O estudo de prevalência do vírus rábico foi realizado em uma amostra de morcegos capturados na Mata Atlântica da região sudeste do Brasil. Os morcegos são um dos principais reservatórios silvestres do vírus rábico. No Brasil existem aproximadamente 144 espécies de morcegos, e pouco se sabe sobre a circulação do vírus rábicos nessas espécies. Foram realizadas estimativas – com duas metodologias de isolamento - para detectar a presença do vírus rábico na população estudada. Os resultados foram obtidos pelo cruzamento entre a variável infectividade (presença de vírus rábico) e as variáveis epidemiológicas (espécies de morcegos, sexo, idade, local de captura ). Observou-se que o método de isolamento que utiliza as células McCoy isolou com maior facilidade vírus de morcegos insetívoros, além de apresentar maior capacidade de detectar a infecção na fase latente (subclínica). Já as células N2A foram mais eficientes na detecção do vírus rábico em morcegos hematófagos D. rotundus. As duas metodologias utilizadas apresentaram maior proporção de isolamento do vírus rábico em morcegos insetívoros, nectarívoros e fitófagos. Tais resultados sugerem que os morcegos insetívoros desempenham importante papel na manutenção do vírus no reservatório cuja população foi estudada. Também foi possível constatar que a circulação do vírus ocorre inter e intra-espécies, mas estudos especificamente desenhados para avaliar esse aspecto devem ser implementados. / The prevalence study of the rabies virus was carried out in a sample of bats captured in the Brazilian southeastern São Paulo. Bats are one of the main wild reservoirs of the rabies virus. Brazil holds 144 species of bats and little is know about the circulation of such virus in these species. Two metodologies were used for the estimates of the presence of the rabies virus in the captured in the Parque Estadual Intervales. The results were obtained crossing the variable (presence of rabies virus) with epidemiological variables (bat species, sex, age, site of capture). The McCoy cell line method proved isolating more easily the virus of insectivorous bats besides presenting more capability of detection of infection in the latent phase (sub-clinic phase). On the other hand the N2A cell line were more efficient in detecting the rabies virus in D. rotundus hematophagous bats. It was also observed that for both cells the insectivorous, nectarivorous and phytophagous bats presented higher rabies virus isolation proportion. These results suggest that insectivorous bats play in important role in the maintence of the virus in this reservoir. Although could also be observed that the circulation of the virus occurs intra and inter species, but studies specially designed to asses this issue must be re-evaluated.

Le cancer colorectal est la deuxième cause de mortalité due au cancer en Europe et aux États-Unis. Etant donné l’efficacité limitée et la toxicité élevée des agents de chimiothérapie, de nouvelles approches sont nécessaires. Le flavanolignane silybine représente le principal constituant actif du chardon-marie (Silybum marianum). Les mécanismes moléculaires des propriétés anticancéreuses de la silybine ont été étudiés dans un modèle cellulaire de progression du cancer colorectal humain : les cellules SW480 issues d’un adénocarcinome, et leurs dérivées métastatiques les cellules SW620. Les effets chimiopréventifs de la silybine ont été étudiés dans un modèle de cancérogenèse colique induite par l’azoxyméthane chez le rat. La silybine induit une mort apoptotique avec activation de la caspase-3 dans les deux lignées. L’expression des récepteurs de mort TRAIL est augmentée, et la caspase-8 activée. Le potentiel mitochondrial est perturbé provoquant une libération du cytochrome c et une activation de la caspase-9. En plus de l’activation des voies apoptotiques extrinsèque et intrinsèque la silybine induit une réponse autophagique. La combinaison de la silybine et de TRAIL, un agent anti-cancéreux prometteur, provoque une mort cellulaire synergique dans les deux lignées. Un effet synergique est aussi observé avec la combinaison de la silybine et des inhibiteurs des histones déacétylases (HDAC) : TSA et SAHA. Dans le modèle chez le rat, la silybine réduit de 50% le nombre des lésions prénéoplasiques. En conclusion, la silybine est un agent naturel intéressant pour la prévention du cancer colorectal et dans le cadre d’une combinaison avec TRAIL/des inhibiteurs d’HDACs. / Colorectal cancer (CRC) is the second most common cause for cancer-related deaths in Europe and in the USA. Because of the limited efficacy and considerable toxicity of chemotherapeutic agents, new approaches are needed. The hepatoprotective flavonolignan silibinin is the major biologically active compound of the milk thistle (Silybum marianum).The molecular mechanisms of the anticancer properties of silibinin in CRC were studied in an in vitro model of cancer progression consisting of the adenocarcinoma cell line SW480 and its derived metastatic cell line SW620. Its chemopreventive effects were assessed in an in vivo model of azoxymethane-induced colon carcinogenesis in the rat. Silibinin induced apoptotic cell death with activation of caspase-3 in both cell lines. The expression of death receptors was upregulated, and caspase-8 was activated. The potential of the mitochondrial membrane was perturbed permitting the release of cytochrome c and the activation of caspase-9. Besides the activation of the extrinsic and the intrinsic apoptotic pathway, silibinin induced an autophagic response. Combination of silibinin and TRAIL, a promising anticancer agent selectively inducing apoptosis in cancer cells, induced synergistic cell death in both cell lines. Synergy in cell death induction was also observed by the combination of silibinin and the histone deacetylase (HDAC) inhibitors TSA and SAHA. In the preclinical model in the rat, silibinin administration was able to reduce by half the number of preneoplastic lesions present in the colon. In conclusion, silibinin is a promising natural agent for colon cancer chemoprevention and for combination therapy with TRAIL/HDAC inhibitors.

A presença de Ad com competência para replicar (RCA, replication-competent adenovirus) nas preparações é um dos maiores problemas para a produção de Ad em larga escala. RCAs são gerados pela recombinação entre seqüência do vetor e seqüência homóloga do gene E1 presente nas células helper. Objetivo: desenvolver uma nova linhagem auxiliar para produção de Ad livre de RCA - LEY79 - derivada da linhagem de retinoblastoma humano Y79, tratando-se da primeira linhagem empacotadora de adenovírus com inativação mutacional da proteína supressora de tumor pRb, que crescem em suspensão. Células Y79 foram infectadas com o retrovírus pCLDE1A/E1BSN, selecionadas com G418. A eficiência de produção de AdeGFP na linhagem LEY79 foi testada e comparada com a HEK293A. Células Y79 foram adaptadas em meio livre de soro. Esperamos com a linhagem LEY79SF inovar no campo de processos para a produção de Ad recombinante. / The presence of Ad with the ability to replicate (RCA, replication-competent adenovirus) in preparations is a major problem in the large-scale production of Ad. RCAs are generated by recombination between the vector sequence and sequence of the homologous gene in E1 helper cells. Objective: To develop a new helper cell line for the production of RCA-free Ad., called LEY79, derived from the Y79 of human retinoblastoma line, the first line Packer adenovirus with mutational inactivation of the tumor suppressor protein pRb, which are adapted to grow in suspension. Y79 cells were infected with the retrovirus pCLDE1A/E1BSN, selected with G418. The efficiency of production of AdeGFP in the LEY79 was tested and compared with the HEK293A. Y79 cells were adapted to grow in serum-free medium. We hope that use of the the LEY79SF cell line will promote innovation in the processing and production of recombinant Ad.

Trophic factors have shown great promise in their potential to treat neurological disease. In particular, glial cell line-derived neurotrophic factor (GDNF) has been identified as a potent neurotrophic factor for midbrain dopamine (DA) neurons in the substantia nigra (SN), which lose function in Parkinson’s disease (PD). GDNF progressed to phase II clinical trials, which did not meet proposed endpoints. The large size and binding characteristics of GDNF have been suspected to contribute to some of the shortcomings of GDNF related to delivery to target brain regions. Smaller peptides derived from GDNF (Dopamine-Neuron Stimulating Peptides – DNSPs) have been recently investigated and appear to demonstrate trophic-like effects comparable to GDNF. In the described studies, a time course study was conducted to determine in vivo DA-release characteristics 1-, 2- and 4- weeks after peptide treatment. These studies determined the effects on DA terminals within striatal sub-regions using microelectrodes. A heterogeneous effect on striatal sub-regions was apparent with the maximum effect in the dorsal striatum – corresponding to terminals originating from the SN. Dysregulation of GDNF or GDNF signaling is believed to contribute to motor dysfunction in aging and PD. Thus, it is hypothesized that GDNF is necessary for the maintenance and function of neurons. To extend this line of investigation, in vivo functional measures (DA-release and -uptake) and behavioral and cellular alterations were investigated in a transgenic mouse model (Gdnf+/-) with reduced GDNF protein levels. The described studies determined that both DA-uptake and -release properties were altered in middle-aged Gdnf+/- mice with only modest reductions in DA neurochemical levels. GDNF levels in Gdnf+/- mice were restored to levels comparable to wild-type (WT) counterparts by treatment with GDNF. GDNF protein supplementation led to enhanced motor behavior and increased markers for DA neurons in the SN of Gdnf+/- mice. Gdnf+/- mice appeared to show a heightened sensitivity to GDNF treatment compared to WT counterparts. Overall, this body of work examines novel synthetic peptides with potential to enhance DA-neuron function and expands upon the current understanding of GDNF’s role in the nigrostriatal pathway.

<p>In this thesis, methods to study gene and protein expression in cells and tissues were developed and utilized in combination with protein-specific antibodies, with the overall objective to attain greater understanding of protein function.</p><p>To analyze protein expression in <i>in vitro</i> cultured cell lines, a cell microarray (CMA) was developed, facilitating antibody-based protein profiling of cell lines using immunohistochemistry (IHC). Staining patterns in cell lines were analyzed using image analysis, developed to automatically identify cells and immunohistochemical staining, providing qualitative and quantitative measurements of protein expression. Quantitative IHC data from CMAs stained with nearly 3000 antibodies was used to evaluate the adequacy of using cell lines as models for cancer tissue. We found that cell lines are homogenous with respect to protein expression profiles, and generally more alike each other, than corresponding cancer cells <i>in vivo</i>. However, we found variability between cell lines in regards to the level of retained tumor phenotypic traits, and identified cell lines with a preserved link to corresponding cancer, suggesting that some cell lines are appropriate model systems for specific tumor types. </p><p>Specific gene expression patterns were analyzed in vitiligo vulgaris and malignant melanoma. Transcriptional profiling of vitiligo melanocytes revealed dysregulation of genes involved in melanin biosynthesis and melanosome function, thus highlighting some mechanisms possibly involved in the pathogenesis of vitiligo. Two new potential markers for infiltrating malignant melanoma, Syntaxin-7 and Discs large homolog 5, were identified using antibody-based protein profiling of melanoma in a tissue microarray format. Both proteins were expressed with high specificity in melanocytic lesions, and loss of Syntaxin-7 expression was associated with more high-grade malignant melanomas.</p><p>In conclusion, the combination of antibody-based proteomics and microarray technology provided valuable information of expression patterns in cells and tissues, which can be used to better understand associations between protein signatures and disease.</p>

Mitochondrial mutations and heteroplasmy have been associated with disease states that result from inadequate cellular energy production. As mitochondrial DNA (mtDNA) encodes many of the polypeptides involved in oxidative phosphorylation (OXPHOS), mtDNA mutations may lower energy production which is required for cell division and sustained ATP synthesis. In order to test the relationship between mtDNA mutations and the rate of cell division, a mammary epithelial cancer cell line, MCF-7, is used as a model. Nine proliferate single cell clones have been isolated from MCF-7. Population doubling times of six single cell clones and the MCF-7 stock have been determined. Clones with distinctly different growth rates were selected for mutational analysis. Growth rates of these clones appeared to be different from each other. Using polymerase chain reaction (PCR) and DNA sequencing, three cases of heteroplasmy have been identified in the mitochondrial genes of the MCF-7 stock and four single cell clones (ATPase C9119T, ND6 T14300G, Cytb G15807A). Heteroplasmy present in the Cytb gene is differs between single cell clones. Differences between the growth rates may be indicative of metabolic variations in these single cell clones. The OXPHOS enzymes encoded by the mutated genes were quantified by standard enzymatic assays. The assays demonstrated significant differences in specific activity between the clones, but were not correlated with mitochondrial heteroplasmy. This thesis determines that the differences in specific activity observed between clones is of nuclear origin.

The availability of murine genetic models is extremely advantageous to studying gastrointestinal function, but the benefits afforded by studying enteric neurons in mice has been hindered by their accessibility. Fetal (E13) and 2 day post-natal (P2) enteric neuron cell lines (IM-FEN and IM-PEN, respectively) were recently developed from the H-2Kb-tsA58 immortomouse. Our goal was to identify the electrophysiological properties of these cell lines and clarify their utility as a model of enteric neurons. IM-PEN cells stained positively for the neuron specific markers βIII-tubulin and PGP9.5 and were negative for the glial cell marker S100. Detection of mRNA for TRPA1, TRPV1, ClCa1, KCa3.1, NaV1.3 and NaV1.9 were present while CaV2.2 and TASK1 were very faint. No significant difference was observed in the passive membrane properties of IM-FEN and IM-PEN. The cells had depolarized resting membrane potentials -29.8 ± 0.9mV (n=30) and high input resistances ranging from 552 ± 104MΩ (IM-FEN, n=6) to 728 ± 128MΩ (IM-PEN, n=20). xiv In current clamp, hyperpolarizing current was given to obtain a holding potential of -60mV or -80mV, yet neither IM-FEN (n=6) nor the IM-PEN cells (n=20) were able to generate action potentials in response to depolarizing pulses. In whole cell voltage clamp depolarization induced an inward current which was identified as an L-type Ca2+ channel. Niflumic acid inhibited the outward current as well as the tail currents indicating a ClCa current supporting the mRNA data. A volume sensitive chloride channel was also identified that was DCPIB sensitive (n=7) and removed when chloride was replaced with gluconate (n=4), displaying characteristics of ICl,swell. As a result IM-PEN cells had a high chloride conductance resulting in a depolarized membrane potential, which is a characteristic of immature neurons. The transcription factor MASH1 has been found to be required for enteric neuron differentiation. Transfection of MASH1 after 4 and 8 days did not alter the electrophysiological characteristics of IM-PEN (n=6). We conclude that IM-PEN may represent immature enteric neurons and are a useful model to examine the effect of factors required for the development of enteric neurons.

0 Abstract Cystic fibrosis is an autosomal recessive disease caused by mutation in CFTR gene coding for a chloride channel in apical membrane of epithelial cells. This disorder leads to the change in ion transport causing the increase in mucus viscosity in airways as well as changes in glycosylation of saccharide structures on the cells. Because of that these cells are the target for bacterial adhesion. Chronic bacterial infections, which lead to gradual decline of lung function and damage of lung tissue, are the major cause of death of patients suffering with cystic fibrosis. Pseudomonas aeruginosa is the main pathogen causing chronic infections in cystic fibrosis patients. This bacterium produces a biofilm protecting them from host immune system and antibiotics. Once the colonization with PA occurs, it is difficult to get rid of this pathogen. The prophylactic treatment with orally administered hen antibodies against the PA virulence structures could be a prevention of chronic PA infections. In this work we tested the antibody against the bacterial lectin PA-IIL, which is suggested to be involved in the adhesion of the pathogen on epithelial cells. First, it was verified that the prepared antibody from egg yolks of a hen immunized with the bacterial lectin PA-IIL recognizes this antigen expressed...

NK buňky (z angl. natural killer cells, přirozeně zabíječské buňky) hrají klíčovou roli při rozpoznávání a ničení nádorových, infikovaných nebo jinak pozměněných buněk. Na svém povrchu nemají antigenně specifické receptory, proto je řadíme mezi složky přirozené imunity. K rozpoznání cílových buněk slouží řada jiných povrchových receptorů. Inhibiční receptory zajišťují buněčnou toleranci, naopak aktivační receptory spouští cytotoxické mechanismy vedoucí k apoptóze a tedy lýzi buňky. Díky této vlastnosti jsou NK buňky intenzivně studovány v souvislosti s imunoterapií nádorových onemocnění. Jedním z aktivačních receptorů je NKp80 rozpoznávající svůj ligand AICL. Oba proteiny patří do rodiny receptorů podobných lektinům C-typu. Tento komplex se účastní nejenom přímé lýze maligních buněk myeloidního charakteru, ale má také důležitou roli v imunomodulaci zánětu. Předmětem této diplomové práce je příprava receptoru NKp80 a jeho ligandu AICL. Receptor NKp80 byl připraven v linii lidských embryonálních ledvinných buněk (HEK 293S GnTI- ). Byly připraveny stabilně transfekované linie produkující protein NKp80 konstitutivně nebo indukovatelně. Zapojení disulfidických můstků a obsazení N-glykosylačních míst proteinu NKp80 bylo ověřeno hmotnostní spektrometrií. Dále byl optimalizován postup Mgr. Jiřího Nového na...

O núcleo é uma estrutura organizada e possui verdadeiras organelas nucleares. Entre elas, estão os nucléolos e os corpúsculos de Cajal (CBs). Estes compartimentos nucleares são estruturas dinâmicas, mantidos pela associação de macromoléculas envolvidas na expressão gênica que interagem entre si delimitando-as. A principal proteína encontrada nos CBs é a p-80-coilin e, portanto, o principal epítopo capaz de marcar essas estruturas. Suas funções específicas ainda tem sido alvo de estudo. Existe uma proteína em comum a ambas as estruturas, a fibrilarina que participa no processamento de rRNA. Estes corpúsculos já foram descritos na periferia dos nucléolos ou mesmo fisicamente ligados a ele. Acredita-se que os corpúsculos de Cajal participem da síntese de rRNA, maturação, transporte e associação das subunidades ribossômicas.Diante esta relação, este trabalho visa estudar a inter-relação entre estas estruturas em células normais e as respectivas linhagens de células tumorais em cultura antes e após tratamentos com actinomicina D. Esta droga se usada em baixas concentrações, bloqueia a transcrição dos genes que foram decodificados pela RNA polimerase I e II, e -amanitin, por sua vez bloqueia a transcrição de genes decodificado pela RNA polimerase II . Além disso, também visa investigar uma relação entre a proliferação das linhagens estudadas e freqüência dos corpúsculos de Cajal nas células controle e tratadas. O microscópio confocal de varredura a laser permitiu o estudo dessas estruturas em preparações imunofluorescência fornecendo uma análise tridimensional destas estruturas quando utilizados anticorpos específicos. Linhagens de células que apresentaram um crescimento mais lento foram aquelas que tinham uma maior freqüência de corpúsculos por núcleo. Por outro lado, aquelas que apresentaram um crescimento mais intenso, foram aquelas que apresentaram maior variação no número de corpúsculos por núcleo. Após o tratamento com inibidores de síntese de RNA, tanto os corpúsculos de Cajal quanto os nucléolos, apresentaram alterações morfológicas, às vezes apresentando um grande acúmulo na região dos corpúsculos ou desorganizando os nucléolos. Mudanças no tamanho e forma também puderam ser destacadas. / The nucleus is a structure that has sub-compartments which can be called nuclear organelles. Among them, may be cited the nucleoli and the Cajal bodies (CBs). These nuclear compartments are dynamic structures, maintained by association and stock of macromolecules involved in gene expression. The main protein found in the CB is a p-80-coilin and therefore the main epitope able to label these structures. Their functions are still to be clarified. There is a protein in common to the nucleolus and Cajal bodies, the fibrillarin that takes part in the processing of rRNA. The CBs can be found at the periphery of the nucleoli or even physically connected to them. It is believed that the CBs may have role in the synthesis of rRNA and maturation, transport and association of ribosome subunits. In view of this relationship between Cajal bodies and nucleoli, this work aims to study the interrelationship between these structures in normal cells and their respective tumor cell line in culture before and after treatments with actinomycin D, which in low concentrations, blocks the transcription of genes that were decoded by the RNA polymerase I and II and -amanitin, which is responsible for blocking the transcription of genes decoded by the RNA polymerase II and find out a relationship between cell proliferation and Cajal bodies frequencies in control and treated cells. The confocal microscope of laser scanning enabled the study of these structures in preparations immunofluorescent providing a three-dimensional analysis of these structures when used specific antibodies before and after treatment. Cell lines that shown low cell grow, appears to have lass CB/ nucleus in the other hand, cell lines that have fastest grow shown nuclei with more Cajal bodies frequencies and more variation in the number of Cajal/nucleus After treatment with inhibitors, both Cajal bodies as nucleoli, made quite clear morphological changes, sometimes giving large accumulation of proteins in organelles and sometimes appeared disorganized in the nucleoplasm. Changes in the size and shape were also highlighted. The tumor cell lines also showed changes compared to their normal cell type.