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Made available in DSpace on 2020-12-12T02:05:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-08-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Objective: To investigate GTN lethality among Brazilian women comparing cases of death by GTN with those who survived, thereby identifying factors associated with GTN lethality. Methods: We retrospectively reviewed medical records of women with GTN treated at ten Brazilian GTN Reference Centers, from January 1960 to December 2017. We evaluated factors associated with death from GTN and used Cox proportional hazards regression models to identify independent variables with significant influence on the risk of death. Results: From 2186 patients with GTN included in this study, 2092 (95.7%) survived and 89 (4%) died due to GTN. When analyzing the relative risk (RR), adjusted for WHO/FIGO score, patients with low risk disease had a significantly higher risk of death if they had choriocarcinoma (RR: 12.40), metastatic disease (RR: 12.57), chemoresistance (RR: 3.18) or initial treatment outside the Reference Center (RR: 12.22). In relation to patients with high-risk GTN, these factors were significantly associated with death due to GTN: the time between the end of antecedent pregnancy and the initiation of chemotherapy (RR: 4.10), metastatic disease (RR: 14.66), especially in brain (RR: 8.73) and liver (RR: 5.76); absence of chemotherapy or initial treatment with single agent chemotherapy (RR: 10.58 and RR: 1.81, respectively), chemoresistance (RR: 3.20) and the initial treatment outside the Reference Center (RR: 28.30). Conclusion: The risk of mortality from low and high-risk GTN can be reduced by referral of these patients to a Reference Center or, if not possible, to involve clinicians in a Reference Center with consultation regarding management. Rio de Janeiro Trophoblastic Disease Center Maternity School of Rio de Janeiro Federal University Antonio Pedro University Hospital of Fluminense Federal University Postgraduate Program in Medical Sciences Fluminense Federal University Postgraduate Program in Perinatal Health Faculty of Medicine Maternity School of Rio de Janeiro Federal University Goiania Trophoblastic Disease Center Clinics Hospital of Goias Federal University Botucatu Trophoblastic Disease Center Clinical Hospital of Botucatu Medical School Department of Gynecology and Obstetrics São Paulo State University-UNESP Porto Alegre Trophoblastic Disease Center Mario Totta Maternity Ward Irmandade da Santa Casa de Misericórdia Hospital Caxias do Sul Trophoblastic Disease Center General Hospital of Caxias do Sul School of Medicine Center for Biological and Health Sciences Caxias do Sul University Campinas Trophoblastic Disease Center University of Campinas Jundiai Trophoblastic Disease Center Jundiai Medical School Santos Trophoblastic Disease Center Guilherme Álvaro Hospital Rio Branco Trophoblastic Disease Center Clinics Hospital of Acre São Paulo Hospital Trophoblastic Disease Center Paulista School of Medicine São Paulo Federal University New England Trophoblastic Disease Center Division of Gynecologic Oncology Department of Obstetrics Gynecology and Reproductive Biology Brigham and Women's Hospital Harvard Medical School Botucatu Trophoblastic Disease Center Clinical Hospital of Botucatu Medical School Department of Gynecology and Obstetrics São Paulo State University-UNESP CAPES: 001

To compare the outcomes of patients with low-risk gestational trophoblastic neoplasia (GTN) treated with 8-day methotrexate (MTX) with two different regimens of folinic acid (FA).Retrospective cohort study of low-risk GTN followed at Rio de Janeiro Federal University, from January/2000-December/2019 with 8-day MTX with FA at 0.1 mg/kg versus 15 mg fixed dose.Among 667 patients with low-risk GTN, 323 were treated with FA at 0.1 mg/kg and 142 with FA at 15 mg fixed dose. The weight-based and fixed dose groups were comparable in terms of clinical profile but did differ in the hCG pretreatment level (8883 versus 5127 IU/L, p0.01) and FIGO risk score 5/6 (3.4% versus 18.3%, p0.01), respectively. Despite this, there was no difference in the remission rate in first-line treatment (76.8 versus 81%, p = 0.33), although FA at 0.1 mg/kg had a significantly higher number of chemotherapy cycles to remission (5 versus 4, p0.01), need to delay chemotherapy due to toxicity (6.8 versus 2.8%, p0.01) and time to remission, (12 versus 8 weeks, p0.01), respectively. A logistic regression analysis showed that the different FA rescue regimens appeared comparable in terms of achieving remission in first-line chemotherapy for low-risk GTN (OR:5.16, CI95%:0.84-31.64, p = 0.08).FA with 15 mg fixed dose as compared to 0.1 mg/kg of FA was associated with similar primary remission rate, relapse or death among low-risk GTN treated with 8-day MTX. This regimen is highly practical, reduces visits to health facilities, appears equally safe and may be preferable with the 8-day MTX regimen in the treatment of low-risk GTN.

Introdução: A neoplasia trofoblástica gestacional (NTG) é um tumor singular, pois surge do tecido gestacional, e é altamente curável na maioria das pacientes utilizando-se apenas quimioterapia (QT). A QT por agente único, capaz de curar 80% das pacientes com NTG e a mais utilizada no Brasil, é o esquema de oito8 dias, sendo o metotrexato (MTX) administrado nos dias 1, 3, 5 e 7 e o ácido folínico (AF) nos dias 2, 4, 6 e 8. O uso do AF visa diminuir os efeitos colaterais do MTX. Entre os protocolos que utilizam oito dias de MTX para o tratamento de NTG, temos dois esquemas de AF mais utilizados no Brasil: a dose de 0,1 mg/kg (habitualmente feita via intramuscular ou fracionando-se os comprimidos) e a dose fixa de 15 mg (utilizando-se a apresentação comercial brasileira nessa posologia, sem necessidade de ajuste). Contudo, há preocupação sobre se maiores doses de AF diminuem o efeito do tratamento com MTX, podendo diminuir a taxa de remissão ou aumentar a ocorrência de recidiva. O presente estudo descreve os resultados de paciente brasileiras com NTG de baixo risco tratadas com esquema de MTX em oito dias e os dois esquemas de AF. Objetivo: Comparar os desfechos das pacientes com NTG de baixo risco tratadas com esquema de MTX em oito dias, com dois regimes de resgate com AF. Pacientes e métodos: Trata-se de um estudo de coorte retrospectiva de pacientes atendidas no Centro de Referência em NTG da Maternidade Escola da Universidade Federal do Rio de Janeiro, entre janeiro de 2000 e dezembro de 2019, com diagnóstico de NTG de baixo risco e tratadas com MTX no esquema de oito dias e AF nas duas doses: 0,1 mg/kg e dose fixa de 15 mg. Resultados: Das 667 pacientes com NTG de baixo risco incluídas no estudo, 323 foram tratadas com AF na dose de 0,1 mg/kg e 142 com AF na dose fixa de 15 mg. Ambos os grupos foram comparáveis no perfil clínico, porém diferiram nos níveis de gonadotrofina coriônica humana (hCG) pré-tratamento e no escore de risco da Federação Internacional de Ginecologia e Obstetrícia (FIGO). Apesar disso, não houve diferença na taxa de remissão, embora a dose de AF de 0,1 mg/kg tenha apresentado maior número de ciclos de QT para remissão, maior necessidade de atrasar a QT em razão da toxicidade e maior tempo para remissão. Apesar de a regressão logística ter mostrado que as duas doses de AF não foram fatores preditivos para a remissão, a regressão multivariada de Cox mostrou que o AF na dose de 0,1 mg/kg esteve significativamente associado com maior tempo para a remissão. Conclusão: O uso da dose fixa de AF não modificou a taxa de remissão primária, recidiva ou morte das pacientes com NTG de baixo risco. O uso da dose fixa é prático (não sendo necessário fracionar o comprimido), reduz as idas à unidade de saúde (pois dispensa a aplicação intramuscular), pode ser feito em casa, é igualmente seguro e pode ser o regime de escolha no esquema de tratamento da NTG de baixo risco com MTX.

Introdução: Pacientes com neoplasia trofoblástica gestacional (NTG) e escore de risco FIGO 5 ou 6 recebem inicialmente quimioterapia de menor toxicidade com agente único. É amplamente aceito que apenas um terço é curado, o restante necessitando de tratamento tóxico com múltiplos agentes para atingir a remissão. Identificar fatores preditivos para resistência a agentes únicos melhoraria a estratificação das pacientes que receberiam quimioterapia com múltiplos agentes, evitando que se inicie esse esquema quimioterápico com maiores efeitos colaterais em todos os casos cujo escore de risco FIGO seja 5 ou 6. Objetivo: Identificar quais pacientes portadoras de NTG com escore de risco FIGO 5 ou 6 são adequadamente tratadas com agente quimioterápico único como primeira linha, e quais requerem terapia inicial com múltiplos agentes. Material e Métodos: Trata-se de um estudo de coorte retrospectivo, multicêntrico, internacional, no qual foi realizada a revisão de prontuários de pacientes portadoras de NTG com escore de risco FIGO 5 ou 6, em três dos maiores centros de referência em NTG do mundo. Foram incluídas pacientes acompanhadas em Londres, no Rio de Janeiro e em Boston, entre janeiro de 1964 e dezembro de 2018. Variáveis significativas associadas à quimiorresistência à terapia com agente único foram testadas em um modelo multivariado. Resultados e Conclusão: Apenas 40,2% (141/351) das pacientes com escore de risco FIGO 5 ou 6 desenvolveram resistência a tratamentos com agente único e necessitaram de quimioterapia com múltiplos agentes para entrar em remissão. A regressão logística univariada e multivariada revelou metástases (odds ratio — OR: 1,6, intervalo de confiança de 95% — IC95% 1,0‒2,6; p=0,044), histopatologia de coriocarcinoma (OR: 3,0, IC95% 1,6‒5,6; p=0,001), gonadotrofina coriônica humana (hCG) pré-tratamento (OR: 2,4, IC95% 1,7‒3,4; p

Made available in DSpace on 2018-12-11T17:18:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-06-01 Fondo Nacional de Desarrollo Científico y Tecnológico Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundação de Amparo à Pesquisa do Estado de Goiás Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Canadian Institutes of Health Research Here, we report that the Neurospora crassa FLB-3 protein, the ortholog of the Aspergillus nidulans FlbC transcription factor, is required for developmental control. Deletion of flb-3 leads to changes in hyphae morphology and affects sexual and asexual development. We identified, as putative FLB-3 targets, the N. crassa aba-1, wet-1 and vos-1 genes, orthologs of the ones involved in A. nidulans asexual development and that work downstream of FlbC (abaA, wetA and vosA). In N. crassa, these three genes require FLB-3 for proper expression; however, they appear not to be required for normal development, as demonstrated by gene expression analyses during vegetative growth and asexual development. Moreover, mutant strains in the three genes conidiate well and produce viable conidia. We also determined FLB-3 DNA-binding preferences via protein-binding microarrays (PBMs) and demonstrated by chromatin immunoprecipitation (ChIP) that FLB-3 binds the aba-1, wet-1 and vos-1 promoters. Our data support an important role for FLB-3 in N. crassa development and highlight differences between the regulatory pathways controlled by this transcription factor in different fungal species. Departamento de Bioquímica e Tecnologia Química Instituto de Química UNESP Universidade Estadual Paulista Millennium Institute for Integrative Systems and Synthetic Biology (MIISSB) Departamento de Genética Molecular y Microbiología Facultad de Ciencias Biológicas Pontificia Universidad Católica de Chile Donnelly Centre University of Toronto Center for Autoimmune Genomics and Etiology (CAGE) and Divisions of Biomedical Informatics and Developmental Biology Cincinnati Children's Hospital Medical Center Department of Pediatrics University of Cincinnati College of Medicine Department of Molecular Genetics University of Toronto Canadian Institutes for Advanced Research Departamento de Bioquímica e Tecnologia Química Instituto de Química UNESP Universidade Estadual Paulista Fondo Nacional de Desarrollo Científico y Tecnológico: 1171151 FAPESP: 2013/24705-3 Fundação de Amparo à Pesquisa do Estado de Goiás: 201710267000110 CNPq: 444068/2014-7 CAPES: 88887.126652/2017-00 Canadian Institutes of Health Research: FDN-148403

Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development.

Summary Aspergillus fumigatus is an opportunistic pathogen and allergen of mammals. Calcium signalling is essential for A. fumigatus pathogenicity and is regulated by the CrzA transcription factor. We used ChIP-seq (Chromatin Immunoprecipitation DNA sequencing) to explore CrzA gene targets in A. fumigatus. In total, 165 potential binding peaks including 102 directly regulated genes were identified, resulting in the prediction of the A[GT][CG]CA[AC][AG] CrzA-binding motif. The 102 CrzA putatively regulated genes exhibited a diverse array of functions. The phkB (Afu3g12530) histidine kinase and the sskB (Afu1g10940) MAP kinase kinase kinase of the HOG (high-osmolarity glycerol response) pathway were regulated by CrzA. Several members of the two-component system (TCS) and the HOG pathway were more sensitive to calcium. CrzA::GFP was translocated to the nucleus upon osmotic stress. CrzA is important for the phosphorylation of the SakA MAPK in response to osmotic shock. The ΔsskB was more sensitive to CaCl2, NaCl, and paraquat stress, while being avirulent in a murine model of invasive pulmonary aspergillosis. The presence of CaCl2 and osmotic stresses resulted in synergistic inhibition of ΔcrzA and ΔsskB growth. These results suggest there is a genetic interaction between the A. fumigatus calcineurin-CrzA and HOG pathway that is essential for full virulence.

Made available in DSpace on 2018-12-11T17:02:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-05-01 When exposed to stress conditions, all cells induce mechanisms resulting in an attempt to adapt to stress that involve proteins which, once activated, trigger cell responses by modulating specific signaling pathways. In this work, using a combination of pulldown assays and mass spectrometry analyses, we identified the Neurospora crassa SEB-1 transcription factor that binds to the Stress Response Element (STRE) under heat stress. Orthologs of SEB-1 have been functionally characterized in a few filamentous fungi as being involved in stress responses; however, the molecular mechanisms mediated by this transcription factor may not be conserved. Here, we provide evidences for the involvement of N. crassa SEB-1 in multiple cellular processes, including response to heat, as well as osmotic and oxidative stress. The Δseb-1 strain displayed reduced growth under these conditions, and genes encoding stress-responsive proteins were differentially regulated in the Δseb-1 strain grown under the same conditions. In addition, the SEB-1-GFP protein translocated from the cytosol to the nucleus under heat, osmotic, and oxidative stress conditions. SEB-1 also regulates the metabolism of the reserve carbohydrates glycogen and trehalose under heat stress, suggesting an interconnection between metabolism control and this environmental condition. We demonstrated that SEB-1 binds in vivo to the promoters of genes encoding glycogen metabolism enzymes and regulates their expression. A genome-wide transcriptional profile of the Δseb-1 strain under heat stress was determined by RNA-seq, and a broad range of cellular processes was identified that suggests a role for SEB-1 as a protein interconnecting these mechanisms. Departamento de Bioquímica e Tecnologia Química Instituto de Química Universidade Estadual Paulista (UNESP) Department of Plant and Microbial Biology University of California Departamento de Química Orgânica Instituto de Química Universidade Estadual de Campinas (UNICAMP) Departamento de Bioquímica e Tecnologia Química Instituto de Química Universidade Estadual Paulista (UNESP)

Made available in DSpace on 2018-12-11T17:06:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-08-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Environmental pH induces a stress response triggering a signaling pathway whose components have been identified and characterized in several fungi. Neurospora crassa shares all six components of the Aspergillus nidulans pH signaling pathway, and we investigate here their regulation during an alkaline pH stress response. We show that the N. crassa pal mutant strains, with the exception of Δpal-9, which is the A. nidulans Pali homolog, exhibit low conidiation and are unable to grow at alkaline pH. Moreover, they accumulate the pigment melanin, most likely via regulation of the tyrosinase gene by the pH signaling components. The PAC-3 transcription factor binds to the tyrosinase promoter and negatively regulates its gene expression. PAC-3 also binds to all pal gene promoters, regulating their expression at normal growth pH and/or alkaline pH, which indicates a feedback regulation of PAC-3 in the pal gene expression. In addition, PAC-3 binds to the pac-3 promoter only at alkaline pH, most likely influencing the pac-3 expression at this pH suggesting that the activation of PAC-3 in N. crassa results from proteolytic processing and gene expression regulation by the pH signaling components. In N. crassa, PAC-3 is proteolytically processed in a single cleavage step predominately at alkaline pH; however, low levels of the processed protein can be observed at normal growth pH. We also demonstrate that PAC-3 preferentially localizes in the nucleus at alkaline pH stress and that the translocation may require the N. crassa importin-α since the PAC-3 nuclear localization signal (NLS) has a strong in vitro affinity with importin-α. The data presented here show that the pH signaling pathway in N. crassa shares all the components with the A. nidulans and S. cerevisiae pathways; however, it exhibits some properties not previously described in either organism. Departamento de Bioquímica e Tecnologia Química Instituto de Química Universidade Estadual Paulista UNESP Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista UNESP Departamento de Bioquímica e Tecnologia Química Instituto de Química Universidade Estadual Paulista UNESP Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista UNESP CNPq: 444068/2014-7

The cAMP–PKA signaling pathway plays an important role in many biological processes including glycogen metabolism. In this work we investigated its role in the Neurospora crassa glycogen metabolism control using mutant strains affected in components of the pathway, the cr-1 strain deficient in adenylyl cyclase activity therefore has the PKA pathway not active, and the mcb strain a temperature-sensitive mutant defective in the regulatory subunit of PKA therefore is a strain with constitutively active PKA. We analyzed the expression of the gene encoding glycogen synthase ( gsn ), the regulatory enzyme in glycogen synthesis as a potential target of the regulation. The cr-1 strain accumulated, during vegetative growth, glycogen levels much higher than the wild type strain indicating a role of the PKA pathway in the glycogen accumulation. The gsn transcript was not increased in this strain but the GSN protein was less phosphorylated “ in vitro ”, and therefore more active, suggesting that the post-translational modification of GSN is likely the main mechanism controlling glycogen accumulation during vegetative growth. Heat shock down-regulates gsn gene transcription in the two mutant strains, as well as in the wild type strain, suggesting that the PKA pathway may not be the only pathway having a direct role in gsn transcription under heat shock. DNA–protein complexes were formed between the STRE motif in the gsn promoter and nuclear proteins from heat-shocked mycelium. However STRE was not able to induce transcription of a reporter gene in Saccharomyces cerevisiae , suggesting that the motif might be involved in a different way of regulation in the N. crassa gene expression under heat shock. The CRE-like DNA elements present in the gsn promoter were shown to be bound by different proteins from the PKA mutant strains. The DNA–protein complexes were observed with proteins from the strains grown under normal condition and under heat shock indicating the functionality of this DNA element. In this work we presented some evidences that the PKA signaling pathway regulates glycogen metabolism in N. crassa in a different way when compared to the well-characterized model of regulation existent in S. cerevisiae.

The gene encoding glycogen synthase in Neurospora crassa (gsn) is transcriptionally down-regulated when mycelium is exposed to a heat shock from 30 to 45 degrees C. The gsn promoter has one stress response element (STRE) motif that is specifically bound by heat shock activated nuclear proteins. In this work, we used biochemical approaches together with mass spectrometric analysis to identify the proteins that bind to the STRE motif and could participate in the gsn transcription regulation during heat shock. Crude nuclear extract of heat-shocked mycelium was prepared and fractionated by affinity chromatography. The fractions exhibiting DNA-binding activity were identified by electrophoretic mobility shift assay (EMSA) using as probe a DNA fragment containing the STRE motif. DNA-protein binding activity was confirmed by Southwestern analysis. The molecular mass (MM) of proteins was estimated by fractionating the crude nuclear extract by SDS-PAGE followed by EMSA analysis of the proteins corresponding to different MM intervals. Binding activity was detected at the 30-50 MM kDa interval. Fractionation of the crude nuclear proteins by IEF followed by EMSA analysis led to the identification of two active fractions belonging to the pIs intervals 3.54-4.08 and 6.77-7.31. The proteins comprising the MM and pI intervals previously identified were excised from a 2-DE gel, and subjected to mass spectrometric analysis (MALDI-TOF/TOF) after tryptic digestion. The proteins were identified by search against the MIPS and MIT N. crassa databases and five promising candidates were identified. Their structural characteristics and putative roles in the gsn transcription regulation are discussed.

Made available in DSpace on 2015-10-21T13:11:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-06-19. Added 1 bitstream(s) on 2015-10-22T09:52:24Z : No. of bitstreams: 1 WOS000356835000027.pdf: 1517523 bytes, checksum: 00ccbb0dd1b8fc729e525abc5c242925 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Neurospora crassa is a filamentous fungus that has been extensively studied as a model organism for eukaryotic biology, providing fundamental insights into cellular processes such as cell signaling, growth and differentiation. To advance in the study of this multicellular organism, an understanding of the specific mechanisms for protein transport into the cell nucleus is essential. Importin-alpha (Imp-alpha) is the receptor for cargo proteins that contain specific nuclear localization signals (NLSs) that play a key role in the classical nuclear import pathway. Structures of Imp-a from different organisms (yeast, rice, mouse, and human) have been determined, revealing that this receptor possesses a conserved structural scaffold. However, recent studies have demonstrated that the Imp alpha mechanism of action may vary significantly for different organisms or for different isoforms from the same organism. Therefore, structural, functional, and biophysical characterization of different Impa proteins is necessary to understand the selectivity of nuclear transport. Here, we determined the first crystal structure of an Impa from a filamentous fungus which is also the highest resolution Impa structure already solved to date (1.75 angstrom). In addition, we performed calorimetric analysis to determine the affinity and thermodynamic parameters of the interaction between Impa and the classical SV40 NLS peptide. The comparison of these data with previous studies on Impa proteins led us to demonstrate that N. crassa Imp-alpha possess specific features that are distinct from mammalian Imp-alpha but exhibit important similarities to rice Imp-alpha, particularly at the minor NLS binding site. Univ Estadual Paulista, UNESP, Inst Biociencias, Dept Fis &Biofis, Botucatu, SP, Brazil Univ Estadual Paulista, UNESP, Inst Quim, Dept Bioquim &Tecnol Quim, Araraquara, SP, Brazil Departamento de Física e Biofísica, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Botucatu, SP, Brazil Departamento de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista, UNESP, Araraquara, SP, Brazil

The transcription factor CreA/Mig1/CRE-1 is a repressor protein that regulates the use of alternative carbon sources via a mechanism known as Carbon Catabolite Repression (CCR). In Saccharomyces cerevisiae, Mig1 recruits the complex Ssn6-Tup1, the Neurospora crassa RCM-1 and RCO-1 orthologous proteins, respectively, to bind to promoters of glucose-repressible genes. We have been studying the regulation of glycogen metabolism in N. crassa and the identification of the RCO-1 corepressor as a regulator led us to investigate the regulatory role of CRE-1 in this process. Glycogen content is misregulated in the rco-1(KO), rcm-1(RIP) and cre-1(KO) strains, and the glycogen synthase phosphorylation is decreased in all strains, showing that CRE-1, RCO-1 and RCM-1 proteins are involved in glycogen accumulation and in the regulation of GSN activity by phosphorylation. We also confirmed the regulatory role of CRE-1 in CCR and its nuclear localization under repressing condition in N. crassa. The expression of all glycogenic genes is misregulated in the cre-1(KO) strain, suggesting that CRE-1 also controls glycogen metabolism by regulating gene expression. The existence of a high number of the Aspergillus nidulans CreA motif (5'-SYGGRG-3') in the glycogenic gene promoters led us to analyze the binding of CRE-1 to some DNA motifs both in vitro by DNA gel shift and in vivo by ChIP-qPCR analysis. CRE-1 bound in vivo to all motifs analyzed demonstrating that it down-regulates glycogen metabolism by controlling gene expression and GSN phosphorylation.

Importin-α recognizes cargo proteins that contain classical nuclear localization sequences (NLS) and, in complex with importin-β, is able to translocate nuclear proteins through the nuclear pore complex. The filamentous fungusNeurospora crassais a well studied organism that has been widely used as a model organism for fundamental aspects of eukaryotic biology, and is important for understanding the specific mechanisms of protein transport to the cell nucleus. In this work, the crystallization and preliminary X-ray diffraction analysis of importin-α fromN. crassa(IMPα-Nc) complexed with a classical NLS peptide (SV40 NLS) are reported. IMPα-Nc–SV40 NLS crystals diffracted X-rays to 2.0 Å resolution and the structure was solved by molecular-replacement techniques, leading to a monomeric structure. The observation of the electron-density map indicated the presence of SV40 NLSs interacting at both the minor and major NLS-binding sites of the protein.

Microorganisms accumulate glycogen as carbon and energy reserves to face adverse environmental conditions during growth and development. The processes of glycogen synthesis and degradation share similarities among different microorganisms. However, the regulation of the metabolism as a whole shows differences, likely due to the environmental conditions to which they individually respond. This chapter aims to present some molecular mechanisms that regulate glycogen metabolism in the fungus Neurospora crassa. The availability of its genome sequence (Galagan et al., Nature 422:859–868, 2003) and a collection of mutant strains, each carrying a deletion in a specific ORF, allowed investigation into the role of specific proteins as regulators of glycogen metabolism to begin. Here we present some biochemical and molecular mechanisms that have already been described for this fungus, and additionally, we focused on more recent findings including the molecular basis underlying the metabolism regulation, mainly at transcriptional level.

Made available in DSpace on 2013-08-28T14:12:09Z (GMT). No. of bitstreams: 1 WOS000308221300070.pdf: 1119022 bytes, checksum: 1b64a3e409634995367640ca01b7a9dc (MD5) Made available in DSpace on 2013-09-30T19:07:41Z (GMT). No. of bitstreams: 2 WOS000308221300070.pdf: 1119022 bytes, checksum: 1b64a3e409634995367640ca01b7a9dc (MD5) WOS000308221300070.pdf.txt: 68626 bytes, checksum: 510051e219ebf1e3f81df629339e28b3 (MD5) Previous issue date: 2012-08-31 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T14:17:50Z No. of bitstreams: 2 WOS000308221300070.pdf: 1119022 bytes, checksum: 1b64a3e409634995367640ca01b7a9dc (MD5) WOS000308221300070.pdf.txt: 68626 bytes, checksum: 510051e219ebf1e3f81df629339e28b3 (MD5) Made available in DSpace on 2014-05-20T14:17:50Z (GMT). No. of bitstreams: 2 WOS000308221300070.pdf: 1119022 bytes, checksum: 1b64a3e409634995367640ca01b7a9dc (MD5) WOS000308221300070.pdf.txt: 68626 bytes, checksum: 510051e219ebf1e3f81df629339e28b3 (MD5) Previous issue date: 2012-08-31 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Glycogen is a polysaccharide widely distributed in microorganisms and animal cells and its metabolism is under intricate regulation. Its accumulation in a specific situation results from the balance between glycogen synthase and glycogen phosphorylase activities that control synthesis and degradation, respectively. These enzymes are highly regulated at transcriptional and post-translational levels. The existence of a DNA motif for the Aspergillus nidulans pH responsive transcription factor PacC in the promoter of the gene encoding glycogen synthase (gsn) in Neurospora crassa prompted us to investigate whether this transcription factor regulates glycogen accumulation. Transcription factors such as PacC in A. nidulans and Rim101p in Saccharomyces cerevisiae play a role in the signaling pathway that mediates adaptation to ambient pH by inducing the expression of alkaline genes and repressing acidic genes. We showed here that at pH 7.8 pacC was overexpressed and gsn was down-regulated in wild-type N. crassa coinciding with low glycogen accumulation. In the pacC(KO) strain the glycogen levels and gsn expression at alkaline pH were, respectively, similar to and higher than the wild-type strain at normal pH (5.8). These results characterize gsn as an acidic gene and suggest a regulatory role for PACC in gsn expression. The truncated recombinant protein, containing the DNA-binding domain specifically bound to a gsn DNA fragment containing the PacC motif. DNA-protein complexes were observed with extracts from cells grown at normal and alkaline pH and confirmed by ChIP-PCR analysis. The PACC present in these extracts showed equal molecular mass, indicating that the protein is already processed at normal pH, in contrast to A. nidulans. Together, these results show that the pH signaling pathway controls glycogen accumulation by regulating gsn expression and suggest the existence of a different mechanism for PACC activation in N. crassa. Univ Estadual Paulista, UNESP, Inst Quim, Dept Bioquim & Tecnol Quim, São Paulo, Brazil Nalco Co, Sugar Land, TX USA Univ Estadual Paulista, UNESP, Inst Quim, Dept Bioquim & Tecnol Quim, São Paulo, Brazil FAPESP: 08/57566-8 FAPESP: 07/07766-8 CNPq: 303973/2009-9

Transcription factors play a key role in transcription regulation as they recognize and directly bind to defined sites in promoter regions of target genes, and thus modulate differential expression. The overall process is extremely dynamic, as they have to move through the nucleus and transiently bind to chromatin in order to regulate gene transcription. To identify transcription factors that affect glycogen accumulation in Neurospora crassa, we performed a systematic screen of a deletion strains set generated by the Neurospora Knockout Project and available at the Fungal Genetics Stock Center. In a wild-type strain of N. crassa, glycogen content reaches a maximal level at the end of the exponential growth phase, but upon heat stress the glycogen content rapidly drops. The gene encoding glycogen synthase (gsn) is transcriptionally down-regulated when the mycelium is exposed to the same stress condition. We identified 17 deleted strains having glycogen accumulation profiles different from that of the wild-type strain under both normal growth and heat stress conditions. Most of the transcription factors identified were annotated as hypothetical protein, however some of them, such as the PacC, XlnR, and NIT2 proteins, were biochemically well-characterized either in N. crassa or in other fungi. The identification of some of the transcription factors was coincident with the presence of DNA-binding motifs specific for the transcription factors in the gsn 5′-flanking region, and some of these DNA-binding motifs were demonstrated to be functional by Electrophoretic Mobility Shift Assay (EMSA) experiments. Strains knocked-out in these transcription factors presented impairment in the regulation of gsn expression, suggesting that the transcription factors regulate glycogen accumulation by directly regulating gsn gene expression. Five selected mutant strains showed defects in cell cycle progression, and two transcription factors were light-regulated. The results indicate that there are connections linking different cellular processes, such as metabolism control, biological clock, and cell cycle progression.

Made available in DSpace on 2013-08-28T14:12:07Z (GMT). No. of bitstreams: 1 WOS000305335800062.pdf: 1062294 bytes, checksum: f286e05f0c5cc5f96d3ea2622d7596c5 (MD5) Made available in DSpace on 2013-09-30T19:07:40Z (GMT). No. of bitstreams: 2 WOS000305335800062.pdf: 1062294 bytes, checksum: f286e05f0c5cc5f96d3ea2622d7596c5 (MD5) WOS000305335800062.pdf.txt: 73864 bytes, checksum: 8502f99cf5690998cef4024bcc752f3c (MD5) Previous issue date: 2012-05-23 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T14:17:49Z No. of bitstreams: 2 WOS000305335800062.pdf: 1062294 bytes, checksum: f286e05f0c5cc5f96d3ea2622d7596c5 (MD5) WOS000305335800062.pdf.txt: 73864 bytes, checksum: 8502f99cf5690998cef4024bcc752f3c (MD5) Made available in DSpace on 2014-05-20T14:17:49Z (GMT). No. of bitstreams: 2 WOS000305335800062.pdf: 1062294 bytes, checksum: f286e05f0c5cc5f96d3ea2622d7596c5 (MD5) WOS000305335800062.pdf.txt: 73864 bytes, checksum: 8502f99cf5690998cef4024bcc752f3c (MD5) Previous issue date: 2012-05-23 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca+2-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca+2-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5'-CACAGCCAC-3' and 5'-CCCTGCCCC-3' sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -beta and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The Delta pmcA and Delta pmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the Delta calA and Delta crzA mutant strains. However, only the A. fumigatus Delta pmcA was avirulent in the murine model of invasive pulmonary aspergillosis. Lab Nacl Ciência & Tecnol Bioetanol CTBE, São Paulo, Brazil Univ São Paulo, Fac Ciencias Farmaceut Ribeirao Preto, São Paulo, Brazil Univ São Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, São Paulo, Brazil Univ São Paulo, Fac Med Ribeirao Preto, São Paulo, Brazil Univ Londrina, Dept Microbiol, Londrina, Parana, Brazil Univ Estadual Paulista, Inst Quim, São Paulo, Brazil Univ Estadual Paulista, Inst Quim, São Paulo, Brazil

Neurospora crassa has been widely used as a model organism and contributed to the development of biochemistry and molecular biology by allowing the identification of many metabolic pathways and mechanisms responsible for gene regulation. Nuclear proteins are synthesized in the cytoplasm and need to be translocated to the nucleus to exert their functions which the importin-α receptor has a key role for the classical nuclear import pathway. In an attempt to get structural information of the nuclear transport process in N. crassa, we present herein the cloning, expression, purification and structural studies with N-terminally truncated IMPα from N. crassa (IMPα-Nc). Circular dichroism analysis revealed that the IMPα-Nc obtained is correctly folded and presents a high structural conservation compared to other importins-α. Dynamic light scattering, analytical size-exclusion chromatography experiments and molecular dynamics simulations indicated that the IMPα-Nc unbound to any ligand may present low stability in solution. The IMPα-Nc theoretical model displayed high similarity of its inner concave surface, which binds the cargo proteins containing the nuclear localization sequences, among IMPα from different species. However, the presence of non-conserved amino acids relatively close to the NLS binding region may influence the binding specificity of IMPα-Nc to cargo proteins.

O fungo Trichoderma reesei é conhecido por sua elevada capacidade de secreção de enzimas celulolíticas que atuam degradando o polímero de celulose em moléculas de glicose, sendo este um ponto chave na produção do etanol de segunda geração. O presente trabalho tem por objetivo contribuir para o entendimento dos mecanismos moleculares envolvidos no processo de desconstrução da biomassa por T. reesei através da identificação de novos fatores de transcrição associados a este processo. Para isso, foi realizado um screening de 14 fatores de transcrição potencialmente envolvidos com a degradação de biomassa e identificação do gene Tr103275, homólogo ao regulador Azf1 de Saccharomyces cerevisiae. Posteriormente, foi feita a construção de um mutante com deleção do gene azf1 em T. reesei. Durante o crescimento em celulose e bagaço de cana, o gene azf1 foi altamente expresso em T. reesei, ao passo que glicose reprimiu a expressão gênica desse fator de transcrição. A ausência de azf1 afetou o crescimento e a esporulação em T. reesei, uma vez que a linhagem mutante produziu esporos menores e em quantidade reduzida em relação ao parental. Além disso, a proteína TrAzf1 atua como um regulador positivo da expressão de enzimas holocelulolíticas, considerando que as os genes que codificam as CBHs, EGs, BGs e xyn4 foram down regulados no mutante Δazf1 em celulose e bagaço de cana. O sítio de ligação para TrAzf1 foi predito nesse trabalho através de análise de bioinformática. Para avaliar a interação de TrAzf1 com esse sítio de ligação no DNA, o motivo dedo de zinco de Azf1 recombinante foi obtido por expressão heteróloga em E. coli e utilizado nas análises de SPR. No entanto, a validação da interação fator de transcrição-DNA por SPR não foi conclusiva e outros experimentos devem ser realizados para validar o motivo de ligação de TrAzf1 em T. reesei. O papel de Azf1 na regulação da degradação da biomassa vegetal foi descrito, pela primeira vez, nesse trabalho. Esse fator de transcrição é um alvo em potencial para manipulação genética de T. reesei, visando o melhoramento da produção de enzimas holocelulolíticas por essa espécie. The fungus Trichoderma reesei is known for its high capacity in secrete cellulolytic enzymes that act in the degradation of the cellulose polymer into glucose molecules, being a key point in the production of second generation ethanol. The present work aims to contribute to the understanding of the molecular mechanisms involved in the process of biomass deconstruction in T. reesei through the identification of new transcription factors associated to this process. For this, we screened 14 transcription factors potentially involved in the degradation of biomass and identified the gene Tr103275, homologous to the Azf1 regulator of Saccharomyces cerevisiae. Posteriorly, a mutant strain with deletion of azf1 was obtained. During growth in cellulose and sugarcane bagasse, azf1 was highly induced in T. reesei, while glucose repressed the gene expression of this transcription factor. The absence of azf1 affected growth and sporulation in T. reesei, since the mutant strain produced less and smaller spores relative to the parental strain. In addition, we have shown that TrAzf1 acts as a positive regulator of the expression of holocellulolytic enzymes, whereas the genes coding for CBHs, EGs, BGs and xyn4 were down-regulated in the Δazf1 mutant in cellulose and sugarcane bagasse. The binding site for TrAzf1 was predicted in this work through bioinformatics analysis. To evaluate the interaction of TrAzf1 with this DNA binding site, the recombinant Azf1 zinc finger motif was obtained by heterologous expression in E. coli and used in the SPR analyzes. However, validation of the transcription factor-DNA interaction by SPR was not conclusive and other approaches should be performed to validate the binding motif of TrAzf1 in T. reesei. The role of Azf1 in regulating the degradation of plant biomass was described for the first time in this work. This transcription factor is a potential target for the genetic manipulation of T. reesei, aiming to improve the production of holocellulolytic enzymes by this species.

O fungo filamentoso Trichoderma reesei é um dos principais fungos utilizados para a produção em larga escala de enzimas devido a sua grande capacidade de produção e secreção de holocelulases para aplicação em processos de sacarificação da biomassa vegetal lignocelulósica. Embora o T. reesei seja utilizado como um dos principais produtores de celulases a nível industrial, diversos processos e estudos são realizados com o objetivo de aprimorar o entendimento de todo o mecanismo de degradação de biomassa vegetal além de prover o aumento da eficiência tanto da produção quanto da atividade das celulases. No presente trabalho foi realizado a construção de uma linhagem mutante para o gene cel74a (codificando para uma xiloglucanase) e a caracterização funcional de CEL74A na regulação gênica de holocelulases durante o cultivo em bagaço de cana-de-açúcar. Os nossos resultados mostraram que deleção de cel74a possivelmente pode estar envolvida no sinergismo da regulação da expressão de holocelulases durante o cultivo em bagaço de cana-de-açúcar. A partir da análise do perfil de expressão gênica, foi possível observar a redução na expressão de todos os genes de celulases testados (cel7a, cel7b e cel6a) embora não tenha afetado a atividade enzimática, ao passo que as hemicelulases (xyn1 e xyn2) apresentaram aumento tanto na expressão quanto na atividade enzimática. Na linhagem ?cel74a, foi observado redução na liberação de glicose, xilose e galactose após a hidrólise de xiloglucano. Além disso, a atividade de CEL74A foi modulada na presença de cálcio e pode ser necessária para a atuação mais eficiente de outras enzimas envolvidas na degradação de xiloglucano. Desta forma, em T. reesei, CEL74A apresenta um papel importante tanto na regulação de genes holocelulolíticos quanto para a degradação eficiente de bagaço de cana-de-açúcar, contribuindo para a elucidação de mecanismos pelos quais este fungo utiliza para a utilização do bagaço de cana-de-açúcar como fonte de carbono The filamentous fungus Trichoderma reesei is one of the main fungi used for the large-scale production of enzymes due to their great capacity of production and secretion of holocellulases for application in saccharification processes of lignocellulosic plant biomass. Although T. reesei is used as one of the main producers of cellulases at industrial level, several processes and studies are carried out with the aim of improving the understanding of the whole plant biomass degradation mechanism, as well as increasing the efficiency of both the production and cellulase activity. In the present work the construction of a mutant lineage for the cel74a gene (coding for a xyloglucanase) and the functional characterization of CEL74A in the gene regulation of holocellulases during the cultivation of sugarcane bagasse were carried out. Our results showed that deletion of cel74a may be involved in the regulation of holocellulase expression during sugarcane bagasse cultivation. From the analysis of the gene expression profile, it was possible to observe the reduction in the expression of all tested cellulase genes (cel7a, cel7b and cel6a), although it did not affect the enzymatic activity, whereas the hemicellulases (xyn1 and xyn2) presented increase in both expression and enzymatic activity. In the ?cel74a strain, a reduction in glucose, xylose and galactose release was observed after xyloglucan hydrolysis. In addition, the activity of CEL74A was modulated in the presence of calcium and may be required for the more efficient performance of other enzymes involved in the degradation of xyloglucan. Thus, in T. reesei, CEL74A plays an important role both in the regulation of holocellulolytic genes and in the efficient degradation of sugarcane bagasse, contributing to the elucidation of mechanisms by which this fungus uses for the use of sugarcane bagasse as source of carbon