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Valéria Ferrer

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Thiago Lopes De Mari

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Papers by Valéria Ferrer

Identificação e avaliação de uma isoforma de hialuronidase (Dietrich's Hyaluronidase) do veneno de Loxosceles intermedia (aranha-marrom)

Aos meus pais e irmãos por sempre terem acreditado no meu potencial e não terem poupado esforços ... more Aos meus pais e irmãos por sempre terem acreditado no meu potencial e não terem poupado esforços para que eu chegasse até a esta tese! AGRADECIMENTOS Primeiramente gostaria de agradecer a Deus, por ter me fornecido saúde, disposição e intelectualidade para o desenvolvimento deste trabalho. Em alguns momentos difíceis que passei somente uma força Divina para fazer continuar e eu só tenho a agradecer. Ao meu orientador desde a iniciação científica, Prof. Dr. Silvio Sanches Veiga, pela oportunidade de fazer parte da equipe do "Laboratório de Matriz Extracelular e Biotecnologia de Venenos", a qual me orgulho tanto! Por ter me confiado este projeto e por meu crescimento científico adquirido durante esses anos. À minha co-orientadora, Profa. Dra. Andrea Senff Ribeiro, pelas várias reuniões de discussão científica, por ter confiado esse projeto a mim e por toda ajuda prestada na burocracia da patente desse trabalho. À minha co-orientadora, não no papel... mas de coração sempre, Profa. Dra. Olga Meiri Chaim! Por ter depositado toda a confiança em mim desde minha iniciação científica! A qualquer hora esteve disposta a ouvir e discutir assuntos relacionados ao laboratório fosse de origem científica ou organizacional. Muito obrigada! Levarei sua amizade sempre comigo! À Dra. Dilza Trevisan Silva! Bem Dil, sem comentários, né?! Companheira e amiga de todas as horas desde que eu entrei no laboratório! Muito obrigada por tudo! Pelas discussões científicas, pelos experimentos, cursos, disciplinas e tarefas realizados juntos... E claro, por sua amizade magnífica! À Profa. Dra. Luiza Helena Gremski! Lu, obrigada por ser este exemplo de cientista e pessoa! Eu só tenho a agradecer por toda a ajuda prestada durante todos esses anos nos experimentos, discussões científicas, correções de inglês, entre diversas outras tarefas! Muito obrigada por você sempre me escutar e ter acreditado no meu potencial! Ao querido e quase mestre Thiago Lopes de Mari! Obrigada por ter me ajudado tanto durante esses anos e também por sua amizade! Tenho muito orgulho de ter contribuído para sua formação científica e torço por uma carreira de muito sucesso pra você! Aos demais amigos do LME: MSc.

Identificação e avaliação de uma isoforma de hialuronidase (Dietrich´s hyaluronidase) do veneno de Loxosceles intermedia (aranha-marrom): da clonagem molecular à caracterização bioquímica e funcional

Aos meus pais e irmãos por sempre terem acreditado no meu potencial e não terem poupado esforços ... more Aos meus pais e irmãos por sempre terem acreditado no meu potencial e não terem poupado esforços para que eu chegasse até a esta tese! AGRADECIMENTOS Primeiramente gostaria de agradecer a Deus, por ter me fornecido saúde, disposição e intelectualidade para o desenvolvimento deste trabalho. Em alguns momentos difíceis que passei somente uma força Divina para fazer continuar e eu só tenho a agradecer. Ao meu orientador desde a iniciação científica, Prof. Dr. Silvio Sanches Veiga, pela oportunidade de fazer parte da equipe do "Laboratório de Matriz Extracelular e Biotecnologia de Venenos", a qual me orgulho tanto! Por ter me confiado este projeto e por meu crescimento científico adquirido durante esses anos. À minha co-orientadora, Profa. Dra. Andrea Senff Ribeiro, pelas várias reuniões de discussão científica, por ter confiado esse projeto a mim e por toda ajuda prestada na burocracia da patente desse trabalho. À minha co-orientadora, não no papel... mas de coração sempre, Profa. Dra. Olga Meiri Chaim! Por ter depositado toda a confiança em mim desde minha iniciação científica! A qualquer hora esteve disposta a ouvir e discutir assuntos relacionados ao laboratório fosse de origem científica ou organizacional. Muito obrigada! Levarei sua amizade sempre comigo! À Dra. Dilza Trevisan Silva! Bem Dil, sem comentários, né?! Companheira e amiga de todas as horas desde que eu entrei no laboratório! Muito obrigada por tudo! Pelas discussões científicas, pelos experimentos, cursos, disciplinas e tarefas realizados juntos... E claro, por sua amizade magnífica! À Profa. Dra. Luiza Helena Gremski! Lu, obrigada por ser este exemplo de cientista e pessoa! Eu só tenho a agradecer por toda a ajuda prestada durante todos esses anos nos experimentos, discussões científicas, correções de inglês, entre diversas outras tarefas! Muito obrigada por você sempre me escutar e ter acreditado no meu potencial! Ao querido e quase mestre Thiago Lopes de Mari! Obrigada por ter me ajudado tanto durante esses anos e também por sua amizade! Tenho muito orgulho de ter contribuído para sua formação científica e torço por uma carreira de muito sucesso pra você! Aos demais amigos do LME: MSc.

Glioblastoma Factors Increase the Migration of Human Brain Endothelial Cells In Vitro by Increasing MMP-9/CXCR4 Levels

Anticancer Research

Background/Aim: Glioblastoma (GB) is the most aggressive type of tumor in the central nervous sys... more Background/Aim: Glioblastoma (GB) is the most aggressive type of tumor in the central nervous system and is characterized by resistance to therapy and abundant vasculature. Tumor vessels contribute to the growth of GB, and the tumor microenvironment is thought to influence tumor vessels. We evaluated the molecular communication between human GB cells and human brain microvascular endothelial cells (HBMEC) in vitro. Materials and Methods: We investigated whether GB-conditioned media (GB-CM) influenced HBMEC proliferation and migration, as well as the levels of MMP-9, CXCL12, CXCR4, CXCR7, VEGFs, VEGFR-2, and WNT5a in HBMEC. Results: Although HBMEC proliferation was not modified, increased HBMEC migration was detected after GB-CM treatment. Furthermore, treatment of HBMEC with GB-CM resulted in increased levels of MMP-9 and CXCR4. The levels of WNT5a, VEGFs and VEGFR-2 were not affected. Conclusion: GB-secreted factors lead to increased endothelial cell migration and to increased levels of MMP-9 and CXCR4. Glioblastoma (GB) is the most common and deadly type of primary brain tumor in adults (1). The hallmarks of GB are its heterogeneity, the high proliferative rate, infiltration into the cerebral parenchyma (2), resistance to treatment (3-5), and high vascularization (6). The modest outcomes of antiangiogenic strategies in GB patients (7) and the lack of understanding of how tumors modify endothelial cells (EC) behavior have increased the interest in studying GB-EC molecular interactions to identify alternative angiogenic targets for the development of new therapeutic perspectives. The blood-brain barrier (BBB) is a complex structure formed by highly specialized brain microvascular endothelial cells (EC) with specific occlusive junctions (8, 9). Like other solid tumors, GB response to hypoxia involves the regulation of growth factors, chemokines and proteases, which induce alterations in the vessel structure by inducing the proliferation and migration of EC to form new blood vessels from preexisting vessels (10). Angiogenesis in GB occurs at a high rate and also results in the aberrant and subfunctional architecture of the tumor vasculature (11, 12). In addition to EC-dependent angiogenesis, vascular mimicry also contributes to GB vascularization, although to a lesser extent (13). These vascular-like channels can be formed independently of EC via the transdifferentiation of GB stem cells (GSCs) into pericytes (PC) or smooth muscle cells (SMC). In contrast, in the presence of EC, GSC-differentiated PC/SMC cells can interact intimately with EC to facilitate angiogenesis and produce more stable vessels, which perfuse tumors more efficiently (14, 15). These angiogenic processes highlight the idiosyncrasies and complexity of GB angiogenesis, which may influence tumor EC in specific ways. Therefore, studies of the behavior of EC in the context of GB angiogenesis are important. The principal and best-described angiogenic factor is vascular endothelial growth factor (VEGF), which has at least 6 isoforms in humans that are important for both embryonic development and tumor growth (16). These growth factors can induce survival, proliferation and migration of EC (17, 18). VEGFs are highly upregulated in GB, and their receptors are overexpressed in tumor vessels (13, 19, 20). VEGF-A is synthesized in massive quantities in gliomas, whereas other members of this family, such as VEGF-C and VEGF-D, are 2725

GBM-Derived Wnt3a Induces M2-Like Phenotype in Microglial Cells Through Wnt/β-Catenin Signaling

Molecular neurobiology, Jan 14, 2018

Glioblastoma is an extremely aggressive and deadly brain tumor known for its striking cellular he... more Glioblastoma is an extremely aggressive and deadly brain tumor known for its striking cellular heterogeneity and capability to communicate with microenvironment components, such as microglia. Microglia-glioblastoma interaction contributes to an increase in tumor invasiveness, and Wnt signaling pathway is one of the main cascades related to tumor progression through changes in cell migration and invasion. However, very little is known about the role of canonical Wnt signaling during microglia-glioblastoma crosstalk. Here, we show for the first time that Wnt3a is one of the factors that regulate interactions between microglia and glioblastoma cells. Wnt3a activates the Wnt/β-catenin signaling of both glioblastoma and microglial cells. Glioblastoma-conditioned medium not only induces nuclear translocation of microglial β-catenin but also increases microglia viability and proliferation as well as Wnt3a, cyclin-D1, and c-myc expression. Moreover, glioblastoma-derived Wnt3a increases micr...

Recent advances in the understanding of brown spider venoms: From the biology of spiders to the molecular mechanisms of toxins

Toxicon, 2014

The Loxosceles genus spiders (the brown spiders) are encountered in all the continents, and the c... more The Loxosceles genus spiders (the brown spiders) are encountered in all the continents, and the clinical manifestations following spider bites include skin necrosis with gravitational lesion spreading and occasional systemic manifestations, such as intravascular hemolysis, thrombocytopenia and acute renal failure. Brown spider venoms are complex mixtures of toxins especially enriched in three molecular families: the phospholipases D, astacin-like metalloproteases and Inhibitor Cystine Knot (ICK) peptides. Other toxins with low level of expression also present in the venom include the serine proteases, serine protease inhibitors, hyaluronidases, allergen factors and translationally controlled tumor protein (TCTP). The mechanisms by which the Loxosceles venoms act and exert their noxious effects are not fully understood. Except for the brown spider venom phospholipase D, which causes dermonecrosis, hemolysis, thrombocytopenia and renal failure, the pathological activities of the other venom toxins remain unclear. The objective of the present review is to provide insights into the brown spider venoms and loxoscelism based on recent results. These insights include the biology of brown spiders, the clinical features of loxoscelism and the diagnosis and therapy of brown spider bites. Regarding the brown spider venom, this review includes a description of the novel toxins revealed by molecular biology and proteomics techniques, the data regarding three-dimensional toxin structures, and the mechanism of action of these molecules. Finally, the biotechnological applications of the

MUC16 mutation is associated with tumor grade, clinical features, and prognosis in glioma patients

ABSTRACTMUC16 is a member of the attached mucin family that encodes cancer antigen 125 (CA-125), ... more ABSTRACTMUC16 is a member of the attached mucin family that encodes cancer antigen 125 (CA-125), but the association of MUC16 status with grade and subtypes of glioma patients has not yet been established. Data for MUC16 mRNA expression in 37 different cancer types were considered, and genomic data from the Cancer Genome Atlas (TCGA) from 1051 low-grade glioma (LGG) patients and 833 glioblastoma (GBM) patients were analyzed. LGG and GBM has low expression of MUC16, but it is frequently mutated in GBM. Kaplan-Meier survival analysis, glioma subtypes, methylation, and isocitrate dehydrogenase (IDH1) status were all performed. We found that mutated-MUC16 in LGG patients is associated with better prognosis considering overall survival (OS), IDH1, methylation, 1p/19q, and 10q status. Conversely, MUC16 mutation were related with worse prognosis in GBM patients upon analyzing those same parameters. Therefore, MUC16 mutations may assist in glioma diagnosis and prognosis and should be furthe...

Clonagem e expressão heterológa de hialuronidase e alérgeno presentes no veneno de aranha marrom (Loxosceles Intermedia)

Endothelial cells and SARS-CoV-2: An intimate relationship

Vascular Pharmacology

GANT-61 Induces Autophagy and Apoptosis in Glioblastoma Cells despite their heterogeneity

Cellular and Molecular Neurobiology

ctDNA as a Cancer Biomarker: A Broad Overview

Circulating tumor DNA (ctDNA) in fluids has gained attention because ctDNA seems to identify tumo... more Circulating tumor DNA (ctDNA) in fluids has gained attention because ctDNA seems to identify tumor-specific abnormalities, which could be used for diagnosis, follow-up of treatment, and prognosis: the so-called liquid biopsy. Liquid biopsy is a minimally invasive approach and presents the sum of ctDNA from primary and secondary tumor sites. It has been possible not only to quantify the amount of ctDNA but also to identify (epi)genetic changes. Specific mutations in genes have been identified in the plasma of patients with several types of cancer, which highlights ctDNA as a possible cancer biomarker. However, achieving detectable concentrations of ctDNA in body fluids is not an easy task. ctDNA fragments present a short half-life, and there are no cut-off values to discriminate high and low ctDNA concentrations. Here, we discuss the use of ctDNA as a cancer biomarker, the main methodologies, the inherent difficulties, and the clinical predictive value of ctDNA.

MicroRNAs, Hypoxia and the Stem-Like State as Contributors to Cancer Aggressiveness

Frontiers in Genetics

MicroRNAs (miRNAs) are small non-coding RNA molecules that play key regulatory roles in cancer ac... more MicroRNAs (miRNAs) are small non-coding RNA molecules that play key regulatory roles in cancer acting as both oncogenes and tumor suppressors. Due to their potential roles in improving cancer prognostic, predictive, diagnostic and therapeutic approaches, they have become an area of intense research focus in recent years. Several studies have demonstrated an altered expression of several miRNAs under hypoxic condition and even shown that the hypoxic microenvironment drives the selection of a more aggressive cancer cell population through cellular adaptations referred as the cancer stem-like cell. These minor fractions of cells are characterized by their self-renewal abilities and their ability to maintain the tumor mass, suggesting their crucial roles in cancer development. This review aims to highlight the interconnected role between miRNAs, hypoxia and the stem-like state in contributing to the cancer aggressiveness as opposed to their independent contributions, and it is based in four aggressive tumors, namely glioblastoma, cervical, prostate, and breast cancers.

Glioma infiltration and extracellular matrix: key players and modulators

Glia

Microglia/Astrocytes-Glioblastoma Crosstalk: Crucial Molecular Mechanisms and Microenvironmental Factors

Frontiers in cellular neuroscience, 2018

In recent years, the functions of glial cells, namely, astrocytes and microglia, have gained prom... more In recent years, the functions of glial cells, namely, astrocytes and microglia, have gained prominence in several diseases of the central nervous system, especially in glioblastoma (GB), the most malignant primary brain tumor that leads to poor clinical outcomes. Studies showed that microglial cells or astrocytes play a critical role in promoting GB growth. Based on the recent findings, the complex network of the interaction between microglial/astrocytes cells and GB may constitute a potential therapeutic target to overcome tumor malignancy. In the present review, we summarize the most important mechanisms and functions of the molecular factors involved in the microglia or astrocytes-GB interactions, which is particularly the alterations that occur in the cell's extracellular matrix and the cytoskeleton. We overview the cytokines, chemokines, neurotrophic, morphogenic, metabolic factors, and non-coding RNAs actions crucial to these interactions. We have also discussed the most ...

$Research paper thumbnail of {"__content__"=>"Recombinant Protein Containing B-Cell Epitopes of Different Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits.", "i"=>{"__content__"=>"Loxosceles"}}$

{"__content__"=>"Recombinant Protein Containing B-Cell Epitopes of Different Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits.", "i"=>{"__content__"=>"Loxosceles"}}

Frontiers in immunology, 2018

Loxoscelism is the most important form of araneism in South America. The treatment of these accid... more Loxoscelism is the most important form of araneism in South America. The treatment of these accidents uses heterologous antivenoms obtained from immunization of production animals with crude loxoscelic venom. Due to the scarcity of this immunogen, new alternatives for its substitution in antivenom production are of medical interest. In the present work, three linear epitopes for Loxosceles astacin-like protease 1 (LALP-1) (SLGRGCTDFGTILHE, ENNTRTIGPFDYDSIMLYGAY, and KLYKCPPVNPYPGGIRPYVNV) and two for hyaluronidase (LiHYAL) (NGGIPQLGDLKAHLEKSAVDI and ILDKSATGLRIIDWEAWR) from spider venom were identified by SPOT-synthesis technique. One formerly characterized linear epitope (DFSGPYLPSLPTLDA) of sphingomyelinase D (SMase D) SMase-I from was also chosen to constitute a new recombinant multiepitopic protein. These epitopes were combined with a previously produced chimeric multiepitopic protein (rCpLi) composed by linear and conformational B-cell epitopes from SMase D from venom, generati...

Dual treatment with shikonin and temozolomide reduces glioblastoma tumor growth, migration and glial-to-mesenchymal transition

Cellular Oncology, 2017

Glioblastomas (GBM) comprise 17% of all primary brain tumors. These tumors are extremely aggressi... more Glioblastomas (GBM) comprise 17% of all primary brain tumors. These tumors are extremely aggressive due to their infiltrative capacity and chemoresistance, with glial-to-mesenchymal transition (GMT) proteins playing a prominent role in tumor invasion. One compound that has recently been used to reduce the expression of these proteins is shikonin (SHK), a naphthoquinone with anti-tumor properties. Temozolomide (TMZ), the most commonly used chemotherapeutic agent in GBM treatment, has so far not been studied in combination with SHK. Here, we investigated the combined effects of these two drugs on the proliferation and motility of GBM-derived cells. The cytotoxic and proliferative effects of SHK and TMZ on human GBM-derived cells were tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Ki67 staining and BrdU incorporation assays. The migration capacities of these cells were evaluated using a scratch wound assay. The expression levels of β3 integrin, metalloproteinases (MMPs) and GMT-associated proteins were determined by Western blotting and immunocytochemistry. We found that GBM-derived cells treated with a combination of SHK and TMZ showed decreases in their proliferation and migration capacities. These decreases were followed by the suppression of GMT through a reduction of β3 integrin, MMP-2, MMP-9, Slug and vimentin expression via inactivation of PI3K/AKT signaling. From our results we conclude that dual treatment with SHK and TMZ may constitute a powerful new tool for GBM treatment by reducing therapy resistance and tumor recurrence.

Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: endothelial cell membrane phospholipids as targets for toxicity

Biochimica et biophysica acta, 2011

Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical ... more Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. The mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin - LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutati...

A Novel Hyaluronidase from Brown Spider (Loxosceles intermedia) Venom (Dietrich's Hyaluronidase): From Cloning to Functional Characterization

PLoS Neglected Tropical Diseases, 2013

Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Cl... more Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. The venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal Histag fusion protein (,45 kDa) in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA) and chondroitin sulfate (CS), while dermatan sulfate (DS) and heparan sulfate (HS) were not affected. Zymograph experiments resulted in ,45 kDa lytic zones in hyaluronic acid (HA) and chondroitin sulfate (CS) substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1). These data support the hypothesis that hyaluronidase is a ''spreading factor''. Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrich's Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans.

Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes

Toxins, 2011

Venomous animals use their venoms as tools for defense or predation. These venoms are complex mix... more Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5-40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the

Brown spider (Loxosceles genus) venom toxins: evaluation of biological conservation by immune cross-reactivity

Toxicon : official journal of the International Society on Toxinology, Jan 13, 2015

Loxosceles spiders are responsible for serious human envenomations worldwide. The collection of s... more Loxosceles spiders are responsible for serious human envenomations worldwide. The collection of symptoms found in victims after accidents is called loxoscelism and is characterized by two clinical conditions: cutaneous loxoscelism and systemic loxocelism. The only specific treatment is serum therapy, in which an antiserum produced with Loxosceles venom is administered to the victims after spider accidents. Our aim was to improve our knowledge, regarding the immunological relationship among toxins from the most epidemiologic important species in Brazil (L. intermedia, L. gaucho and L. laeta). Immunoassays using spider venoms and L. intermedia recombinant toxins were performed and their cross-reactivity assessed. The biological conservation of the main Loxosceles toxins (Phospholipases-D, Astacin-like metalloproteases, Hyaluronidase, ICK-insecticide peptide and TCTP-histamine releasing factor) were investigated. An in silico analysis of the putative epitopes was performed and is discu...

Clonagem e expressão heterológica de hialuronidase e alérgeno presentes no veneno de aranha marrom (Loxosceles Intermedia)

Loxoscelism is the term used to describe the clinical symptoms evoked in victims of accidents wit... more Loxoscelism is the term used to describe the clinical symptoms evoked in victims of accidents with spiders of the Loxosceles genus. In the State of Parana, accidents involving this genus are a public health problem, especially in the city of Curitiba and metropolitan region, which are considered endemic areas for loxoscelism. Loxosceles venom is a mixture of toxins, essentially proteins, capable of causing dermonecrotic lesions with gravitational spreading and systemic manifestations such as platelet aggregation, hemolytic anemia, acute renal failure and cutaneous rash. Toxins as phospholipases-D, serineproteases, metalloproteases, hyaluronidases, among others were already described in the venom of spiders of the genus. Biochemical studies showed hyaluronidase activities at 41 and 43KDa in Loxosceles intermedia whole venom. Hyaluronidases are enzymes that degrade extracellular matrix components and may act increasing the spread of other toxins in adjacent tissues of the inoculation site of venom. Due to this activity, hyaluronidades are popularly known as "spreading factors". In venom of Loxosceles intemedia other components aside from histamine are reported be responsible for mast cell degranulation and inflammatory events. In order to expand the knowledge of loxoscelism this work has aimed the cloning and expression of two new toxins of Loxosceles intermedia venom: hyaluronidase and allergen. Analysis of cDNA library of L. intermedia venom gland identified a partial sequence encoding an allergen. By means of molecular biology techniques and by designing specific primers for this sequence it was possible to obtain the complete sequence of the allergen, which was then subcloned into the expression vector pET-14b using primers with restriction sites for XhoI and BamHI enzymes. In the case of hyaluronidase, primers were designed by analyzing the sequences of hyaluronidases from several species. Then, using specific techniques it was possible to obtain the full sequence of this enzyme, which was cloned with primers with restriction sites for NdeI and BamHI enzymes. After induction trials for this recombinant protein in E. coli BL21(DE3)pLysS it was standardized an expression condition of 0,05mM IPTG during 3,5h. However between 16ºC-30ºC temperatures the protein was expressed in the form of inclusion bodies. A small scale expression in a different strain (E. coli AD494(DE3)) using induction parameters of 0,05mM-0,4mM of IPTG during 5h at 30ºC resulted in part of recombinant hyaluronidase in the soluble form. The data of this work give conditions for expression of hyaluronidase and allergen in large scale as well as purification in affinity chromatography so that will allow the evaluation of their biological activities.

Identificação e avaliação de uma isoforma de hialuronidase (Dietrich's Hyaluronidase) do veneno de Loxosceles intermedia (aranha-marrom)

Aos meus pais e irmãos por sempre terem acreditado no meu potencial e não terem poupado esforços ... more Aos meus pais e irmãos por sempre terem acreditado no meu potencial e não terem poupado esforços para que eu chegasse até a esta tese! AGRADECIMENTOS Primeiramente gostaria de agradecer a Deus, por ter me fornecido saúde, disposição e intelectualidade para o desenvolvimento deste trabalho. Em alguns momentos difíceis que passei somente uma força Divina para fazer continuar e eu só tenho a agradecer. Ao meu orientador desde a iniciação científica, Prof. Dr. Silvio Sanches Veiga, pela oportunidade de fazer parte da equipe do "Laboratório de Matriz Extracelular e Biotecnologia de Venenos", a qual me orgulho tanto! Por ter me confiado este projeto e por meu crescimento científico adquirido durante esses anos. À minha co-orientadora, Profa. Dra. Andrea Senff Ribeiro, pelas várias reuniões de discussão científica, por ter confiado esse projeto a mim e por toda ajuda prestada na burocracia da patente desse trabalho. À minha co-orientadora, não no papel... mas de coração sempre, Profa. Dra. Olga Meiri Chaim! Por ter depositado toda a confiança em mim desde minha iniciação científica! A qualquer hora esteve disposta a ouvir e discutir assuntos relacionados ao laboratório fosse de origem científica ou organizacional. Muito obrigada! Levarei sua amizade sempre comigo! À Dra. Dilza Trevisan Silva! Bem Dil, sem comentários, né?! Companheira e amiga de todas as horas desde que eu entrei no laboratório! Muito obrigada por tudo! Pelas discussões científicas, pelos experimentos, cursos, disciplinas e tarefas realizados juntos... E claro, por sua amizade magnífica! À Profa. Dra. Luiza Helena Gremski! Lu, obrigada por ser este exemplo de cientista e pessoa! Eu só tenho a agradecer por toda a ajuda prestada durante todos esses anos nos experimentos, discussões científicas, correções de inglês, entre diversas outras tarefas! Muito obrigada por você sempre me escutar e ter acreditado no meu potencial! Ao querido e quase mestre Thiago Lopes de Mari! Obrigada por ter me ajudado tanto durante esses anos e também por sua amizade! Tenho muito orgulho de ter contribuído para sua formação científica e torço por uma carreira de muito sucesso pra você! Aos demais amigos do LME: MSc.

Identificação e avaliação de uma isoforma de hialuronidase (Dietrich´s hyaluronidase) do veneno de Loxosceles intermedia (aranha-marrom): da clonagem molecular à caracterização bioquímica e funcional

Aos meus pais e irmãos por sempre terem acreditado no meu potencial e não terem poupado esforços ... more Aos meus pais e irmãos por sempre terem acreditado no meu potencial e não terem poupado esforços para que eu chegasse até a esta tese! AGRADECIMENTOS Primeiramente gostaria de agradecer a Deus, por ter me fornecido saúde, disposição e intelectualidade para o desenvolvimento deste trabalho. Em alguns momentos difíceis que passei somente uma força Divina para fazer continuar e eu só tenho a agradecer. Ao meu orientador desde a iniciação científica, Prof. Dr. Silvio Sanches Veiga, pela oportunidade de fazer parte da equipe do "Laboratório de Matriz Extracelular e Biotecnologia de Venenos", a qual me orgulho tanto! Por ter me confiado este projeto e por meu crescimento científico adquirido durante esses anos. À minha co-orientadora, Profa. Dra. Andrea Senff Ribeiro, pelas várias reuniões de discussão científica, por ter confiado esse projeto a mim e por toda ajuda prestada na burocracia da patente desse trabalho. À minha co-orientadora, não no papel... mas de coração sempre, Profa. Dra. Olga Meiri Chaim! Por ter depositado toda a confiança em mim desde minha iniciação científica! A qualquer hora esteve disposta a ouvir e discutir assuntos relacionados ao laboratório fosse de origem científica ou organizacional. Muito obrigada! Levarei sua amizade sempre comigo! À Dra. Dilza Trevisan Silva! Bem Dil, sem comentários, né?! Companheira e amiga de todas as horas desde que eu entrei no laboratório! Muito obrigada por tudo! Pelas discussões científicas, pelos experimentos, cursos, disciplinas e tarefas realizados juntos... E claro, por sua amizade magnífica! À Profa. Dra. Luiza Helena Gremski! Lu, obrigada por ser este exemplo de cientista e pessoa! Eu só tenho a agradecer por toda a ajuda prestada durante todos esses anos nos experimentos, discussões científicas, correções de inglês, entre diversas outras tarefas! Muito obrigada por você sempre me escutar e ter acreditado no meu potencial! Ao querido e quase mestre Thiago Lopes de Mari! Obrigada por ter me ajudado tanto durante esses anos e também por sua amizade! Tenho muito orgulho de ter contribuído para sua formação científica e torço por uma carreira de muito sucesso pra você! Aos demais amigos do LME: MSc.

Glioblastoma Factors Increase the Migration of Human Brain Endothelial Cells In Vitro by Increasing MMP-9/CXCR4 Levels

Anticancer Research

Background/Aim: Glioblastoma (GB) is the most aggressive type of tumor in the central nervous sys... more Background/Aim: Glioblastoma (GB) is the most aggressive type of tumor in the central nervous system and is characterized by resistance to therapy and abundant vasculature. Tumor vessels contribute to the growth of GB, and the tumor microenvironment is thought to influence tumor vessels. We evaluated the molecular communication between human GB cells and human brain microvascular endothelial cells (HBMEC) in vitro. Materials and Methods: We investigated whether GB-conditioned media (GB-CM) influenced HBMEC proliferation and migration, as well as the levels of MMP-9, CXCL12, CXCR4, CXCR7, VEGFs, VEGFR-2, and WNT5a in HBMEC. Results: Although HBMEC proliferation was not modified, increased HBMEC migration was detected after GB-CM treatment. Furthermore, treatment of HBMEC with GB-CM resulted in increased levels of MMP-9 and CXCR4. The levels of WNT5a, VEGFs and VEGFR-2 were not affected. Conclusion: GB-secreted factors lead to increased endothelial cell migration and to increased levels of MMP-9 and CXCR4. Glioblastoma (GB) is the most common and deadly type of primary brain tumor in adults (1). The hallmarks of GB are its heterogeneity, the high proliferative rate, infiltration into the cerebral parenchyma (2), resistance to treatment (3-5), and high vascularization (6). The modest outcomes of antiangiogenic strategies in GB patients (7) and the lack of understanding of how tumors modify endothelial cells (EC) behavior have increased the interest in studying GB-EC molecular interactions to identify alternative angiogenic targets for the development of new therapeutic perspectives. The blood-brain barrier (BBB) is a complex structure formed by highly specialized brain microvascular endothelial cells (EC) with specific occlusive junctions (8, 9). Like other solid tumors, GB response to hypoxia involves the regulation of growth factors, chemokines and proteases, which induce alterations in the vessel structure by inducing the proliferation and migration of EC to form new blood vessels from preexisting vessels (10). Angiogenesis in GB occurs at a high rate and also results in the aberrant and subfunctional architecture of the tumor vasculature (11, 12). In addition to EC-dependent angiogenesis, vascular mimicry also contributes to GB vascularization, although to a lesser extent (13). These vascular-like channels can be formed independently of EC via the transdifferentiation of GB stem cells (GSCs) into pericytes (PC) or smooth muscle cells (SMC). In contrast, in the presence of EC, GSC-differentiated PC/SMC cells can interact intimately with EC to facilitate angiogenesis and produce more stable vessels, which perfuse tumors more efficiently (14, 15). These angiogenic processes highlight the idiosyncrasies and complexity of GB angiogenesis, which may influence tumor EC in specific ways. Therefore, studies of the behavior of EC in the context of GB angiogenesis are important. The principal and best-described angiogenic factor is vascular endothelial growth factor (VEGF), which has at least 6 isoforms in humans that are important for both embryonic development and tumor growth (16). These growth factors can induce survival, proliferation and migration of EC (17, 18). VEGFs are highly upregulated in GB, and their receptors are overexpressed in tumor vessels (13, 19, 20). VEGF-A is synthesized in massive quantities in gliomas, whereas other members of this family, such as VEGF-C and VEGF-D, are 2725

GBM-Derived Wnt3a Induces M2-Like Phenotype in Microglial Cells Through Wnt/β-Catenin Signaling

Molecular neurobiology, Jan 14, 2018

Glioblastoma is an extremely aggressive and deadly brain tumor known for its striking cellular he... more Glioblastoma is an extremely aggressive and deadly brain tumor known for its striking cellular heterogeneity and capability to communicate with microenvironment components, such as microglia. Microglia-glioblastoma interaction contributes to an increase in tumor invasiveness, and Wnt signaling pathway is one of the main cascades related to tumor progression through changes in cell migration and invasion. However, very little is known about the role of canonical Wnt signaling during microglia-glioblastoma crosstalk. Here, we show for the first time that Wnt3a is one of the factors that regulate interactions between microglia and glioblastoma cells. Wnt3a activates the Wnt/β-catenin signaling of both glioblastoma and microglial cells. Glioblastoma-conditioned medium not only induces nuclear translocation of microglial β-catenin but also increases microglia viability and proliferation as well as Wnt3a, cyclin-D1, and c-myc expression. Moreover, glioblastoma-derived Wnt3a increases micr...

Recent advances in the understanding of brown spider venoms: From the biology of spiders to the molecular mechanisms of toxins

Toxicon, 2014

The Loxosceles genus spiders (the brown spiders) are encountered in all the continents, and the c... more The Loxosceles genus spiders (the brown spiders) are encountered in all the continents, and the clinical manifestations following spider bites include skin necrosis with gravitational lesion spreading and occasional systemic manifestations, such as intravascular hemolysis, thrombocytopenia and acute renal failure. Brown spider venoms are complex mixtures of toxins especially enriched in three molecular families: the phospholipases D, astacin-like metalloproteases and Inhibitor Cystine Knot (ICK) peptides. Other toxins with low level of expression also present in the venom include the serine proteases, serine protease inhibitors, hyaluronidases, allergen factors and translationally controlled tumor protein (TCTP). The mechanisms by which the Loxosceles venoms act and exert their noxious effects are not fully understood. Except for the brown spider venom phospholipase D, which causes dermonecrosis, hemolysis, thrombocytopenia and renal failure, the pathological activities of the other venom toxins remain unclear. The objective of the present review is to provide insights into the brown spider venoms and loxoscelism based on recent results. These insights include the biology of brown spiders, the clinical features of loxoscelism and the diagnosis and therapy of brown spider bites. Regarding the brown spider venom, this review includes a description of the novel toxins revealed by molecular biology and proteomics techniques, the data regarding three-dimensional toxin structures, and the mechanism of action of these molecules. Finally, the biotechnological applications of the

MUC16 mutation is associated with tumor grade, clinical features, and prognosis in glioma patients

ABSTRACTMUC16 is a member of the attached mucin family that encodes cancer antigen 125 (CA-125), ... more ABSTRACTMUC16 is a member of the attached mucin family that encodes cancer antigen 125 (CA-125), but the association of MUC16 status with grade and subtypes of glioma patients has not yet been established. Data for MUC16 mRNA expression in 37 different cancer types were considered, and genomic data from the Cancer Genome Atlas (TCGA) from 1051 low-grade glioma (LGG) patients and 833 glioblastoma (GBM) patients were analyzed. LGG and GBM has low expression of MUC16, but it is frequently mutated in GBM. Kaplan-Meier survival analysis, glioma subtypes, methylation, and isocitrate dehydrogenase (IDH1) status were all performed. We found that mutated-MUC16 in LGG patients is associated with better prognosis considering overall survival (OS), IDH1, methylation, 1p/19q, and 10q status. Conversely, MUC16 mutation were related with worse prognosis in GBM patients upon analyzing those same parameters. Therefore, MUC16 mutations may assist in glioma diagnosis and prognosis and should be furthe...

Clonagem e expressão heterológa de hialuronidase e alérgeno presentes no veneno de aranha marrom (Loxosceles Intermedia)

Endothelial cells and SARS-CoV-2: An intimate relationship

Vascular Pharmacology

GANT-61 Induces Autophagy and Apoptosis in Glioblastoma Cells despite their heterogeneity

Cellular and Molecular Neurobiology

ctDNA as a Cancer Biomarker: A Broad Overview

Circulating tumor DNA (ctDNA) in fluids has gained attention because ctDNA seems to identify tumo... more Circulating tumor DNA (ctDNA) in fluids has gained attention because ctDNA seems to identify tumor-specific abnormalities, which could be used for diagnosis, follow-up of treatment, and prognosis: the so-called liquid biopsy. Liquid biopsy is a minimally invasive approach and presents the sum of ctDNA from primary and secondary tumor sites. It has been possible not only to quantify the amount of ctDNA but also to identify (epi)genetic changes. Specific mutations in genes have been identified in the plasma of patients with several types of cancer, which highlights ctDNA as a possible cancer biomarker. However, achieving detectable concentrations of ctDNA in body fluids is not an easy task. ctDNA fragments present a short half-life, and there are no cut-off values to discriminate high and low ctDNA concentrations. Here, we discuss the use of ctDNA as a cancer biomarker, the main methodologies, the inherent difficulties, and the clinical predictive value of ctDNA.

MicroRNAs, Hypoxia and the Stem-Like State as Contributors to Cancer Aggressiveness

Frontiers in Genetics

MicroRNAs (miRNAs) are small non-coding RNA molecules that play key regulatory roles in cancer ac... more MicroRNAs (miRNAs) are small non-coding RNA molecules that play key regulatory roles in cancer acting as both oncogenes and tumor suppressors. Due to their potential roles in improving cancer prognostic, predictive, diagnostic and therapeutic approaches, they have become an area of intense research focus in recent years. Several studies have demonstrated an altered expression of several miRNAs under hypoxic condition and even shown that the hypoxic microenvironment drives the selection of a more aggressive cancer cell population through cellular adaptations referred as the cancer stem-like cell. These minor fractions of cells are characterized by their self-renewal abilities and their ability to maintain the tumor mass, suggesting their crucial roles in cancer development. This review aims to highlight the interconnected role between miRNAs, hypoxia and the stem-like state in contributing to the cancer aggressiveness as opposed to their independent contributions, and it is based in four aggressive tumors, namely glioblastoma, cervical, prostate, and breast cancers.

Glioma infiltration and extracellular matrix: key players and modulators

Glia

Microglia/Astrocytes-Glioblastoma Crosstalk: Crucial Molecular Mechanisms and Microenvironmental Factors

Frontiers in cellular neuroscience, 2018

In recent years, the functions of glial cells, namely, astrocytes and microglia, have gained prom... more In recent years, the functions of glial cells, namely, astrocytes and microglia, have gained prominence in several diseases of the central nervous system, especially in glioblastoma (GB), the most malignant primary brain tumor that leads to poor clinical outcomes. Studies showed that microglial cells or astrocytes play a critical role in promoting GB growth. Based on the recent findings, the complex network of the interaction between microglial/astrocytes cells and GB may constitute a potential therapeutic target to overcome tumor malignancy. In the present review, we summarize the most important mechanisms and functions of the molecular factors involved in the microglia or astrocytes-GB interactions, which is particularly the alterations that occur in the cell's extracellular matrix and the cytoskeleton. We overview the cytokines, chemokines, neurotrophic, morphogenic, metabolic factors, and non-coding RNAs actions crucial to these interactions. We have also discussed the most ...

$Research paper thumbnail of {"__content__"=>"Recombinant Protein Containing B-Cell Epitopes of Different Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits.", "i"=>{"__content__"=>"Loxosceles"}}$

{"__content__"=>"Recombinant Protein Containing B-Cell Epitopes of Different Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits.", "i"=>{"__content__"=>"Loxosceles"}}

Frontiers in immunology, 2018

Loxoscelism is the most important form of araneism in South America. The treatment of these accid... more Loxoscelism is the most important form of araneism in South America. The treatment of these accidents uses heterologous antivenoms obtained from immunization of production animals with crude loxoscelic venom. Due to the scarcity of this immunogen, new alternatives for its substitution in antivenom production are of medical interest. In the present work, three linear epitopes for Loxosceles astacin-like protease 1 (LALP-1) (SLGRGCTDFGTILHE, ENNTRTIGPFDYDSIMLYGAY, and KLYKCPPVNPYPGGIRPYVNV) and two for hyaluronidase (LiHYAL) (NGGIPQLGDLKAHLEKSAVDI and ILDKSATGLRIIDWEAWR) from spider venom were identified by SPOT-synthesis technique. One formerly characterized linear epitope (DFSGPYLPSLPTLDA) of sphingomyelinase D (SMase D) SMase-I from was also chosen to constitute a new recombinant multiepitopic protein. These epitopes were combined with a previously produced chimeric multiepitopic protein (rCpLi) composed by linear and conformational B-cell epitopes from SMase D from venom, generati...

Dual treatment with shikonin and temozolomide reduces glioblastoma tumor growth, migration and glial-to-mesenchymal transition

Cellular Oncology, 2017

Glioblastomas (GBM) comprise 17% of all primary brain tumors. These tumors are extremely aggressi... more Glioblastomas (GBM) comprise 17% of all primary brain tumors. These tumors are extremely aggressive due to their infiltrative capacity and chemoresistance, with glial-to-mesenchymal transition (GMT) proteins playing a prominent role in tumor invasion. One compound that has recently been used to reduce the expression of these proteins is shikonin (SHK), a naphthoquinone with anti-tumor properties. Temozolomide (TMZ), the most commonly used chemotherapeutic agent in GBM treatment, has so far not been studied in combination with SHK. Here, we investigated the combined effects of these two drugs on the proliferation and motility of GBM-derived cells. The cytotoxic and proliferative effects of SHK and TMZ on human GBM-derived cells were tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Ki67 staining and BrdU incorporation assays. The migration capacities of these cells were evaluated using a scratch wound assay. The expression levels of β3 integrin, metalloproteinases (MMPs) and GMT-associated proteins were determined by Western blotting and immunocytochemistry. We found that GBM-derived cells treated with a combination of SHK and TMZ showed decreases in their proliferation and migration capacities. These decreases were followed by the suppression of GMT through a reduction of β3 integrin, MMP-2, MMP-9, Slug and vimentin expression via inactivation of PI3K/AKT signaling. From our results we conclude that dual treatment with SHK and TMZ may constitute a powerful new tool for GBM treatment by reducing therapy resistance and tumor recurrence.

Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: endothelial cell membrane phospholipids as targets for toxicity

Biochimica et biophysica acta, 2011

Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical ... more Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. The mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin - LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutati...

A Novel Hyaluronidase from Brown Spider (Loxosceles intermedia) Venom (Dietrich's Hyaluronidase): From Cloning to Functional Characterization

PLoS Neglected Tropical Diseases, 2013

Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Cl... more Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. The venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal Histag fusion protein (,45 kDa) in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA) and chondroitin sulfate (CS), while dermatan sulfate (DS) and heparan sulfate (HS) were not affected. Zymograph experiments resulted in ,45 kDa lytic zones in hyaluronic acid (HA) and chondroitin sulfate (CS) substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1). These data support the hypothesis that hyaluronidase is a ''spreading factor''. Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrich's Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans.

Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes

Toxins, 2011

Venomous animals use their venoms as tools for defense or predation. These venoms are complex mix... more Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5-40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the

Brown spider (Loxosceles genus) venom toxins: evaluation of biological conservation by immune cross-reactivity

Toxicon : official journal of the International Society on Toxinology, Jan 13, 2015

Loxosceles spiders are responsible for serious human envenomations worldwide. The collection of s... more Loxosceles spiders are responsible for serious human envenomations worldwide. The collection of symptoms found in victims after accidents is called loxoscelism and is characterized by two clinical conditions: cutaneous loxoscelism and systemic loxocelism. The only specific treatment is serum therapy, in which an antiserum produced with Loxosceles venom is administered to the victims after spider accidents. Our aim was to improve our knowledge, regarding the immunological relationship among toxins from the most epidemiologic important species in Brazil (L. intermedia, L. gaucho and L. laeta). Immunoassays using spider venoms and L. intermedia recombinant toxins were performed and their cross-reactivity assessed. The biological conservation of the main Loxosceles toxins (Phospholipases-D, Astacin-like metalloproteases, Hyaluronidase, ICK-insecticide peptide and TCTP-histamine releasing factor) were investigated. An in silico analysis of the putative epitopes was performed and is discu...

Clonagem e expressão heterológica de hialuronidase e alérgeno presentes no veneno de aranha marrom (Loxosceles Intermedia)

Loxoscelism is the term used to describe the clinical symptoms evoked in victims of accidents wit... more Loxoscelism is the term used to describe the clinical symptoms evoked in victims of accidents with spiders of the Loxosceles genus. In the State of Parana, accidents involving this genus are a public health problem, especially in the city of Curitiba and metropolitan region, which are considered endemic areas for loxoscelism. Loxosceles venom is a mixture of toxins, essentially proteins, capable of causing dermonecrotic lesions with gravitational spreading and systemic manifestations such as platelet aggregation, hemolytic anemia, acute renal failure and cutaneous rash. Toxins as phospholipases-D, serineproteases, metalloproteases, hyaluronidases, among others were already described in the venom of spiders of the genus. Biochemical studies showed hyaluronidase activities at 41 and 43KDa in Loxosceles intermedia whole venom. Hyaluronidases are enzymes that degrade extracellular matrix components and may act increasing the spread of other toxins in adjacent tissues of the inoculation site of venom. Due to this activity, hyaluronidades are popularly known as "spreading factors". In venom of Loxosceles intemedia other components aside from histamine are reported be responsible for mast cell degranulation and inflammatory events. In order to expand the knowledge of loxoscelism this work has aimed the cloning and expression of two new toxins of Loxosceles intermedia venom: hyaluronidase and allergen. Analysis of cDNA library of L. intermedia venom gland identified a partial sequence encoding an allergen. By means of molecular biology techniques and by designing specific primers for this sequence it was possible to obtain the complete sequence of the allergen, which was then subcloned into the expression vector pET-14b using primers with restriction sites for XhoI and BamHI enzymes. In the case of hyaluronidase, primers were designed by analyzing the sequences of hyaluronidases from several species. Then, using specific techniques it was possible to obtain the full sequence of this enzyme, which was cloned with primers with restriction sites for NdeI and BamHI enzymes. After induction trials for this recombinant protein in E. coli BL21(DE3)pLysS it was standardized an expression condition of 0,05mM IPTG during 3,5h. However between 16ºC-30ºC temperatures the protein was expressed in the form of inclusion bodies. A small scale expression in a different strain (E. coli AD494(DE3)) using induction parameters of 0,05mM-0,4mM of IPTG during 5h at 30ºC resulted in part of recombinant hyaluronidase in the soluble form. The data of this work give conditions for expression of hyaluronidase and allergen in large scale as well as purification in affinity chromatography so that will allow the evaluation of their biological activities.