Intimina é o principal fator de virulência envolvido na lesão attaching/effacing em E. coli enteropatogênica (EPEC) e E. coli enterohemorrágica (EHEC), que são responsáveis pela diarreia infantil e colite hemorrágica/síndrome hemolítico-urêmica, respectivamente. Nos países em desenvolvimento, o diagnóstico desses patógenos ou é demorado e caro, ou não é realizado em laboratórios de rotina. No entanto, a detecção é essencial para definição do tratamento da infecção. O objetivo do presente trabalho foi definir o vetor de expressão mais apropriado e avaliar a sensibilidade e especificidade do scFv-intimina obtido. O gene scFv-intimina anteriormente obtido foi clonado nos vetores de expressão pAE e pSMT3, transformados em uma E. coli BL21 (DE3). A expressão foi induzida com 1 mM de IPTG. Em ambos os vetores, a proteína expressa foi direcionada para o citoplasma bacteriano, dessa forma a construção pAE-scFv-intimina foi utilizada na obtenção do anticorpo recombinante. Após cromatografia de afinidade ao níquel o scFv-intimina foi empregado no ensaio de imunofluorescência indireta para definir um teste de ensaio rápido e confiável, analisando-se 199 isolados de E. coli e isolados de outras Enterobactérias, dos quais 132 resultaram em isolados eae-positivos e 67 isolados eae-negativos. O teste de imunofluorescência mostrou a detecção de 132 isolados eae-positivos (100% de sensibilidade) e a não detecção de 67 isolados eae-negativos (100% especificidade). Assim, o ensaio de imunofluorescência é um método eficaz e rápido, e o scFv-intimina é uma excelente ferramenta para o imunodiagnóstico de EPEC e EHEC. / Intimin is the main virulence factor involved in the attaching/effacing lesion of enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), which are responsible for infantile diarrhea and hemorrhagic colitis/hemolytic uremic syndrome, respectively. In developing countries, their diagnosis is either time-consuming and expensive or is not done in the routine laboratory. Nevertheless, their detection is essential in defining the infection treatment. The aim of the study was to define the most suitable expression vector and evaluate the sensitivity and specificity of the scFv-intimin obtained. The scFv-intimin gene previously obtained was cloned into pAE and pSMT3 expression vectors and transformed into an E. coli BL21 (DE3) strain. Expression was induced with 1 mM IPTG. In both vectors, recombinant protein was expressed as inclusion bodies, so pAE was chosen as the expression vector for nickel affinity chromatography. Once purified, scFv-intimin was employed in an indirect immunofluorescence assay to define a rapid and reliable assay testing 199 E. coli strains and other Enterobacteriaceae isolates, of which 132 isolates were eae-positive and 67 isolates were eae-negative. The immunofluorescence assay showed the detection of 132 eae-positive isolates (100% sensitive) and no detection in the 67 eae-negative isolates (100% specific). Thus, immunofluorescence is an effective and rapid method and scFv-intimin an excellent tool for the immunodiagnosis of EPEC and EHEC.

Anticorpos recombinantes vêm sendo utilizados como ferramenta diagnóstica por serem produzidos com baixo custo e em larga escala. Partiu-se de um gene sintético que codifica um fragmento de anticorpo (scFv) específico contra a toxina termoestável, com otimização de códons para expressão em Escherichia coli. Esse gene foi amplificado no vetor de clonagem e subclonado em vetor de expressão pET28a. Células E. coli BL21(DE3) foram transformadas com o plasmídeo recombinante e induzidas em meio de expressão. O fragmento de anticorpo obtido estava contido na fração insolúvel, portanto foi submetido a purificado por cromatografia de afinidade ao níquel na presença de ureia, seguido de renaturação. A molécula se apresentou funcional e sem reatividade com inespecífica por ensaios de imunofluorescência e ELISA. Além disso, mostrou-se estável quando armazenada a 4ºC, sendo assim uma ferramenta promissora para ser utilizada no diagnóstico de ETEC para detecção da toxina ST. / Recombinant antibodies have been used as diagnostic tools since they can be produced at low cost and on a large scale. A synthetic gene encoding an antibody fragment (scFv) specific for the heat-stable toxin (ST) with optimized codon for Escherichia coli expression was employed. This gene was amplified in the cloning vector and subcloned into pET28a expression vector. E. coli BL21(DE3) cells were transformed with the recombinant plasmid and induced. Large amounts of antibody fragment were found in the insoluble fraction. Thus it is submitted to nickel-affinity chromatography in urea presence, followed by refolding step. By immunofluorescence assay and ELISA, the obtained antibody showed to be functional with no cross-reaction to the negative controls. Furthermore, it was stable when stored at 4 °C, therefore a promising tool for ETEC diagnosis detecting the ST toxin.

All cells carry the genetic machinery required to commit cell suicide; a process known as programmed cell death (PCD). While the ability to initiate PCD serves a number of useful purposes during development and homeostasis, misregulation of PCD is the underlying basis of most human diseases, including cancer, autoimmunity disorders and neurodegeneration. Using the tobacco hawkmoth Manduca sexta as a model organism, the Schwartz lab at UMass has demonstrated that PCD requires de novo gene expression and has cloned many death-associated genes. One of these genes encodes a novel protein that was named Acheron after one of the rivers of the Underworld in Greek mythology. Acheron (also known as Lupus Antigen Related Protein 6; Larp6) is an RNA binding protein that mediates a number of cellular processes, including cell survival, angiogenesis, migration, and differentiation. The molecular mechanisms that mediate Acheron’s diverse roles are poorly understood, but several lines of evidence suggest that it is mediated in part by protein protein interactions and motifs that target it to different cellular compartments. In this thesis, I employed immunofluorescence and confocal microscopy to conduct two studies employing mammalian cells. The first study was to determine the subcellular localization for Acheron in normal cells and cells treated with growth factors. I found that Acheron predominantly coincides with the microtubule cytoskeleton. In our second study, I tested the hypothesis that Acheron’s binding partners BAD, Human Homolog of Ariadne-1 (HHARI) and Calcium/Calmodulin-dependent Serine Protein Kinase (CASK) colocalize with Acheron. As part of this analysis, I sought to determine if Acheron could facilitate the translocation of CASK to the nucleus. I found that Acheron localizes predominantly to microtubules, with some expression in the cytoplasm and nucleus. When Acheron and its partners are co-labeled in cycling mouse C2C12 myoblasts or human U2-OS osteosarcoma cells, Acheron did not colocalize with BAD, HHARI or CASK on microtubules. However, I found that when Acheron is driven into the nucleus with high levels of growth factors, CASK also appeared to translocate to both the nucleus and to microtubules, where it colocalized with Acheron. The data acquired through these studies should provide not only insights into the subcellular distribution of Acheron and its potential binding partners but may also help elucidate its roles in programmed cell death, differentiation, and pathogenesis in mammalian models.

Charles University Faculty of Pharmacy in Hradec Králové Department od Biochemical Sciences Candidate: Růžena Melicharová Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of thesis: The detection of protein covalent complexes with DNA using fluorescent microscopy Anthracycline antibiotics are present one of the most potent antineoplastic drugs. The mechanism of their action is complex. They are reported to intercalate to DNA, form DNA adducts and interact with topoisomerase II (TopII) as its poisons. Catalytic cycle of TopII is interrupted when anthracyclines stabilize the covalent complex of DNA and TopII and that causes cell damage. However, using of anthracyclines is limited by several adverse effects e. g. myelotoxicity and cardiotoxicity. The mechanism of cardiotoxicity is still unclear but may be associated with poisoning of the TopIIβ isoform. Unlike the TopIIα, TopIIβ is present mostly in quiescent cells as cardiomyocytes. Furthermore, the only clinically approved cardioprotective drug dexrazoxane belongs to TopII catalytic inhibitors. Nevertheless, the details of the dexrazoxane-afforded protection are unclear. This thesis was aimed to optimize the TARDIS (trapped in agarose DNA immunostaining) assay to detect and quantify covalent cleavage complexes, compare different ways for analysis of...

The synthesis of iron-sulfur clusters is an essential cellular process, which depends on complex biosynthetic pathways. In model eukaryotes, these pathways are the ISC pathway in the mitochondria and the CIA pathway in the cytosol. A recent genome and transcriptome analysis showed, that an amitochondriate protist Monocercomonoides exilis lacks the canonical ISC pathway, which has been replaced by a bacterial SUF pathway. A close free-living relative of M. exilis, Paratrimastix pyriformis possesses a mitochondrion-related organelle, yet also possesses a SUF pathway instead of ISC. The acquisition of the SUF pathway has been suggested as the primordial cause for mitochondrial loss in M. exilis, which is the first documented eukaryotic organism without a mitochondrion. The SUF pathway has been the subject of numerous studies in bacteria, however, its role as the core provider of iron-sulfur clusters for eukaryotic cells has been reported in merely a handful of eukaryotes and was based predominantly on genomic data. This thesis focuses on the putative ATPase SufC and the putative scaffold protein SufB. Both proteins were successfully produced in recombinant forms. SufC has been found to possess ATPase activity in vitro, which was increased upon interaction with SufB. The conditions for theATPase...

This thesis describes the use of antibodies and immunofluorescence for subcellular localization of proteins. The key objective is the creation of an open-source atlas with information on the subcellular location of every human protein. Knowledge of the spatial distribution and the precise location of a protein within a cell is important for its functional characterization, and describing the human proteome in terms of compartment proteomes is important to decipher cellular organization and function.   Immunofluorescence and confocal microscopy of cultured cells were used for high-resolution detection of proteins on a high-throughput scale. Critical to immunofluorescence results are sample preparation and specific antibodies. Antibody staining of cells requires fixation and permeabilization, both of which can result in loss or redistribution of proteins and masking of epitopes. A high-throughput approach demands a standardized protocol suitable for the majority of proteins across cellular compartments. Paper I presents an evaluation of sample preparation techniques from which such a single fixation and permeabilization protocol was optimized. Paper II describes the results from applying this protocol to 4000 human proteins in three cell lines of different origin.   Paper III presents a strategy for application-specific antibody validation. Antibodies are the key reagents in immunofluorescence, but all antibodies have potential for off-target binding and should be validated thoroughly. Antibody performance varies across sample types and applications due to the competition present and the effect of the sample preparation on antigen accessibility. In this paper application-specific validation for immunofluorescence was conducted using colocalization with fluorescently tagged protein in transgenic cell lines. / <p>QC 20160509</p>

To understand the role of nonmuscle myosin II in cardiac and skeletal muscle, we used a number of polyclonal antibodies, three detecting nonmuscle myosin heavy chain II-B (NMHC II-B) and two detecting NMHC II-A, to examine the localization of these two proteins in fresh-frozen, acetone-fixed sections of normal human and mouse hearts and human skeletal muscles. Results were similar in both species and were confirmed by examination of fresh- frozen sections of human hearts subjected to no fixation or to treatment with either 4% p-formaldehyde or 50% glycerol. NMHC II-B was diffusely distributed in the cytoplasm of cardiac myocytes during development, but after birth it was localized to the Z-lines and intercalated discs. Dual labeling showed almost complete colocalization of NMHC II-B with α-actinin. Whereas endothelial cells, smooth muscle cells and fibroblasts showed strong immunoreactivity for NMHC II-A and NMHC II-B, cardiac myocytes only showed reactivity for the latter. The Z-lines of human skeletal muscle cells, in contrast to those of cardiac myocytes, gave positive reactions for both NMHC II-A and NMHC II-B. The presence of a motor protein in the Z-lines and intercalated discs raises the possibility that these structures may play a more dynamic role in the contraction/relaxation mechanism of cardiac and skeletal muscle than has been previously suspected.

Parallel detection of antibodies with different specificity has many potential applications in epidemiological research, vaccine development and in the diagnosis of allergies, autoimmune and infectious diseases. Although ELISA-based tests are suitable for this purpose, available assays can be time consuming and require large quantities of both sample and reagents thus limiting their application for mass screening. / Revisión por pares

La présence du système endocannabinoïde (eCB), en particulier le récepteur CB1 (CB1R), dans la rétine, le corps genouillé latéral, et l’aire visuelle primaire (V1) du singe a récemment été mise en évidence. Cependant, aucune étude n’a démontré la présence de ce système dans l’aire visuelle secondaire V2, une région qui reçoit la plupart des efférences de V1. Comme V1 exprime ce récepteur, nous faisons l’hypothèse que l’aire V2 l’exprime également. Le but de notre étude est donc de caractériser l’expression et la localisation cellulaire de ce système dans l’aire V2 du singe vervet. Des cerveaux de cinq singes vervets (Chlorocebus sabeus) adultes ont été utilisés. Les marqueurs cellulaires NeuN, SMI-32, et PV ont été employés pour caractériser et identifier les différentes couches de V2. En localisant ces derniers, nous déterminons la distribution de CB1R et des enzymes de synthèse (NAPE-PLD) et de dégradation (FAAH) du système eCB en utilisant des techniques d’immunofluorescence. De plus, l’organisation laminaire en six couches de V2 a été mise en évidence par nos marqueurs cellulaires. Nos résultats démontrent la présence de CB1R dans les fibres axonales aux extrémités de V2, c’est-à-dire dans les couches superficielles (1-3) et profondes (5-6). CB1R est peu ou pas exprimé dans la couche 4. CB1R entoure, mais n’est pas exprimé par les cellules positives- NeuN, SMI-32 et PV. Cependant, les enzymes NAPE-PLD et FAAH sont présentes dans les cellules pyramidales SMI-32 et les cellules interneurones PV -positives. Ces données indiquent que CB1R, NAPE-PLD et FAAH sont présentes dans V2 et pourraient moduler l’information visuelle provenant de V1 et se dirigeant vers les aires V4 et V5, et probablement, influencer la perception visuelle. / The presence of the endocannabinoid system in the retina, the lateral geniculate body, and the primary visual area (V1) of the monkey has recently been established. However, no study has demonstrated the presence of this system in area V2, a region that receives most of the afferents from V1. As V1 expresses this system, we assume that the area V2 also expresses it. The aim of our study is to characterize the expression and cellular localization of this system in the visual cortex V2 of the vervet monkey. The brains of 5 adult monkeys were used in this project. Cellular markers NeuN, SMI-32, and PV were used to characterize and identify the layers of V2. Using immunofluorescence, these markers were also localized in order to study the distribution of CB1R, the enzyme of synthesis (NAPE-PLD) and of degradation (FAAH) of eCB ligands. The six-layer organization of V2 was also identified by our cellular markers. Our results show the presence of the eCB system in area V2. Furthermore, we found that CB1R immunoreactivity is present in the axonal fibers at the ends of V2; in the superficial (L1-3) and deep (L5-6) layers. CB1R expression was low to non-existence in layer 4. CB1R surrounds but does not co-localize with NeuN-, SMI-32-, and PV- positive cells. On the other hand, NAPE-PLD and FAAH enzymes were co-localized with SMI-32-positive pyramidal cells and PV-positive interneuron cells. These data, therefore, indicate that CB1R, NAPE-PLD and FAAH are present in V2 and their presence can modulate visual information coming from V1 and going to V4 and V5, and probably, influence visual perception.

Giardia is a parasite that causes disease in both humans and animals all around the world. Approximately 200 million people are infected each year. The parasite is spread mainly through contact with contaminated feces but can also be spread through food and water contaminated with infectious cysts. Symptoms that may occur during infection could be diarrhea and abdominal pain. Not all host organisms get symptoms and there are lots of asymptomatic carriers. The aim of this report was to compare and evaluate the performance of three different methods used to detect Giardia in dogs and cats. Today the gold standard is detecting cysts with immunofluorescence, which was one of the methods evaluated. One cyst visible was enough to say the test was positive. The other two methods were an ELISA and a rapid test, and both detected soluble Giardia antigen. For both methods, color development occurred in case of positive results. The ELISA wells were read spectrophotometrically at 450 nm. During this project, 294 tests from randomly chosen dogs and cats were analyzed. Of all these tests, 103 were analyzed with all three methods, whereas 191 tests were only analyzed with immunofluorescence and ELISA. When analyzing the results, the number of positive test results was very similar between the three methods and there was no statistically significant difference between them. Therefore, when price, time and results were taken into consideration, the method recommended was ELISA.