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Made available in DSpace on 2022-04-29T08:32:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The requirement to achieve natural looking restorations is one of the most challenging aspects in dentistry. Although zirconia has provided new opportunities for achieving superior aesthetics and physicochemical outcomes, very little has been achieved for its cellular and molecular performance, especially considering angiogenesis and osteogenesis. As angiogenesis is a secondary event and concomitant to osteogenesis, an indirect effect of dental implant on endothelial cells could be the release of active molecules such as those already reported affecting osteoblasts. To better address this issue, we challenged human endothelial cells (HUVECs) with zirconia-conditioned medium up to 72 h to allow analysis specific gene expression and protein pattern of mediators of epigenetic machinery in full. Our data shows involvement of zirconia in triggering intracellular signaling through MAPK-ERK activation, leading the signal to activate histone deacetylase HDAC6 likely with concomitant well-modulated DNA methylation profile by DNMTs and TETs. These signaling pathways seem to culminate in cytoskeleton rearrangement of endothelial cells, an important prerequisite to cell migration expected in angiogenesis. Collectively, this study demonstrates for the first time epigenetic-related molecular mechanism involved in endothelial cells responding to zirconia, revealing a repertoire of signaling molecules capable of executing the reprogramming process of gene expression, which are necessary to drive cell proliferation, migration, and consequently angiogenesis. This set of data can further studies using gene editing approaches to better elucidate functional roles. Lab. of Bioassays and Cellular Dynamics Department of Chemistry and Biochemistry Institute of Biosciences UNESP – São Paulo State University Department of Dentistry University of Taubaté Program in Environmental and Experimental Pathology Paulista University Lab. of Bioassays and Cellular Dynamics Department of Chemistry and Biochemistry Institute of Biosciences UNESP – São Paulo State University

Made available in DSpace on 2020-12-12T01:08:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Modifications on shear stress-based mechanical forces are associated with pathophysiological susceptibility and their effect on endothelial cells (EC) needs to be better addressed looking for comprehending the cellular and molecular mechanisms. This prompted us to better evaluate the effects of shear stress in human primary venous EC obtained from the umbilical cord, using an in vitro model to mimic the laminar blood flow, reaching an intensity 1–4 Pa. First, our data shows there is a significant up-expression of phosphatidylinositol 3-kinase (PI3K) in shear-stressed cells culminating downstream with an up-phosphorylation of AKT and up-expression of MAPK-ERK, concomitant to a dynamic cytoskeleton rearrangement upon integrin subunits (α4 and ß 3) requirements. Importantly, the results show there is significant involvement of nitric oxide synthase (eNOS), nNOS, and vascular endothelial growth factors receptor 2 (VEGFR2) in shear-stressed EC, while cell cycle-related events seem to being changed. Additionally, although diminution of 5-hydroxymethylcytosine in shear-stressed EC, suggesting a global repression of genes transcription, the promoters of PI3K and eNOS genes were significantly hydroxymethylated corroborating with their respective transcriptional profiles. Finally, to better address, the pivotal role of PI3K in shear-stressed EC we have revisited these biological issues by wortmannin targeting PI3K signaling and the data shows a dependency of PI3K signaling in controlling the expression of VGFR1, VGFR2, VEGF, and eNOS, once these genes were significantly suppressed in the presence of the inhibitor, as well as transcripts from Ki67 and CDK2 genes. Finally, our data still shows a coupling between PI3K and the epigenetic landscape of shear-stressed cells, once wortmannin promotes a significant suppression of ten-11 translocation 1 (TET1), TET2, and TET3 genes, evidencing that PI3K signaling is a necessary upstream pathway to modulate TET-related genes. In this study we determined the major mechanotransduction pathway by which blood flow driven shear stress activates PI3K which plays a pivotal role on guaranteeing endothelial cell phenotype and vascular homeostasis, opening novel perspectives to understand the molecular basis of pathophysiological disorders related with the vascular system. Bioassays and Cell Dynamics Laboratory Department of Chemistry and Biochemistry Bioscience Institute UNESP Department of Biology Dental School University of Taubaté Bioassays and Cell Dynamics Laboratory Department of Chemistry and Biochemistry Bioscience Institute UNESP FAPESP: 2014/22689-3 FAPESP: 2016/22270-8

Made available in DSpace on 2019-10-06T17:13:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-10-10 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação Oswaldo Cruz Universidade Estadual de Campinas In recent years, forest breeding programs have increased Eucalyptus production for commercial purposes; however, high-performing Eucalyptus clones have had problems with propagation, especially when rooting. Nevertheless, studies have shown that inoculation of microorganisms producing indole-3-acetic acid (IAA) is an especially productive procedure to help these clones breed. In this study, we evaluated the production of IAA and analogues in 16 microbial strains. For the first time, a high IAA production was described in the strain Aspergillus flavipes (ATCC® 16814™), and A. flavipes was shown by LC-MS/MS to produce IAA through a tryptophan-dependent biosynthetic pathway. A. flavipes reached the highest IAA production when cultivated under solid-state fermentation in an optimized medium composed of soybean bran, water and tryptophan. We mixed the fermentation products in solid form (SF) and liquid form (LF) with the substrate Carolina I® and then planted the cuttings of the hybrid Eucalyptus grandis x E. urophylla (clone IPB2). In fact, treatments with 40–120 mg kg−1 of SF increased the adventitious rooting rate, root length and both root fresh and dry mass, while 120 mg kg−1 of LF increased root length and dry mass. Additionally, there was no toxicity on fibroblasts (NIH/3t3), and, therefore, the plant biostimulant was confirmed as a novel, non-toxic, and eco-friendly solution. São Paulo State University (UNESP) Institute of Biosciences São Paulo State University (UNESP) School of Agriculture Technische Universität Dresden Institute of Botany São Paulo State University (UNESP) Institute of Biosciences São Paulo State University (UNESP) School of Agriculture CAPES: 001 CAPES: 88881.133019/2016–01

Made available in DSpace on 2019-10-06T16:30:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-08-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The HOXA gene cluster is generally recognized as a pivotal mediator of positional identity in the skeletal system, expression of different orthologues conferring alternative locational phenotype of the vertebrate bone. Strikingly, however, the molecular mechanisms that regulate orthologue-specific expression of different HOXA cluster members in gestating osteoblasts remain largely obscure, but in analogy to the processes observed in acute lymphatic leukemia it is assumed that alternative methylation of HOXA promoter regions drives position specific expression patterns. In an effort to understand HOXA cluster gene expression in osteogenesis we characterize both expression and the epigenetic landscape of the HOXA gene cluster during in vitro osteoblast formation from mesenchymal precursors. We observe that osteoblast formation per se provokes strong upregulation of HOXA gene cluster expression, in particular of midcluster genes, and paradoxal downregulation of HOXA7 and HOXA10. These differences in expression appear related to promoter methylation. LnRNAs HOTAIR and HOTTIP, known to modulate HOXA expression, are also regulated by their promoter methylation processing, but do not correlate with HOXA cluster expression profile. We thus conclude that HOXA expression is profoundly regulated during osteoblast differentiation through canonical methylation-dependent mechanisms but not through the flanking lnRNAs. Laboratory of Bioassays and Cellular Dynamics Department of Chemistry and Biochemistry Institute of Biosciences São Paulo State University - UNESP Department of Gastroenterology and Hepatology Erasmus MC University Medical Center Rotterdam Department of Biological Sciences Bauru School of Dentistry University of São Paulo, Al. Octávio Pinheiro Brisolla, 9-75 Laboratory of Bioassays and Cellular Dynamics Department of Chemistry and Biochemistry Institute of Biosciences São Paulo State University - UNESP FAPESP: 2014/22689-3 FAPESP: 2016/01139-0 FAPESP: 2017/01046-5

Made available in DSpace on 2021-06-25T10:20:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Although osseointegration and clinical success of titanium (Ti)-implanted materials depend on neovascularization in the reactional peri-implant tissue, very little has been achieved considering the Ti-molecules release on the behavior of endothelial cells. To address this issue, we challenged endothelial cells (HUVECs) with Ti-enriched medium obtained from two types of commercial titanium surfaces [presenting or not dual-acid etching (DAE)] up to 72 h to allow molecular machinery analysis. Our data show that the Ti-enriched medium provokes significant stimulus of angiogenesis-related machinery in endothelial cells by upexpressing VEGFR1, VEGFR2, VEGF, eNOS, and iNOS genes, while the PI3K/Akt signaling pathway was also significantly enhanced. As PI3K/AKT signaling was related to angiogenesis in response to vascular endothelial growth factor (VEGF), we addressed the importance of PI3K/Akt upon Ti-enriched medium responses by concomitantly treating the cells with wortmannin, a well-known PI3K inhibitor. Wortmannin suppressed the angiogenic factors, because VEGF, VEGFR1, and eNOS genes were downregulated in those cells, highlighting the importance of PI3K/AKT signaling on driving angiogenic phenotype and angiogenesis performance within the peri-implant tissue reaction. In conjunction, these data reinforce that titanium-implantable devices modify the metabolism of surrounding cells, such as endothelial cells, probably coupling osteogenesis and angiogenesis processes in peri-implant tissue and then contributing to successfully osseointegration of biomedical titanium-based devices. [Figure not available: see fulltext.]. Institute of Biosciences of Botucatu Department of Chemical and Biological Sciences UNESP – São Paulo State University Institute of Biosciences of Botucatu Department of Chemical and Biological Sciences UNESP – São Paulo State University

The aim of this study was to show that abundant and inexpensive plant oils can be biotransformed to increase biological activity (antioxidant, antimicrobial and cytotoxicity) through hydrolysis reaction catalysed by lipases. We tested homemade and commercial lipases through the biotransformation of nine different plant oils in forty different combinations. First, the chemical composition of the samples was investigated. Thereafter, biological tests were conducted to assess the antioxidant and antimicrobial activity of the sampled biotransformation products, as well as analyzed their influence on the viability of healthy and cancer cells. Summarising, sunflower, corn and olive oils modified by orange waste‐obtained lipases presented the most promising results, reaching up to 90% of antioxidant activity increase and significant growth inhibition of bacteria colonies belonging to genera Escherichia, Listeria, Staphylococcus, Pseudomonas and Salmonella. In addition, those compounds affected human oral squamous carcinoma cells. The bioconversion of plant oils through lipases improves their biological properties and might be an option for biotechnological application.

Significant health concerns have been raised by the high levels of Cr and Co ions into whole blood as resulted of corrosion process released from biomedical implants, but very little is known about their biological behavior in governing cell metabolism. Thus, we prompted to address this issue by exploring the effects of CoCr enriched medium on both fibroblast and preosteoblast (pre-Ob) cells. First, we showed there is a significant difference in Co and Cr releasing dependent on engineered surface, it being even more released in dual acid-etching treating surface (named w/DAE) than the machined surfaces (named wo/DAE). Thereafter, we showed CoCr affects pre-osteoblast and fibroblast metabolism by dynamically modulating integrin-based downstream signaling (FAK, Src, Rac1, and Cofilin). Specifically on this matter, we have shown there is dynamic β1-integrin gene activation up 24 h in both preosteoblast and fibroblast. Our analysis showed also that both pre-Ob and fibroblast are important resource of proinflammatory cytokines when responding to CoCr enriched medium. In addition, survival-related signaling pathway was also affected interfering on survival and proliferating signal, mainly affecting CDK2, mapk-Erk and mapk-p38 phosphorylations, while AKT/PKB-related gene remained active. In addition, during cell adhesion PP2A (an important Ser/Thr phosphatase) was inactive in both cell lineages and it seems be a CoCr's molecular fingerprint, regulating specific metabolic pathways involved with cytoskeleton rearrangement. Altogether, our results showed for the first time CoCr affects cellular performance in vitro by modulating integrin activation-based downstream signaling and requiring a reprograming of inflammatory genes activations in vitro. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 839-849, 2018.

Made available in DSpace on 2022-04-28T19:42:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-09-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Cobalt-chromium (CoCr)-based alloys have emerged as an interesting biomaterial within biomedical field, mainly considering their biocompatibility, resistance to corrosion and absence of magnetism; however, its effect on cell metabolism is barely known and this prompted us better evaluating whether CoCr-enriched medium affects the metabolism of both osteoblast and endothelial cells, and also if there is a coupling between them. This is also considered here the already-known effect of Cobalt (Co) as a hypoxic element. Firstly, discs of CoCr [subjecting (W) or not (Wo) to dual acid-etched (DAE)] were incubated into FBS-free cell culture medium up to 24 h (37 °C). This CoCr-enriched medium was further used to treat shear-stressed endothelial cells cultures up to 72 h. Thereafter, the conditioned medium containing metabolites of shear-stressed endothelial cells in response to CoCr-enriched medium was further used to subject osteoblast's cultures, when the samples were properly harvested to allow the analysis of the molecular issues. Our data shows that CoCr-enriched medium contains 1.5 ng–2.0 ng/mL of Co, which was captured by endothelial cells and osteoblasts in about 30% in amount and it seems modulate their metabolic pathways: shear-stressed endothelial cells expressed higher profile of HIF1α, VEGF and nNOS genes, while their global profile of protein carbonylation was lower than the control cultures, suggesting lower oxidative stress commitment. Additionally, osteoblasts responding to metabolites of CoCr-challenged endothelial cells show dynamic expression of marker genes in osteogenic differentiation, with alkaline phosphatase (ALP), osteocalcin, and bone sialoprotein (BSP) genes being significantly increased. Additionally, tensional shear-stress forces decrease the stimulus for ColA1gene expression in osteoblasts responding to endothelial cells metabolites, as well as modifying the extracellular matrix remodeling related genes. Analyzing the activities of matrix metalloproteinases (MMPs), the data shows that shear-stressed endothelial cells metabolites increase the activities of both MMP9 and MMP2 in osteoblasts. Altogether, our data shows for the first time that shear-stressed endothelial metabolites responding to CoCr discs contribute to osteogenic phenotype in vitro, and this predicts an active crosstalk between angiogenesis and osteogenesis during osseointegration of CoCr alloy and bone healing, maybe guided by the Co-induced hypoxic condition. Lab. of Bioassays and Cell Dynamics Department of Chemical and Biological Sciences Institute of Biosciences UNESP - Universidade Estadual Paulista Lab. of Bioassays and Cell Dynamics Department of Chemical and Biological Sciences Institute of Biosciences UNESP - Universidade Estadual Paulista FAPESP: 2019/26854-2 CNPq: PQ2

Made available in DSpace on 2020-12-12T01:48:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-02-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The understanding of vascular calcification-based mechanism is an urgent pending task in vascular biology and this prompted us to better address this issue by investigating whether DNA methylation mechanism might drive osteogenic marker genes modulation in primary human vascular smooth muscle cells (VSMCs) responding to calcium and phosphate levels overload up to 72 h. Firstly, our data shows this calcifying process recapitulates the molecular repertory of osteogenic biomarkers and specifically requiring RUNX2, Osterix and ALP, BSP genes activations along 72 h in vitro, and this behavior was validated here using other lineages. Conversely, both BMPs 4 and 7 were significantly overexpressed, maybe already as a mechanism in response to RUNX2 and Osterix genes activities identified earlier in response to the calcifying condition, and taken into maintain the calcifying phenotype of VSMCs. Additionally, survival signaling was maintained active and accompanied by a dynamic cytoskeleton rearrangement signaling requiring MAPK and AKT phosphorylations. Moreover, during the contractile-to-calcifying transition phenotype of VSMCs, epigenetic machinery was finely modulated, requiring the translocation of DNMT3B and TET2 into nucleus and this prompted us evaluating whether the profile of osteogenic-related gene promoters’ methylation might contribute with this process. By firstly estimating 5meC/5 hmeC ratio changes, we further specifically show the significance of the epigenetic modulation of Osterix and Bone sialoprotein related gene promoters, presenting a positive correlation between the epigenetic signature of their gene promoters and transcriptional patterns. Altogether, our results show for the first time the importance of epigenetic mechanism on modulating osteogenic gene markers reprogramming during calcifying VSMCs phenotype acquisition, which might drive the genesis of vascular ectopic calcification. Laboratory of Bioassays and Cellular Dynamics of the Department of Chemistry and Biochemistry Institute of Biosciences São Paulo State University - UNESP, Botucatu Department of Biology Dental School University of Taubaté, Taubaté Laboratory of Bioassays and Cellular Dynamics of the Department of Chemistry and Biochemistry Institute of Biosciences São Paulo State University - UNESP, Botucatu

Made available in DSpace on 2018-12-11T17:12:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-10-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The reciprocal and adaptive interactions between cells and substrates governing morphological transitions in the osteoblast compartment remain largely obscure. Here we show that osteoblast cultured in basement membrane matrix (Matrigel™) exhibits significant morphological changes after ten days of culture, and we decided to exploit this situation to investigate the molecular mechanisms responsible for guiding osteoblast morphological transitions. As almost all aspects of cellular physiology are under control of kinases, we generated more or less comprehensive cellular kinome profiles employing PepChip peptide arrays that contain over 1000 consensus substrates of kinase peptide. The results obtained were used to construct interactomes, and these revealed an important role for FoxO in mediating morphological changes of osteoblast, which was validated by Western blot technology when FoxO was significantly up-expressed in response to Matrigel™. As FoxO is a critical protein in canonical hedgehog signalling, we decided to explore the possible involvement of hedgehog signalling during osteoblast morphological changes. It appeared that osteoblast culture in Matrigel™ stimulates release of a substantial amounts Shh while concomitantly inducing upregulation of the expression of the bona fide hedgehog target genes Gli-1 and Patched. Functional confirmation of the relevance of these results for osteoblast morphological transitions came from experiments in which Shh hedgehog signalling was inhibited using the well-established pathway inhibitor cyclopamine (Cyc). In the presence of Cyc, culture of osteoblasts in Matrigel™ is not capable of inducing morphological changes but appears to provoke a proliferative response as evident from the upregulation of Cyclin D3 and cdk4. The most straightforward interpretation of our results is that hedgehog signalling is both necessary and sufficient for membrane matrix-based morphological transitions. Lab. de Bioensaios e Dinâmica Celular Depto de Química e Bioquímica Instituto de Biociências Universidade Estadual Paulista - UNESP, campus Botucatu Laboratory of Bioassays and Signal Transduction Departamento de Bioquímica Instituto de Biologia Universidade Estadual de Campinas (Unicamp), 6109 Instituto Nacional de Metrologia Normalização e Qualidade Industrial (INMETRO) Life Sciences Applied Metrology (Dimav)/Bioengineering Group Erasmus MC Cancer Institute Erasmus MC Erasmus University of Rotterdam Lab. de Bioensaios e Dinâmica Celular Depto de Química e Bioquímica Instituto de Biociências Universidade Estadual Paulista - UNESP, campus Botucatu FAPESP: 2014/22689-3 CNPq: 301966/2015-0 CNPq: 477452/2012-4

Made available in DSpace on 2019-10-06T16:06:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-02-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Although layered double hydroxides (LDH) have been listed as promising nanomaterials in human healthcare, very little has been achieved on osteoblast inflammatory signaling. Thus, osteoblasts were challenged with two LDHs (Mg 2 Al-Cl and Zn 2 Al-Cl, at 0.002 mg/mL) up to 24 h, establishing an acute inflammatory mechanism, as well as identifying whether Sonic hedgehog (Shh) signaling has an influence. Functional experiments were performed by previously treating (2 h) semiconfluent osteoblast cultures with cyclopamine molecule (cyc), a widely used Shh inhibitor. Considering inflammasome complex, the asc1 gene was significantly up-expressed in response to Zn 2 Al-Cl - LDHs, as well as the nrlp3 gene. By treating the osteoblast with cyc, the asc1 gene presented an even higher profile. Our results found a down-modulation of major pro-inflammatory cytokines-related genes, when tnfα and il1ß were significantly down-modulated in response to LDHs. Conversely, anti-inflammatory cytokines were up-modulated considering the same experimental procedures. Except the il6, the other il13, il10, and tgfß genes were up modulated. Additionally, Shh signaling seems to modulate this repertory as both the il13 and il10 genes were significantly up-modulated when the Shh signaling was inhibited. Altogether, our results reveal for the first time the exigency of Shh-dependent anti-inflammatory signals in LDH-induced osteoblast responses. Lab. de Bioensaios e Dinâmica Celular Departamento de Química e Bioquímica Instituto de Biociências Universidade Estadual Paulista - UNESP campus Botucatu Lab. de Engenharia Celular Hemocentro Hospital das Clínicas Faculdade de Medicina de Botucatu Departamento de Cirurgia Universidade Federal de São Paulo-UNIFESP, Rua Botucatu 740 Departamento de Química Fundamental Instituto de Química Universidade de São Paulo-USP, Av. Prof. Lineu Prestes 748 Lab. de Bioensaios e Dinâmica Celular Departamento de Química e Bioquímica Instituto de Biociências Universidade Estadual Paulista - UNESP campus Botucatu Lab. de Engenharia Celular Hemocentro Hospital das Clínicas Faculdade de Medicina de Botucatu FAPESP: 2011/50318-1 FAPESP: 2014/22689-3

Made available in DSpace on 2018-12-11T17:21:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-12-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Titanium is widely used for biomedical applications, but little information is being delivered regarding the cellular/molecular mechanisms explaining their efficacy, mainly considering the effects of the Ti-released trace elements on pre-osteoblasts. We addressed this issue by investigating decisive intracellular signal transduction able to modulate cytoskeleton rearrangement, proliferative phenotype and extracellular matrix (ECM) remodeling. We considered titanium grades IV and V, submitted or not to dual acid-etching (w/DAE or wo/DAE, respectively). Our results showed there is no cytotoxicity, preserving AKT involvement. Additionally, Ti-enriched medium promoted a diminution of the downstream signaling upon integrin activation (phosphorylating Rac1 and cofilin), guaranteeing a dynamic cytoskeleton rearrangement. Moreover, the low profile of ECM remodeling obtained in response to trace molecules released by Ti-based devices seems contributing to the osteoblast performance in mediating extracellular support to cell anchorage. This hypothesis was validated by the up-expression of ß1-integrin, src and Focal adhesion kinase (fak) genes, mainly in response to titanium grade V. Proliferative phenotype showed an unbalance between cyclin-dependent kinases (CDKs) and p15INK4b/p21Cip1. In conjunction, we showed for the first time that trace elements from Ti-based biomedical devices provoke important modulation of the osteoblast biology, driving cell anchoring, viability, and proliferative phenotype. Certainly, these biological outcomes compromise implant osseointegration. Dept. of Chemistry and Biochemistry Bioscience Institute São Paulo State University UNESP Campus Botucatu Electron Microscopy Center IBB UNESP Dept. of Chemistry and Biochemistry Bioscience Institute São Paulo State University UNESP Campus Botucatu Electron Microscopy Center IBB UNESP CNPq: # 477452/2012-4 FAPESP: #2014/22689-3 CNPq: #301966/2015-0

Molecular mechanism governing inflammatory scenario in response to titanium (Ti)-nanotexturing surfaces needs to be better addressed. Thus, we subjected pre-osteoblast to different Ti-texturing surfaces, as follows: machined (Mac), double acid-etching (DAE), and nanoscaled hydroxyapatite-blasted titanium surface (nHA), considering the cells chronically responding either directly (when the cells were cultured onto the surfaces) or indirectly (when the cells were challenged with the conditioned medium by the surfaces), up to 10 days. Our results showed that there is a dynamic requirement of inflammatory-related genes activation in response to nHA by up expressing IL1s, IL6, IL10, and IL33 (direct condition) and IL6, IL10, IL18 (indirect condition). Importantly, our data show that there is inflammasome involvement, once NLRP3, ASC1, and CASP1 genes were also required. As we found a strong signal of IL10, an anti-inflammatory cytokine, we further investigated Sonic Hedgehog (Shh) signaling cascade. Surprisingly, Shh ligand and Smoothened (Smo) genes were up-modulated in response to nHA, while Patched (Ptc) was down-modulated. Finally, an interactome was built using bioinformatics reinforcing Shh signaling cascade on modulating IL10 transcripts by Src mediating this process and this prevalence of anti-inflammatory picture might explain the low profile of RANKL transcripts in response to nHA, compromising the osteoclastogenesis surrounding the implants. Taking our results into account, our data show that the inflammatory landscape promoted by nHA is strictly modulated by Shh signaling promoted anti-inflammatory pathways. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1597-1604, 2019.

Lipases are an economic important group of biocatalysts that can be produced by some fungal under solid-state fermentation. Orange wastes are source of lipases and potential substrates for lipases production. This work assessed 19 fugal strains cultivated in Citrus sinensis cv. Hamlin orange wastes (peel, frit and core) for production of lipases in order to generate compounds with antioxidant, antimicrobial and cytotoxic properties. Fifteen of those fungi grew and produced lipases, mainly the Aspergillus brasiliensis [National Institute of Quality Control (INCQS) 40036]/frit system, which showed 99.58 U/g total lipase. The substrate with the highest production of lipase was frit with 26.67 and 78.91 U/g of total lipases produced on average by the 15 microorganisms. Aspergillus niger 01/frit (33.53 U/g) and Aspergillus niger (INCQS 40015)/frit (34.76 U/g) systems showed the highest specificity values in all the herein tested synthetic substrates with 4, 12 and 16 carbons. Analysis of the fatty acid profile of hydrolysis products obtained in the most prominent systems applied to corn and sunflower oils showed: palmitic acid, linoleic acid, oleic acid, and stearic acid. These acids showed antioxidant capacity of up to 58% DPPH (2,2-diphenyl-1-pierylhydrazyl) radical reduction and antibacterial activity against Escherichia coli, Listeria monocytogenes, Pseudomonas aureginosa, Salmonella Enteritidis and Staphylococcus aureus, as well as cytotoxicity to SCC9 cells (squamous cancer cells).

Made available in DSpace on 2018-12-11T16:52:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-04-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The biological response to zirconia (ZrO2) is not completely understood, which prompted us to address its effect on pre-osteoblastic cells in both direct and indirect manner. Our results showed that zirconia triggers important intracellular signaling mainly by governing survival signals which leads to cell adhesion and proliferation by modulating signaling cascade responsible for dynamic cytoskeleton rearrangement, as observed by fluorescence microscopy. The phosphorylations of Focal Adhesion Kinase (FAK) and Rac1 decreased in response to ZrO2 enriched medium. This corroborates the result of the crystal violet assay, which indicated a significant decrease of pre-osteoblast adhesion in responding to ZrO2 enriched medium. However, we credit this decrease on pre-osteoblast adhesion to the need to govern intracellular repertory of intracellular pathways involved with cell cycle progression, because we found a significant up-phosphorylation of Mitogen-Activated Protein Kinase (MAPK)-p38 and Cyclin-dependent kinase 2 (CDK2), while p15 (a cell cycle suppressor) decreased. Importantly, Protein phosphatase 2 A (PP2A) activity decreased, guaranteeing the significant up-phosphorylation of MAPK -p38 in response to ZrO2 enriched medium. Complementarily, there was a regulation of Matrix Metalloproteinases (MMPs) in response to Zirconia and this remodeling could affect cell phenotype by interfering on cell anchorage. Altogether, our results show a repertory of signaling molecules, which suggests that ECM remodel as a pre-requisite to pre-osteoblast phenotype by affecting their anchoring in responding to zirconia. Bioassays and Cell Dynamics Lab Dept. of Chemistry and Biochemistry Bioscience Institute Universidade Estadual Paulista - UNESP Bioassays and Cell Dynamics Lab Dept. of Chemistry and Biochemistry Bioscience Institute Universidade Estadual Paulista - UNESP FAPESP: 2014/22689-3

Made available in DSpace on 2018-12-11T17:21:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-10-01 Background: The placenta is a multifunctional organ that can suffer with imbalances between pro- and antioxidant molecules, contributing for inflammatory imbalance. The inflammation generated by oxidative stress may induce inflammasome activation, an essential complex for pro-inflammatory cytokine production. Objective: The aim of this study was to evaluate whether hydrogen peroxide (H2O2) mediated oxidative stress induces inflammasome activation on placental explants. Study design: Tissue cultures of placental explants obtained from normotensive pregnant women were performed in different concentrations of H2O2. Gene expressions of NLRP3, caspase-1, IL-1β TNF-α and IL-10 were evaluated by qPCR. Superoxide dismutase (SOD), catalase, Heat shock protein 70 (Hsp70), Caspase-1, TNF-α IL-1β IL-10 and human Chorionic Gonadotropin (hCG) were determined by ELISA. Results: Concentrations of catalase, Hsp70, hCG and SOD were higher in cultures with 100 and 1000 µM H2O2 compared to controls. Gene and protein expressions of TNF-α and IL-1β were elevated in cultures with 1000 μM H2O2 compared to controls. This concentration led to inflammasome activation, by increasing gene expressions of NLRP3, caspase-1 and IL-1β. In contrast, gene and protein expressions of IL-10 were reduced at 100 and 1000 μM H2O2. Protein expression of caspase-1 was higher in cultures of 100 μM H2O2 compared to controls. Treatment with Glybenclamide at 200 μM was used to prevent NLRP3 inflammasome activation. This concentration reduced protein expression of caspase-1 compared to culture with only H2O2 and control cultures. Conclusions: Our results confirm that H2O2 induces oxidative stress on placental explants and demonstrate that cell responses to this stress involve inflammasome activation. Department of Gynaecology and Obstetrics Medical School Botucatu Sao Paulo State University (Unesp) Department of Microbiology and Immunology Institute of Biosciences Botucatu Sao Paulo State University (Unesp) Department of Gynaecology and Obstetrics Medical School Botucatu Sao Paulo State University (Unesp) Department of Microbiology and Immunology Institute of Biosciences Botucatu Sao Paulo State University (Unesp)

Made available in DSpace on 2018-12-11T17:19:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-03-01 Over the last several years, we have focused on the importance of intracellular signaling pathways in dynamically governing the biointerface between pre-osteoblast and surface of biomaterial. Thus, this study investigates the molecular hallmarks involved in the pre-osteoblast relationship with different topography considering Machined (Mc), Dual Acid-Etching (DAE), and nano hydroxyapatite-blasted (nHA) groups. There was substantial differences in topography of titanium surface, considering Atomic Force Microscopy and water contact angle (Mc = 81.41 ± 0.01; DAE = 97.18 ± 0.01; nHA = 40.95 ± 0.02). Later, to investigate their topography differences on biological responses, pre-osteoblast was seeded on the different surfaces and biological samples were collected after 24 h (to consider adhesion signaling) and 10 days (to consider differentiation signaling). Preliminary results evidenced significant differences in morphological changes of pre-osteoblasts mainly resulting from the interaction with the DAE and nHA, distinguishing cellular adaptation. These results pushed us to analyze activation of specific genes by exploring qPCR technology. In sequence, we showed that Src performs crucial roles during cell adhesion and later differentiation of the pre-osteoblast in relationship with titanium-based biomaterials, as our results confirmed strong feedback of the Src activity on the integrin-based pathway, because integrin-ß1 (∼5-fold changes), FAK (∼12-fold changes), and Src (∼3.5-fold changes) were significantly up-expressed when Src was chemically inhibited by PP1 (5 μM). Moreover, ECM-related genes were rigorously reprogrammed in response to the different surfaces, resulting on Matrix Metalloproteinase (MMP) activities concomitant to a significant decrease of MMP inhibitors. In parallel, we showed PP1-based Src inhibition promotes a significant increase of MMP activity. Taking all our results into account, we showed for the first time nano hydroxyapatite-blasted titanium surface creates a biointerface able to govern Src-dependent osteoblast metabolism as pre-requisite to ECM remodeling Department of Chemistry and Biochemistry Bioscience Institute São Paulo State University UNESP Campus Botucatu Electron Microscopy Center IBB UNESP Department of Chemistry and Biochemistry Bioscience Institute São Paulo State University UNESP Campus Botucatu Electron Microscopy Center IBB UNESP

Made available in DSpace on 2018-12-11T16:47:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-12-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) We hypothesized that a crosstalk between osteoblast and fibroblast (FB) exists, which contributes to bone as a dynamic tissue. Cell-free supernatants were harvested from fibroblast cultures and later subject pre-osteoblasts to investigate there capacity to modulate cell viability and differentiation mechanisms, reporting the possible involvement of Shh signaling as a paracrine mechanism. By exploring immunoblotting technology, we have shown that FB-released factors interfere with osteoblast metabolism by up-regulating the phosphorylation of FAK and Rac-1 proteins at the early stage and later contribute to osteoblast differentiation by up-modulating alkaline phosphatase (ALP) and in vitro mineralization. We also found that Shh signaling was not required during osteoblastic differentiation promoted by the FB-released factors as well as MAPK-ERK phosphorylation, while pre-osteoblast cultures subjected to osteogenic medium (O.M.) require downstream transducers of Shh, such as Patched and Gli-1, and MAPK-ERK. Altogether, our results indicate for the first time a possible mechanism involved in the crosstalk between fibroblasts and osteoblasts, as it was possible to observe trophic factors released by fibroblasts interfering decisively in osteoblast metabolism in a Shh-independent manner. This study collaborates the body of work that indicates paracrine signaling molecules participate in the crosstalk among bone-resident cells and explains, at least partially, the biological mechanisms responsible for bone tissue dynamism, opening new avenues to understand etiologies of bone diseases. Bioassays and Cell Dynamics Lab Department of Chemistry and Biochemistry Bioscience Institute UNESP Bioassays and Cell Dynamics Lab Department of Chemistry and Biochemistry Bioscience Institute UNESP FAPESP: #2014/22689-3 FAPESP: #2015/00581-9

Made available in DSpace on 2018-12-11T17:34:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-10-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases. Chemistry and Biochemistry Department Institute of Biosciences São Paulo State University (UNESP) Agronomic Institute (IAC) CEP Chemistry and Biochemistry Department Institute of Biosciences São Paulo State University (UNESP) FAPESP: 2014/ 22689-3 FAPESP: 2014/10962-7 FAPESP: 2015/01753-8