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Made available in DSpace on 2022-04-29T08:38:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-23 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background: Coronavirus disease 2019 (COVID-19) is a viral respiratory disease that spreads rapidly, reaching pandemic status, causing the collapse of numerous health systems, and a strong economic and social impact. The treatment so far has not been well established and there are several clinical trials testing known drugs that have antiviral activity, due to the urgency that the global situation imposes. Drugs with specific mechanisms of action can take years to be discovered, while vaccines may also take a long time to be widely distributed while new virus variants emerge. Thus, drug repositioning has been shown to be a good strategy for defining new therapeutic approaches. Studies of the effect of enriched heparin in the replication of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) in vitro assays justify the advance for clinical tests. Methods and analysis: A phase I/II triple-blind parallel clinical trial will be conducted. Fifty participants with radiological diagnosis of grade IIA pneumonia will be selected, which will be allocated in 2 arms. Participants allocated in Group 1 (placebo) will receive nebulized 0.9% saline. Participants allocated in Group 2 (intervention) will receive nebulized enriched heparin (2.5 mg/mL 0.9% saline). Both groups will receive the respective solutions on a 4/4 hour basis, for 7 days. The main outcomes of interest will be safety (absence of serious adverse events) and efficacy (measured by the viral load). Protocols will be filled on a daily basis, ranging from day 0 (diagnosis) until day 8. Department of Surgery and Orthopedics São Paulo State University – UNESP Botucatu Medical School, SP Applied Biotechnology Laboratory Research Nucleus of Clinical Hospital São Paulo State University – UNESP Botucatu Medical School, SP Department of Bioprocess and Biotechnology São Paulo State University – UNESP School of Pharmaceutical Sciences, SP Internal Medicine Division São Paulo State University – UNESP Botucatu Medical School, SP Department of Microbiology Institute of Biomedical Sciences University of São Paulo – USP, SP Scientific Platform Pasteur University of São Paulo – USP, SP Bioprocessing and Biotechnology Department São Paulo State University – UNESP School of Agriculture, SP Molecular & Structural Biosciences School of Life Sciences Keele University, Newcastle-Under-Lyme Department of Biochemistry Federal University of São Paulo – UNIFESP, SP Institute of Science and Technology Federal University of São Paulo – UNIFESP, SP Department of Infectious Diseases São Paulo State University – UNESP Botucatu Medical School, SP Clinical Research Unit São Paulo State University – UNESP Botucatu Medical School, SP Biosciences Institute São Paulo State University UNESP, SP Quality Control Laboratory Cellavita Scientific Research, SP Department of Orthopedics and Traumatology University of Campinas – UNICAMP School of Medical Sciences, SP Department of Surgery and Orthopedics São Paulo State University – UNESP Botucatu Medical School, SP Applied Biotechnology Laboratory Research Nucleus of Clinical Hospital São Paulo State University – UNESP Botucatu Medical School, SP Department of Bioprocess and Biotechnology São Paulo State University – UNESP School of Pharmaceutical Sciences, SP Internal Medicine Division São Paulo State University – UNESP Botucatu Medical School, SP Bioprocessing and Biotechnology Department São Paulo State University – UNESP School of Agriculture, SP Department of Infectious Diseases São Paulo State University – UNESP Botucatu Medical School, SP Clinical Research Unit São Paulo State University – UNESP Botucatu Medical School, SP Biosciences Institute São Paulo State University UNESP, SP FAPESP: 2020/12165-8

Made available in DSpace on 2022-05-01T09:31:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) The interaction between bacteriophages and integrins has been reported in different cancer cell lines, and efforts have been undertaken to understand these interactions in tumor cells along with their possible role in gene alterations, with the aim to develop new cancer therapies. Here, we report that the non-specific interaction of T4 and M13 bacteriophages with human PC-3 cells results in differential migration and varied expression of different integrins. PC-3 tumor cells (at 70% confluence) were exposed to 1 × 107 pfu/mL of either lytic T4 bacteriophage or filamentous M13 bacteriophage. After 24 h of exposure, cells were processed for a histochemical analysis, wound-healing migration assay, and gene expression profile using quantitative real-time PCR (qPCR). qPCR was performed to analyze the expression profiles of integrins ITGAV, ITGA5, ITGB1, ITGB3, and ITGB5. Our findings revealed that PC-3 cells interacted with T4 and M13 bacteriophages, with significant upregulation of ITGAV, ITGA5, ITGB3, ITGB5 genes after phage exposure. PC-3 cells also exhibited reduced migration activity when exposed to either T4 or M13 phages. These results suggest that wildtype bacteriophages interact non-specifically with PC-3 cells, thereby modulating the expression of integrin genes and affecting cell migration. Therefore, bacteriophages have future potential applications in anticancer therapies. Laboratory of Extracellular Matrix Biology Department of Structural and Functional Biology Institute of Biosciences of Botucatu Sao Paulo State University (UNESP) Laboratory of Extracellular Matrix and Gene Regulation Department of Structural and Functional Biology Institute of Biology State University of Campinas Department of Histology Embryology and Cell Biology Institute of Biological Sciences Federal University of Goiás (UFG) Laboratory of Monoclonal Antibodies Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences Sao Paulo State University (UNESP) Laboratory of Extracellular Matrix Biology Department of Structural and Functional Biology Institute of Biosciences of Botucatu Sao Paulo State University (UNESP) Laboratory of Monoclonal Antibodies Department of Bioprocess and Biotechnology School of Pharmaceutical Sciences Sao Paulo State University (UNESP) FAPESP: #2019/19644-1 CNPq: #310805/2018-0 CAPES: #963-14-2

As an emerging global health challenge, COVID-19 requires international knowledge to reach novel possible therapeutic strategies, especially for intensive-care patients. During the early stages of infection, pneumocytes II are the primary infected cells, harming the respiratory system. We have previous evidence in murine models that MSc's secretome can be used to treat pulmonary injuries induced with bleomycin, due to its content: growth factors, extracellular vesicles, and exosomes. We hypothesize and strongly recommend MSc secretome testing and production, in xenofree conditions, to be used as an alternative approach in SARS-Cov-2 patients in critical conditions.

Made available in DSpace on 2021-06-25T15:07:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Venous thromboembolism (VTE) is a serious clinical condition which early and accurate diagnosis may contribute to the reduction of associated morbidity and mortality. VTE occurs when a blood clot (thrombus) blocks the vein blood flow causing deep vein thrombosis (DVT) and, when it migrates to the lungs, it may clog the pulmonary arteries characterizing pulmonary embolism (PE). Analysis using fibrin degradation products or D-dimer and coagulation factor VIII may assist early diagnosis of VTE. Thus, two immunosensors were built using layer-by-layer (LbL) films technique, one containing the anti-D-dimer immobilized on polyethylene imine (PEI) and another the anti-FVIII on silk fibroin (SF). Immunosensor response, the antigen-antibody specific interaction, was investigated using cyclic voltammetry. When immunosensors, PEI/anti-D-dimer and SF/anti-FVIII, were exposed to antigens, D-dimer and Factor VIII, the voltammograms area and current were significantly increased with increasing specific antigen concentration. The specific interaction was confirmed with control experiments, electrodes containing only PEI or SF, that no significant changes in the voltammogram responses were observed and principal component analysis confirmed these results. The films formation and response were verified using scanning electronic microscopy (SEM). The developed immunosensor seems to be a promising and effective early complementary exam to assist in the VTE diagnosis, through the combined response of two biomarkers very sensible. Univ Fed Sao Paulo, Inst Ciencia & Tecnol, Sao Jose Dos Campos, SP, Brazil Univ Sao Paulo, Inst Fis Sao Carlos, Sao Carlos, SP, Brazil Univ Estadual Paulista, Inst Quim, Araraquara, SP, Brazil Univ Estadual Paulista, Hemoctr Botucatu, Botucatu, SP, Brazil Univ Estadual Paulista, Inst Quim, Araraquara, SP, Brazil Univ Estadual Paulista, Hemoctr Botucatu, Botucatu, SP, Brazil FAPESP: 2014/03145-2 CNPq: 480896/2013-5

Despite fast advances in genomics and proteomics, monoclonal antibodies (mAbs) are still a valuable tool for areas such as the evolution of basic research in stem cells and cancer, for immunophenotyping cell populations, diagnosing and prognosis of diseases, and for immunotherapy. To summarize different subtractive immunization approaches successfully used for the production of highly specific antibodies, we identified scientific articles in NCBI PubMed using the following search terms: subtractive immunization, monoclonal antibody, tolerization, neonatal, high-zone tolerance, masking immunization. Patent records were also consulted. From the list of results, we included all available reports, from 1985 to present, that used any enhanced immunization technique to produce either polyclonal or monoclonal antibodies. Our examination yielded direct evidence that these enhanced immunization techniques are efficient in obtaining specific antibodies to rare epitopes, with different applications, such as to identify food contaminants or tumor cells.

The original article [1] contained a minor error regarding the mean diameter of the alginate microcapsules described in relation to Fig. 4 in the Results section. The microcapsules had an actual mean diameter of 3000 μm instead of 1000 μm as mistakenly mentioned in the original article.

Made available in DSpace on 2018-12-11T17:18:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-03-27 Background: Mesenchymal stem cells derived from the synovial membrane (MSCSM) have a greater potential for joint regeneration, besides the capacity for chondrogenic differentiation, since they are a source closer to the chondrocytes. This study aimed to cultivate and evaluate viability and differentiation of MSCSM encapsulated in a three-dimensional alginate hydrogel (HA) scaffold. Samples of the synovial membrane of the metatarsophalangeal joint of 4 horses were collected by astroscopic surgery. These were subjected to enzymatic digestion, isolated mesenchymal cells, cultured in monolayers and encapsulated at various concentrations, 104; 204; 504; 105; 205 cells in 1.5% sodium alginate solution. The gelatinization process was carried out and cultured for 4 weeks. Viability and cell proliferation were performed by dissolving the microcapsules and counting with trypan blue. The ratio of live cells and total live cells at intervals 0, 7, 14, 21 and 28 days was analyzed. Results: For the evaluation of differentiation, histological sections stained with hematoxylin and eosin and toluidine blue were performed. There was no statistical difference in the proportion of live cells between groups over the 28 days. The group of 105 cells obtained a higher total number of living cells at the end of the experiment. Through the histological analysis it was possible to observe at 7 days a low amount of spherical cells with chondrocyte characteristics. On day 21, chondrogenic differentiation became evident, with pericellular and territorial matrix production. Conclusions: This study demonstrated the efficiency of HA as a scaffold for MSCSM and the chondrogenic differentiation, promising for use in the treatment of joint injuries in horses. Department of Veterinary Surgery and Anesthesiology University of Veterinary Medicine and Animal Science UNESP, Rubião Júnior, s / n Institute of Biosciences UNESP Departament of Statistics, Rubião Júnior, s / n Departament of Graduate Program in Research and Development Medical Biotechnology (Professional Master's) from the Blood Center of UNESP Blood Centre Division, Rubião Júnior, s/no - University of Medicine UNESP District of Rubião Junior s/no Blood Centre Division - Laboratory of Cellular Engineering Departament of Urology Departament of Bioprocesses and Biotechnology FCFAR - UNESP, Rodovia Araraquara Jaú, KM 01 Department of Veterinary Surgery and Anesthesiology University of Veterinary Medicine and Animal Science UNESP, Rubião Júnior, s / n Institute of Biosciences UNESP Departament of Statistics, Rubião Júnior, s / n Departament of Graduate Program in Research and Development Medical Biotechnology (Professional Master's) from the Blood Center of UNESP Blood Centre Division, Rubião Júnior, s/no - University of Medicine UNESP District of Rubião Junior s/no Blood Centre Division - Laboratory of Cellular Engineering Departament of Urology Departament of Bioprocesses and Biotechnology FCFAR - UNESP, Rodovia Araraquara Jaú, KM 01

Matrix metalloproteinases (MMPs) are zinc (Zn(2+)) and calcium (Ca(2+)) dependant endopeptidases, capable of degradation of numerous components of the extracellular matrix. Cadmium (Cd(2+)) is a well known environmental contaminant which could impair the activity of MMPs. In this sense, this study was conducted to evaluate if Cd(2+) intake inhibits these endopeptidases activities at the rat prostate and testicles and if it directly inhibits the activity of MMP2 and MMP9 at gelatinolytic assays when present in the incubation buffer. To investigate this hypothesis, Wistar rats (5 weeks old), were given tap water (untreated, n = 9), or 15 ppm CdCl2 diluted in drinking water, during 10 weeks (n = 9) and 20 weeks (n = 9). The animals were euthanized and their ventral prostate, dorsal prostate, and testicles were removed. These tissue samples were processed for protein extraction and subjected to gelatin zymography evaluation. Additionally, we performed an experiment of gelatin zymography in which 5 μM or 2 mM cadmium chloride (CdCl2) was directly dissolved at the incubation buffer, using the prostatic tissue samples from untreated animals that exhibited the highest MMP2 and MMP9 activities in the previous experiment. We have found that CdCl2 intake in the drinking water led to the inhibition of 35% and 30% of MMP2 and MMP9 (p < 0.05) at the ventral prostate and testis, respectively, in Cd(2+) treated animals when compared to controls. Moreover, the activities of the referred enzymes were 80% and 100% inhibited by 5 μM and 2 mM of CdCl2, respectively, even in the presence of 10 mM of CaCl2 within the incubation buffer solution. These important findings demonstrate that environmental cadmium contamination may deregulate the natural balance in the extracellular matrix turnover, through MMPs downregulation, which could contribute to the toxic effects observed in prostatic and testicular tissue after its exposure.

This study aimed to investigate the stimulatory effect of glucagon-like peptide 1 (GLP-1) receptor agonist (GLP-1RA) on the apoptosis of hepatic stellate cells (HSCs) activated by high glucose, and to explore the underlying molecular mechanism with a focus on the c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) signaling pathways. Human HSCs were cultured in-vitro and their morphological features were identified. HSC samples were randomly collected and divided into a control group, GLP-1RA group, GLP-1RA+JNK blockade group, and ERK blockade group. The apoptosis of HSCs in each group were analyzed by fluorescence-activated cell sorting (FACS) after 120 h of culture. Phosphorylated JNK and ERK (p-JNK and p-ERK) proteins were detected by western blotting. p-JNK expression was higher in the GLP-1RA group compared with that of the control group and the GLP-1RA+JNK blockade group (both P

The purpose of this study was to evaluate the effect of intralesional Mesenchymal Stem Cells (MSC) on the treatment of experimentally induced articular chondral defects in horses, emphasizing the benefits of this application in veterinary medicine. Chondral defects were induced in the medial femoral trochlea of both hind limbs of four horses. Thirty days post induction; the horses were divided into two groups. The G1 was submitted to treatment with MSC and the G2 was the control group. Clinical evaluations, synovial fluid analysis and synovial Prostaglandin E2 (PGE2) assessment were performed prior to defects and fortnightly up to 120 and 150 days. Macroscopic, histopathological and histochemical evaluations were performed at the end of the experiment. The treatment with MSC reduced the intraarticular inflammatory process. The G1 showed lower PGE2 concentrations in the synovial fluid and greater percentage of mononuclear cells and lower percentages of lymphocytes and neutrophils. The treatment improved the macro and microscopic aspects of repair tissue. No difference was observed in the scores of lameness between the G1 and G2. The use of MSC in the treatment of chondral defects minimized joint inflammation, as confirmed by synovial fluid analysis. The treatment resulted in an improved repair tissue, verified by macroscopic examination, histochemical and histopathological analysis.

The aim of this study was to investigate the effects of caffeine (20 mg/L) intake on cadmium (15 mg/L) accumulation in the rat blood, testes, epididymis and prostate as well as cadmium-induced changes to the antioxidant defense system of the epididymis. Caffeine reduced the cadmium concentration in all tissues analyzed. Meanwhile, cadmium reduced catalase activity and increased superoxide dismutase (SOD) activity in the epididymis. Caffeine increased SOD activity, catalase and glutathione tissue expression and sustains the cadmium's effect on catalase and GSP-Px activity. No differences in the expression of metallothionein and lipid peroxidation were observed among the different treatments in the epididymis. In conclusion, low doses of cadmium alter the antioxidant enzymatic profile of the epididymis, but not induced oxidative lipid damage. Caffeine intake reduces overall cadmium accumulation in the organism and enhances the levels of antioxidant protein expression in the epididymis, thus exerting a protective effect against this metal.

Coffee intake has been associated with a low risk of developing cancer, including prostate cancer, which is one of the most commonly diagnosed cancer in men. However, few studies have evaluated the chronic effects of caffeine, which is the most abundant methylxanthine in coffee, on prostate morphology and physiology. In the present study, we investigated the effects of chronic, low-dose caffeine intake on rat prostate morphology from puberty to adulthood. Five-week-old male Wistar rats were randomized into two experimental groups: caffeine-treated (20 ppm in drinking water, n = 12) and control (n = 12). The ventral and dorsolateral prostates were dissected, weighted and submitted to morphological, morphometrical and immunohistochemical analysis of cellular proliferation, apoptosis and androgen receptor (AR) tissue expression. The testosterone (T) and dihydrotestosterone (DHT) concentrations were measured in the plasma. Our results show that caffeine intake increased the concentrations of T and DHT, organ weight, epithelial cell proliferation and AR tissue expression in the ventral prostatic lobe. All the ventral prostates from the caffeine-treated animals presented various degrees of epithelial and stromal hyperplasia. Our results suggest that chronic caffeine intake from puberty increases androgenic signalling and cell proliferation in the rat prostate gland and can be related to the development of benign prostatic hyperplasia.

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n = 5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1 × 10(5)) were than encapsulated inside 60 µl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI.

Made available in DSpace on 2015-12-07T15:30:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2014. Added 1 bitstream(s) on 2015-12-07T15:52:57Z : No. of bitstreams: 1 PMC4301449.pdf: 250793 bytes, checksum: fd6d64423e5748e0d797dd97a3fc85dd (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The prevalence of chronic venous disease is high and occurs more frequently in females. According to the clinical, etiological, anatomical, and pathological classification (CEAP) definition, the reticular veins are included in the C1 class and are mainly associated with aesthetic complaints. Several invasive techniques are used for treatment, including mini phlebectomy, laser ablation, and radiofrequency ablation. However, a wide range of sclerosing agents may serve as minimally invasive alternatives, promoting chemical sclerosis of the vein wall. Although this technique is routinely performed around the world, there is no consensus on the most efficacious and safe chemical agent to be used. Inclusion criteria are women between 18 and 69 years old with at least 10 cm long reticular veins in the lower limbs, on the outer side of the leg/thigh. Patients with CEAP 2 to 6, or with allergies, pregnancy, performing breastfeeding, or with any dermatologic or clinical problems will be excluded. Patients with venous ultrasound mapping showing involvement of saphenous trunks and/or a deep venous system will also be excluded. Patients will be randomized into two groups, one receiving 75% pure glucose and the other group receiving 0.2% polidocanol diluted in 70% glucose. Just one limb and one session per patient will be performed. The sclerosing agent volume will not exceed 5 mL. Clinical follow-up will include visits on days 7 and 60, always with photographic documentation. This project aims to enroll 96 patients and subject them to a double-blind treatment after the randomization process. The design is intended to evaluate efficacy through a primary end point and safety through a secondary end point. Forty-eight patients have currently been enrolled. Preliminary results for these patients showed that 25 received treatment, 2 were excluded, and 22 returned after 7 days and showed no greater adverse events. To date, establishing efficacy criteria has not been possible, and no patients have reached the 60-day return point. These data may help doctors choose the best chemical agent for the treatment of reticular veins. ClinicalTrials.gov Identifier: NCT02054325, 3/02/2014. Universidade Estadual Paulista, Departamento de Cirurgia e Ortopedia, Faculdade de Medicina de Botucatu FAPESP: 2012/11034-0

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:28:13Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:28:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-01-25 High-grade prostate cancers express high levels of matrix metalloproteinases (MMPs), major enzymes involved in tumor invasion and metastasis. However, the tumor cell lines commonly employed for prostate cancer research express only small amounts of MMPs when cultivated as monolayer cultures, in common culture media. The present study was conducted to ascertain whether culture conditions that include fibronectin can alter MMP2 and MMP9 expression by the human prostatic epithelial cell lines RWPE-1, LNCaP and PC-3. These cells were individually seeded at 2×104cells/cm2, cultivated until they reached 80% confluence, and then exposed for 4h to fibronectin, after which the conditioned medium was analyzed by gelatin zymography. Untreated cells were given common medium. Only RWPE-1 cells express detectable amounts of MMP9 when cultivated in common medium, whereas the addition of fibronectin induced high expression levels of pro and active forms of MMP2 in all tested cell lines. Our findings demonstrate that normal and tumor prostate cell lines express MMP2 activity when in contact with extracellular matrix components or blood plasma proteins such as fibronectin. Future studies of transcriptomes and proteomes in prostate cancer research using these cell lines should not neglect these important conclusions. © 2012 Elsevier Inc.. Univ Estadual Paulista - UNESP Institute of Biosciences Department of Morphology, Botucatu, SP Univ Estadual Paulista - UNESP Botucatu Medical School, Blood Transfusion Center, Cell Engineering Lab., Botucatu, SP Univ Estadual Paulista - UNESP Botucatu Medical School Department of Urology, Botucatu, SP Univ Estadual Paulista - UNESP Institute of Biosciences Department of Morphology, Botucatu, SP Univ Estadual Paulista - UNESP Botucatu Medical School, Blood Transfusion Center, Cell Engineering Lab., Botucatu, SP Univ Estadual Paulista - UNESP Botucatu Medical School Department of Urology, Botucatu, SP

Made available in DSpace on 2015-02-02T12:39:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-08-01Bitstream added on 2015-02-02T13:07:53Z : No. of bitstreams: 1 S0102-86502014000800485.pdf: 1113648 bytes, checksum: 73fe0eda2fb268c8b1975cad67b071b8 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) PURPOSE:To obtain a decellularized tracheal scaffold associating traditional approaches with the novel light-emitting diode (LED) proposal.METHODS: This study was performed with New Zealand adult rabbits weighing 3.0 - 4.0 kg. Different protocols (22) were used combining physical (agitation and LED irradiation), chemical (SDS and Triton X-100 detergents), and enzymatic methods (DNase and RNase).RESULTS: Generally, the cells surrounding soft tissues were successfully removed, but none protocol removed cells from the tracheal cartilage. However, longer protocols were more effective. The cost-benefits relation of the enzymatic processes was not favorable. It was possible to find out that the cartilaginous tissue submitted to the irradiation with LED 630nm and 475 nm showed an increased number of gaps without cells, but several cells were observed to be still present.CONCLUSION: The light-emitting diode is a promising tool for decellularization of soft tissues. Paulista State University Medical School Department of Surgery and Orthopedy Hermínio Ometto University Center UNESP School of Pharmaceutical Sciences Department of Bioprocess and Biotechnology UNESP Medical School Blood Transfusion Center Paulista University Department of Health Science UNESP Bioscience Institute Department of Morphology UNESP Faculty of Science Department of Science Biology UNESP Medical School Department of Surgery and Orthopedy UNESP Medical School Department of Urology Paulista State University Medical School Department of Surgery and Orthopedy UNESP School of Pharmaceutical Sciences Department of Bioprocess and Biotechnology UNESP Medical School Blood Transfusion Center UNESP Bioscience Institute Department of Morphology UNESP Faculty of Science Department of Science Biology UNESP Medical School Department of Surgery and Orthopedy UNESP Medical School Department of Urology

Cell Engineering Lab, Blood Transfusion Center, Botucatu Medical School Univ Estadual Paulista, UNESP, District of Rubiao Junior S/N, Botucatu, 18618-970

BackgroundCardiovascular diseases remain leaders as the major causes of mortality in Western society. Restoration of the circulation through construction of bypass surgical treatment is regarded as the gold standard treatment of peripheral vascular diseases, and grafts are necessary for this purpose. The great saphenous vein is often not available and synthetic grafts have their limitations. Therefore, new techniques to produce alternative grafts have been developed and, in this sense, tissue engineering is a promising alternative to provide biocompatible grafts. This study objective was to reconstruct the endothelium layer of decellularized vein scaffolds, using mesenchymal stem cells (MSCs) and growth factors obtained from platelets.MethodsFifteen nonpregnant female adult rabbits were used for all experiments. Adipose tissue and vena cava were obtained and subjected to MSCs isolation and tissue decellularization, respectively. MSCs were subjected to differentiation using endothelial inductor growth factor (EIGF) obtained from human platelet lysates. Immunofluorescence, histological and immunohistochemical analyses were employed for the final characterization of the obtained blood vessel substitute.ResultsThe scaffolds were successfully decellularized with sodium dodecyl sulfate. MSCs actively adhered at the scaffolds, and through stimulation with EIGF were differentiated into functional endothelial cells, secreting significantly higher quantities of von Willebrand factor (0.85 μg/mL; P < .05) than cells cultivated under the same conditions, without EIGF (0.085 μg/mL). Cells with evident morphologic characteristics of endothelium were seen at the lumen of the scaffolds. These cells also stained positive for fascin protein, which is highly expressed by differentiated endothelial cells.ConclusionsTaken together, the use of decellularized bioscaffold and subcutaneous MSCs seems to be a potential approach to obtain bioengineered blood vessels, in the presence of EIGF supplementation.Clinical RelevanceThe reported outcomes and techniques on this paper may be employed to constitute decellularized heterologous blood vessel banks. These specimens may be seeded with the patients' own mesenchymal stem cells, which can be differentiated into functional endothelial cells that secrete von Willebrand factor. This is done through a novel approach using a concentrate of growth factors obtained from the patient's own platelets, also described in this article. Extra steps would be the seeding of smooth muscle cells and adventitial fibroblasts, which also could be differentiated from the patient's mesenchymal stem cells. Finally, an autologous blood vessel substitute can be obtained for cardiovascular peripheral disease graft treatment. These grafts will not induce tissue rejection, as the donor cells are completely removed and replaced by the patient's cells.

Background Platelet-rich plasma has been largely used as a therapeutic option for the treatment of chronic wounds of different etiologies. The enhanced regeneration observed after the use of platelet-rich plasma has been systematically attributed to the growth factors that are present inside platelets' granules. Aim We hypothesize that the remaining plasma and platelet-bound fibronectin may act as a further bioactive protein in platelet-rich plasma preparations. Methods Recent reports were analyzed and presented as direct evidences of this hypotheses. Results Fibronectin may directly influence the extracellular matrix remodeling during wound repair. This effect is probably through matrix metalloproteinase expression, thus exerting an extra effect on chronic wound regeneration. Conclusions Physicians should be well aware of the possible fibronectin-induced effects in their future endeavors with PRP in chronic wound treatment.

The purpose of this work was to isolate and cultivate mesenchymal stem cells (MSC) derived from equine adipose tissue and conduct cellular characterization with the following markers: CD90, CD44 and CD13. Adipose tissue collection was performed at the base of the horses' tails, followed by immediate isolation and cultivation of the MSC and posterior characterization by flow cytometry for the interspecies reaction test using mouse anti-rat CD90 monoclonal antibody (mAb), fluorescein isothiocyanate (FITC), and tests with specific mAb mouse anti-horse CD13 and mouse anti-horse CD44. The technique used for isolation and cell cultivation proved to be safe and viable. The CD90 mAb expressed cross-reaction with MSC derived from equine adipose tissue and CD44 showed greater expression in cells as the number of culture passages increased. Although marker CD13 expresses reaction in other studies involving MSC in different species, it presented no expression in the experiment realized. The results obtained revealed the immunophenotypic characterization of the surface of isolated and cultivated MSC, classifying these cells as a promising type of progenitor cells that can be applied in equine cellular therapy.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:03:50Z No. of bitstreams: 1 S1413-78522009000200008.pdf: 673400 bytes, checksum: 437dce8bbb937457952ceb01d774ef5e (MD5) Made available in DSpace on 2013-08-22T19:03:50Z (GMT). No. of bitstreams: 1 S1413-78522009000200008.pdf: 673400 bytes, checksum: 437dce8bbb937457952ceb01d774ef5e (MD5) Previous issue date: 2009-01-01 Made available in DSpace on 2013-09-30T20:06:11Z (GMT). No. of bitstreams: 2 S1413-78522009000200008.pdf: 673400 bytes, checksum: 437dce8bbb937457952ceb01d774ef5e (MD5) S1413-78522009000200008.pdf.txt: 16570 bytes, checksum: e552694f718d211afa142a014a922115 (MD5) Previous issue date: 2009-01-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:51:42Z No. of bitstreams: 2 S1413-78522009000200008.pdf: 673400 bytes, checksum: 437dce8bbb937457952ceb01d774ef5e (MD5) S1413-78522009000200008.pdf.txt: 16570 bytes, checksum: e552694f718d211afa142a014a922115 (MD5) Made available in DSpace on 2014-05-20T13:51:42Z (GMT). No. of bitstreams: 2 S1413-78522009000200008.pdf: 673400 bytes, checksum: 437dce8bbb937457952ceb01d774ef5e (MD5) S1413-78522009000200008.pdf.txt: 16570 bytes, checksum: e552694f718d211afa142a014a922115 (MD5) Previous issue date: 2009-01-01 INTRODUÇÃO: O reparo tissular é o objetivo final da cirurgia. A cultura celular requer arcabouço mecânico que dê suporte ao crescimento celular e difusão dos nutrientes. O uso do plasma rico em plaquetas (PRP) como um arcabouço 3D possui diversas vantagens: é material biológico, de fácil absorção pós-transplante, rico em fatores de crescimento, em especial PDGF- ββ e TGF-β que estimula síntese de matriz extracelular na cartilagem. OBJETIVO: Desenvolver arcabouço 3D à base de PRP. MATERIAIS E MÉTODOS: Duas formas foram idealizadas: Sphere e Carpet. Condições estéreis foram utilizadas. O gel de plaquetas permaneceu em cultura celular, observado diariamente em microscópio invertido. RESULTADOS: Ambos arcabouços obtiveram sucesso, com aspectos positivos e negativos. DISCUSSÃO: A forma Sphere não aderiu ao plástico. Observou-se retração do gel e investigação ao microscópio dificultada devido às áreas opacas no campo visual. A forma Carpet não aderiu ao plástico e apresentou-se translúcida. O tempo de estudo foi de 20 dias. CONCLUSÕES: A produção de um arcabouço 3D PRP foi um sucesso, e trata-se de uma alternativa que necessita ser mais utilizado e investigado para que se consolide em uma rota eficiente e confiável na tecnologia de engenharia tissular, particularmente em cultura de tecido cartilaginoso. INTRODUCTION: Tissue repair has been the ultimate goal of surgery. Cell culture requires a mechanical scaffold that supports cell growth and nutrient diffusion. Using platelet-rich plasma (PRP) as a 3D scaffold presents various advantages: it is a biological material, easily absorbed after transplantation, rich in growth factors, in particular, PDGF-ββ and TGF-β that stimulate extracellular matrix synthesis in cartilage culture. OBJECTIVE: To develop a PRP 3D scaffold. Material and METHODS: Two forms were idealized: Sphere and Carpet. Sterile conditions were used. The platelet gel remained in culture conditions, observed at an inverted microscope on a daily basis. RESULTS: Both forms were successful because they produced a 3D environment that supports cell growth, with positive and negative features. DISCUSSION: The Sphere form didn't attach to the plate. Gel retraction was observed and the investigation at the microscope was difficult, because of the opaque areas in the optical field. The Carpet form didn't retract, and didn't produce opaque areas. Follow-up time was 20 days. CONCLUSIONS: The production of a PRP 3D scaffold was successful, and this is an alternative requiring further investigation in order to establish an efficient and reliable route in tissue engineering technology, particularly in cartilage tissue culture. Universidade Estadual Paulista Faculdade de Medicina de Botucatu Universidade Estadual Paulista Faculdade de Medicina de Botucatu

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:03:50Z No. of bitstreams: 1 S1413-78522009000400011.pdf: 698392 bytes, checksum: e2c2bc376d0c064ee17dea6df08437d2 (MD5) Made available in DSpace on 2013-08-22T19:03:50Z (GMT). No. of bitstreams: 1 S1413-78522009000400011.pdf: 698392 bytes, checksum: e2c2bc376d0c064ee17dea6df08437d2 (MD5) Previous issue date: 2009-01-01 Made available in DSpace on 2013-09-30T20:06:11Z (GMT). No. of bitstreams: 2 S1413-78522009000400011.pdf: 698392 bytes, checksum: e2c2bc376d0c064ee17dea6df08437d2 (MD5) S1413-78522009000400011.pdf.txt: 23096 bytes, checksum: 2d9ca931d656ea65870c8cb659ead8ab (MD5) Previous issue date: 2009-01-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:32:23Z No. of bitstreams: 2 S1413-78522009000400011.pdf: 698392 bytes, checksum: e2c2bc376d0c064ee17dea6df08437d2 (MD5) S1413-78522009000400011.pdf.txt: 23096 bytes, checksum: 2d9ca931d656ea65870c8cb659ead8ab (MD5) Made available in DSpace on 2014-05-20T13:32:23Z (GMT). No. of bitstreams: 2 S1413-78522009000400011.pdf: 698392 bytes, checksum: e2c2bc376d0c064ee17dea6df08437d2 (MD5) S1413-78522009000400011.pdf.txt: 23096 bytes, checksum: 2d9ca931d656ea65870c8cb659ead8ab (MD5) Previous issue date: 2009-01-01 OBJETIVOS: O presente estudo teve como objetivo cultivar condrócitos retirados da articulação do joelho de coelhos encapsulados em hidrogel de alginato (HA) e caracterizar a produção de matriz extracelular (ECM). MÉTODOS: A cartilagem articular foi removida do joelho de coelhos, com três a seis meses, fragmentada em pedaços de 1mm e submetida à digestão enzimática. Uma concentração de 1x106 céls/mL foram ressuspensas em uma solução de alginato de sódio a 1,5% (w/v), em seguida fez-se o processo de gelatinização em CaCl2 (102 mM), permitindo a formação do HA e cultivo em meio DMEM-F12 durante quatro semanas. A distribuição das células e a ECM foram acessadas através das secções histológicas coradas com e azul de toluidina hematoxilina e eosina (HE). RESULTADOS: Houve um aumento no número e na viabilidade dos condrócitos durante as quatro semanas de cultura. Através das análises histológicas dos HAs corados com azul de toluidina e HE foi possível observar a distribuição definida dos condrócitos no hidrogel, assemelhando-se a grupos isógenos e formação de matriz territorial. CONCLUSÃO: Este estudo demonstrou a eficiência do HA como arcabouço para ser usado na cultura de condrócitos, constituindo uma alternativa no reparo de lesões na cartilagem articular. OBJECTIVES: The aim of this study was to culture chondrocytes from knee joint cartilage of rabbits encapsulated in alginate hydrogel (HA) and to characterize the production of extracelular matrix (ECM). METHODS: Joint cartilage was obtained from rabbits' knees, three to six months old, fragmented into 1-mm pieces and submitted to enzymatic digestion. A concentration of 1x106 cells/mL were re-suspended into a 1.5% (w/v) sodium alginate solution, followed by gel formation process with CaCl2 (102 mM), allowing HA to build for culturing it into a DMEM-F12 medium for four weeks. The distribution of cells and ECM were assessed from histological slices stained toluidine blue and hematoxyline-eosin (HE). RESULTS: There was an increase of the number and viability of the chondrocytes during the four weeks of culture. By assessing the histological sections stained with toluidine blue and HE, we could note the definitive distribution of chondrocytes in the hydrogel, similarly to isogenous groups and territorial matrix formation. CONCLUSION: In this study, the alginate was shown to be an effective scaffold for use in chondrocytes culture, constituting an alternative for repairing joint cartilage defects. UNESP Faculdade de Medicina de Botucatu UNESP Instituto de Biociências UNESP Faculdade de Medicina de Botucatu UNESP Instituto de Biociências

The subject of the article is the field research of the folk tradition of the northern Russian area (the region of Kargopol, Archangelskaya oblast), led by the folklore and ethnolinguistic expedition of the Russian State University for the Humanities (RSUH) since 1993 until now, and the expedition data archive. The expeditions are organized 1–3 times a year, the participants include professors, postgraduate and undergraduate students of the RSUH. Several groups take part in the expedition, each consisting of 10–15 people who investigate one village during 2–4 weeks. The expedition is aimed at collecting ethnographic, ethnolinguistic and folkloric data, which could give a researcher the opportunity to get as detailed as possible a description of a modern folk culture state in the region. The main method of the field research used during the expedition work is interviewing a considerable part of the village inhabitants – about 20-60% – using 27 questionnaires that concern different spheres of the traditional culture, the audio records are later deciphered, and deciphered texts make the card index. The computer version of the archive has been created in the Laboratory of Folklore since 1997. The multimedia database “Traditional culture of Northern Russia (Kargopol region)” contains written texts, audio records of some fragments of interviews and graphic data (photos, scanned manuscripts, containing incantations, prayers, songs, etc). The database includes now about 20% of all archive materials. The card index contains about 120000 cards, while the database – about 23000 cards.

Tissue engineering has evolved rapidly over the past 10 years and hold great promise for the treatment of several health injuries, especially due to the accumulated knowledge in stem cell biology, ...

Made available in DSpace on 2015-03-18T15:54:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-08-01Bitstream added on 2015-03-18T16:29:06Z : No. of bitstreams: 1 WOS000339704600010.pdf: 9970113 bytes, checksum: 710e5a5adb00b18600557c9e6530c69e (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Clinical experience for peripheral arterial disease treatment shows poor results when synthetic grafts are used to approach infrapopliteal arterial segments. However, tissue engineering may be an option to yield surrogate biocompatible neovessels. Thus, biological decellularized scaffolds could provide natural tissue architecture to use in tissue engineering, when the absence of ideal autologous veins reduces surgical options. The goal of this study was to evaluate different chemical induced decellularization protocols of the inferior vena cava of rabbits. They were decellularized with Triton X100 (TX100), sodium dodecyl sulfate (SDS) or sodium deoxycholate (DS). Afterwards, we assessed the remaining extracellular matrix (ECM) integrity, residual toxicity and the biomechanical resistance of the scaffolds. Our results showed that TX100 was not effective to remove the cells, while protocols using SDS 1% for 2 h and DS 2% for 1 h, efficiently removed the cells and were better characterized. These scaffolds preserved the original organization of ECM. In addition, the residual toxicity assessment did not reveal statistically significant changes while decellularized scaffolds retained the equivalent biomechanical properties when compared with the control. Our results concluded that protocols using SDS and DS were effective at obtaining decellularized scaffolds, which may be useful for blood vessel tissue engineering. (C) 2014 Published by Elsevier Inc. Sao Paulo State Univ UNESP, Botucatu Med Sch, Dept Surg & Orthoped, Vasc Lab, BR-18618970 Botucatu, SP, Brazil Sao Paulo State Univ UNESP, Botucatu Med Sch, Cell Engn Lab, Blood Transfus Ctr, Botucatu, SP, Brazil Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Bioproc & Biotechnol, Araraquara, SP, Brazil Sao Paulo State Univ, Botucatu Biosci Inst, Extracellular Matrix Lab, Dept Morphol, Botucatu, SP, Brazil Sao Paulo State Univ UNESP, Botucatu Med Sch, Flow Cytometry Lab, Ctr Blood Transfus, Botucatu, SP, Brazil Sao Paulo State Univ, Botucatu Med Sch, Immunohistochem Lab, Dept Pathol, Botucatu, SP, Brazil Scio Paulo State Univ UNESP, Botucatu Med Sch, Dept Urol, Botucatu, SP, Brazil Sao Paulo State Univ UNESP, Botucatu Med Sch, Dept Surg & Orthoped, Vasc Lab, BR-18618970 Botucatu, SP, Brazil Sao Paulo State Univ UNESP, Botucatu Med Sch, Cell Engn Lab, Blood Transfus Ctr, Botucatu, SP, Brazil Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Bioproc & Biotechnol, Araraquara, SP, Brazil Sao Paulo State Univ, Botucatu Biosci Inst, Extracellular Matrix Lab, Dept Morphol, Botucatu, SP, Brazil Sao Paulo State Univ UNESP, Botucatu Med Sch, Flow Cytometry Lab, Ctr Blood Transfus, Botucatu, SP, Brazil Sao Paulo State Univ, Botucatu Med Sch, Immunohistochem Lab, Dept Pathol, Botucatu, SP, Brazil Scio Paulo State Univ UNESP, Botucatu Med Sch, Dept Urol, Botucatu, SP, Brazil FAPESP: 10/52549-8

Made available in DSpace on 2016-01-28T16:55:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2012. Added 1 bitstream(s) on 2016-01-28T17:02:25Z : No. of bitstreams: 1 ISSN2176-9745-2012-58-04-687-693.pdf: 1001577 bytes, checksum: e6eedc36309a1929325724d8194f0eef (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Introdução: MicroRNAs são pequenos RNAs não codificantes, que atuam como reguladores pós-transcricionais de RNAs mensageiros-alvo. Estudos recentes demonstram que a expressão diferencial de alguns microRNAs associa-se com o desenvolvimento, invasão e metástase de vários tipos de câncer, incluindo o câncer de próstata. Os avanços nas técnicas de detecção e descoberta de novos marcadores moleculares são ferramentas importantes para o diagnóstico precoce e tratamento individual dos pacientes. Objetivo: Realizar revisão científica sobre os principais microRNAs alterados no câncer de próstata. Método: O PubMed foi a base de dados escolhida para a pesquisa e as palavras-chave utilizadas foram microRNA e câncer de próstata. A descoberta e o estudo dos microRNAs envolvidos na progressão do câncer são temas científicos recentes e, das 260 publicações encontradas, 25 foram selecionadas para compor esta revisão. Resultados: Muitos microRNAs são classificados como promotores da sobrevivência celular e do crescimento tumoral. Os trabalhos selecionados para esta revisão descrevem expressão diferenciada de microRNAs em amostras de tumores prostáticos e em células tumorais prostáticas estudadas in vitro. A alteração de microRNAs no plasma, na urina e no tecido tumoral mostrou-se uma ferramenta interessante para distinguir pacientes com câncer de próstata de pacientes saudáveis, classificando essas moléculas como biomarcadores promissores. Conclusão: A excelente qualidade das publicações evidencia a importância da regulação pós-transcricional exercida pelos microRNAs na progressão do câncer de próstata. Apesar de o tema ser recente, técnicas moleculares de última geração estão sendo empregadas para a descoberta de novos microRNAs e para a caracterização da função daqueles já existentes. Introduction: MicroRNAs are small non-coding RNAs that act as post-transcriptional regulators of mRNA targets. Recent studies suggest that differential expression of some microRNAs are associated with cancer development, invasion and metastasis, including prostate cancer. Improvements in detection techniques and findings of new molecular markers are important tools for early diagnosis and treatment of individual patients. Objective: To carry out a scientific review about the main microRNAs altered in prostate cancer. Methods: The PubMed database was chosen for the research using the key words microRNA and prostate cancer. The discovery and study of microRNAs involved in cancer progression are recent scientific topics and, within the 260 publications found, 25 were selected for this review. Results: Many microRNAs are classified as promoters of cell survival and tumor growth. The papers selected for this review describe differential expression of microRNAs in samples of prostate tumors and prostatic tumor cells studied in vitro. Alterations of microRNAs in plasma, urine and tumor tissue were found to be interesting tools to distinguish patients with prostate cancer from healthy individuals, classifying these molecules as promising biomarkers. Conclusion: The excellent quality of publications highlights the importance of post-transcriptional regulation exerted by microRNAs in prostate cancer progression. Despite being a recent topic, modern molecular techniques are being employed to unveil novel microRNAs and to characterize their mechanisms of action. Introducción: Los MicroARNs son pequeños ARNs no codificantes que actúan como reguladores post-transcripcionales de ARNs mensajeros blancos. Estudios recientes demostraron que la expresión diferencial de algunos MicroARNs está asociada con el desarrollo, invasión y metástasis de varios tipos de cánceres, entre ellos el cáncer de próstata. Los avances en las técnicas de detección y hallazgo de nuevos marcadores moleculares son herramientas importantes para el diagnóstico precoz y tratamiento individualizado de los pacientes. Objetivo: Realizar revisión científica sobre los principales MicroARNs alterados en el cáncer de próstata. Método: La base de datos PubMed fue elegida para el estudio y las palabras claves utilizadas han sido MicroARNs y cáncer de próstata. El descubrimiento y el estudio de los MicroARNs involucrados en la progresión del cáncer son temas científicos recientes, y de las 260 publicaciones encontradas, se seleccionaron 25 para esta la revisión. Resultados: Muchos MicroARNs son clasificados como promotores de la supervivencia celular y del crecimiento tumoral. Los trabajos seleccionados describen una expresión distinta de MicroARNs en muestras de tumores de próstata y en células tumorales de la próstata estudiadas in vitro. La alteración de los niveles de expresión de los microRNAs en plasma, orina y en los tejidos tumorales, resultó ser una interesante herramienta para distinguir a los pacientes con cáncer de próstata de pacientes sanos, lo cual clasifica estas moléculas como biomarcadores prometedores. Conclusión: La excelente calidad de las publicaciones destaca la importancia de la regulación post transcripcional ejercida por los MicroARNs en la progresión del cáncer de próstata. A pesar de ser un tema reciente, las técnicas moleculares de última generación se están empleando en el hallazgo de nuevos MicroARNs y para la caracterización de la función de aquellos que ya existen. Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Instituto de Biociências de Botucatu (IBB), Departamento de Morfologia, Distrito de Rubião Júnior, s/n., CEP 18618-000, Botucatu, SP, Brasil Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Instituto de Biociências de Botucatu (IBB), Departamento de Morfologia, Distrito de Rubião Júnior, s/n., CEP 18618-000, Botucatu, SP, Brasil FAPESP: 2010/05042-5 FAPESP: 2010/16671-3