar havendo um mecanismo auto-regulatório para evitar produção em excesso de NO. A existência do perfil Th1 e Th2 pode ser explicado pelo estágio de desenvolvimento da doença. Assim, pelo fato de a LTA apresentar uma resposta Th1 multifacetada torna-se necessário estudos complementares para verificar o papel de outros tipos celulares e citocinas na progressão e cura da doença.