Patrick Silva | FACCAT - Academia.edu (original) (raw)

Papers by Patrick Silva

Anais Do Simposio Brasileiro De Informatica Na Educacao, 2014

Resumo. O déficit de aprendizado da matemática é um dos grandes desafios que as escolas têm enfre... more Resumo. O déficit de aprendizado da matemática é um dos grandes desafios que as escolas têm enfrentado. Neste contexto, os jogos digitais, mostram-se excelentes alternativas a fim de potencializar a abstração de conhecimento de uma geração que nasceu em meio à tecnologia. Este trabalho visou desenvolver um jogo digital, para crianças, entre 7 a 9 anos, que as permitisse exercitar os conceitos de adição e subtração. O software, que foi aplicado em uma escola, apresentou resultados positivos e foi bem recebido pelas crianças que se sentiram motivadas em calcular.

Speech processing is a data-driven technology that relies on public corpora and associated resour... more Speech processing is a data-driven technology that relies on public corpora and associated resources. In contrast to languages such as English, there are few resources for Brazilian Portuguese (BP). This work describes efforts toward decreasing such gap and presents systems for speech recognition in BP using two public corpora: Spoltech and OGI-22. The following resources are made available: ATK and HTK scripts, pronunciation dictionary, language and acoustic models. The work discusses the baseline results obtained with these resources.

J Biol Chem, 2000

Diphtheria toxin (DT) is a disulfide linked AB-toxin consisting of a catalytic domain (C), a memb... more Diphtheria toxin (DT) is a disulfide linked AB-toxin consisting of a catalytic domain (C), a membrane-inserting domain (T), and a receptor-binding domain (R). It gains entry into cells by receptor-mediated endocytosis. The low pH ( approximately 5.5) inside the endosomes induces a conformational change in the toxin leading to insertion of the toxin in the membrane and subsequent translocation of the C domain into the cell, where it inactivates protein synthesis ultimately leading to cell death. We have used a highly reactive hydrophobic photoactivable reagent, DAF, to identify the segments of DT that interact with the membrane at pH 5.2. This reagent readily partitions into membranes and, on photolysis, indiscriminately inserts into lipids and membrane-inserted domains of proteins. Subsequent chemical and/or enzymatic fragmentation followed by peptide sequencing allows for identification of the modified residues. Using this approach it was observed that T domain helices, TH1, TH8, and TH9 insert into the membrane. Furthermore, the disulfide link was found on the trans side leaving part of the C domain on the trans side. This domain then comes out to the cis side via a highly hydrophobic patch corresponding to residues 134-141, originally corresponding to a beta-strand in the solution structure of DT. It appears that the three helices of the T domain could participate in the formation of a channel from a DT-oligomer, thus providing the transport route to the C domain after the disulfide reductase separates the two chains.

Protein Sci, 1999

Recent advances in attempts to unravel the protein folding mechanism have indicated the need to i... more Recent advances in attempts to unravel the protein folding mechanism have indicated the need to identify the folding intermediates. Despite their transient nature, in a number of cases it has been possible to detect and characterize some of the equilibrium intermediates, for example, the molten globule~MG! state. The key features of the MG state are retention of substantial secondary structure of the native state, considerable loss of tertiary structure leading to increased hydrophobic exposure, and a compact structure. NMR, circular dichroism, and fluorescence spectroscopies have been most useful in characterizing such intermediates. We report here a new method for structural characterization of the MG state that involves probing the exposed hydrophobic sites with a hydrophobic photoactivable reagent-2@ 3 H#diazofluorene. This carbene-based reagent binds to hydrophobic sites, and on photolysis covalently attaches itself to the neighboring amino acid side chains. The reagent photolabels a-lactalbumin as a function of pH~3-7.4!, the labeling at neutral pH being negligible and maximal at pH 3. Chemical and proteolytic fragmentation of the photolabeled protein followed by peptide sequencing permitted identification of the labeled residues. The results obtained indicate that the sequence corresponding to B~23-34! and C~86-98! helix of the native structure are extensively labeled. The small b-domaiñ 40-50! is poorly labeled, Val42 being the only residue that is significantly labeled. Our data, like NMR data, indicate that in the MG state of a-lactalbumin, the a-domain has a greater degree of persistent structure than the b-domain. However, unlike the NMR method, the photolabeling method is not limited by the size of the protein and can provide information on several new residues, for example, Leu115. The current method using DAF thus allows identification of stable and hydrophobic exposed regions in folding intermediates as the reagent binds and on photolysis covalently links to these regions.

American Journal of Plant Sciences, 2014

Microorganism resistance to the existing products is yet another difficulty that agriculturalists... more Microorganism resistance to the existing products is yet another difficulty that agriculturalists have to deal with. In this context, the search for new agricultural products that can fight phytopathogens has become increasingly important. Plants have played an important role in this process, because they can serve as a source of new compounds for drug discovery. Plants belonging to the genus Pinus produce an oleoresin that protects the plant against herbivores and pathogens. With a view to developing products that can combat fungal pathogens without harming the environment, this work aimed to evaluate the antifungal activity of the oleoresins and fractions of Pinus elliottii Engelm and Pinus tropicalis against phytopathogens. The methodology based on NCCLS M38-A standards aided antifungal activity assessment. The microdilution method helped to determine the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC). The oleoresins of P. elliottii and P. tropicalis afforded the most significant results-they displayed fungicidal activity against all the tested species. MIC values were promising, especially the MIC of the oleoresin of P. elliottii against S. rolfsii (1.95 µg•mL −1 ). The MIC values of the oleoresins of P. elliottii and P. tropicalis ranged from 1.95 to 1000 µg•mL −1 and from 31.25 to 250 μg•mL −1 , respectively. Fraction PT2 of P. tropicalis furnished the best results among all the assayed fractions: MIC values lay between 125 and 500 µg•mL −1 . In conclusion, the oleoresin of P. tropicalis is a promising source of new antifungal agents for application in the treatment of phytopathogenic infections.

Speech processing is a data-driven technology that relies on public corpora and associated resour... more Speech processing is a data-driven technology that relies on public corpora and associated resources. In contrast to languages such as English, there are few resources for Brazilian Portuguese (BP). This work describes efforts toward decreasing such gap and presents systems for speech recognition in BP using two public corpora: Spoltech and OGI-22. The following resources are made available: HTK scripts, pronunciation dictionary, language and acoustic models. The work discusses the baseline results obtained with these resources.

Collected Essays on Learning and Teaching, Jun 17, 2013

1 Humber College has two writing streams, ESL and COMM, into which students are streamed by means... more 1 Humber College has two writing streams, ESL and COMM, into which students are streamed by means of an English placement test. "If the writing sample displays ESL characteristics, the student will be placed in an appropriate level ESL course" ("Entrance/ Placement Testing," n.d.). Students streamed into the ESL branch of the program complete their course work within the ESL stream for the remainder of their program.

Mol Cell, 2005

Communication between the ATPase and substrate binding domains of Hsp70 is critical for regulated... more Communication between the ATPase and substrate binding domains of Hsp70 is critical for regulated interaction between this molecular chaperone and its client proteins. In this issue of Molecular Cell, Jiang et al. (2005) report the structure of an intact Hsp70, revealing critical interactions between the two domains.

Molecular biology of the cell, 2008

Import of proteins across the inner mitochondrial membrane through the Tim23:Tim17 translocase re... more Import of proteins across the inner mitochondrial membrane through the Tim23:Tim17 translocase requires the function of an essential import motor having mitochondrial 70-kDa heat-shock protein (mtHsp70) at its core. The heterodimer composed of Pam18, the J-protein partner of mtHsp70, and the related protein Pam16 is a critical component of this motor. We report that three interactions contribute to association of the heterodimer with the translocon: the N terminus of Pam16 with the matrix side of the translocon, the inner membrane space domain of Pam18 (Pam18(IMS)) with Tim17, and the direct interaction of the J-domain of Pam18 with the J-like domain of Pam16. Pam16 plays a major role in translocon association, as alterations affecting the stability of the Pam18:Pam16 heterodimer dramatically affect association of Pam18, but not Pam16, with the translocon. Suppressors of the growth defects caused by alterations in the N terminus of Pam16 were isolated and found to be due to mutation...

Lecture Notes in Computer Science, 2008

Speech processing is a data-driven technology that relies on public corpora and associated resour... more Speech processing is a data-driven technology that relies on public corpora and associated resources. In contrast to languages such as English, there are few resources for Brazilian Portuguese (BP). This work describes efforts toward decreasing such gap and presents systems for speech recognition in BP using two public corpora: Spoltech and OGI-22. The following resources are made available: HTK scripts, pronunciation dictionary, language and acoustic models. The work discusses the baseline results obtained with these resources.

Lecture Notes in Computer Science, 2010

This work is part of the effort to develop a speech recognition system for Brazilian Portuguese. ... more This work is part of the effort to develop a speech recognition system for Brazilian Portuguese. The resources for the training and test stages of this system, such as corpora, pronunciation dictionary, language and acoustic models, are publicly available. Here, an application programming interface is proposed in order to facilitate using the opensource Julius speech decoder. Performance tests are presented, comparing the developed systems with a commercial software.

Nature Structural & Molecular Biology, 2004

Preproteins synthesized on cytosolic ribosomes, but destined for the mitochondrial matrix, pass t... more Preproteins synthesized on cytosolic ribosomes, but destined for the mitochondrial matrix, pass through the presequence translocase of the inner membrane. Translocation is driven by the import motor, having at its core the essential chaperone mtHsp70 (Ssc1 in yeast). MtHsp70 is tethered to the translocon channel at the matrix side of the inner membrane by the peripheral membrane protein Tim44. A key question in mitochondrial import is how the mtHsp70-Tim44 interaction is regulated. Here we report that Tim44 interacts with both the ATPase and peptide-binding domains of mtHsp70. Disruption of these interactions upon binding of polypeptide substrates requires concerted conformational changes involving both domains of mtHsp70. Our results fit a model in which regulated interactions between Tim44 and mtHsp70, controlled by polypeptide binding, are required for efficient translocation across the mitochondrial inner membrane in vivo.

Molecular and Cellular Biology, 2013

Tim23 is an essential channel-forming subunit of the presequence translocase recruiting multiple ... more Tim23 is an essential channel-forming subunit of the presequence translocase recruiting multiple components for assembly of the core complex, thereby regulating the protein translocation process. However, understanding of the precise interaction of subunits associating with Tim23 remains largely elusive. Our findings highlight that transmembrane helix 1 (TM1) is required for homodimerization of Tim23, while, together with TM2, it is involved in preprotein binding within the channel. Based on our evidence, we predict that the TM1 and TM2 from each dimer are involved in the formation of the central translocation pore, aided by Tim17. Furthermore, TM2 is also involved in the recruitment of Tim21 and the presequence-associated motor (PAM) subcomplex to the Tim23 channel, while the matrix-exposed loop L1 generates specificity in their association with the core complex. Strikingly, our findings indicate that the C-terminal sequence of Tim23 is dispensable for growth and functions as an inhibitor for binding of Tim21. Our model conceptually explains the cooperative function between Tam41 and Pam17 subunits, while the antagonistic activity of Tim21 predominantly determines the bound and free forms of the PAM subcomplex during import.

Molecular and Cellular Biology, 2014

Mitochondria are indispensable organelles implicated in multiple aspects of cellular 22 processes... more Mitochondria are indispensable organelles implicated in multiple aspects of cellular 22 processes, including tumorigenesis. Heat-Shock Proteins play a critical regulatory role in 23 accurately delivering the nuclear encoded proteins through membrane-bound presequence 24 translocase (Tim23-complex) machinery. Although, altered expression of mammalian 25 presequence translocase components had been previously associated with a malignant 26 phenotype, the overall organization of Tim23-complexes is still unsolved. In this report, we 27

Journal of Biological Chemistry, 1998

We report here the use of a hydrophobic photoactivable reagent, 2-[ 3 H]diazofluorene (DAF), to m... more We report here the use of a hydrophobic photoactivable reagent, 2-[ 3 H]diazofluorene (DAF), to map the hydrophobic sites exposed when the pH is lowered in diphtheria toxin (DT). The reagent binds to DT, and on photolysis with light of wavelength >350 nm, it covalently attaches itself to DT. The labeling was observed to increase considerably when the pH was lowered from 7.4 to 5.2. Although both A-and B-chains were labeled to a similar degree at pH 7.4, at lower pH (5.2), B-chain was labeled to a much higher extent. Subsequent chemical and enzymatic fragmentation of DT followed by separation indicated that the putative transmembrane domain was labeled to its maximum extent at pH 5.2, with the bulk of labeling associated with residues 340 -459. Protein sequencing analysis indicated that the two buried hydrophobic helices, identified in the crystal structure and suggested to insert and span the membrane bilayer, corresponding to residues 326 -347 and 358 -376, are strongly labeled. The Pro-345 residue was observed to be labeled maximally at lower pH values. Finally, the DAF labeling pattern indicated that the parent structural motifs are retained at low pH, suggesting that the low pH conformation of DT corresponds to an equilibrium molten globule state.

Human Molecular Genetics, 2010

Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in p... more Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in prostate carcinomas and cells exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF). Reduced Magmas expression resulted in decreased proliferative rates in cultured cells. However, the cellular function of Magmas is still elusive. In this report, we have showed that human Magmas is an ortholog of Saccharomyces cerevisiae Pam16 having similar functions and is critical for protein translocation across mitochondrial inner membrane. Human Magmas shows a complete growth complementation of Dpam16 yeast cells at all temperatures. On the basis of our analysis, we report that Magmas localizes into mitochondria and is peripherally associated with inner mitochondrial membrane in yeast and humans. Magmas forms a stable subcomplex with J-protein Pam18 or DnaJC19 through its C-terminal region and is tethered to TIM23 complex of yeast and humans. Importantly, amino acid alterations in Magmas leads to reduced stability of the subcomplex with Pam18 that results in temperature sensitivity and in vivo protein translocation defects in yeast cells. These observations highlight the central role of Magmas in protein import and mitochondria biogenesis. In humans, absence of a functional DnaJC19 leads to dilated cardiac myophathic syndrome (DCM), a genetic disorder with characteristic features of cardiac myophathy and neurodegeneration. We propose that the mutations resulting in decreased stability of functional Magmas:DnaJC19 subcomplex at human TIM23 channel leads to impaired protein import and cellular respiration in DCM patients. Together, we propose a model showing how Magmas:DnaJC19 subcomplex is associated with TIM23 complex and thus regulates mitochondrial import process.

Functional & Integrative Genomics, 2009

Plants are sessile organisms that have evolved a variety of mechanisms to maintain their cellular... more Plants are sessile organisms that have evolved a variety of mechanisms to maintain their cellular homeostasis under stressful environmental conditions. Survival of plants under abiotic stress conditions requires specialized group of heat shock protein machinery, belonging to Hsp70:J-protein family. These heat shock proteins are most ubiquitous types of chaperone machineries involved in diverse cellular processes including protein folding, translocation across cell membranes, and protein degradation. They play a crucial role in maintaining the protein homeostasis by reestablishing functional native conformations under environmental stress conditions, thus providing protection to the cell. J-proteins are co-chaperones of Hsp70 machine, which play a critical role by stimulating Hsp70s ATPase activity, thereby stabilizing its interaction with client proteins. Using genome-wide analysis of Arabidopsis thaliana, here we have outlined identification and systematic classification of J-protein co-chaperones which are key regulators of Hsp70s function. In comparison with Saccharomyces cerevisiae model system, a comprehensive domain structural organization, cellular localization, and functional diversity of A. thaliana J-proteins have also been summarized.

Environmental Geochemistry and Health, 2013

In this study, we investigated the toxic effects of water-soluble elements from a contaminated so... more In this study, we investigated the toxic effects of water-soluble elements from a contaminated soil via gavage in a single dose, simulating a geophagy event. The contaminated soil was collected in a field located in an industrial complex, and the control soil was collected in a reference area. Metabolic and behavioral parameters in Wistar male rats were measured after 24 and 96 h of gavage. After 96 h, the major organs were weighed, blood was collected to check hematological parameters, the bone marrow was taken for the micronucleus test, and the liver was used for evaluating the total antioxidant capacity, lipoperoxidation and protein carbonylation. Animals exposed to contaminated soil presented a few significant alterations by comparison with control animals: TBARS and protein carbonyl levels increased, the relative weight of the kidneys increased, metabolic parameters (body weight gain, food intake, water consumption, urine and feces production) depressed and there was behavioral alteration. These findings suggest that soils impacted by atmospheric contaminants can affect the organism physiological status jeopardizing the health of populations living in industrial areas. Finally, this study reassures that ingestion of potentially contaminated soils, even for short periods of time, can cause health risks.

Bioinformatics, 2012

Heat shock protein information resource (HSPIR) is a concerted database of six major heat shock p... more Heat shock protein information resource (HSPIR) is a concerted database of six major heat shock proteins (HSPs), namely, Hsp70, Hsp40, Hsp60, Hsp90, Hsp100 and small HSP. The HSPs are essential for the survival of all living organisms, as they protect the conformations of proteins on exposure to various stress conditions. They are a highly conserved group of proteins involved in diverse physiological functions, including de novo folding, disaggregation and protein trafficking. Moreover, their critical role in the control of disease progression made them a prime target of research. Presently, limited information is available on HSPs in reference to their identification and structural classification across genera. To that extent, HSPIR provides manually curated information on sequence, structure, classification, ontology, domain organization, localization and possible biological functions extracted from UniProt, GenBank, Protein Data Bank and the literature. The database offers interactive search with incorporated tools, which enhances the analysis. HSPIR is a reliable resource for researchers exploring structure, function and evolution of HSPs.

Anais Do Simposio Brasileiro De Informatica Na Educacao, 2014

Resumo. O déficit de aprendizado da matemática é um dos grandes desafios que as escolas têm enfre... more Resumo. O déficit de aprendizado da matemática é um dos grandes desafios que as escolas têm enfrentado. Neste contexto, os jogos digitais, mostram-se excelentes alternativas a fim de potencializar a abstração de conhecimento de uma geração que nasceu em meio à tecnologia. Este trabalho visou desenvolver um jogo digital, para crianças, entre 7 a 9 anos, que as permitisse exercitar os conceitos de adição e subtração. O software, que foi aplicado em uma escola, apresentou resultados positivos e foi bem recebido pelas crianças que se sentiram motivadas em calcular.

Speech processing is a data-driven technology that relies on public corpora and associated resour... more Speech processing is a data-driven technology that relies on public corpora and associated resources. In contrast to languages such as English, there are few resources for Brazilian Portuguese (BP). This work describes efforts toward decreasing such gap and presents systems for speech recognition in BP using two public corpora: Spoltech and OGI-22. The following resources are made available: ATK and HTK scripts, pronunciation dictionary, language and acoustic models. The work discusses the baseline results obtained with these resources.

J Biol Chem, 2000

Diphtheria toxin (DT) is a disulfide linked AB-toxin consisting of a catalytic domain (C), a memb... more Diphtheria toxin (DT) is a disulfide linked AB-toxin consisting of a catalytic domain (C), a membrane-inserting domain (T), and a receptor-binding domain (R). It gains entry into cells by receptor-mediated endocytosis. The low pH ( approximately 5.5) inside the endosomes induces a conformational change in the toxin leading to insertion of the toxin in the membrane and subsequent translocation of the C domain into the cell, where it inactivates protein synthesis ultimately leading to cell death. We have used a highly reactive hydrophobic photoactivable reagent, DAF, to identify the segments of DT that interact with the membrane at pH 5.2. This reagent readily partitions into membranes and, on photolysis, indiscriminately inserts into lipids and membrane-inserted domains of proteins. Subsequent chemical and/or enzymatic fragmentation followed by peptide sequencing allows for identification of the modified residues. Using this approach it was observed that T domain helices, TH1, TH8, and TH9 insert into the membrane. Furthermore, the disulfide link was found on the trans side leaving part of the C domain on the trans side. This domain then comes out to the cis side via a highly hydrophobic patch corresponding to residues 134-141, originally corresponding to a beta-strand in the solution structure of DT. It appears that the three helices of the T domain could participate in the formation of a channel from a DT-oligomer, thus providing the transport route to the C domain after the disulfide reductase separates the two chains.

Protein Sci, 1999

Recent advances in attempts to unravel the protein folding mechanism have indicated the need to i... more Recent advances in attempts to unravel the protein folding mechanism have indicated the need to identify the folding intermediates. Despite their transient nature, in a number of cases it has been possible to detect and characterize some of the equilibrium intermediates, for example, the molten globule~MG! state. The key features of the MG state are retention of substantial secondary structure of the native state, considerable loss of tertiary structure leading to increased hydrophobic exposure, and a compact structure. NMR, circular dichroism, and fluorescence spectroscopies have been most useful in characterizing such intermediates. We report here a new method for structural characterization of the MG state that involves probing the exposed hydrophobic sites with a hydrophobic photoactivable reagent-2@ 3 H#diazofluorene. This carbene-based reagent binds to hydrophobic sites, and on photolysis covalently attaches itself to the neighboring amino acid side chains. The reagent photolabels a-lactalbumin as a function of pH~3-7.4!, the labeling at neutral pH being negligible and maximal at pH 3. Chemical and proteolytic fragmentation of the photolabeled protein followed by peptide sequencing permitted identification of the labeled residues. The results obtained indicate that the sequence corresponding to B~23-34! and C~86-98! helix of the native structure are extensively labeled. The small b-domaiñ 40-50! is poorly labeled, Val42 being the only residue that is significantly labeled. Our data, like NMR data, indicate that in the MG state of a-lactalbumin, the a-domain has a greater degree of persistent structure than the b-domain. However, unlike the NMR method, the photolabeling method is not limited by the size of the protein and can provide information on several new residues, for example, Leu115. The current method using DAF thus allows identification of stable and hydrophobic exposed regions in folding intermediates as the reagent binds and on photolysis covalently links to these regions.

American Journal of Plant Sciences, 2014

Microorganism resistance to the existing products is yet another difficulty that agriculturalists... more Microorganism resistance to the existing products is yet another difficulty that agriculturalists have to deal with. In this context, the search for new agricultural products that can fight phytopathogens has become increasingly important. Plants have played an important role in this process, because they can serve as a source of new compounds for drug discovery. Plants belonging to the genus Pinus produce an oleoresin that protects the plant against herbivores and pathogens. With a view to developing products that can combat fungal pathogens without harming the environment, this work aimed to evaluate the antifungal activity of the oleoresins and fractions of Pinus elliottii Engelm and Pinus tropicalis against phytopathogens. The methodology based on NCCLS M38-A standards aided antifungal activity assessment. The microdilution method helped to determine the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC). The oleoresins of P. elliottii and P. tropicalis afforded the most significant results-they displayed fungicidal activity against all the tested species. MIC values were promising, especially the MIC of the oleoresin of P. elliottii against S. rolfsii (1.95 µg•mL −1 ). The MIC values of the oleoresins of P. elliottii and P. tropicalis ranged from 1.95 to 1000 µg•mL −1 and from 31.25 to 250 μg•mL −1 , respectively. Fraction PT2 of P. tropicalis furnished the best results among all the assayed fractions: MIC values lay between 125 and 500 µg•mL −1 . In conclusion, the oleoresin of P. tropicalis is a promising source of new antifungal agents for application in the treatment of phytopathogenic infections.

Speech processing is a data-driven technology that relies on public corpora and associated resour... more Speech processing is a data-driven technology that relies on public corpora and associated resources. In contrast to languages such as English, there are few resources for Brazilian Portuguese (BP). This work describes efforts toward decreasing such gap and presents systems for speech recognition in BP using two public corpora: Spoltech and OGI-22. The following resources are made available: HTK scripts, pronunciation dictionary, language and acoustic models. The work discusses the baseline results obtained with these resources.

Collected Essays on Learning and Teaching, Jun 17, 2013

1 Humber College has two writing streams, ESL and COMM, into which students are streamed by means... more 1 Humber College has two writing streams, ESL and COMM, into which students are streamed by means of an English placement test. "If the writing sample displays ESL characteristics, the student will be placed in an appropriate level ESL course" ("Entrance/ Placement Testing," n.d.). Students streamed into the ESL branch of the program complete their course work within the ESL stream for the remainder of their program.

Mol Cell, 2005

Communication between the ATPase and substrate binding domains of Hsp70 is critical for regulated... more Communication between the ATPase and substrate binding domains of Hsp70 is critical for regulated interaction between this molecular chaperone and its client proteins. In this issue of Molecular Cell, Jiang et al. (2005) report the structure of an intact Hsp70, revealing critical interactions between the two domains.

Molecular biology of the cell, 2008

Import of proteins across the inner mitochondrial membrane through the Tim23:Tim17 translocase re... more Import of proteins across the inner mitochondrial membrane through the Tim23:Tim17 translocase requires the function of an essential import motor having mitochondrial 70-kDa heat-shock protein (mtHsp70) at its core. The heterodimer composed of Pam18, the J-protein partner of mtHsp70, and the related protein Pam16 is a critical component of this motor. We report that three interactions contribute to association of the heterodimer with the translocon: the N terminus of Pam16 with the matrix side of the translocon, the inner membrane space domain of Pam18 (Pam18(IMS)) with Tim17, and the direct interaction of the J-domain of Pam18 with the J-like domain of Pam16. Pam16 plays a major role in translocon association, as alterations affecting the stability of the Pam18:Pam16 heterodimer dramatically affect association of Pam18, but not Pam16, with the translocon. Suppressors of the growth defects caused by alterations in the N terminus of Pam16 were isolated and found to be due to mutation...

Lecture Notes in Computer Science, 2008

Speech processing is a data-driven technology that relies on public corpora and associated resour... more Speech processing is a data-driven technology that relies on public corpora and associated resources. In contrast to languages such as English, there are few resources for Brazilian Portuguese (BP). This work describes efforts toward decreasing such gap and presents systems for speech recognition in BP using two public corpora: Spoltech and OGI-22. The following resources are made available: HTK scripts, pronunciation dictionary, language and acoustic models. The work discusses the baseline results obtained with these resources.

Lecture Notes in Computer Science, 2010

This work is part of the effort to develop a speech recognition system for Brazilian Portuguese. ... more This work is part of the effort to develop a speech recognition system for Brazilian Portuguese. The resources for the training and test stages of this system, such as corpora, pronunciation dictionary, language and acoustic models, are publicly available. Here, an application programming interface is proposed in order to facilitate using the opensource Julius speech decoder. Performance tests are presented, comparing the developed systems with a commercial software.

Nature Structural & Molecular Biology, 2004

Preproteins synthesized on cytosolic ribosomes, but destined for the mitochondrial matrix, pass t... more Preproteins synthesized on cytosolic ribosomes, but destined for the mitochondrial matrix, pass through the presequence translocase of the inner membrane. Translocation is driven by the import motor, having at its core the essential chaperone mtHsp70 (Ssc1 in yeast). MtHsp70 is tethered to the translocon channel at the matrix side of the inner membrane by the peripheral membrane protein Tim44. A key question in mitochondrial import is how the mtHsp70-Tim44 interaction is regulated. Here we report that Tim44 interacts with both the ATPase and peptide-binding domains of mtHsp70. Disruption of these interactions upon binding of polypeptide substrates requires concerted conformational changes involving both domains of mtHsp70. Our results fit a model in which regulated interactions between Tim44 and mtHsp70, controlled by polypeptide binding, are required for efficient translocation across the mitochondrial inner membrane in vivo.

Molecular and Cellular Biology, 2013

Tim23 is an essential channel-forming subunit of the presequence translocase recruiting multiple ... more Tim23 is an essential channel-forming subunit of the presequence translocase recruiting multiple components for assembly of the core complex, thereby regulating the protein translocation process. However, understanding of the precise interaction of subunits associating with Tim23 remains largely elusive. Our findings highlight that transmembrane helix 1 (TM1) is required for homodimerization of Tim23, while, together with TM2, it is involved in preprotein binding within the channel. Based on our evidence, we predict that the TM1 and TM2 from each dimer are involved in the formation of the central translocation pore, aided by Tim17. Furthermore, TM2 is also involved in the recruitment of Tim21 and the presequence-associated motor (PAM) subcomplex to the Tim23 channel, while the matrix-exposed loop L1 generates specificity in their association with the core complex. Strikingly, our findings indicate that the C-terminal sequence of Tim23 is dispensable for growth and functions as an inhibitor for binding of Tim21. Our model conceptually explains the cooperative function between Tam41 and Pam17 subunits, while the antagonistic activity of Tim21 predominantly determines the bound and free forms of the PAM subcomplex during import.

Molecular and Cellular Biology, 2014

Mitochondria are indispensable organelles implicated in multiple aspects of cellular 22 processes... more Mitochondria are indispensable organelles implicated in multiple aspects of cellular 22 processes, including tumorigenesis. Heat-Shock Proteins play a critical regulatory role in 23 accurately delivering the nuclear encoded proteins through membrane-bound presequence 24 translocase (Tim23-complex) machinery. Although, altered expression of mammalian 25 presequence translocase components had been previously associated with a malignant 26 phenotype, the overall organization of Tim23-complexes is still unsolved. In this report, we 27

Journal of Biological Chemistry, 1998

We report here the use of a hydrophobic photoactivable reagent, 2-[ 3 H]diazofluorene (DAF), to m... more We report here the use of a hydrophobic photoactivable reagent, 2-[ 3 H]diazofluorene (DAF), to map the hydrophobic sites exposed when the pH is lowered in diphtheria toxin (DT). The reagent binds to DT, and on photolysis with light of wavelength >350 nm, it covalently attaches itself to DT. The labeling was observed to increase considerably when the pH was lowered from 7.4 to 5.2. Although both A-and B-chains were labeled to a similar degree at pH 7.4, at lower pH (5.2), B-chain was labeled to a much higher extent. Subsequent chemical and enzymatic fragmentation of DT followed by separation indicated that the putative transmembrane domain was labeled to its maximum extent at pH 5.2, with the bulk of labeling associated with residues 340 -459. Protein sequencing analysis indicated that the two buried hydrophobic helices, identified in the crystal structure and suggested to insert and span the membrane bilayer, corresponding to residues 326 -347 and 358 -376, are strongly labeled. The Pro-345 residue was observed to be labeled maximally at lower pH values. Finally, the DAF labeling pattern indicated that the parent structural motifs are retained at low pH, suggesting that the low pH conformation of DT corresponds to an equilibrium molten globule state.

Human Molecular Genetics, 2010

Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in p... more Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in prostate carcinomas and cells exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF). Reduced Magmas expression resulted in decreased proliferative rates in cultured cells. However, the cellular function of Magmas is still elusive. In this report, we have showed that human Magmas is an ortholog of Saccharomyces cerevisiae Pam16 having similar functions and is critical for protein translocation across mitochondrial inner membrane. Human Magmas shows a complete growth complementation of Dpam16 yeast cells at all temperatures. On the basis of our analysis, we report that Magmas localizes into mitochondria and is peripherally associated with inner mitochondrial membrane in yeast and humans. Magmas forms a stable subcomplex with J-protein Pam18 or DnaJC19 through its C-terminal region and is tethered to TIM23 complex of yeast and humans. Importantly, amino acid alterations in Magmas leads to reduced stability of the subcomplex with Pam18 that results in temperature sensitivity and in vivo protein translocation defects in yeast cells. These observations highlight the central role of Magmas in protein import and mitochondria biogenesis. In humans, absence of a functional DnaJC19 leads to dilated cardiac myophathic syndrome (DCM), a genetic disorder with characteristic features of cardiac myophathy and neurodegeneration. We propose that the mutations resulting in decreased stability of functional Magmas:DnaJC19 subcomplex at human TIM23 channel leads to impaired protein import and cellular respiration in DCM patients. Together, we propose a model showing how Magmas:DnaJC19 subcomplex is associated with TIM23 complex and thus regulates mitochondrial import process.

Functional & Integrative Genomics, 2009

Plants are sessile organisms that have evolved a variety of mechanisms to maintain their cellular... more Plants are sessile organisms that have evolved a variety of mechanisms to maintain their cellular homeostasis under stressful environmental conditions. Survival of plants under abiotic stress conditions requires specialized group of heat shock protein machinery, belonging to Hsp70:J-protein family. These heat shock proteins are most ubiquitous types of chaperone machineries involved in diverse cellular processes including protein folding, translocation across cell membranes, and protein degradation. They play a crucial role in maintaining the protein homeostasis by reestablishing functional native conformations under environmental stress conditions, thus providing protection to the cell. J-proteins are co-chaperones of Hsp70 machine, which play a critical role by stimulating Hsp70s ATPase activity, thereby stabilizing its interaction with client proteins. Using genome-wide analysis of Arabidopsis thaliana, here we have outlined identification and systematic classification of J-protein co-chaperones which are key regulators of Hsp70s function. In comparison with Saccharomyces cerevisiae model system, a comprehensive domain structural organization, cellular localization, and functional diversity of A. thaliana J-proteins have also been summarized.

Environmental Geochemistry and Health, 2013

In this study, we investigated the toxic effects of water-soluble elements from a contaminated so... more In this study, we investigated the toxic effects of water-soluble elements from a contaminated soil via gavage in a single dose, simulating a geophagy event. The contaminated soil was collected in a field located in an industrial complex, and the control soil was collected in a reference area. Metabolic and behavioral parameters in Wistar male rats were measured after 24 and 96 h of gavage. After 96 h, the major organs were weighed, blood was collected to check hematological parameters, the bone marrow was taken for the micronucleus test, and the liver was used for evaluating the total antioxidant capacity, lipoperoxidation and protein carbonylation. Animals exposed to contaminated soil presented a few significant alterations by comparison with control animals: TBARS and protein carbonyl levels increased, the relative weight of the kidneys increased, metabolic parameters (body weight gain, food intake, water consumption, urine and feces production) depressed and there was behavioral alteration. These findings suggest that soils impacted by atmospheric contaminants can affect the organism physiological status jeopardizing the health of populations living in industrial areas. Finally, this study reassures that ingestion of potentially contaminated soils, even for short periods of time, can cause health risks.

Bioinformatics, 2012

Heat shock protein information resource (HSPIR) is a concerted database of six major heat shock p... more Heat shock protein information resource (HSPIR) is a concerted database of six major heat shock proteins (HSPs), namely, Hsp70, Hsp40, Hsp60, Hsp90, Hsp100 and small HSP. The HSPs are essential for the survival of all living organisms, as they protect the conformations of proteins on exposure to various stress conditions. They are a highly conserved group of proteins involved in diverse physiological functions, including de novo folding, disaggregation and protein trafficking. Moreover, their critical role in the control of disease progression made them a prime target of research. Presently, limited information is available on HSPs in reference to their identification and structural classification across genera. To that extent, HSPIR provides manually curated information on sequence, structure, classification, ontology, domain organization, localization and possible biological functions extracted from UniProt, GenBank, Protein Data Bank and the literature. The database offers interactive search with incorporated tools, which enhances the analysis. HSPIR is a reliable resource for researchers exploring structure, function and evolution of HSPs.