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Aim: To investigate the antifungal potential of Macrocybe titans extracts against Candida albicans. Material & methods: Extracts were obtained as aqueous (EfraMat-22 and EfraMat-45) and methanolic/ethyl acetate fractions. Results: Broth microdilution and disk diffusion assays showed that EfraMat-45 provided the best results in terms of minimum inhibitory concentration. Scanning electron microscopy analysis revealed morphological changes and slight damage on the surfaces of cells exposed to EfraMat-45 at the MIC. Fluorescence microscopy analysis of the yeasts showed cell elongation. EfraMat-45 presented high levels of phenolic compounds and flavonoids, high antioxidant activity and absence of in vitro cytotoxicity. Conclusion: The results indicated that the aqueous extract of M. titans is highly promising as an antifungal agent.

The objective of this study is to compare the direct fecal smear (DFS) and centrifugal sedimentation (CS) methods in the detection of Cryptosporidium spp. oocysts in fecal samples of dairy calves. One hundred and fourteen fecal samples were collected from calves aged up to six months from 10 dairy farms located in Palotina and Francisco Alves, Paraná, Brazil. The microscopic analysis revealed the presence of Cryptosporidium spp. oocysts in 51.75% (59/114) of the samples in both methods. In CS, 48.25% (55/114) of the samples were positive, while in DFS slides, only 6.14% (7/114) were positive. Only 4 samples were positive exclusively in DFS. To ensure that there were no false-negative results in the microscopic analysis, the 55 samples that were negative in both DFS and CS were selected for molecular analysis using the nested PCR (nPCR). Of these 55 samples, 24% (13/55) were positive and forwarded for sequencing part of the genome, which made it possible to identify C. parvum, C. bovis and C. ryanae. Besides the characterization of the Cryptosporidium species, it was possible to identify bacteria of the genus Acinetobacter interfering directly in the analyzed samples. The microscopic analysis also revealed higher sensitivity when CS was used to make the fecal smears. However, some samples that were negative in this technique had positive PCR results. Thus, molecular analysis is indicated to confirm cases of Cryptosporidium spp. Further studies are necessary to prove the specificities of the used primers since the results obtained in nPCR were positive for the protozoan but, when genetic sequencing was performed, Acinetobacter spp. was identified. O objetivo deste trabalho foi comparar os métodos de esfregaço fecal direto (DFS) e centrífugo sedimentação (CS) para a pesquisa de oocistos de Cryptosporidium spp. em amostras fecais de bezerros leiteiros. Foram coletadas 114 amostras fecais de bezerros com até seis meses de idade, provenientes de dez propriedades leiteiras localizadas nos municípios de Palotina e Francisco Alves, Paraná. Por meio da análise microscópica, foi possível observar oocistos de Cryptosporidium spp. em 51,75% (59/114) das amostras. Pelo método da CS foi identificada positividade em 48,25% (55/114) das amostras, enquanto nas lâminas pelo método do esfregaço fecal direto (DFS), apenas 6,14% (7/114) foram positivas. Somente 4 amostras foram positivas exclusivamente no método do DFS. Para assegurar que na análise microscópica não houvesse resultados falso-negativos, as 55 amostras negativas para os dois métodos de confecção de lâminas (DFS e CS) foram selecionadas para análise molecular por meio da técnica de Nested PCR. Das 55 amostras submetidas a nPCR, 24% (13/55) apresentaram-se positivas e foram encaminhadas para o sequenciamento genético de uma porção do genoma, o qual possibilitou identificar as espécies de C. parvum, C. bovis e C. ryanae. Além da caracterização das espécies de Cryptosporidium, foi possível identificar a presença de bactérias do gênero Acinetobacter interferindo diretamente nas amostras analisadas. A análise microscópica revelou maior sensibilidade quando o método de CS foi usado para a confecção dos esfregaços fecais, entretanto, amostras negativas por meio dessa metodologia apresentaram resultados positivos na nPCR. Desta forma, para a confirmação dos casos de Cryptosporidium spp., é indicado a realização da análise molecular. Novos estudos são necessários para se comprovar a especificidade dos primers utilizados, uma vez que na nPCR o resultado obtido foi positivo, porém ao realizar o sequenciamento genético houve a identificação de Acinetobacter spp.

This work aimed to perform the molecular identification of fungi mango fruits, in addition to verifying the effect of the essential oils for microbial control. Pericarp segments containing injured and healthy areas of mango fruits were initially disinfested, and then incubated in PDA culture medium at 25 °C. From all isolated material, UDC03, UDC04, UDC09 and UDC12 were preselected, which presented homogeneous cultures. The UDC04 and UDC12 isolates were submitted to the sequencing of the ITS region for presenting mycelial growth both greater and morphologically distinct of fungi from Colletotrichum genus. When BLAST is used in species identification, it was observed that UDC04 and UDC12 belong to Botryosphaeria dothidea and Neofusicoccum parvum, respectively. Regarding the control of UDC04, it was observed that BDA culture medium containing 1.5 to 2% of lemongrass and eucalyptus oils resulted in complete inhibition. For UDC12, mycelial growth inhibition was detected with the use of 1% and 1.5% essential oils of eucalyptus and lemongrass, respectively. The chia essential oil was not effective for the isolates control. Finally, the results reveal the importance of molecular identification of these phytopathogens associated with the establishment of an alternative control of fruits due to problems of contamination with chemical products.

This work explores the production of biohydrogen from brewery wastewater using as inoculum a culture produced by natural fermentation of synthetic wastewater and Klebsiella pneumoniae isolated from the environment. Klebsiella pneumoniae showed good performance as inoculum, as evaluated using assays of between 9 and 16 cycles, with durations of 12 and 24 h, carbohydrate concentrations from 2.79 to 7.22 g L−1, and applied volumetric organic loads from 2.6 to 12.6 g carbohydrate L−1 day−1. The best results were achieved with applied volumetric organic loads of 12.6 g carbohydrate L−1 day−1 and cycle length of 12 h, resulting in mean volumetric productivity of 0.88 L H2 L−1 day−1, maximum molar flow of 10.80 mmol H2 h−1, and mean yield of 0.70 mol H2 mol−1 glucose consumed. The biogas H2 content was between 18 and 42%, while the mean organic compounds removal and carbohydrate conversion efficiencies were 23 and 81%, respectively. The inoculum produced by natural fermentation was not viable.

This paper presents a strategy for isolation, identification and evaluation of microalgae strains synthesizing biologically active compounds (BAC) with antimicrobial activity against food-borne and other pathogens. Water samples containing microalgae were initially submitted to the enrichment process, followed by standard procedure for obtaining pure culture. Purified DNA from the microalgae isolates was used for sequencing the amplified 18S rDNA genomic region, which resulted in the identification of Poterioochromonas malhamensis, Chlorella sp., Micractinium sp., Tetradesmus sp. and Desmodesmus sp. All isolated strains were cultivated in 1-L flat-plate PBRs, with the Poterioochromonas malhamensis being the only one to show antibacterial activity in agar diffusion assay. Further, PPEQ-01strain was cultured under different types and scales of PBR, intensity and spectrum of the incident light, in order to study and prove the antibacterial potential of the strain. Ethanolic and methanolic supernatants, or hydrophilic and methanolic extracts produced from biomass cultivated in different conditions led to formation of bacterial growth inhibition halos around the impregnated disks used in the agar diffusion test. The hydrophilic extract, obtained from biomass cultured under green illumination, exhibited stronger activity against all tested pathogenic bacteria as compared to the other extracts. The results obtained with the new isolated Poterioochromonas malhamensis strain proved efficiency of the innovative complex approach applied to BAC aiming the identification of microalgae that present antimicrobial activity.

Plant growth-promoting bacteria are ecological alternatives for fertilization, mainly for gramineous. Since plant x bacteria interaction is genotype and strain dependent, searching for new strains may contribute to the development of new biofertilizers. We aim to characterize plant growth-promoting capacity of Leclercia adecarboxylata strain Palotina, formerly isolated by our group in corn. A single isolated colony was taken and its genome was sequenced using Illumina technology. The whole genome was compared to other Leclercia adecarboxylata strains, and their biological and growth-promoting traits, such as P solubilization and auxin production, were tested. Following that, a 4.8 Mb genome of L. adecarboxylata strain Palotina was assembled and the functional annotation was carried out. This paper is the first to report the genes associated with plant growth promotion demonstrating in vitro indole acid production by this strain. These results project the endophyte as a potential biofertilizer for further commercial exploitation.

O genero Scopulariopsis spp. engloba fungos cosmopolitas que normalmente sao encontrados na poeira domestica ou no solo. Raramente causam infeccao em humanos, todavia podem ocasionar onicomicose, otomicose e panoftalmite, se introduzido nos olhos devido a feridas penetrantes. Em determinadas situacoes podem resultar em micoses cutâneas, profundas e potencialmente infeccoes sistemicas, estas normalmente associadas a pacientes imunodeprimidos (COX; IRVING, 1993). Relatar a ocorrencia de Scopulariopsis spp. proveniente de swab lesional de um felino ( Felis catus domesticus ) em tratamento para esporotricose na cidade de Guaira, estado do Parana, Brasil. Um felino macho, nao-castrado, escore corporal de 2,5/5 e dois anos de idade, foi submetido a colheita de material lesional para isolamento de colonia de Sporothrix spp. O mesmo residia em uma casa com outros 50 gatos, quatro galinaceos, tres caes e possuia acesso a rua, contudo ha uma semana se encontrava isolado juntamente com outros quatro felinos, tambem com esporotricose, em um comodo dentro do ambiente interno da casa da tutora que tambem estava em tratamento para a doenca. O animal apresentava lesoes ulcerativas em ponta de orelha, na regiao interescapular, regiao do metacarpo esquerdo, face e testiculo e se encontrava em tratamento utilizando itraconazol 100mg, iodeto de potassio 30mg e silimarina 100mg. Procedeu-se a colheita do material com swab nasal, swab de unhas e swab lesional, todas as amostras foram armazenadas em solucao salina 0,9% esteril e refrigerados, ate o momento do processamento. O material foi estriado em placa contendo agar Sabouraud com gentamicina, e submetido a crescimento em temperatura ambiente. Apos sete dias, verificou-se o crescimento de uma colonia fungica de coloracao brancacenta, irregular e algodonosa no estriado proveniente de swab lesional. Procedeu-se entao o teste da fita adesiva corada com lactophenol cotton blue e analisada por meio de microscopia optica. Na microscopia foi possivel verificar hifas septadas, conidioforos com aneis hialinos, conidios piriformes com base truncada, de parede lisa em corrente, sendo caracteristico do genero Scopulariopsis spp. Esses dados demonstram que mais estudos referentes aos mecanismos de patogenicidade deste fungo devem ser realizados, visto que a presenca deste no local da lesao causada pela multiplicacao do Sporothrix spp. pode estar relacionada ao desenvolvimento de micoses cutâneas, o que pode favorecer o aumento das lesoes ocasionadas pelo Sporothrix , levando ao questionamento sobre a eficacia do tratamento no paciente felino.

Background Yeasts of the Candida parapsilosis complex have frequently been reported as agents of fungal infection in Brazil and worldwide, most of the cases are related to hospital-acquired infection. C. parapsilosis is the third most common cause of candidemia, and the hands of hospital workers as well as hospital surfaces have been suggested as possible sources. Objectives In this study we verified the frequency of C. parapsilosis on the hands of workers and on surfaces in the adult intensive care unit (AICU) of a tertiary hospital in Parana-Brazil. Methods Surface samples were collected with swabs moistened with saline, and a plastic bag with distilled water was used to collect samples from hands. The yeasts were identified by morphology, MALDI-TOF mass spectrometry and PCR-RFLP of the secondary alcohol dehydrogenase-encoding gene (SADH) after digestion with the restriction enzyme BanI. Results and conclusions A total of 223 yeast were found, of which 101 (45.29%) were identified as C. parapsilosis sensu stricto. Of these, 46.66% (n = 35) were found on surfaces and 44.59% (n = 66) on the hands of the employees. The analysis of C. parapsilosis strains by microsatellite loci (CP1, CP4, CP6 and B5) showed 80 different genotypes. Their antifungal susceptibility profile, evaluated by the microdilution broth method, revealed that C. parapsilosis was sensitive to amphotericin B, fluconazole and voriconazole, but not to micafungin. The results revealed the heterogeneity of the yeast population, suggesting that there is no common source of contamination in the AICU of this hospital.

This work aimed to use ultraviolet (UV) radiation to prevent microbial growth in diesel fuel with different compositions. Commercial samples of ultra-low-sulfur diesel (ULSD) and high-sulfur diesel (HSD) fuels with 11% of biodiesel were used as purchased. These samples were incorporated of 20% biodiesel and different amounts of free water (no addition, 2500, and 10,000 mL.m−3). All samples were stored for ten days and subjected to UV-C radiation treatment (55 W, 253 nm) for 360 min to reduce the number of microorganisms. Physicochemical parameters, such as water content, kinematic viscosity, density, and oxidative stability, were measured before and after UV-C treatment. The presence of microorganisms was detected by UV–vis spectrophotometry (600 nm) during the simulated storage, before and after UV-C treatment. An increase in the number of microorganisms was observed during the storage period due to the water content. The UV-C radiation reduced the microorganisms, especially in samples with high water content. Low sulfur levels did not affect the efficiency of the treatment. Significant variations in kinematic viscosity and oxidative stability were observed after the treatment. The presence of microorganisms was reduced by more than 50% after the incidence of UV-C radiation. Therefore, UV-C radiation is a promising technique for microbiological control in diesel fuel. These effects can last up to ten days after the first incidence of radiation.

Aim: To evaluate if radiation used in radiotherapy can cause changes in the virulence potential of Candida tropicalis ATCC 750. Materials & methods: C. tropicalis was exposed in vitro to identical dose and scheme of irradiation would be used in patients with head and neck cancer. Some virulence parameters were analyzed before and after irradiation. Results: Colony morphologies were irreversibly affected by irradiation. Increase in growth rate, filamentation, adhesion on cell lines and phagocytosis process were also observed. Overall the irradiated C. tropicalis cells became more efficient at causing systemic infection in mice. Conclusion: γ-radiation induced important changes in C. tropicalis increasing its virulence profile, which could directly affect the relationship between yeasts and hosts.

Vulvovaginal candidiasis (VVC) is one of the most frequent female genital disorders and Candida glabrata is the second most common agent. Current study was aimed to study the susceptibility to antifungal agents of C. glabrata isolated from vaginal samples and some virulence attributes in order to better understand why this species is emerging as the main VVC agents. A total of 60 C. glabrata vaginal isolates were included in this study. Firstly they were screened by susceptibility tests to antifungal agents. The isolates that showed sensitivity or resistance to fluconazole were evaluated for their virulence potential, including ability to adhere to polystyrene and vaginal ring, cell surface hydrophobicity (CSH) and capacity to form biofilm. Candida glabrata isolates varied significantly in adherence capacity, biofilm formation and CSH. However, it was possible to observe that isolates resistant to fluconazole adhered more efficiently to the vaginal ring and were statistically more able to form biofilm. These results allow hypothesizing that C. glabrata is an emergent agent in VVC probably because the treatment with fluconazole selects this species. But once adhered, yeasts remain on biotic or abiotic surfaces causing colonization or VVC symptomatology.

As bactérias são os mais abundantes organismos em nosso planeta, são encontradas em diversos habitats e podem ser usadas para o benefício da humanidade. Dentre elas, as bactérias celulolíticas que apresentam um metabolismo capaz de realizar a degradação da celulose, por meio da produção de enzimas celulases tem grande potencial no setor industrial. Para a identificação de novas estirpes de bactérias celulolíticas deve-se estabelecer um protocolo qualitativo. Neste trabalho, foi empregada uma metodologia modificada baseada no método de Theater e Wood (1989), obtendo-se estirpes bacterianas isoladas de solo na região oeste do Paraná. Como resultados, foi obtida a caracterização de estirpes positivas para a degradação da celulose, demostrando a interação da bactéria com a celulose (CMC) com a formação de halos de degradação.

Little is known about the use of microbial lipases for the treatment of beef industry effluent. It is essential to develop techniques for the sustainable management of this waste, given its high polluting potential, especially due to the high concentrations of oils and greases (O&G) present. Therefore, this study investigates the pretreatment of slaughterhouse effluent using lipase-producing microorganisms isolated from the waste itself. Firstly, a physicochemical characterization of the effluent was performed, followed by isolation and identification of a lipase-producing bacterium, which was used in a pretreatment step. A central composite rotational design (CCRD) was applied, considering the independent variables pH, temperature, and inoculum concentration, resulting in 17 assays, with lipolytic activity, O&G, and free fatty acids (measured during 72 h) as the response variables. The slaughterhouse effluent presented high polluting potential, with an O&G concentration of 2553.3 mg L−1 and COD of 8914.4 mg L−1. The isolated bacterium was identified as Aeromonas hydrophila. After 72 h of the experiment, lipolytic activity of 1.40 U mL−1, 59% O&G removal (pH: 7.0, temperature: 30.0 °C and inoculum concentration: 10%), and production of 36.0 μmol mL−1 of free fatty acids were obtained. It could be concluded that cattle slaughterhouse effluent is an excellent source of lipase-producing microorganisms and that the use of enzymes for the treatment of this waste is a promising technology.

Several waste sources have been studied as substrate sources for the production of biogas rich in hydrogen and for the isolation of bacteria capable of fermenting several substrates for the same purpose. Nonetheless, to simplify the process and minimize production costs, it is important to seek alternatives both for the use of microbial consortia using crude waste and for the use of substrates also in their crude form, without the need for purification. The aim of this study was to use only waste as inoculum and substrate for the biological production of hydrogen. Thus, samples from anaerobic ponds of a poultry slaughterhouse were used as inoculum. Sucrose, pure glycerol (in initial tests) and crude glycerol (inserted in blends with pure glycerol) were used as substrates. H2 production experiments were conducted in batches, using a reactor kept in an anaerobic environment for 11 days, at 35°C, under orbital agitation at 150 rpm. To analyse the composition of the biogas and the presence of soluble metabolic products (SMPs), samples of the headspace gases generated and of the reaction medium were collected. The results using sucrose as substrate indicated that the inoculum under study has potential for bio-H2 production, as it produced CH4-free biogas containing 50–60% H2. The inoculum was also shown to be adaptable to the use of glycerine as a substrate, producing biogas with similar characteristics to those obtained from sucrose degradation; however, it required a longer acclimatization period, and thus more in-depth study is required.

Background: In recent years, very few effective drugs against Mycobacterium tuberculosis have emerged, which motivates the research with drugs already used in the treatment of tuberculosis. Ethambutol is a bacteriostatic drug that affects cell wall integrity, but the effects of this drug on bacilli are not fully exploited. Objective: Based on the need to better investigate the complex mechanism of action of ethambutol, our study presented the proteome profile of M. tuberculosis after different times of ethambutol exposure, aiming to comprehend the dynamics of bacilli response to its effects. M. tuberculosis was exposed to ½ MIC of ethambutol at 24 and 48 hours. The proteins were identified by MALDI-TOF/TOF. Results: The main protein changes occurred in metabolic proteins as dihydrolipoyl dehydrogenase (Rv0462), glutamine synthetase1 (Rv2220), electron transfer flavoprotein subunit beta (Rv3029c) and adenosylhomocysteinase (Rv3248c). Conclusion: Considering the functions of these proteins, our results support that the intermediary metabolism and respiration were affected by ethambutol and this disturbance provided proteins that could be explored as additional targets for this drug.

The effect of a meropenem-ciprofloxacin combination (MCC) on the susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa (MRPA) clinical isolates was determined using checkerboard and time-kill curve techniques. Structural changes and differential gene expression that resulted from the synergistic action of the MCC against one of the P. aeruginosa isolates (1071-MRPA]) were evaluated using electron microscopy and representational difference analysis (RDA), respectively. The differentially expressed, SOS response-associated, and resistance-associated genes in 1071-MRPA exposed to meropenem, ciprofloxacin, and the MCC were monitored by quantitative PCR. The MCC was synergistic against 25% and 40.6% of MDR P. aeruginosa isolates as shown by the checkerboard and time-kill curves, respectively. The morphological and structural changes that resulted from the synergistic action of the MCC against 1071-MRPA were a summation of the effects observed with each antimicrobial alone. One exception included outer membrane vesicles, which were seen in a greater amount upon ciprofloxacin exposure but were significantly inhibited upon MCC exposure. Cell wall- and DNA repair-associated genes were differentially expressed in 1071-MRPA exposed to meropenem, ciprofloxacin, and the MCC. However, some of the RDA-detected, resistance-associated, and SOS response-associated genes were expressed at significantly lower levels in 1071-MRPA exposed to the MCC. The MCC may be an alternative for the treatment of MDR P. aeruginosa . The effect of this antimicrobial combination may be not only the result of a summation of the effects of meropenem and ciprofloxacin but also a result of differential action that likely inhibits protective mechanisms in the bacteria.

UNLABELLED This study evaluated the genetic similarity and prevalence of the stx1, stx2, eae, and ehxA genes in Escherichia coli isolated from pasteurized cow milk. Eighty-seven E. coli isolates from pasteurized cow milk from 22 dairies located in northwestern Parana state, Brazil, were analyzed. Genetic similarity was evaluated using enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) and repetitive extragenic palindromic sequence PCR (REP-PCR). E. coli isolates were also analyzed by PCR to investigate the presence of the stx1, stx2, eae, and ehxA genes. ERIC-PCR and REP-PCR clustered 87 bacterial isolates in 76 and 81 genomic profiles, respectively. Both techniques revealed high genetic diversity among the E. coli isolates, confirming the possibility of their use in epidemiological studies. The stx1, stx2, eae, and ehxA virulence genes were not detected in E. coli isolates, indicating a low prevalence of Shiga toxin-producing E. coli in milk produced in the region studied. PRACTICAL APPLICATION Knowledge about the presence of diarrheagenic Escherichia coli in pasteurized milk is important developing and implementing control measures in milk and dairy production.

Since abiotic and biotic factors can compromise the survival of bacteria and their viability, encapsulation of cells in biodegradable gel matrices, a biological macromolecule, is one alternative to have their shelf life extended. Here, it was developed a gel-based formulation of the bioinoculant Azospirillum brasilense strain AbV5 and determined the effect of trehalose and humic acid supplementation in viability and survival of bacteria. For each 2 ml of sodium alginate solution (3%), 1 ml of the inoculum was extruded in a solution containing sodium alginate complexed with calcium chloride, forming calcium alginate beads. Supplements were used in a ratio of 2:2:1. Treatments were peat; alginate; alginate + humic acid; alginate + trehalose 0.1 M; alginate + trehalose 1 M. Morphometric aspects, survival rate and viability were determined in 9 storage periods (3, 5, 7, 14, 21, 30, 45, 60, 90 days). As results, beads were able to sustain the growth of A. brasilense for 90 days. Shelf life quality decreased in all treatments and peat remained the best carrier. Encapsulation, despite promoting the greatest losses in the survival of bacteria in the first days, ensured better cell viability. Trehalose in low concentrations (0.1M) improved cell viability during storage, optimizing plant inoculation.

Identification of the nucleotide consensus sequence in mammalian replication origins is a difficult and controversial problem. The hypothesis that local DNA topology could be involved in recognition by replication proteins is an exciting possibility. Secondary DNA structures, including intrinsically bent DNA, can be easily detected, and they may indicate a specific pattern in or near mammalian replication origins. This work presents the entire mapping of the intrinsically bent DNA sites (IBDSs), using in silico analysis and a circular permutation assay, of the DNA replication origins oriGNAI3, oriC, oriB, and oriA in the mammalian amplified AMPD2 gene domain. The results show that each origin presents an IBDS that flanks the straight core of these DNA replication sites. In addition, the in silico prediction of the nucleosome positioning reveals a strong indication that the center of an IBDS is localized in a nucleosome-free region (NFR). The structure of each of these curved sites is presented together with their helical parameters and topology. Together, the data that we present here indicate that the oriGNAI3 origin where preferential firing to the replication initiation events in the amplified AMPD2 domain occurs is the only origin that presents a straight, narrow region that is flanked on both sides by two intrinsically bent DNA sites within a short distance (~300 bp); however, all of the origins present at least one IBDS, which is localized in the NFR region. These results indicate that structural features could be implicated in the mammalian DNA replication origin and support the possibility of detecting and characterizing these segments.

DNA replication is a crucial process for species survival, nevertheless it is not clear which factors define origin selection in multicellular eukaryotes. Developmental gene amplification systems, such as the one described during ovarian follicles development in Drosophila melanogaster, are useful tools for studying of DNA replication process in these organisms. We previously described that the well characterized third chromosome amplified domain of D. melanogater displays three intrinsically bent DNA sites: b1, localized at an amplification control element (ACE3), b2 and b3, both localized at the preferential origin ori-β. This proposal aimed to construct a Drosophila transformation vector, which contains a short deletion at the ACE3, in order to reduce the intrinsically bent DNA site b1, and analyze the functional role of this site in the gene amplification process. Through a series of cloning steps, we obtained a Big Parent vector derivative, containing a deletion at the positions 176-180 bp, inside the ACE3. The generation of a Drosophila transformation vector displays a reduced intrinsically bent DNA site in the third chromosome amplified domain, it will allow the analysis of the functional role of this curvature in developmental gene amplification, providing new insights on replication initiation in D. melanogaster and the function of intrinsically bent DNA sites.

Aim: To study the proteomic and morphological changes in Mycobacterium tuberculosis H37Rv exposed to subinhibitory concentration of isoniazid (INH). Materials & methods: The bacillus was exposed to ½ MIC of INH at 12, 24 and 48 h. The samples’ cells were submitted to scanning electron microscopy. The proteins were separated by 2D gel electrophoresis and identified by MS. Results: INH exposure was able to alter the format, the multiplication and causing a cell swelling in the bacillus. The major altered proteins were related to the virulence, detoxification, adaptation, intermediary metabolism and lipid metabolism. Conclusion: The protein and morphological changes in M. tuberculosis induced by ½ MIC INH were related to defense mechanism of the bacillus or the action of INH therein.

HMGB-like proteins are architectural chromatin factors, and their function is heavily dependent on their ability to interact with DNA (especially non-canonical DNA structures). HMGB1 is involved in many DNA processes, and dysregulation of HMGB protein expression has profound effects on cellular transcription, resulting in severe developmental defects as well as cancer. During DNA replication, elements that form the origin are still not well defined in metazoans. Sites with A (adenine) or T (thymine) repeats cause intrinsic curvatures in the DNA and are described to be involved in the replication machinery by providing binding sites to replication proteins. As a result, the DNA molecule shows intrinsically bent DNA sites, caused by periodic repeats of 2 or more As/Ts (dA/dT) as well as intrinsically non-bent DNA sites (INBDs), due to a succession of curvatures that cancel each other. In the present study, we mapped 11 INBDSs present in the AMPD2 gene that are related to each replication origin (oriGNAI3, oriC, oriB, and oriA). Following characterization of INBDSs, we tested the ability of HMGB1 to bind to the bent (b1, b2, b4a, b4b, b5, b6, b7, and b8) and non-bent DNA fragments (nb7, nb11, nb1, nb2, nb4, and nb5) via electrophoretic mobility shift assays. All fragments showed efficient binding to HMGB1. However, the non-bent DNA fragments nb2, nb4, and nb5 showed slightly reduced binding efficiency.

Objectives To determine the frequency of yeast in the oral cavity of patients with chronic renal failure, undergoing hemodialysis (PCRFH); identification and antifungal susceptibility profile of yeast and demographic profile of patients. Methods We performed mouthwash in 146 PCRFH; the rinse fluid was collected and cultured, yeasts grown were identified by phenotypic and molecular methods. The antifungal susceptibility profile was determined against nystatin, amphotericin B, fluconazole, voriconazole, and caspofungin based in Clinical and Laboratory Standards Institute (document M27-A3). Results Positive culture was observed in 39% of patients, of whom 53% were women; the median of dialysis time was 2.9 years. The age of the colonized patients varied between 26 and 84 years, with a median of 52.5 years. PCRFH over 45 years were significantly more colonized (P = 0.0108) as well as denture wearers (84.0%). We isolated 81 yeasts, predominantly Candida albicans (63%) followed by Candida glabrata. In general, yeasts were sensitive to the evaluated antifungal agents, but there was significant variation in the minimum inhibitory concentration, especially among non-C. albicans Candida (NCAC) compared to fluconazole, caspofungin, and amphotericin B. NCAC required significantly higher concentrations of fluconazole (P

Previous reports demonstrated that actin is necessary for nucleocapsid transport and viral gene expression during nucleopolyhedrovirus infection of Bombyx mori. The first intron of B. mori A3 actin contains a cryptic promoter that drives expression of a rare isoform. We detected differences in the size and nucleotide composition of the first intron of the A3 actin gene from B. mori strain C24A, which is more resistant to nucleopolyhedrovirus than the M11A strain (22 and 95% lethality, respectively). We sought to determine if resistance to BmMNPV infection and the A3 actin promoter structure are correlated. Intrinsically bent DNA sites in these sequences, which determine curved structures, were analyzed by electrophoretic mobility assays and the helical parameters ENDS ratio, roll and twist. We found both fragments to have non-centralized bent DNA sites with distinct ENDS ratio values, nucleotide positions and two-dimensional structures. Additionally, a conformational-sensitive gel electrophoresis assay identified an allelic variation found in strain M11A that is absent in strain C24A. These data suggest that A3 actin intronic sequence variations impair virus propagation and are markers of BmMNPV-resistant populations.

The aim of this work was to determine whether intrinsically bent DNA sites are present at, or close to, the mammalian replication origins oriGNAI3 and oriB in the Chinese hamster AMPD2 locus. Using an electrophoretic mobility shift assay and in silico analysis, we located four intrinsically bent DNA sites (b1 to b4) in a fragment that contains the oriGNAI3 and one site (b5) proximal to oriB. The helical parameters show that each bent DNA site is curved in a left-handed superhelical writhe. A 2D projection of 3D fragment trajectories revealed that oriGNAI3 is located in a relatively straight segment flanked by bent sites b1 and b2, which map in previously identified Scaffold/Matrix Attachment Region. Sites b3 and b4 are located approximately 2 kb downstream and force the fragment into a strong closed loop structure. The b5 site is also located in an S/MAR that is found just downstream of oriB. [BMB reports 2010; 43(11): 744-749]

Multiple sequence alignments of the Bombyx mori fibroin light chain gene (fib-L) from hybrids and from Chinese and Japanese strains demonstrated that 51.6% of the fib-L third intron is conserved. One of these conserved segments, 41 bp long, contains the sequence CGTTATTATACATATT, which is duplicated in the B. mori Nd-s(D) mutant. In the present work, electrophoretic mobility assays and computational analyses revealed a major peak of intrinsic bent DNA within the segment that undergoes breakage in the previously-described Nd-s(D) mutation. This result suggested that this intrinsically-curved region might mediate DNA cleavage and enhance recombination events in the third intron of the Bombyx mori fib-L gene.

The naturally high minimum inhibitory concentration exhibited by echinocandins against Candida parapsilosis has been known since the first introduction of these antifungal agents. Despite this awareness, clinical failures have not been reported; consequently, the resistance of C. parapsilosis to echinocandins remains unexplored. We exposed 30 isolates of C. parapsilosis to echinocandins (caspofungin, micafungin, and anidulafungin) in vitro and studied the effects of this exposure. After 60 exposures, 80, 67, and 60 % of the isolates changed from susceptible to non-susceptible to caspofungin, micafungin, and anidulafungin, respectively. In addition, four strains exhibited cross-resistance to all three echinocandins. Based on the M27-A3 (CLSI, 2008) and M27-S4 (CLSI, 2012) techniques, the susceptibility of the resistant strains to other antifungal agents was assayed. All of the tested echinocandin-resistant strains were susceptible to amphotericin B, and the resistance rate to fluconazole, voriconazole, and flucytosine was 73.3, 43.3, and 20 %, respectively. The exposure of C. parapsilosis to the three echinocandins generated cross-resistant strains and an unexpected in vitro resistance to azoles and flucytosine.

A agricultura é um dos fatores mais rentáveis no Brasil, além de ser uma das maneiras de sustento das famílias rurais e urbanas. As culturas mais cultivadas no Brasil são soja, milho e arroz. Fungos do gênero Aspergillus podem contaminar grãos de soja e milho durante seu armazenamento e pode produzir micotoxinas, como as aflatoxinas, prejudiciais a saúde de quem os consome. O objetivo desse trabalho foi identificar, através de técnicas moleculares, fungos da espécie Aspergillus flavus e estabelecer uma correlação do tempo de armazenamento dos grãos com a contaminação fúngica. Foram coletadas sementes de milho e soja em diferentes silos da região de Maringá-PR, e posteriormente realizada cultura para fungos em meio de cultivo específico. Após o isolamento e caracterização dos fungos em meio de cultura, foi realizada a extração do DNA genômico e amplificação, pela Reação em Cadeia da Polimerase (PCR), das regiões IGS e β-tubulina. Os produtos de amplificação da região IGS foram submetidos a clivagem com a enzima de restrição BgIII, para a confirmação da espécie, pela técnica de PCR-RFLP. Das amostras coletadas 68,75% estavam contaminadas por Aspergillus spp., seguindo análises microbiológicas. Destas, 31,25% resultou positivo para a espécie Aspergillus flavus. Os outros 37,5% foram dadas como negativas, por vários fatores, um deles a sensibilidade da técnica de PCR. Foi possível concluir que um pouco menos da metade das amostras encontrava-se contaminada por Aspergillus spp.

Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC) and sub-minimal inhibitory concentration (sub-MIC) of the butanolic extract (BUTE) of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1), amino acid metabolism (ILV5, PDC11) and protein synthesis (ASC1) pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria (e.g., saponins and sesquiterpene oligoglycosides), it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds.

Recent approaches have failed to detect nucleotide sequence motifs in Scaffold/Matrix Attachment Regions (S/MARs). The lack of any known motifs, together with the confirmation that some S/MARs are not associated to any peculiar sequence, indicates that some structural elements, such as DNA curvature, have a role in chromatin organization and on their efficiency in protein binding. Similar to DNA curvature, S/MARs are located close to promoters, replication origins, and multiple nuclear processes like recombination and breakpoint sites. The chromatin structure in these regulatory regions is important to chromosome organization for accurate regulation of nuclear processes. In this article we review the biological importance of the co-localization between bent DNA sites and S/MARs.

We have identified bent DNA sites in the distal and proximal DNA puff BhC4-1 amplified gene promoter region of Bradysia hygida. The 2D modeling of the 3D DNA path and the ENDS ratio values calculated in this promoter region resulted in the identification of ten pronounced bent sites named BhC4Bˇ 9t oa 1. The 1847 bp fragment (ˇ 3697 toˇ 1850) in relation to the transcription start site shows multiple bending sites, BhC4Bˇ 9t oBhC4Bˇ 4, with periodicity300 bp. The analysis of the other identified bent region, starting at positionˇ 957, reveals that the BhC4B a 1 bent site colocalizes with the putative BhC4-1 minimal promoter. The sequence analysis of bent site BhC4Bˇ 4 shows a distribution of dA.dT at10 bp intervals between the middle of each tract, but intervals with more than one turn,20 bp, two helix turns, were detected in the other bent sites described here. The bent sites BhC4Bˇ 6 and BhC4B ˇ 4, contain two consensus sequences, with 60 bp each. The apparent molecular weight of fragments in the BhC4-1 promoter region were estimated in agarose gels and compared with the data obtained in polyacrylamide gels without and with ethidium bromide. The mobility reduction ratios (R-values) were determined, and a high R-value, 1.80, for a 1215 bp fragment in the distal promoter region and a 1.23 significant R-value for a 662 bp fragment in the proximal segment were found. To further analyze the predicted bent DNA sites in these fragments, the 2D trajectories of the 3D DNA path and other parameters, AT percentage, roll angle, ENDS ratio and DG, were determined. The role of these bent sites in the BhC4-1 transcription regulation is discussed. J. Cell. Biochem. 83: 1-13, 2001. fl 2001 Wiley-Liss, Inc.

This study evaluated the genetic similarity and prevalence of the stx1, stx2, eae, and ehxA genes in Escherichia coli isolated from pasteurized cow milk. Eighty-seven E. coli isolates from pasteurized cow milk from 22 dairies located in northwestern Paraná state, Brazil, were analyzed. Genetic similarity was evaluated using enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) and repetitive extragenic palindromic sequence PCR (REP-PCR). E. coli isolates were also analyzed by PCR to investigate the presence of the stx1, stx2, eae, and ehxA genes. ERIC-PCR and REP-PCR clustered 87 bacterial isolates in 76 and 81 genomic profiles, respectively. Both techniques revealed high genetic diversity among the E. coli isolates, confirming the possibility of their use in epidemiological studies. The stx1, stx2, eae, and ehxA virulence genes were not detected in E. coli isolates, indicating a low prevalence of Shiga toxin-producing E. coli in milk produced in the region studied.Knowledge about the presence of diarrheagenic Escherichia coli in pasteurized milk is important developing and implementing control measures in milk and dairy production.

Background Candida tropicalis is an emerging major human pathogen in nosocomial infections, and it is considered the second or third species of Candida most isolated from urine cultures. Aims The study aimed at characterizing genotypically C. tropicalis strains from patients with candiduria in a university hospital, and assessed the antifungal susceptibility profile. Methods The study was conducted with hospitalized patients who developed urinary tract infection from C. tropicalis from June 2010 to June 2011 at the Grande Dourados University Hospital of the Federal University, Dourados, MS, Brazil. Susceptibility to the antifungal agents amphotericin B and fluconazole was determined by broth microdilution. The genotypic variability of isolates of C. tropicalis was analyzed by microsatellite markers and RAPD-PCR. Results Only one isolate was resistant to amphotericin B (MIC → 16 μg/ml); the others were susceptible to fluconazole and amphotericin B. The genotypic variability by RAPD-PCR resulted in distinct profiles for RAPD markers. A total of 10 alleles were observed for the microsatellite loci, URA3 and CT14 , which were grouped differently, and four associations were observed for locus URA3 and eight for locus CT14 . Conclusions C. tropicalis isolates from urine were susceptible to the antifungal agents tested. The genotyping techniques make possible proving the similarity and genetic diversity among isolates of C. tropicalis involved in nosocomial infections. This knowledge is important for the control and prevention of nosocomial infections caused by this yeast species.

SUMMARY Introduction: The majority of nosocomial fungal infections are caused by Candida spp. where C. albicans is the species most commonly identified. Molecular methods are important tools for assessing the origin of the yeasts isolated in hospitals. Methods: This is a study on the genetic profifiles of 39 nosocomial clinical isolates of C. albicans using two typing methods: random amplifified polymorphic DNA (RAPD) and microsatellite, two different primers for each technique were used. Results: RAPD provided 10 and 11 different profiles with values for SAB of 0.84 ± 0.126 and 0.88 ± 0.08 for primers M2 and P4, respectively. Microsatellite using two markers, CDC3 and HIS3, allowed the observation of six and seven different alleles, respectively, with combined discriminatory power of 0.91. Conclusions: Although genetic variability is clear, it was possible to identify high similarity, suggesting a common origin for at least a part of isolates. It is important to emphasize that common origin was proven from yeasts isolated from colonization (urine, catheter or endotracheal secretions) and blood culture from the same patient, indicating that the candidemia must have started from a site of colonization. The combination of RAPD and microsatellite provides a quick and efficient analysis for investigation of similarity among nosocomial isolates of C. albicans. RESUMO Introdução: A maioria das infecções fúngicas hospitalares são causadas por Candida spp. e C. albicans é a espécie mais comumente identificada. Métodos moleculares são ferramentas importantes para a avaliação da origem das leveduras isoladas em hospitais. Métodos: Este é um estudo sobre o perfil genético de 39 isolados clínicos nosocomiais de C. albicans através das técnicas de RAPD e microssatélite, foram usados dois diferentes iniciadores para cada técnica. Resultados: RAPD forneceu 10 e 11 diferentes perfis com valores de SAB 0,84 ± 0,126 e 0,88 ± 0,08 para os primers M2 e P4, respectivamente. A análise de microssatélites, usando os marcadores CDC3 e HIS3 permitiu a observação de seis e sete diferentes alelos respectivamente, com poder discriminatório combinado de 0,91. Conclusões: Embora seja clara a variabilidade genética, foi possível identificar alta similaridade, sugerindo origem comum para pelo menos parte deles. É importante enfatizar que foi comprovada origem comum de leveduras isoladas de colonização (urina, cateter ou secreção orotraqueal) e hemocultura do mesmo paciente, indicando que a candidemia deve ter iniciado a partir de um sítio de colonização. A combinação das técnicas RAPD e microssatélites fornece uma análise rápida e eficiente para investigação de similaridade entre isolados nosocomiais de C. albicans.

DNA replication is an essential cellular process for the propagation of life. Prokaryotic cells are examples of organisms that display a fast and precise replication process (Robinson & Bell, 2005; Wang & Sugden, 2005). Although the consensus nucleotide sequence in the bacterial replication origin exhibits variation in number and size between different bacterial species (DoriC, a database of the oriC region in bacterial genomes; Gimenes et al., 2008a), it is capable of directing the association of specific proteins in the initiator chromosomal site and thereby the success of the replicative process in eubacteria (reviewed in Mott & Berger, 2007). However, the identification of hundreds to thousands of replication initiation sites in eukaryotes is more complex than locating clearly defined sites with stretches of consensus sequences, as observed in prokaryotes. While complexity exists, some parallels can be drawn between prokaryotic and eukaryotic systems. Less derived eukaryotes, such as the unicellular yeast Saccharomyces cerevisiae, exhibit similar replicative processes to prokaryotes. Replication initiation sites have approximately 120-200 base pairs (bp), termed ARS (Autonomously Replicating Sequences). Although there is sequence diversity among the ARS sites, these sites present a short consensus sequence of 10-11 bp that is rich in A/T residues and called the ACS (ARS Consensus Sequence) and a more divergent motif known as domain B (review in Bell & Dutta, 2002). Although not all of ARS sites are efficiently used, it has been shown that they can be initiate replication if necessary (Sharma et al., 2001). However, S. cerevisiae and closely related species appear to be the only eukaryotic organisms that present consensus elements in their replication initiation sites. Schizosaccharomyces pombe has initiation replication sequences from 600 to 800 bp, which display numerous A/Trich regions, but these replication initiation sites in S. pombe have a low level of similarity to the ARS of S. cerevisiae, and we have not been able to identify a consensus sequence within this region (Dubey et al., 1996; Zhu et al., 1994). The presence of conserved sequences in S. cerevisiae was important for the isolation of the origin recognition complex (ORC; Bell & Stillman, 1992). The existence of this complex revealed that eukaryotes depend on the binding of specific proteins to identify and activate specific genomic regions to initiate replication. The ORC complex, which is composed of six closely related proteins, Orc1-6, is conserved through evolution and has been identified in all analyzed eukaryotic cells (reviewed in Bell, 2002). Before replication begins, numerous other

Bent DNA sites promote the curvature of DNA in both eukaryotic and prokaryotic chromosomes. Here, we investigate the localization and structure of intrinsically bent DNA sites in the extensively characterized Drosophila melanogaster third chromosome DAFC-66D segment (Drosophila amplicon in the follicle cells). This region contains the amplification control element ACE3, which is a replication enhancer that acts in cis to activate the major replication origin ori-beta. Through both electrophoretic and in silico analysis, we have identified three major bent DNA sites in DAFC-66D. The bent DNA site (b1) is localized in the ACE3 element, whereas the other two bent DNA sites (b2 and b3) are localized in the ori-beta region. Four additional bent DNA sites were identified in the intron of the S18 gene and near the TATA box of the S15, S19, and S16 genes. The identification of DNA bent sites in genomic regions previously characterized as functionally relevant for DNA amplification further supports a function for DNA bent sites in DNA replication in eukaryotes.

Intrinsically bent DNA is an alternative conformation of the DNA molecule caused by the presence of dA/dT tracts, 2 to 6 bp long, in a helical turn phase DNA or with multiple intervals of 10 to 11 bp. Other than flexibility, intrinsic bending sites induce DNA curvature in particular chromosome regions such as replication origins and promoters. Intrinsically bent DNA sites are important in initiating DNA replication, and are sometimes found near to regions associated with the nuclear matrix. Many methods have been developed to localize bent sites, for example, circular permutation, computational analysis, and atomic force microscopy. This review discusses intrinsically bent DNA sites associated with replication origins and gene promoter regions in prokaryote and eukaryote cells. We also describe methods for identifying bent DNA sites for circular permutation and computational analysis.

The Rhynchosciara americana C3-22 gene is located in an amplified domain and is developmentally expressed. The aim of the present work was to identify intrinsically bent DNA sites in a segment containing the gene promoter and downstream sequence. The results indicated that this gene is flanked by intrinsically bent DNA sites. Three bent DNA sites (b(-3), b(-2), and b(-1)) were localized in the promoter, and one was localized downstream of the gene (b(+1)). These sites had helical parameters that confirmed the curved structure, as well as segments with left-handed superhelical writhe. In silico analysis of the promoters of four other insect genes, which encode secreted polypeptides, showed that they all had curved structures and similar helical parameters. Correlation with other results indicates that the detected intrinsically bent DNA sites that flank the C3-22 gene might be a consensus feature of the gene structure in the amplified domains.

Intrinsic bent DNA sites were identified in the 4289 bp segment encompassing the replication zone which directs DNA amplification and transcription of the C3-22 gene of Rhynchosciara americana. Restriction fragments showed reduced electrophoretic mobility in polyacrylamide gels. The 2D modeling of the 3D DNA path and the ENDS ratio values obtained from the dinucleotide wedge model of Trifonov revealed the presence of four major bent sites, positioned at nucleotides -6753, -5433, -5133 and -4757. Sequence analysis showed that these bends are composed of 2-6 bp dA.dT tracts in phase with the DNA helical repeat. The circular permutation analysis permitted the verification that the fragments containing the bending sites promote curvature in other sequence contexts. Computer analyses of the 4289 bp sequence revealed low helical stability (DeltaG values), negative roll angles indicating a narrow minor groove and a putative matrix attachment region. The data presented in this paper add to information about the structural features involved in this amplified segment.

Orientador: Prof.ª Dr.ª Maria Aparecida Fernandez Tese (doutorado em Ciências Biológicas)--Universidade Estadual de Maringá, 2018 Resumo: Melanoma é a forma mais agressiva de câncer de pele. Por ser uma doença muito variável ao nível molecular, sendo que a detecção e o tratamento do melanoma, podem ser considerados um dos principais desafios na área da oncologia, com centenas de diagnósticos e terapias. A transformação dos melanócitos em células de melanomas é um processo de muitas etapas e com vários mecanismos possíveis, sendo um ponto chave para a compreensão desta doença. As proteínas que estão associadas com o reparo do DNA estão entre os alvos favoritos dos estudos com tumores malignos, e entre elas está a proteína KIN (kin17). Descrita como provável envolvida nos processos de iniciação de replicação, recombinação e reparo do DNA em mamíferos, e pode ser ativada pelo stress provocado por radiação gama ou irradiação com UVC. Comparando a proteína KIN entre murinos e humanos, apresenta homologia de 92,4%, contendo 391 e 393 aminoácidos e massa molecular de 45 e 47 kDa, respectivamente e mesmo ponto isoelétrico teórico 9,3. A sua estrutura consiste em um domínio dedo de zinco, que confere afinidade à ácidos nucleicos; um domínio hélice alada, que é um mediador de interação com DNA e colabora com a interação proteína-proteina; um domínio de homologia de 15 aminoácidos com proteína RecA bacteriana; um sinal de localização nuclear; um motivo KOW na extremidade C-terminal, que está relacionada com interação RNA-proteína. A proteína KIN é descrita como superexpressa em vários tipos de câncer, sendo estudada como um potencial biomarcador para diagnóstico de câncer de mama e marcador para resposta a quimioterapia em câncer colorretal. Estudos mais aprofundados estão sendo realizados para determinar se essa proteína pode ser associada como potencial marcador para outros tipos de canceres. Como em células de melanoma ainda não há relatos da expressão da proteína KIN, o objetivo do primeiro artigo foi confirmar se as linhagens de melanomas tinham o potencial metastático e não metastático e se havia expressão diferencial entre os tipos celulares. Foi observado que as linhagens de melanoma derivadas da B16F10-Nex2 por diluição limitante, a B16-8HR ao injetada intravenosamente, formam mais nódulos nos pulmões do que a B16-10CR, caracterizando esta linhagem como metastática. Já quando as células são injetadas subcutâneamente, a linhagem B16-10CR, forma tumores subcutâneos, caracterizando como linhagem não-metastática. Com relação a localização da KIN, em todas as linhagens a KIN foi detectada no núcleo, não havendo diferença significativa de intensidade de detecção. Analisamos, por western blot se havia diferença de detecção da KIN de acordo com a fração proteica extraída das células. Na fração nuclear, quase não houve diferença de detecção da KIN entre as linhagens. Já na fração associada a cromatina houve uma maior detecção na linhagem B16-10CR (não-metastática). Neste trabalho, pudemos observar que há uma diferença de detecção da KIN entre as linhagens metastática e não-metastática, indicando uma diferenciação entre elas o que pode colaborar futuramente com estudos mais aprofundados em um indicador não invasivo de diagnóstico de melanomas. Uma classe complexa em sua estrutura e função de ácidos nucleicos são RNAs não codificantes (ncRNAs), embora sejam uma classe de RNAs que não sejam traduzidos, são altamente transcritos e são responsáveis por diferentes funções celulares. Esses tipos de RNAs são classificados como pequenos (sncRNAs) possuindo menos de 400 nucleotídeos. Neste grupo estão contidos os pequenos RNAs de interferência (siRNAs), Piwi-RNAs de interação e ligação (piRNAs) e os RNAs Y. A principal função celular associada aos RNAs Y é o envolvimento no processo de iniciação da replicação do DNA. Os mecanismos envolvidos na participação destes RNAs na iniciação da replicação do DNA em vertebrados ainda não são estabelecidos, embora a interação dessas moléculas com proteínas envolvidas na replicação tenha sido relatada. Como os RNAs Y são descritos como envolvidos na replicação do DNA e a proteína KIN é descrita como super expressa em tecidos tumorais, o objetivo do segundo trabalho foi detectar proteína KIN e RNAs Y em tumores de melanoma de camundongo, se ambos estão superexpressos nos tecidos estudados e se há diferenças entre os tumores metastáticos e não metastáticos. Para analisar essas alterações foram usadas as técnicas de qPCR para células em cultura, para as amostras de tumores de camundongos qPCR e western blot. A linhagem EJ30 (carcinoma de bexiga humano) já bem estabelecida na análise dos RNAs Y, foi utilizada como padrão de expressão. Na linhagem metastática (B16-8HR) a expressão da proteína KIN foi maior, comparada com as outras linhagens de melanoma. Comparando com a EJ30 ambas tiveram expressão bem semelhante. Os RNAs Y analisados foram o 1 e 3, para ambos a linhagem B1610-CR (não-metastática) teve a maior expressão entre os melanomas e similar a EJ30. Na análise de qPCR dos tecidos, a expressão da KIN entre as linhagens metastática e não-metastática foi bem parecida. Com relação aos RNAs Y, os tecidos injetados com a linhagem B16-10CR apresentaram a maior expressão tanto do RNA Y1 como de Y3. Na análise por western blot dos tecidos, a detecção da proteína KIN, foi semelhante entre os tecidos injetados com as células B16-8HR e B16-10CR, não havendo diferença com relação ao controle. A imunolocalização objetivou complementar o que foi observado anteriormente e confirmar se a KIN se localiza próxima a regiões de ribogênese, como relatados em outros trabalhos do nosso grupo. Detectamos a KIN e a proteína fibrilarina que é um marcador de nucléolo e observamos que nas linhagens, são observados os acúmulos da proteína KIN eles estão próximos a regiões nucleolares. Nossos resultados confirmaram estudos anteriores que descreveram sobre a proteína KIN em melanoma e outros tipos de câncer e que esta proteína é mais expressa em células tumorais e tumores. Além disso, a acumulação de KIN ocorre próximo às proteínas relacionadas ao nucléolo. No que diz respeito aos RNAs Y, eles são mais expressos em células que têm grande intensidade de divisão celular. Nos tecidos injetados com as células B16-8HR e B16-10CR, não houve diferença significativa de quantidade e de tipo de RNA Y. Estudos adicionais devem ser realizados para confirmar a relação da presença de RNAs Y nos tumores de melanoma. Estudos adicionais devem ser realizados com células e tumores de melanoma humano para confirmar se a proteína KIN e os RNAs Y podem estar associados ao fenótipo tumoral Abstract: Melanoma is the most aggressive form of skin cancer. It being a very variable disease at the molecular level. The detection and treatment of melanoma can be considered a major challenge in oncology with hundreds of diagnoses and therapies. The transformation of melanocytes into melanoma cells is a multi-step process with several possible mechanisms, being a key point for understanding this disease. Proteins associated with DNA repair are among the favorite targets of studies with malignant tumors, including KIN protein (kin17). Described as putative in the processes of initiation of replication, recombination and DNA repair in mammals, it can be activated by the stress caused by gamma radiation or irradiation with UVC. Comparing KIN protein between murine and human, it has a homology of 92.4%, containing 391 and 393 amino acids and molecular mass of 45 and 47 kDa, respectively, and the same theoretical isoelectric point 9,3. Its structure consisted of a zinc finger domain, which imparts affinity to nucleic acids; a winged helix domain, which is a mediator of DNA interaction and collaborates with the protein-protein interaction; a 15 amino acids homology domain with bacterial RecA protein; a nuclear localization signal; a KOW motif at the C-terminus, which is related to RNA-protein interaction. The KIN protein is described as overexpressed in various cancers, being studied with a potential biomarker for diagnosis of breast cancer, a marker for response to chemotherapy in colorectal cancer. More in-depth studies are underway to determine whether this protein may be associated as a potential marker for other cancers. As in melanoma cells there are still no reports of KIN protein expression, the aim of the first article was to confirm whether melanoma cell lines had metastatic and non-metastatic potential and whether there was differential expression between cell types. In B16F10-Nex2-derived cell lines by limiting dilution, the B16-8HR when injected intravenously, form more nodules in the lungs than B16-10CR, characterizing this lineage as metastatic. When the cells are injected subcutaneously, the lineage B16-10CR, forms subcutaneous tumors, characterizing as non-metastatic lineage. Regarding the location of KIN, in all strains analyzed, KIN was detected in the nucleus, with no significant difference in detection intensity. We analyzed whether there was difference in detection of KIN according to the protein fraction extracted from the cells by Western blot. In the nuclear fraction, there was no difference in KIN detection between the cell lines. In the associated chromatin fraction, there was a greater detection in the B16-10CR (non-metastatic). In this work, we can observe that there is a difference in detection of KIN between the metastatic and non-metastatic cell lines, which may collaborate in the future with further studies on a noninvasive indicator of diagnosis of melanomas. A complex class in its structure and function of nucleic acids are non-coding RNAs (ncRNAs), although they are a class of RNAs that are not translated, are highly transcribed and are responsible for different cellular functions. These types of RNAs are classified as small RNAs (sncRNAs) having fewer than 400 nucleotides, and they are the small interfering RNAs (siRNAs), Piwi-RNAs of interaction and binding (piRNAs) and the YRNAs. The main cellular function associated with YRNAs is its involvement in the process of initiation of DNA replication. The mechanisms involved in the participation of these RNAs in the activation of DNA replication in vertebrates are not well understood yet, although the interaction of these molecules with proteins involved in replication has been reported. As YRNAs are described as involved in DNA replication and KIN protein is described as super expressed in tumor tissues, the aim of the second work was to detect KIN protein and YRNAs in mouse melanoma tumor, if both are overexpressed in tissues studied and whether there are differences between metastatic and non-metastatic tumor. To analyze these differences, qPCR techniques were used for cells culture, for mouse tumors samples qPCR and Western blot. The EJ30 cell line (human bladder carcinoma), already well established in the analysis of YRNAs, was used as the expression standard. In the metastatic line (B16-8HR) the expression of KIN protein was higher, compared to the other melanoma lines, however, compared to EJ30, both had very similar expression. The YRNAs analyzed were 1 and 3, and B16-10CR (non-metastatic) had the highest expression among melanomas and like EJ30. In the tissue qPCR analysis, KIN expression between metastatic and non-metastatic lines was very similar. The Y RNAs in the tissues injected with B16-10CR had the highest expression of both Y1 and Y3 RNA. In the tissues analyzed by Western blot, the detection of the KIN protein was very similar between the tissues injected with the B16-8HR and B16-10CR cells, with no difference in relation to the control. The immunolocalization complemented the previous work and to confirm if the KIN is located near regions of ribosome biogenesis, as reported in other works of our group. We detected KIN and the fibrillarin protein which is a nucleolus marker and we observed that in the lines that are observed the KIN protein accumulations are close to nucleolar regions. Our results confirmed what previous studies have described on KIN protein in melanoma and other cancers, that this protein is more expressed in tumor cells and tumors. In addition, KIN accumulation is close to nucleolus-related proteins. With respect to Y RNAs, they are more expressed in cells that have high intensity of cell division. In tissues injected with B16-8HR and B16-10CR cells, there was no significant difference in amount and type of RNA Y. Additional studies should be performed to confirm the relationship of the presence of YRNAs in melanoma tumor. Further studies should be performed with human melanoma cells and tumors to confirm whether KIN protein and Y RNAs may be associated with the tumor phenotype

Orientador: Prof.ª Dr.ª Maria Aparecida Fernandez Coorientador: Prof.ª Dr.ª Anelise Cardoso Ramos Dissertação (mestrado em Ciências Biológicas)--Universidade Estadual de Maringá, 2018 Resumo: A detecção e o tratamento do melanoma são considerados um dos principais desafios da oncologia devido à variabilidade no nível molecular e à heterogeneidade celular dessa doença. Acredita-se que as instabilidades estruturais e genéticas, características marcantes do câncer, sejam a força no desenvolvimento dessa heterogeneidade de células tumorais que proporcionam ao tumor uma variedade de subclones para seleção de resistência a muitas formas de terapia. A estrutura nuclear pode ser utilizada como uma ferramenta de diagnóstico para distinguir uma célula normal de uma célula cancerígena, principalmente em relação à análise da subestrutura nuclear dinâmica, a matriz nuclear. A matriz nuclear desempenha um papel na organização da cromatina, além de apresentar um papel funcional em eventos que levam à instabilidade genômica e mudanças no conteúdo de DNA. Acredita-se que a organização do DNA em interfase seja realizada pela interação do DNA em locais específicos com um sistema de matriz nuclear que, como subcomponente estrutural dinâmico do núcleo, direciona a organização tridimensional do DNA em domínios de alças de DNA (loops) e fornece sítios para o controle específico de transporte de proteínas e ácidos nucleicos intranucleares. Esta estrutura é essencialmente desprovida de histonas e lipídios, e nas células tumorais, a composição proteica da matriz nuclear é alterada, alterações que podem ser úteis marcadores tumorais. Dentre as proteínas encontradas superexpressas em células tumorais, o estudo da função da proteína KIN torna-se importante. KIN, anteriormente denominada kin17, foi originalmente identificada em células de ratos com base na reatividade cruzada de anticorpos contra a proteína RecA de Escherichia coli, que está envolvida na estabilidade do genoma como mediador da via de reparo de DNA, conhecida como resposta SOS. KIN é uma proteína de ligação de DNA e RNA de aproximadamente 45 kDa, expressa de forma housekeeping em mamíferos, e inicialmente descrita como associada à iniciação da replicação, recombinação e reparo do DNA. A proteína KIN foi relatada como sendo superexpressa na maioria das linhagens tumorais analisadas e está localizada principalmente no núcleo, envolvida em processos celulares complexos, como replicação do DNA e resposta celular ao dano do DNA. Além disso, estudos anteriores mostraram que a regulação positiva de KIN está associada à proliferação, quimiorresistência e radiorresistência em tumores. No entanto, o papel dessa proteína na invasão de tumores e metástases ainda é desconhecido. Através de técnicas de imunofluorescência e microscopia eletrônica, KIN mostrou-se associada à cromatina, sugerindo um papel dessa proteína na replicação do DNA. Além disso, KIN também foi identificada entre as proteínas encontradas no spliceosoma humano, sugerindo um possível papel no metabolismo do RNA devido aos seus domínios de ligação ao RNA. Foi demonstrado que a superexpressão desta proteína poderia levar a alterações conformacionais na cromatina, confirmando sua associação com cromatina e principalmente com DNA curvo. O gene KIN é conservado em diferentes espécies, entre eles: Homo sapiens, Mus musculus, Arabidopsis thaliana, Drosophila melanogaster, Brugia malayi, Schizosaccharomyces pombe, entre outros, indicando uma atividade funcional básica. Nessa área de estudo propomos descrever a provável localização da proteína KIN na matriz nuclear através da utilização de técnicas de imunofluorescência e Western Blot, em linhagens celulares de murinos, sendo elas, a linhagem primária de melanoma B16F10-Nex2 e seus dois clones B168HR (melanoma metastático) e B1610CR (melanoma não metastático), e também a linhagem GMA32 (fibroblasto de pulmão de hamster chinês) usada como um controle positivo. A matriz nuclear foi extraída através de protocolos padrões com LIS (diiodosalicylato de lítio) e alta concentração de sal, e para a detecção da proteína KIN utilizamos o anticorpo contra a região RecA da proteína. Como controles foram utilizados anticorpos específicos marcadores de proteínas nucleares e da matriz nuclear. Os resultados obtidos neste trabalho confirmam a co-localização da proteína KIN associada ao núcleo e à matriz nuclear em todas as linhagens celulares analisadas. Através da análise por Western Blot, em amostras de fração nuclear, a expressão dessa proteína foi significativamente maior no clone B1610CR, e nas amostras de matriz nuclear, nos clones de melanoma B168HR e B1610CR. Por ser um tipo heterogêneo de câncer e devido as linhagens tumorais serem instáveis in vitro, passagens celulares dão origem a clones com características únicas. Desse modo, a análise em células de melanoma é importante para determinar se o nível da proteína KIN pode ser usado como biomarcador de acordo com sua agressividade Abstract: The detection and treatment of melanoma are considered one of the main challenges of oncology due to the variability in the molecular level and the cellular heterogeneity of this disease. Structural and genetic instabilities, a hallmark of cancer, are believed to be the force in the development of this heterogeneity of tumor cells that provide the tumor with a variety of subclones for selection of resistance to many forms of therapy. The nuclear structure is used as a diagnostic tool to distinguish a normal cell from a cancer cell, mainly in relation to the analysis of the dynamic nuclear substructure, the nuclear matrix. The nuclear matrix plays a role in chromatin organization, as well as to play a functional role in events leading to genomic instability and changes in DNA content. It is believed that the interphase DNA organization is performed by the interaction of the DNA at specific sites with a nuclear matrix system which, as a dynamic structural subcomponent of the nucleus, directs the three-dimensional organization of DNA in loop domains and provides sites for the specific control of transport of proteins and intranuclear nucleic acids. This structure is essentially devoid of histones and lipids, and in tumor cells, the proteinaceous composition of the nuclear matrix is altered, changes that may be useful tumor markers. Among the proteins found to be overexpressed in tumor cells, the study of KIN protein function becomes important. KIN, previously called kin17, was originally identified in mice cells based on cross-reactivity to antibodies against the RecA protein from Escherichia coli, which is involved in the genome stability as a mediator of the DNA repair pathway known as the SOS response. KIN is a approximately 45 kDa DNA and RNA binding protein expressed as housekeeping in mammals and initially described as associated with the initiation of DNA replication, recombination and repair. KIN protein was reported to be overexpressed in most of the tumor lines analyzed and is mainly located in the nucleus involved in complex cellular processes such as DNA replication and cellular response to DNA damage. In addition, previous studies have shown that the positive regulation of KIN is associated with proliferation, chemoresistance and radioresistance in tumors. However, the role of KIN17 in tumor invasion and metastasis is still unknown. Through immunofluorescence techniques and electron microscopy, KIN has been shown to be associated with chromatin, suggesting a role of this protein in DNA replication. In addition, KIN was also identified among the proteins found in the human spliceosome, suggesting a possible role in RNA metabolism due to its RNA binding domains. It has been shown that overexpression of this protein could lead to conformational changes in the chromatin, confirming its association with chromatin and mainly with curved DNA. The KIN gene is conserved in different species, among them: Homo sapiens, Mus musculus, Arabidopsis thaliana, Drosophila melanogaster, Brugia malayi, Schizosaccharomyces pombe, among others, indicating a basic functional activity. In this area of study, we propose to describe the probable location of the KIN protein in the nuclear matrix through the use of immunofluorescence and Western Blotting techniques in murine cell lines, being the primary lineage of melanoma B16F10- Nex 2 and its two clones B16-8HR (metastatic melanoma) and B16-10CR (nonmetastatic melanoma), as well as the GMA32 (Chinese hamster lung fibroblast) cell line used as a positive control. The nuclear matrix was extracted by standard protocols with LIS (lithium diiodosalicylate) and high salt concentration, and for detection of KIN protein, we used the antibody against the RecA region of the protein. As controls, specific antibodies to nucleic and nuclear matrix markers were used. The results obtained in this work confirm the co-localization of the KIN protein associated with the nucleus and the nuclear matrix in all cell lines analyzed. Through Western Blotting analysis, in nuclear fraction samples, in the B1610CR clone, and in nuclear matrix samples, the higher signal was revealed in the B168HR and B1610CR melanoma clones. For being a heterogeneous type of cancer and due the tumoral lineages are unstable in vitro, cell passages originate clones with unique characteristics. Thus, analysis in melanoma cells is important in determining if the level of the KIN protein can be used as a biomarker according to its aggressiveness