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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.

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.

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.

The COVID‐19 pandemic has led to the adoption of social quarantine measures in numerous countries. In this context, both COVID‐19 and quarantine can have a substantial impact in the mental health of affected populations. Health care services have had to adapt themselves to this new reality. Fil: Dorman, Guido. Instituto de Neurología Cognitiva; Argentina Fil: Alvarez Dengra, Agustín. Instituto de Neurología Cognitiva; Argentina Fil: Fiorini, Adriana. Instituto de Neurología Cognitiva; Argentina Fil: Failla, Belén. Instituto de Neurología Cognitiva; Argentina Fil: Vallejos, Florencia. Instituto de Neurología Cognitiva; Argentina Fil: Pontello, Noelia. Instituto de Neurología Cognitiva; Argentina Fil: Roca, María. Instituto de Neurología Cognitiva; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bustin, Julian. Instituto de Neurología Cognitiva; Argentina

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.

Staphylococcus aureus is one of the most common microorganisms responsible for high morbidity and mortality in humans and animals. Methicillin-resistant S. aureus are responsible for several outbreaks worldwide and therapeutic arsenal has become scarce. The present investigation verified the epidemiological profile of S. aureus strains isolated from the veterinary hospital staff, from dairy cattle workers and also from milk samples of dairy cattle presenting mastitis. Samples were characterized phenotypically by antibiogram, catalase, and coagulase tests, and also by Voges-Proskauer test. The isolated strains were characterized genotypically by specific Polymerase Chain Reaction and Amplified Ribosomal DNA Restriction Analysis (ARDRA). From the 218 isolated strains, 27 were identified as S. aureus (12%), four of them were resistant to oxacillin and two of them were classified as MRSA (Methicillin-resistant S. aureus). The prevalence of isolated strains among animal personnel care was low (2%) but all MRSA isolates were found among the clinical staff. Results of ARDRA pointed out that S. aureus strains isolated from different animal care personnel were grouped in the same cluster when HindIII and HinfII restriction enzymes were used. When ARDRA was performed with HaeIII enzyme, the formation of two clusters was observed, but the isolated strains were not correlated. The prevalence of S. aureus strains isolated was higher in clinical staff and the biochemical and molecular assays of them presented 100% of correlation. Staphylococcus aureus está entre os microrganismos que apresentam as maiores taxas de morbidade e mortalidade em seres humanos e animais. Linhagens de S. aureus resistentes a meticilina podem causar surtos de infecção em todo o mundo, o que contribui para a escassez de arsenal terapêutico. Este trabalho analisou o perfil epidemiológico de estirpes de S. aureus isoladas de pessoas que trabalham em contato com animais em um hospital veterinário com gado leiteiro e também em amostras de leite de vacas acometidas por mastite. As estirpes de S. aureus isoladas foram caracterizadas fenotipicamente por meio de antibiograma, testes de catalase e coagulase, e pelo teste de Voges-Proskauer. As amostras também foram caracterizadas genotipicamente pela técnica de Analise de Restricao de DNA Ribossomico Amplificado (ARDRA-PCR). Das 218 estirpes isoladas, 27 foram identificados como S. aureus (12%). Entre essas, quatro estirpes foram resistentes a oxacilina e duas classificadas como SARM (S. aureus resistente a meticilina). A ocorrência de estirpes de S.aureus isoladas entre o pessoal que trabalha em contato com os animais foi baixa (2%), mas estirpes identificadas como SARM foram encontradas na equipe clínica. As análises de ARDRA realizadas com as enzimas de restrição HindIII e HinfII demonstraram que S. aureus isolados de diferentes indivíduos que trabalhavam com animais foram agrupados no mesmo cluster. Quando a ARDRA foi realizada com HaeIII foi observada formação de dois grupos, mas os isolados não se correlacionaram. Conclusão: a ocorrência de estirpes de S. aureus isoladas foi maior na equipe clínica, apresentando também correlação de 100% nos ensaios bioquímicos e moleculares.

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.

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.

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.

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 features of the nucleotide sequences in both replication and promoter regions have been investigated in many organisms. Intrinsically bent DNA sites associated with transcription have been described in several prokaryotic organisms. The aim of the present study was to investigate intrinsic bent DNA sites in the segment that holds the chromosomal replication origin, oriC, of Xylella fastidiosa 9a5c. Electrophoretic behavior analyses, as well as in silico analyses of both the 2-D projection and helical parameters, were performed. The chromosomal segment analyzed contains the initial sequence of the rpmH gene, an intergenic region, the dnaA gene, the oriC sequence, and the 5' partial sequence of the dnaN gene. The analysis revealed fragments with reduced electrophoretic mobility, which indicates the presence of curved DNA segments. The analysis of the helical parameter ENDS ratio revealed three bent DNA sites (b1, b2, and b3) located in the rpmH-dnaA intergenic region, the dnaA gene, and the oriC 5' end, respectively. The chromosomal segment of X. fastidiosa analyzed here is rich in phased AT tracts and in CAnT motifs. The 2-D projection indicated a segment whose structure was determined by the cumulative effect of all bent DNA sites. Further, the in silico analysis of the three different bacterial oriC sequences indicated similar negative roll and twist >34.00 degrees values. The DnaA box sequences, and other motifs in them, may be associated with the intrinsic DNA curvature.

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.

O objetivo desse trabalho foi verificar os efeitos da desnutrição protéica sobre a morfologia, a morfometria e a densidade dos neurônios do plexo mientérico. Foi utilizado o íleo de 10 ratos. Para este estudo, cinco ratos com 90 dias receberam, durante 120 dias, ração hipoprotéica (grupo desnutrido) e 5 ratos (grupo de controle) receberam ração com teor protéico normal. Segmentos do íleo foram coletados e submetidos à elaboração de preparados de membrana, corados por Giemsa (Barbosa, 1978), e, para tratamento histológico de rotina, corados por HE. Os neurônios agrupavam-se formando gânglios localizados entre os estratos circular e longitudinal da túnica muscular. Com base nos comprimentos dos maiores eixos longitudinal e transversal, os neurônios foram classificados em três grupos: pequenos (14,44 a 22,32μm), médios (22,60 a 39,40) e grandes (40,70 a 63,02). A densidade neuronal no íleo de ratos em uma área de 7,08mm² foi em média 1.482 e 2.515 neurônios, respectivamente, nos grupos de controle e desnutrido. Os dados obttidos sugerem que a desnutrição protéica não provocou alteração na densidade neuronal.This paper deals with the effects of proteic desnutrition on the morphology, morphometry and density of the neurons of the myenteric plexus. The ileum of 10 rats was used. For this study five rats aging 90 days were fed during 120 days with hypoproteic ration (experimental group) and five rats (control group) received ration with normal proteic level. Segments of the ileum were obtained for the method of whole-mount preparation, stained with Giemsa (Barbosa, 1978) and for histological routine treatment stained with HE. The neurons were clustered in ganglia located between the circular and longitudinal layers of the muscular tunica. Based on the lengths of the major longitudinal and transverse axes, the neurons were selected in three groups: small, medium and large. The neuronal density on the ileum of rats corresponding to an area of 7.08mm2 was 1,482 and 2,515 neurons on the control and experimental groups respectively. The data suggested that proteic desnutrition did not cause alteration to the neuronal density.

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

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.

This study evaluated the reactivity of membrane antigens of Leishmania (Viannia) braziliensis for the diagnosis of ACL by enzyme immunoassay (EIA). Promastigotes of L. (V.) braziliensis were grown in medium 199 and lysed in a sonicator. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting showed that specific proteins of L. (V.) braziliensis (apparent molecular weights 36 kDa and 48–56 kDa) were recognized by sera from ACL patients. These proteins were eluted from the SDS-PAGE and tested in EIA-IgG with sera from ACL patients, healthy individuals, patients with toxoplasmosis, paracoccidioidomycosis, syphilis, tuberculosis, leprosy, and Chagas disease. The EIA-IgG with membrane antigens allowed us to distinguish patients with ACL from healthy individuals and patients with other diseases (P < 0.0001), and showed a sensitivity of 93.3% and specificity of 90.8%, not including Chagas disease patients. 2D-SDS-PAGE followed by Western blotting was performed to improve the characterization of the antigens, and showed a component with isoelectric points near the acid pH side and apparent molecular weights of 48–56 kDa. The results showed good sensitivity and specificity of EIA-IgG with membrane antigens, indicating their potential use for diagnosis of ACL, as well as seroepidemiological surveys and follow-up of clinically cured patients.

Submitted by Neusa Fagundes (neusa.fagundes@unioeste.br) on 2020-10-06T13:17:09Z No. of bitstreams: 2 Marcos_Polinarski2020.pdf: 1212402 bytes, checksum: 9a7f7aa44de76d6ba51228eb9dcab4e6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Made available in DSpace on 2020-10-06T13:17:09Z (GMT). No. of bitstreams: 2 Marcos_Polinarski2020.pdf: 1212402 bytes, checksum: 9a7f7aa44de76d6ba51228eb9dcab4e6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2020-02-28 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES The presence of microorganisms in diesel oil, biodiesel and their blends, is an issue that has been well documented in the literature, especially due to the negative effects that it tends to cause, for example, the sludge formation, clogging of filters and valves and storage tanks corrosion. With the gradual increase in the levels of biodiesel to commercial diesel, the precautions during fuel storage should be greater, since this biofuel has a hygroscopic character, which facilitates contamination due to the water absorption capacity. One of the ways to avoid microbiological contamination is the application of techniques such as periodic cleaning of storage tanks or the use of biocides. However, their use may be associated with changes in the fuel physicochemical parameters. The aim of this work was to evaluate the presence of microorganisms in diesel fuel B11 and B20 with variations in the sulfur (10 ppm and 500 ppm) and water (without addition, 2500 ppm and 10000 ppm) content, and to propose the use of ultraviolet radiation as alternative to microbial control and to verify if the UV use promotes changes in the fuel physical-chemical characteristics. The fuel samples were stored for 10 days, where the presence of microorganisms was monitored from the absorbance reading. After storage, the fuels were exposed to ultraviolet radiation for 360 minutes, and the microorganisms presence was monitored every 45 minutes. Water content, viscosity, density and oxidative stability were evaluated at the beginning and end of the treatment. After the treatment the microorganisms presence was followed during 30 days. The results showed that during simulated storage, fuels with water addition were the most susceptible microorganisms growth, while fuels without alteration the most significant changes were related to a higher sulfur content. The ultraviolet radiation treatment showed efficiency higher than 90% for 2 of the 12 samples analyzed, while only 4 samples had an efficiency lower than 50% in the microorganisms presence reduction. Regarding the physical-chemical parameters, it was found that the use of ultraviolet radiation promoted statistically significant changes but did not change the final quality of the fuel. The UV treatment proved to be stable for up to 10 days after its application. The results have showed the potential of ultraviolet radiation as a method of microbial control and reduction of sulfur levels does not increase microbial contamination in the fuel, with the presence of water being the main reason for contamination. A presença de microrganismos em óleo diesel, biodiesel e suas misturas, é uma pauta que tem sido bastante documentada pela literatura, sobretudo pelos efeitos negativos que tende a causar, como por exemplo, a formação de borras, entupimento de filtros e válvulas e corrosão de tanques de armazenamento. Com o aumento gradativo dos teores de biodiesel ao diesel comercializado, os cuidados durante a estocagem do combustível devem ser ainda maiores, uma vez que esse biocombustível possui caráter higroscópico, o que facilita a contaminação devido a capacidade de absorção de água. Uma das formas de evitar e/ou diminuir a contaminação microbiológica é a aplicação de técnicas como limpeza periódica dos tanques de armazenamento ou o uso de biocidas. Contudo seu uso pode estar associado a mudanças nos parâmetros físico-químicos do combustível. O objetivo desse trabalho foi avaliar a presença de microrganismos em combustível diesel B11 e B20 com variações quanto ao teor de enxofre (10 ppm e 500 ppm) e água (sem adição, 2500 ppm e 10000 ppm), propor o uso da radiação ultravioleta como alternativa ao controle microbiano e verificar se seu promove alterações em suas características físico-químicas. Para tanto, as amostras de combustíveis foram estocadas por 10 dias, onde a presença de microrganismos foi acompanhada a partir da leitura de absorbância. Após a estocagem, os combustíveis foram expostos à radiação ultravioleta durante 360 minutos, nos quais a presença de microrganismos foi acompanhada a cada 45 minutos. Características como teor de água, viscosidade, massa específica e estabilidade oxidativa foram avaliadas no início e final do tratamento. Após o tratamento a presença de microrganismos foi acompanhada a cada 10 dias por um período de 30 dias a fim de verificar sua eficácia. Os resultados apontaram que durante a estocagem simulada os combustíveis com adição de água foram os mais susceptíveis ao crescimento de microrganismos, enquanto para os combustíveis sem alteração as mudanças mais expressivas foram em relação ao combustível com maior teor de enxofre. O tratamento com radiação ultravioleta demonstrou eficiência acima de 90% para duas das doze amostras analisadas, enquanto apenas 4 amostras tiveram eficiência menor que 50% na redução da presença de microrganismos caracterizada pela leitura da absorbância. Tratando-se dos parâmetros físico-químicos, foi verificado que a utilização da radiação ultravioleta promoveu mudanças estatisticamente significativas, porém não alteraram a qualidade final do combustível. O uso da radiação ultravioleta para controle da presença de microrganismos mostrou-se estável por até 10 dias após sua aplicação. Os resultados encontrados apontam o potencial da radiação ultravioleta como método de controle microbiano, além disso, foi verificado que a redução dos teores de enxofre não aumenta, por si só, a contaminação microbiana no combustível, sendo a presença de água a principal razão da contaminação.

Submitted by Helena Bejio (helena.bejio@unioeste.br) on 2019-09-18T12:27:59Z No. of bitstreams: 2 Silvio_Ferreira_2019.pdf: 3155182 bytes, checksum: 36c9c4ce42428468f7d0787bb98a5bc3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Made available in DSpace on 2019-09-18T12:27:59Z (GMT). No. of bitstreams: 2 Silvio_Ferreira_2019.pdf: 3155182 bytes, checksum: 36c9c4ce42428468f7d0787bb98a5bc3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2019-06-13 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES The Digitaria insularis has been distinguished by glyphosate resistance, biological alterations, difficult control and also the damage caused to soybean and corn. Therefore, if there is a change in the biological factors that act as resistance mechanisms in resistant D. insularis populations, there may be a change in the of Economic Threshold Level of this biotype in soybean and safrinha corn crop. Given the above, the objective of this work was to evaluate the absorption and translocation of glyphosate herbicide, the accumulation of shikimic acid, the anatomical characteristics of the leaves and the Economic Threshold Level in soybean and safrinha corn of two populations of D. insularis (resistant and susceptible) collected in agricultural areas in Paraná state. First the test was performed to prove the resistance and the Resistance Factor (FR80). In the study of the absorption and translocation of glyphosate herbicide and the accumulation of shikimic acid, a randomized complete block design in a 2x9 factorial scheme with four replications was used. The first factor was D. insularis biotypes (resistant and susceptible) and the second factor was the time of herbicide contact with the leaves of D. insularis plants m-2 (0; 2; 4; 8; 12; 24, 36, 48 and 72 hours). To determine the absorption, the percentage of control of the plants was verified. Shikimic acid accumulation in D. insularis biotypes was determined on harvested leaves that did not receive spraying. For the Economic Threshold Level experiments on soybean and safrinha corn, a randomized complete block design was used, in a 2x5 factorial scheme, with four replications. The first factor corresponded to D. insularis biotypes (susceptible and resistant) and the second factor, the densities of 0; 7; 14; 28 and 56 D. insularis plants m-2. The preliminary test to confirm the degree of resistance of D. insularis biotypes indicated the resistance of the biotype collected at PUC Toledo - PR experimental station, with FR80 equal to 19.3. The control of the resistant biotype observed at 28 DAA was considered satisfactory (80%) when the herbicide remained for 48 hours in contact with the applied leaf. In the susceptible biotype, satisfactory control occurred after eight hours of herbicide contact with the leaf. In resistant biotype, shikimic acid accumulation increased by 1.8 times from 0 to 12 HAA. In the susceptible it was verified that the accumulation increased twice. There was no change in the Economic Threshold Level on soybean crop among glyphosate resistant and insects susceptible D. insularis biotypes. Considering the low cost of D. insularis control and the high selling price of soybean for the 2017/18 crop, it was possible to determine the Economic Threshold Level of 0.86 plants m-2 of D. insularis. In safrinha corn crop, Economic Threshold Levels were different for glyphosate resistant and susceptible D. insularis biotypes. The differences may have occurred mainly due to the environmental conditions verified between the evaluated years. Therefore, considering the average values of grains and control cost for the year 2017 the Economic Threshold Level was 17.4 and 22.9 plants m-2 of the resistant and susceptible biotype, respectively and in 2018 the Economic Threshold Level. It was 13.7 plants m-2 for the resistant biotype and 11.6 plants m-2 for the susceptible biotype. A Digitaria insularis tem se destacado pela resistência ao glyphosate, alterações biológicas, difícil controle e também pelos prejuízos causados na cultura da soja e do milho. Portanto, se ocorre alteração dos fatores biológicos que funcionam como mecanismos de resistências em populações de D. insularis resistente, pode ocorrer alteração no nível de dano econômico deste biótipo na cultura da soja e do milho safrinha. Diante do exposto o trabalho teve como objetivos avaliar a absorção e translocação do herbicida glyphosate, o acúmulo do ácido chiquímico, as características anatômicas das folhas e o Nível de Dano Econômico na cultura da soja e do milho safrinha de duas populações de D. insularis (resistente e suscetível) coletadas em áreas agrícolas no Paraná. Primeiramente realizou-se o teste para comprovar a resistência e o Fator da Resistência (FR80). No estudo da absorção e translocação do herbicida glyphosate e o acúmulo do ácido chiquímico, utilizou-se o delineamento experimental em blocos casualizados, em esquema fatorial 2x9, com quatro repetições. O primeiro fator foi constituído pelos biótipos de D. insularis (resistente e suscetível) e o segundo fator, pelo tempo de contato do herbicida com as folhas das plantas m-2 de D. insularis (0; 2; 4; 8; 12; 24; 36; 48 e 72 horas). Para determinar a absorção, verificou-se o percentual de controle das plantas. O acúmulo de ácido chiquímico nos biótipos de D. insularis foi determinado nas folhas colhidas que não receberam a pulverização. Para os experimentos do Nível de Dano Econômico na cultura da soja e milho safrinha foi utilizado o delineamento em blocos casualizados, no esquema fatorial 2x5, com quatro repetições. O primeiro fator correspondeu aos biótipos de D. insularis (suscetível e resistente) e segundo fator, as densidades de 0; 7; 14; 28 e 56 plantas m-2 de D. insularis. O teste preliminar da confirmação do grau de resistência dos biótipos de D. insularis, indicaram a resistência do biótipo coletado na estação experimental da PUC Toledo – PR, com FR80 igual a 19,3. O controle do biótipo resistente observado as 28 DAA foi considerado satisfatório (80%) quando o herbicida permaneceu por 48 horas em contato com a folha aplicada. Enquanto, no biótipo suscetível o controle satisfatório ocorreu a partir das oito horas de contato do herbicida com a folha. No biótipo resistente, o acúmulo de ácido chiquímico aumentou em 1,8 vezes do 0 para as 12 HAA. No suscetível foi verificado que o acúmulo aumentou duas vezes. Não houve alteração no Nível de Dano Econômico na cultura da soja entre os biótipos de D. insularis resistente e suscetível ao glyphosate. Ao considerar o baixo custo de controle da D. insularis e o elevado preço de venda da soja para a safra 2017/18, foi possível determinar o Nível de Dano Econômico de 0,86 plantas m-2 da D. insularis. Na cultura do milho safrinha, os Níveis de Dano Econômicos foram diferentes para os biótipos de Digitaria insularis resistente e suscetível ao glyphosate. As diferenças podem ter ocorrido principalmente em virtude das condições ambientais verificadas entre os anos avaliados. Portanto, considerando os valores médios dos grãos e do custo de controle para o ano 2017 o Nível de Dano Econômico foi de 17,4 e 22,9 plantas m-2 do biótipo resistente e suscetível, respectivamente e em 2018 o Nível de Dano Econômico ficou em 13,7 plantas m-2 do biótipo resistente e 11,6 plantas m-2 para o biótipo suscetível.

Submitted by Neusa Fagundes (neusa.fagundes@unioeste.br) on 2021-05-14T14:22:24Z No. of bitstreams: 2 Ana Paula_Mallmann2019.pdf: 805123 bytes, checksum: cfe945c0e82f83d5f6d6ba577f9d1e69 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Made available in DSpace on 2021-05-14T14:22:24Z (GMT). No. of bitstreams: 2 Ana Paula_Mallmann2019.pdf: 805123 bytes, checksum: cfe945c0e82f83d5f6d6ba577f9d1e69 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2019-02-20 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES Brazil has a great biodiversity, because of that the products originating from the secondary metabolism of plants arouse great interest for its variety and for being able to promote biological activities of great importance. The species Ilex brevicuspis is a tree native to southern Brazil, known as caúna da serrá, congonha ou orelha de mico, and it was used in this work being investigated the phytochemical composition, antimicrobial activities, by broth microdilution technique, antioxidant activity, by method 2,2-difenil-1-picril-hidrazila (DPPH) e acaricidal activity against Dermanyssus gallinae (Degeer, 1778), of plant extracts extracted from the leaves by ethanol (EE), methanol (EM), ethyl acetate (EAE), distilled water (Eaq), hexanic (EH) and acetonic (EA). Phytochemistry revealed the presence of saponins, steroids, tannins, flavonoids and triterpenoids. The extracts showed inhibition results against all strains used, both Gram-positive and Gram-negative, including Salmonella serotypes. The methanol extract showed a high acaricidal activity of 94% and a moderate antioxidant capacity of 81.45%. Antioxidant activity above 70% was also verified for hexane and acetone extracts. Therefore, the vegetable extracts of I. brevicuspis presented a secondary metabolism compound capable of promoting important biological activities and being an alternative for the development of natural products. O Brasil possui uma grande biodiversidade, diante disso os produtos oriundos do metabolismo secundário das plantas despertam grande interesse pela sua variedade e por ser capaz de promover atividades biológicas de grande importância. A espécie Ilex brevicuspis é uma arvore nativa do sul do Brasil, conhecida como caúna da serrá, congonha ou orelha de mico, e foi utilizada nesse trabalho sendo investigada a composição fitoquimica, as atividades antimicrobianas, pela técnica de microdiluição em caldo, atividade antioxidante, pelo método 2,2-difenil-1-picril-hidrazila (DPPH) e atividade acaricida frente a Dermanyssus gallinae (Degeer, 1778) dos extratos vegetais extraídos das folhas da mesma por etanol (EE), metanol (EM), acetato de etila (EAE), água destilada (Eaq), hexânico (EH) e acetônico (EA). A fitoquimica revelou presença de compostos saponinas, esteroides, taninos, flavonoides e triterpenóides. Os extratos apresentaram resultados de inibição frente a todas as cepas ultizadas, tanto Gram positivas e Gram-negativas, incluindo sorotipos de Salmonella. O extrato metanolico apresentou elevada atividade acaricida de 94% e moderada capacidade antioxidante de 81,45%. Também foi verificada atividade antioxidante acima de 70% para os extratos hexânico e acetônico. Portanto, os extratos vegetais de I. brevicuspis apresentaram composto do metabolismo secundário capaz de promover atividade biológicas importantes e ser uma alternativa para o desenvolvimento de produtos naturais.

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

Orientadora: Profª. Drª. Terezinha Inez Estivalet Svidzinski Coorientadora: Profª. Drª. Érika Seki Kioshima Tese (doutorado em Biociências e Fisiopatologia) - Universidade Estadual de Maringá, 2018 RESUMO: Nas últimas décadas as doenças fúngicas invasivas (DFI) destacaram-se mundialmente como uma das principais causas de infecção em humanos, principalmente em pacientes imunocomprometidos. Neste contexto, as infecções por Candida spp. tornaram-se uma das etiologias mais comuns com quadros de alta gravidade e Candida albicans continua sendo a espécie mais prevalente. Porém, outros gêneros de fungos como Paracoccidioides e Cryptococcus também podem ser responsabilizados pelas DFI. Considerando as restritas opções de antifúngicos comercialmente disponíveis, toxicidade/efeitos colaterais e o aumento de cepas resistentes, o desenvolvimento de novos fármacos se faz necessário. Estratégias objetivando diminuir custos e tempo na produção de novos medicamentos, comparado com as tradicionais, têm sido amplamente investigadas, e avanços nas técnicas computacionais e hardware têm habilitado métodos in silico para acelerar a identificação e otimização de possíveis novos fármacos. Recentemente em nosso grupo, utilizando o modelo da tioredoxina redutase de C. albicans e a varredura virtual em quimiotecas, foram selecionados onze compostos que interagem com esse alvo. De acordo com o primeiro screening in vitro de atividade antifúngica, dois deles (LMM5 e LMM11) foram selecionados com melhor atividade contra C. albicans. O presente trabalho objetivou utilizar técnicas in vitro e in vivo para caracterização da atividade antifúngica em C. albicans desses dois compostos inéditos. Além disso, foi realizada a otimização in silico de LMM5 e LMM11 para a obtenção de novos candidatos ao desenvolvimento de drogas antifúngicas direcionadas para o tratamento de doenças fúngicas invasivas. Com os resultados obtidos através dessas metodologias, três artigos foram gerados. O primeiro demonstrando a eficiente atividade fungistática de LMM5 e LMM11 contra C. albicans, apresentando baixa citotoxicidade e diminuição da carga fúngica em modelo experimental de candidíase sistêmica murina. O segundo trabalho utilizou abordagem in silico de reposicionamento de drogas complementados por ensaios in vitro e in vivo, resultando no antiviral raltegravir com promissora atividade antifúngica para tratamento de paracoccidioidomicose. Também com atividade anti-Paracoccidioides, no terceiro artigo, apresentamos os compostos ebselen e butein, que foram obtidos in silico por semelhança de sitio de ligação com a tioredoxina redutase de P. lutzii e confirmados através de ensaios in vitro. ABSTRACT: Over the past decades, invasive fungal diseases (IFD) have emerged as one of the main causes of human infection worldwide, mainly in immunocompromised patients. In this context, Candida spp. have become of the most common etiologies in more severe cases, and Candida albicans remains the most prevalent species. Despite, other genera of fungi such as Paracoccidioides and Cryptococcus also have caused IFD. Considering the restricted options of commercially available antifungal, toxicity/side effects and the increase of resistant strains, the development of new drugs is necessary. Strategies aimed at reducing costs and time in the production of new drugs, compared to traditional methodologies, have been widely investigated. Advances in computer techniques and hardware have allowed in silico methods to accelerate the identification and optimization of possible new drugs. Recently in our group, using the thioredoxin reductase model of C. albicans and virtual screening of chemical library, were selected eleven compounds that interact with this target. According to the first in vitro screening of antifungal activity, two of them (LMM5 and LMM11) were selected with better activity against C. albicans. In this sense, the present study aimed, through in vitro and in vivo assays to characterize the antifungal activity against C. albicans of these two unpublished compounds. In addition, in silico optimization of LMM5 and LMM11 to obtain new candidates for the development of antifungal drugs addressed the treatment of invasive fungal diseases. In accordance with the results obtained through these methodologies, three articles were generated. The first study demonstrated the efficient fungistatic activity of LMM5 and LMM11 against C. albicans, presenting low cytotoxicity and decreased fungal burden in a murine model of systemic candidiasis. The second, used in silico approach of drug repositioning supplemented by in vitro and in vivo assays, resulting in antiviral raltegravir with promising antifungal activity for treatment of paracoccidioidomycosis. Also with anti-Paracoccidioides activity, in the third article, we present the compounds ebselen and butein, which were obtained in silico by similarity of ligand binding site with thioredoxin reductase from P. lutzii and confirmed by in vitro assays. 103 f. : il. (algumas col.).

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

The treatment of vulvovaginal candidiasis (VVC) is still unsatisfactory, especially in pregnant women, being promising to the utilization of alternative therapies. Propolis extract (PES) has demonstrated antifungal efficacy and low toxicity. In addition, the subproduct of propolis extract (SPES) is produced during the process of preparing PES and is usually discarded, but can still submit substances responsible for biological effects, such as the polyphenols, responsible for the therapeutic activity of propolis. SPES have not been investigated or used as an antimicrobial agent. Thus, the objective of the present study was to investigate the effect of PES and SPES on Candida spp. isolated from the vaginal material of pregnant women. Vaginal samples from 291 pregnant women were collected and cultivated for yeasts, which were identified by the classical method and performing susceptibility tests against PES, SPES and conventional antifungal agents. The anti-biofilm effect and cytotoxicity tests of the PES and SPES were evaluated. In 38.48% (112/291) of the women the culture was positive for Candida spp. being 82.61% C. albicans; 6.08% C. glabrata; 5.22% C. tropicalis; 5.22% C. parapsilosis and 0.87% C. krusei. PES and SPES were effective, even against isolates resistant to conventional antifungal and reduced about 62% of the biofilm of Candida spp., besides presenting its low toxicity in the concentrations of fungicides. Thus, besides PES the SPPS is also a promising alternative for the CVV treatment, especially in this population. O tratamento da candidíase vulvovaginal (CVV) é ainda insatisfatório, especialmente em gestantes, sendo promissor a utilização de terapias alternativas. O extrato de própolis (PES) tem demonstrado eficácia antifúngica e baixa toxicidade. Além disso, o subproduto da extração de própolis (SPES) é produzido durante o processo de preparação do extrato de própolis e costuma ser descartado, mas pode apresentar ainda substâncias responsáveis por efeitos biológicos, tais como os polifenóis, responsáveis pelas atividades terapêuticas da própolis, entretanto, SPES não tem sido utilizado ou investigado como agente antimicrobiano. Assim, o objetivo do presente estudo foi verificar o efeito do PES e SPES sobre Candida spp. isoladas de material vaginal de gestantes. Foram coletadas amostras vaginais de 291 gestantes e cultivadas para leveduras, as quais foram identificadas pelo método clássico e realizados testes de suscetibilidade frente a PES, SPES e antifúngicos convencionais. Foi avaliado o efeito anti-biofilme e testes de citotoxicidade em células Hela do PES e SPES. Em 38,48% (112/291) das gestantes a cultura foi positiva para Candida spp. sendo 82,61% C. albicans; 6,08% C. glabrata; 5,22% C. tropicalis; 5,22% C. parapsilosis e 0,87% C. krusei. PES e SPES foram efetivos, inclusive frente aisolados resistentes aos antifúngicos convencionais e reduziram cerca de 62% do biofilme de Candida spp., além de apresentarem baixa toxicidade frente a células Hela nas concentrações fungicidas. Assim, além do PES o SPPS também é uma alternativa promissora para o tratamento de CVV, principalmente nesse grupo populacional. 45 f

Salmonella spp. is the most important foodborne pathogen, leading to consequences, especially to public health and food industry. The effect of cinnamaldehyde against Salmonella enterica serotype Typhimurium ATCC 14028 planktonic and biofilm cells on polypropylene was evaluated. Additionally, differential protein pattern expressed by planktonic and biofilm bacteria, as well protein expression on biofilms treated with cinnamaldehyde, was also evaluated. Minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of cinnamaldehyde were 312 and 624 μg/mL, respectively. Time-kill curve assay demonstrated that cinnamaldehyde presented bacteriostatic activity at sub-MIC and MIC concentrations and completely eliminated bacterial cells at 624 μg/mL after 12 h of exposure. S. Typhimurium formed biofilm on polypropylene, observed by colony counts (8.03 log CFU/cm2) and scanning electron microscopy. Cinnamaldehyde at 234 μg/mL (0.75 x MIC) decreased 1.3 log CFU/cm2 during 48 h of biofilm formation, while treatment for 1 h with cinnamaldehyde at 624 μg/mL reduced 2.33 log CFU/cm2 of 2-days-old biofilm. In proteomic analysis of biofilm compared to planktonic cells, 16 proteins were up and 5 were down-regulated. Lipoprotein Lpp, 30S ribosomal protein S2 and 50S ribosomal protein L3 were indentified only in biofilm cells, suggesting their importance on biofilm formation. In cinnamaldehyde-treated biofilm, it was identified 8 up and 11 down-regulated proteins. Among down-regulated, serine hydroxymethyltransferase presented the major decrease (23.8-fold). Cinnamaldehyde showed antimicrobial activity against planktonic cells, nevertheless it was not effective to completely eliminate the biofilm on polypropylene. Proteomic analysis showed that biofilm cells presented differentially expressed proteins compared to its planktonic counterparts, especially proteins involved in cell metabolism. Moreover, it was also observed that proteomic profile of biofilm-treated cells is affect by cinnamaldehyde. Salmonella spp. é o patógeno transmitido por alimentos mais importante, causando consequências, especialmente para saúde pública e para indústria de alimentos. O efeito do cinamaldeído contra células planctônicas e em biofilme de Salmonella Typhimurium em polipropileno foi avaliado. Além disso, a diferença no padrão de expressão proteica por bactérias planctônicas e em biofilme, bem como a expressão de proteínas de biofilmes tratados com cinamaldeído também foram avaliados. A concentração inibitória mínima (CIM) e concentração bactericida mínima sobre células planctônicas foi 312 e 624 μg/mL, respectivamente. O ensaio de curva de morte mostrou que o cinamaldeído apresenta atividade bacteriostática em concentrações subinibitórias e concentração inibitória mínima, eliminando completamente as células bacterianas com concentração de 624 μg/mL após 12 h de exposição. S. Typhimurium formou biofilme em polipropileno, observado pelas contagens de colônia (8.03 log UFC/cm2) e microscopia eletrônica de varredura. O cinamaldeído a 234 μg/mL (0.75 x CIM) reduziu 1.3 log UFC/cm2 durante 48 h de formação de biofilme, enquanto que o tratamento por 1 h com cinamaldeído a 624 μg/mL reduziu 2.33 log UFC/cm2 em biofilme formado há dois dias. Nas análises de proteômica, comparando células em biofilme e planctônicas, 16 proteínas foram positivamente reguladas e 5 foram negativamente reguladas. As Lipoproteína Lpp, proteína 30S ribossômica e proteína 50S ribossomal L3 foram identificadas apenas em células sésseis, sugerindo a importância delas na formação de biofilme. No biofilme tratado com cinamaldeído, foram identificadas 8 e 11 proteínas reguladas positivamente e negativamente, respectivamente. Dentre as proteínas reguladas negativamente, serina hidroximetil transferase apresentou a maior redução de expressão (23.8-fold). O cinamaldeído apresentou atividade antimicrobiana contra células planctônicas, no entanto não foi efetivo para eliminar completamente o biofilme em polipropileno. As análises de proteômica mostraram que as células em biofilme expressam diferencialmente proteínas comparadas às células planctônicas homólogas, especialmente proteínas envolvidas no metabolismo celular. Além disso, foi observado que o perfil proteico de células em biofilme é afetado pelo cinamaldeído. 56 f

The Tuberculosis (TB) is an infectious disease caused by the Mycobacterium tuberculosis Complex, and M. tuberculosis is, clinically, the most important specie. According to the World Health Organization (WHO), took place in 2011 8.7 million new cases worldwide. The traditional chemotherapy of TB has limitations due resistant strains and the side effects of some drugs. These problems have been exacerbates by outbreaks with multi-resistant strains (MDR-TB). An alternative for the treatment of TB is the association of new compounds with the drugs already used, obtaining synergic effect and consequent decrease of doses. The checkerboard method is used to evaluate the antimicrobial combination and check whether the effect of the drugs combined is significantly greater than the results of drugs used separately.The amoxicillin / clavulanate potassium (AMO / CLAV) has already been studied in some papers, and present an interesting result and suggests further research to better interpretation. Thus, the objective of this study was to evaluate the in vitro antimicrobial activity of AMO / CLAV in combination with the drug isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and linezolid (LINE) against M. tuberculosis and verify the synergistic action of these compounds. In the combination of AMO / CLAV with INH, there was synergism in eight clinical isolates (33.33%). Between RIF and AMO / CLAV there was synergism in nineteen clinical isolates (79.16%). In the interaction with the EMB, there was synergism in reference strain H37Rv and clinical isolates with nineteen (79.16%). Already in association with LINE, there was synergism in just five clinical isolates (20.83%). Our studies show that AMO / CLAV is very promising for future studies in treatment of anti-TB drugs and aid in the treatment of MDR-TB. Similarly to the results of the interaction of AMO / CLAV with anti-TB drugs, their use in combination in treatment could contribute to reducing the dosage of INH, RIF and especially the EMB showing important side effects for the patient. A tuberculose (TB) é uma doença infecciosa causada por bactérias do Complexo Mycobacterium tuberculosis, sendo M. tuberculosis a espécie mais importante clinicamente. De acordo com a Organização Mundial da Saúde (OMS), em 2011 aconteceram 8,7 milhões de novos casos em todo o mundo. A quimioterapia tradicional da TB tem limitações devido a cepas resistentes e aos efeitos secundários de alguns fármacos. Estes problemas têm sido exacerbados por surtos com TB multidrogas resistentes (MDR-TB) e a co-infecção AIDS/TB. Uma alternativa para o tratamento da TB é a associação de fármacos já utilizados para outras bactérias, obtendo efeito sinérgico e consequente diminuição de doses. O método checkerboard é usado para avaliar a combinação antimicrobiana e verificar se o efeito dos fármacos combinados é significativamente maior do que resultados dos fármacos empregados separadamente. A amoxacilina/clavulanato de potássio (AMO/CLAV) já vem sendo estudada em alguns trabalhos, que apresentam resultados promissores e sugerem novas pesquisas para melhor interpretação. Diante disso, o objetivo do presente trabalho foi avaliar a ação sinérgica, in vitro, da AMO/CLAV em combinação com os fármacos isoniazida (INH), rifampicina (RIF), etambutol (EMB) e linezolida (LINE) contra a cepa de referência e 23 isolados de M. tuberculosis. Na combinação da AMO/CLAV com INH, houve sinergismo em oito isolados clínicos (33,33%). Entre a RIF e a AMO/CLAV houve sinergismo em dezenove isolados clínicos (79,16%). Na interação com o EMB, houve sinergismo na cepa de referência H37Rv e com dezenove isolados clínicos (79,16%). Já em associação a LINE, houve sinergismo em apenas cinco isolados clínicos (20,83%). Nossos estudos mostram que AMO/CLAV é bastante promissor para estudos futuros como auxiliar no tratamento de cepas resistente de M. tuberculosis. Da mesma forma com os resultados obtidos da interação da AMO/CLAV com os fármacos anti-TB, seu uso em associação no tratamento também poderia contribuir para a redução da dosagem de INH, RIF e principalmente do EMB que apresentam importantes efeitos colaterais para o paciente. 37 f

Although milk is a highly consumed food, its contamination and the development of pathogenic microorganisms due to its intrinsic features are rife. Escherichia coli not only indicates contamination from fecal sources but may also be pathogenic. Among the several pathotypes of E. coli that may cause diarrhea in humans, E. coli as a producer of Shiga toxic (STEC) causes food-transmitted diseases worldwide. Serious implications to health may occur in humans and may range from mild diarrhea to hemorrhagic colitis to severe extra-intestine complication, such as Uremic Hemolytic Syndrome (UHS) and Thrombotic Thrombocytopenic Purpura. Virulence factors, such as Shiga toxins, intimine and enterohemolisin, are involved in STEC pathogenesis and several types of food from animal or vegetal origin cause STEC episodes. Although most food contamination episodes caused by STEC are linked to non-pasteurized milk consumption and its derivates, episodes related to the consumption of pasteurized milk have been reported. Genotype methods such as Polymerase Chain Reaction (PCR) have been employed to study E. coli epidemiology and to research on virulent, fast and sensitive genes.AIMS. Current research evaluates the genetic similarity and research genes stx1, stx2, eae, ehxA in E. coli isolated from pasteurized cattle milk. Eighty-seven isolates of E. coli from pasteurized bovine milk from 22 dairies in the northwestern region of the state of Paraná, Brazil, were analyzed. Isolates were stored in Tryptic Soy Broth (TSB, Difco) with glycerol at -20 ° C, the samples were identified biochemically before extraction of DNA, which was in agreement with Swanenburg et al. (1998), with modifications. Technical enterobacterial repetitive sequence Intergenic Consensus (ERIC-PCR) and sequence repetitive extragenic palindromic (REP-PCR) were used for the genetic similarity. Values equal to or greater than 98% similarity were considered related. The discriminatory power (D) of ERIC-PCR and REP-PCR was calculated using Simpson's diversity index. For the detection of virulence genes was used PCR, primers and amplification conditions were specific to each gene stx1, stx2, eae, ehxA. ERIC-PCR and REP-PCR differentiated the isolates of E. coli genomic profiles 76 and 81, respectively. Analysis of ERIC-PCR and REP-PCR showed high genetic diversity among the isolates of E. coli could be explained by different sources of contamination by E. coli in bovine milk. REP-PCR mustered only bacterial isolates from the same samples of milk, ERIC PCR showed that the isolates from different samples of milk were similar. Both techniques not grouped bacterial isolates from processed milk in dairy different. Were not detected virulence genes stx1, stx2, eae, ehxA in isolates of E. coli analyzed, possibly by the low incidence of these pathogens in pasteurized dairy products. ERIC and REP-PCR revealed a high genetic diversity of E. coli isolated from bovine milk pasteurized and non-detection of genes stx1, stx2, eae, ehxA normally present in STEC in bacterial isolates. Knowledge of the epidemiology of. diarrheagenic E. coli in pasteurized milk is highly relevant to understanding the transmission and provides information for the development and implementation of control measures throughout the production chain. The research on virulence factors in other pathotypes of E. coli is important to evaluate the potential of pasteurized commercially available as vector bacterium analyzed. O leite é um alimento muito consumido pela população, porém devido suas características intrínsecas ele se torna propicio à contaminação e desenvolvimento de micro-organismos patogênicos, dentre eles, destaca-se Escherichia coli, que além de indicar contaminação de origem fecal pode ser patogênica. Entre os diferentes patotipos de E. coli que podem causar diarréia em humanos, E. coli produtora de toxina de Shiga (STEC) é responsável por surtos de Doença Transmitida por Alimentos (DTA) em diferentes partes do mundo. Em humanos trazem sérias implicações à saúde, podendo causar desde diarréia branda à colite hemorrágica, até complicações extra-intestinais severas como Síndrome Hemolítica Urêmica (SHU) e Púrpura Trombocitopênica Trombótica. Fatores de virulência como toxinas de Shiga, intimina e enterohemolisina estão envolvidos na patogênese de STEC. Diversos alimentos, tanto de origem animal quanto vegetal, já foram incriminados em surtos de STEC. Em se tratando de leite, embora grande parte dos surtos alimentares causados por STEC estejam ligados ao consumo de leite não pasteurizado e de seus derivados, surtos relacionados ao consumo de leite pasteurizado foram relatados. Métodos genotípicos como Reação em Cadeia de Polimerase (PCR) tem sido utilizados para estudar a epidemiologia de E. coli bem como para a pesquisa de genes de virulência por serem rápidos e sensíveis. Avaliar a similaridade genética e pesquisar os genes stx1, stx2, eae, ehxA em E. coli isoladas de leite bovino pasteurizado. Foram analisados 87 isolados de E. coli oriundos de leite bovino pasteurizado proveniente de 22 laticínios localizados na região Noroeste do estado do Paraná. Os isolados bacterianos estavam armazenados a -20ºC em Tryptic Soy Broth (TSB) com glicerol e tiveram a identificação bioquímica confirmada antes da extração do DNA, que seguiu o preconizado por Swanenburg et al. (1998) com modificações. Para o estudo de similaridade genética empregou-se as técnicas ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus Sequence) e REP-PCR (Repetitive Extragenic Palindromic Sequence). Valores iguais ou maiores que 98% representaram amostras geneticamente idênticas. O poder discriminatório (D) de ERIC-PCR e REP-PCR foi calculado com base no índice de diversidade de Simpson. Para a pesquisa dos genes de virulência utilizou-se a PCR, onde os iniciadores e as condições de amplificação foram específicos para cada gene stx1, stx2, eae, ehxA. ERIC-PCR e REP-PCR diferenciaram os isolados de E. coli em 76 e 81 perfis genômicos respectivamente. Ambos os métodos mostraram elevada diversidade genética entre os isolados bacterianos, o que pode ser justificado pelas diversas fontes de contaminação do leite bovino por E. coli. REP-PCR agrupou apenas isolados bacterianos provenientes de mesmas amostras de leite, enquanto por ERIC PCR, isolados de amostras de leite diferentes foram considerados idênticos. Ambas as técnicas não agruparam isolados bacterianos provenientes de leite processado em laticínios diferentes. Não foram detectados os genes de virulência stx1, stx2, eae, ehxA nos isolados de E. coli analisados, possivelmente pela baixa incidência desse patógeno em produtos lácteos pasteurizados. Tanto ERIC quanto REP-PCR mostraram alta diversidade genética de E. coli isolada de leite bovino pasteurizado e os isolados bacterianos não apresentaram os genes stx1, stx2, eae, ehxA, normalmente presentes em STEC. O conhecimento da epidemiologia de E. coli diarreiogênicas em leite pasteurizado é de fundamental importância para o entendimento da transmissão, além de dar subsídios para o desenvolvimento e a implantação de medidas de controle ao longo da cadeia produtiva. A pesquisa de fatores de virulência de outros patotipos de E. coli é importante para avaliar o potencial do leite pasteurizado comercialmente disponível como veículo de transmissão desta bactéria. 41 f

The identification of high-risk (HR) Human Papillomavirus (HPV) types offers the possibility of improving the efficiency of cervical screening programs. Therefore, there is a continuing need for the identification of factors that modulate the risk of disease progression, and which might serve as predictive biomarkers. The aim of the present study was determine by polymerase chain reaction (PCR) the distribution of HPV genotypes and the E2 gene disruption of the HPV16/18 to assess whether viral pure integration is correlated with the degree of cervical abnormalities in South Brazilian women. A total of 137 women were studied between November 2009 and August 2011, cervical samples were collected in the colposcopy clinic at União da Vitória city, south/Brazil. The mean age was 33.60±30.72 years (range of 16-78 years), and those with invasive carcinoma (IC) were older (p < 0.05). The prevalence of total DNA- HPV, HR-HPV and multiple infections in all women was 56.2%, 38.7% and 12.4%, respectively and were higher detected in those with high-grade lesions (HSIL) and invasive carcinomas (IC) (p < 0.05). The most common HPV detected types was HPV-16 (17.5%; p < 0.05). The low-risk (LR)-HPV types were detected in 29.2%, being HPV-72 the most common (15.3%) and HPV-6 and -11 were not detected. For multiple infections, the most common occurred between HPV-72 and 16 (64.7% the all co-infections and 8.0% of the women). The overall HPV-18 E2 gene disruption was 100.0% and this infection was evenly distributed in atypia (ASC-H), low-grade lesions (LSIL), HSIL and IC (0.7% each). For HPV-16 E2 gene, the overall disruption was 54.2%, and occurred in 50.0% in ASC, 66.7% in LSIL, 53.8% in HSIL and 50% in IC (p < 0.05). Considering the similar results for pure integration of HPV-16 among different degrees of cytological abnormalities since ASC to IC, the use of PCR does not seem to be applicable to distinguish women with greater propensity for progression of the lesions. A identificação dos tipos de Papilomavirus Humano (HPV) de alto risco (HR) oferece a possibilidade de melhorar a eficiência dos programas de rastreamento do câncer cervical. A identificação de fatores que modulam o risco de progressão da doença podem servir como biomarcadores preditivos. O objetivo do presente estudo foi determinar através da técnica da reação em cadeia pela polimerase (PCR), a distribuição de genótipos do HPV e a interrupção do gene E2 dos HPV16/18 para avaliar se a integração viral total está correlacionada com o grau de anormalidades cervicais em mulheres Sul do Brasil. Um total de 137 mulheres foram estudadas entre novembro de 2009 e agosto de 2011. As amostras foram coletadas no ambulatório de colposcopia de União da Vitória-PR. A idade média foi de 33,60 ± 30,72 anos (variação 16-78 anos), sendo as mulheres com carcinoma invasivo (IC) relativamente mais velhas (p

Species of the Zungaro genus, popularly known as jaú , have great economical importance on the fishing industry. They are among the largest migratory fishes in Brazil, occurring throughout the Amazon and Paraná-Paraguay basins. They have usually been relatively abundant in the Paraná-Paraguay River Basin, but after the enclosure of several dams, a considerable decline on population size was recorded. In this study, Zungaro populations of Paraná-Paraguay River and Amazonic basins were molecularly described (variability and genetic structuration) through mitochondrial genome. The analyses of mitochondrial sequences of D-loop and ATPase 6 gene regions evidenced a genetical difference corresponding to intimately related species, as recorded in literature. Therefore, it supports the conclusion that the Zungaro population of the Paraná-Paraguay River basin (Zungaro jahu) does not belong to the same species found in the Amazonic basin (Zungaro zungaro). When molecular analyses of sub-populations of the Paraná-Paraguay basin were made, AMOVA and FST detected the identicalness of both subpopulations, demonstrating that they are not genetically different, i.e., present a very low genetical variability. This fact may be related to the founder or bottleneck effects. As Z. jahu is an endemic species of the Paraná-Paraguay basin, these results indicate that should this species disappear from Paraná River, due to reservoir building, remaining individuals will be located only in Paraguay River, subject to many survival hardships caused by low genetical diversity. O peixe conhecido popularmente por jaú pertence ao gênero Zungaro, tem grande importância pesqueira e econômica. Além de ser um dos maiores peixes migradores do Brasil, tem sua distribuição ao longo das bacias Amazônica e do Paraná-Paraguai. Este sempre foi um peixe encontrado com relativa abundância na bacia do Paraná-Paraguai, mas após a construção de uma série de barragens no rio Paraná tem-se registrado uma diminuição do tamanho efetivo dessa população. Neste trabalho foram caracterizadas molecularmente (variabilidade e estruturação genética), através do genoma mitocondrial, populações de jaú das bacias do rio Paraná-Paraguai e Amazônica. As análises das seqüências mitocondriais das regiões D-loop e do gene da ATPase 6 evidenciaram uma diferenciação genética, entre os indivíduos das duas bacias, em níveis equivalentes ao registrado na literatura entre muitas espécies próximas entre si. Portanto, é inevitável concluir que a população de Zungaro da bacia do Paraná-Paraguai não pertence à espécie de Zungaro zungaro da bacia Amazônica, mas sim à espécie Z. jahu. Quando feitas as análises moleculares entre as subpopulações da bacia do Paraná-Paraguai, a AMOVA e o FST detectaram a identidade dessas populações, que não estão diferenciadas geneticamente, ou seja, apresentam uma variabilidade genética extremamente baixa. Esse fato deve estar relacionado ou ao efeito fundador ou ao efeito bottleneck. Como Z. jahu é endêmico da bacia Paraná-Paraguai, esses resultados indicam que com a extinção local no rio Paraná (causada pela drástica diminuição dos estoques), só restarão indivíduos dessa espécie no rio Paraguai, com grandes problemas de sobrevivência causados pela falta de diversidade genética. 59 f