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Tesis por compendio / ‘Candidatus Liberibacter solanacearum’ is a α-Proteo bacteria, Gram-negative, restricted to plant phloem and to the haemolymph of psyllids that act as vectors. This emerging bacterium has been associated with different diseases in different hosts and associated with carrot (Daucus carota) in Spain. Vegetative disorders of unknown etiology have also been observed in celery (Apium graveolens) since 2008. A real-time PCR protocol specific to ‘Ca. L. solanacearum’ detection, using TaqMan probe and direct sample preparation methods have been developed. This technology has been validated in an intra-laboratory study (sensitivity 1, specificity 1 and accuracy 100%) and is available commercially as a complete kit. It has been demonstrated that ‘Ca. L. solanacearum’ is associated with the observed syndrome in celery and a new bacterium haplotype (E) has been identified. With these results it is concluded that celery is a new host of ‘Ca. L. solanacearum’ (Teresani et al., 2014a). Using the newly developed real-time PCR protocol ‘Ca. L. solanacearum’ has been detected in 42,6% of the carrot seeds lots tested and in individual seeds. The number of cells/seeds has been estimated in 4.8±3.3 to 210±6.7, which only 5% were viable. After 150 days post-germination, 12% of seedlings showed symptoms and tested positive for ‘Ca. L. solanacearum’. Liberibacter-like cells were observed in the phloem sieve elements of the seed coat and in the phloem of carrot leaf midrib from seedlings. These results demonstrated that ‘Ca. L. solanacearum’ is transmitted by carrot seeds (Bertolini et al., 2014b). The collected arthropods were classified into families, and the superfamily Psylloidea was identified to the species level resulting Bactericera trigonica, B. tremblayi and B. nigricornis the main identified species. The population dynamics of different psyllids species visiting carrot, celery and potato has been determined, concluding that the highest populations are captured during summer. The bacterium has been detected in the different Bactericera species previous cited additionally to Bactericera sp. The psyllid species carrying the bacteria can be considered as possible vectors of the bacterium (Teresani et al. 2014b). Electrical Penetration Graphs showed that B. trigonica was able to feed in the phloem of carrot, celery and potato but not in the phloem of tomato plants. Experimental transmission showed that B. trigonica transmitted ‘Ca. L. solanacearum’ from carrot to carrot, celery, potato and tomato. More efficient transmission occurred with ten individuals, and the transmission rates were 100% in celery, 80% in carrot and 10% in potato and tomato. The experimental transmission to potatoes threatens this crop (Teresani et al., 2014c). These combined results have built a scientific foundation of the biological and epidemiological aspects of ‘Ca. L. solanacearum’ contributing to new scientific information that is key in cultivation of celery and carrot to establish bacteria control strategies. The use of bacteria-free carrot seed lots will definitely contribute to mitigate damage and reduce risks of transmission to solanaceous crops. / Ribeiro Teresani, G. (2014). Candidatus Liberibacter solanacearum: detection, characterization, new hosts and epidemiology in Spain [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48459 / Compendio

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia and Anaplasma species are rickettsial organisms which infect a variety of mammalian species. The organisms are transmitted from ticks and are maintained in reservoir hosts. Several pathogens have been identified in recent years as the causative agents for emerging infections in people. One of the primary reservoir hosts for the pathogens is the white tailed deer. In this study, 147 deer blood samples and 37 ticks were evaluated for the prevalence of Ehrlichia/Anaplasma species by TaqMan-based real time amplification assay and DNA sequence analysis. One hundred and thirteen (74%) samples tested positive with the Ehrlichia/Anaplasma genera-specific probe. Further analysis of the samples with the probes specific for human ehrlichiosis agents, E. chaffeensis and E. ewingii identified 4 (2.7%) and 7 (4.7%) positives, respectively. Test positives from 24 randomly selected samples were further evaluated by sequence analysis targeting to a 450 bp segment of 16S rRNA gene. All 24 samples were confirmed as positive for the Ehrlichia GA isolate # 4 (GenBank #U27104.1). DNAs from 37 pools of ticks collected from the white tailed deer were also evaluated. The TaqMan-based real time PCR assay with Anaplasma/Ehrlichia common probe identified 29 (78%) tick pools as positives whereas E. chaffeensis- and E. ewingii-specific probes identified three (8%) and one (3%) positives, respectively. The PCR and sequence analysis of tick samples identified Gram-negative bacteria species which included one endosymbiont of Rickettsia species (one tick pool), one Alcaligenes faecalis strain (three tick pools), five different Pseudomonas species (9 tick pools) and five different uncultured bacteria organisms (7 tick pools). Although the pathogenic potential of the white-tailed deer isolates of Anaplasma and Ehrlichia agents remains to be established, their high prevalence and the presence of human ehrlichiosis pathogens in white-tailed deer is similar to earlier findings. The high prevalence of the deer isolates of Anaplasma and Ehrlichia species demonstrates the need for further assessment of the pathogenic potential of these organisms to people and domestic animals.

Mislabeling of ground meat products is a form of food fraud that can lead to economic deception and interfere with dietary restrictions related to allergens or religious beliefs. In various parts of the world, including Ireland, Mexico and Turkey, high levels of meat mislabeling have been reported between 2000-2015. However, there is currently a lack of information regarding this practice in the United States. Therefore, the objective of this study was to test a variety of ground meat products sold on the U.S. commercial market for the presence of potential mislabeling. Forty-eight ground meat samples were purchased from online and local retail sources, including both supermarkets and specialty meat retailers. DNA was extracted from each sample in duplicate and tested using DNA barcoding of the cytochrome c oxidase subunit I (COI) gene. The resulting sequences were identified at the species level using the Barcode of Life Database (BOLD). Any samples that failed DNA barcoding went through repeat extraction and sequencing. Due to the possibility of a species mixture, these samples were also tested with real-time polymerase chain reaction (PCR) targeting beef, chicken, lamb, turkey, pork and horse. Of the 48 products analyzed in this study, 10 were found to be mislabeled, with nine containing multiple meat species. Meat samples purchased from online specialty meat distributors had a higher rate of being mislabeled (35%) compared to samples purchased from a local butcher (18%) and samples purchased at local vii supermarkets (5.8%). Horsemeat, which is illegal to sell on the U.S. commercial market, was detected in two of the samples acquired from online specialty meat distributors. Overall, the mislabeling detected in this study appears to be due to reasons such as intentional mixing of lower-cost meat species into higher cost products or unintentional mixing of meat species due to cross-contamination during processing.

The increasing incidence of children suffering from allergic diseases could be caused by sensitization of immature immune system during the intrauterine development. Several important scientific papers have demonstrated the ability of cord blood cells to respond by elevated proliferation activity after stimulation by common allergens. Following these findings, present study follows the production of cytokines which play a role in the pro- and anti-allergenic tuning of the immune system. Umbilical cord blood cells were stimulated with polyclonal activators (phytohaemagglutinin) and common allergens (ovalbumin, timothy grass, birch, mite). Subsequently, cytokine production was monitored using selected methods that reflect different stages of cell activation - at the level of mRNA by quantitative real time PCR (qRT-PCR), by flow cytometry detection of the presence of intracellular cytokines in different cell subpopulations and by ELISA measurement of cytokines in CBMC culture supernatants. The results obtained point to a very weak ability of these common allergens (timothy grass, birch, mite, ovalbumin) to stimulate CBMC to produce cytokines observed by all of these methodological procedures. Although we did not observe significant differences in CBMC cytokine production (IL-2, IL-4, IL-10, IL-12,...

Pluripotence je schopnost buňky diferencovat do jakéhokoliv buněčného typu. Formuje se během časného embryonálního vývoje u savců a její vznik je spojen s reprogramací genové exprese na globální úrovni. Proces přirozeného vzniku pluripotence není stále zcela pochopen. Pro získání nového pohledu na události, které vedou ke vzniku pluripotence u savců, studovali jsme změny v genové expresi během oocyt-zygotického přechodu u myši. V tomto modelovém systému, oplodněné vajíčko podstoupí reprogramaci, která vede k vytvoření pluripotentních blastomer. Tyto blastomery zakládají samotné embryo. Cílem mé diplomové práce bylo analyzovat aktivaci transkripce během časného vývoje a vyvinout metodu pro monitorování exprese genů v oocytech, časných embryích a embryonálních kmenových buňkách. Metoda využívá kvantitativní PCR a umožnuje změřit expresi až 48 vybraných genů, které slouží jako markery pro maternální degradaci, aktivaci pluripotentního programu a diferenciaci do zárodečných linií. Dále ukazujeme, že náš systém monitoruje dynamiku transkriptomu během oocyt-zygotického přechodu, a získané výsledky jsou srovnatelné s daty naměřenými pomocí jiných metod. Díky našemu bioinformatickému přístupu jsme navíc identifikovali nové oocyt-specifické a zygotické nekódující RNA. Klíčová slova: pluripotence,...

MicroRNAs (miRNAs) are small noncoding RNAs of length 18 to 25 nucleotides that regulate gene expression posttranscriptionally. Expression of some miRNAs is tissue specific. I assumed that pregnancy induced complications associated with placental insufficiency could be characterized by a unique profil of placental-specific miRNAs in maternal circulation. I measured concentration and gene expression of selected miRNAs (miR-516-5p, miR-517*, miR-518b, miR-520a*, miR-520h, miR-525 and miR-526a) in the plasma of patients with preeclampsia (PE), fetal growth restriction (FGR) and gestational hypertension (GH). The control group consisted of patients with a normal course of pregnancy (FG). I processed 168 plasma samples, the representation of individual diagnosis were as follows: PE 63, FGR 27, GH 23, FG 55. Detection and quantification was carried out by quantitative real-time PCR. I identified three miRNAs with elevated levels in a group of preeclamptic patients: miR-517*, miR-520a* and miR-525. The severity of the PE, which was characterized by a form of the disease (mild or severe PE) and term (before or after the 34th week of pregnancy), did not have a statistically significant effect on the levels of miRNAs. More than a quarter of patients had PE superimposed on previous hypertension. Previous history of...

O aumento da longevidade da população mundial vem acompanhado do aumento da incidência de doenças crônicas. Várias são as doenças que podem acometer esta população e entre estas, a osteoporose, que compromete a resistência e qualidade do tecido ósseo, predispondo a fraturas. Além disso, a osteoporose pode dificultar a reparação óssea. Uma técnica importante e que tem por objetivo a neoformação óssea é a regeneração óssea guiada (ROG), que utiliza uma membrana que atua como uma barreira mecânica, permitindo a criação de um espaço protegido em torno do defeito ósseo. Embora a membrana de politetrafluoretileno (PTFE) seja uma das mais utilizadas na ROG, novas membranas têm sido desenvolvidas dentre elas a membrana obtida pela associação do polímero de poli(vinilideno-trifluoretileno) e da cerâmica de titanato de bário (P(VDF-TrFE)/BT). Estudos in vitro demonstraram resultados favoráveis à membrana de P(VDF-TrFE)/BT. Desta forma, o objetivo deste estudo foi avaliar in vivo, o efeito da membrana de poli(vinilidenotrifluoretileno)/titanato de bário (P(VDF-TrFE)/BT), e como controle a de PTFE no reparo ósseo em ratas com modelo experimental para a osteoporose. Foram utilizados 30 animais, sendo 5 pertencentes ao grupo controle - Grupo 1 (G1) e 25 que foram ovariectomizados bilateralmente (OVX). Após 150 dias foram confeccionados defeitos ósseos (5 mm) na calvária e os animais OVX foram distribuídos em três grupos com relação à utilização ou não de membranas nos defeitos ósseos: Grupo 2 - nenhum tipo de membrana; Grupo 3 - membrana de PTFE e Grupo 4 - membrana de P(VDF-TrFE)/BT. Após 4 semanas, os animais foram eutanasiados e as calvárias foram coletadas para as análises histológica, histomorfométrica por micro-CT e de expressão gênica por PCR em tempo real. A análise histomorfométrica mostrou que os animais que receberam a membrana de P(VDF-TrFE)/BT apresentaram parâmetros morfométricos semelhantes ou até melhores quando comparados com os animais que receberam a membrana de PTFE. A comparação dos grupos que receberam as membranas mostrou para o grupo P(VDF-TrFE)/BT uma menor expressão para os genes RUNX2, BSP, OPN, OSX e RANKL; uma expressão semelhante para os genes ALP, OC, RANK e CTSK e uma maior expressão dos genes OPG, CALCR e MMP9. Estes resultados evidenciam que a membrana de P(VDF-TrFE)/BT pode ser considerada um biomaterial promissor para a reparação óssea em condições de osteoporose / The worldwide population age is increasing accomplished by chronic disease. The most common disease which involves the population is the osteoporosis. The bone resistance and quality of bone matrix are compromised and the fractures risks are higher. Nonetheless, the osteoporosis can avoid the bone healing. The important technique to achieve the bone neoformation is the Bone Guided Regeneration (BGR), which used a membrane as mechanic barrier to allow the new gap protection around the bone defect. Although the politetrafluoetilene (PTFE) is the most used in BGR, newest membranes have been developed as the polyvinylidene-trifluoroethylene polymer and barium titanate ceramics (P(VDF-TrFE)/BT). In vitro assays showed favorable results to P(VDF-TrFE)/BT membrane. The aim of the study was to evaluate the poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) effects, and the PTFE has been used as control on the bone repair in the osteoporosis experimental rats model. It was used 30 animals, 5 on the control group- Group 1 (G1) and 25 were ovariectomized bilaterally (OVX). Bone gaps were done 5mm size on the rats calvaria´s and the (OVX) were housed in three different groups concerned by membranes applied or not at the bone gap. Group 2 no membrane applied; Group 3 PTFE membrane and Group 4 P(VDF-TrFE)/BT membrane. Four weeks later, the animals were euthanized and the calvarias were collected to the histological analysis, histomorphometric assays by micro CT and the gene expression by the real time PCR. The histomorphometric analysis showed that the animals which received P(VDF-TrFE)/BT membrane presented similar morphometric parameters better than PTFE. The P(VDF-TrFE)/BT showed a minor expression to RUNX2, BSP, OPN, OSX e RANKL genes; the similar were to RANK e CTSK and the higher expression were to OPG, CALCR e MMP9. These results evidence that the P(VDF-TrFE)/BT could be used as promising biomaterial to bone healing under osteoporosis

O objetivo do trabalho foi avaliar a qualidade, a quantidade e a capacidade de amplificação por PCR de DNA extraído de grãos de soja e milho, seus derivados e produtos acabados contendo como ingredientes obtidos desses grãos, com vistas à detecção de resíduos de organismos geneticamente modificados em alimentos. Para a amplificação de DNA pela PCR convencional, não houve melhor adequação de um protocolo de extração. Ambos métodos, CTAB e coluna de sílica tiveram desempenho comparável para as 32 matrizes avaliadas. A técnica de PCR em tempo real se mostrou mais sensível à qualidade do DNA testado e nesse contexto, o método CTAB se mostrou mais eficiente do que o método de coluna de sílica. Independentemente do método de extração utilizado não foi possível detectar DNA em óleos de soja e milho e em alguns derivados de amido, sugerindo que a aplicabilidade da lei de rotulagem pode esbarrar num entrave técnico no caso de algumas matrizes alimentares altamente processadas. / The aim of the study was to evaluate the quality, the quantity and the amplifiability by PCR of DNA isolated from soybean and maize grains and their by-products targeting the detection of genetically modified organisms in food. PCR amplification of DNA samples isolated either from CTAB and silica-column extraction methods achieved comparable performances. Both extraction methods showed similar results for the 32 tested matrices. The DNA amplification by real time PCR appeared to be affected by the quality of the isolated DNA. In this context, the CTAB extraction method showed to be more suitable when compared to the silica-column method. No DNA was amplified from soy and maize oils, as well as from some starch by-products, regardless the DNA extraction method used. It suggests that, the labeling requirement may rely on technical issues considering some high processed foodstuffs.