Thyroid hormone status interferes with estrogen target gene expression in breast cancer samples in menopausal women

We investigated thyroid hormone levels in menopausal BrC patients and verified the action of triiodothyronine on genes regulated by estrogen and by triiodothyronine itself in BrC tissues. We selected 15 postmenopausal BrC patients and a control group of 18 postmenopausal women without BrC. We measured serum TPO-AB, TSH, FT4, and estradiol, before and after surgery, and used immunohistochemistry to examine estrogen and progesterone receptors. BrC primary tissue cultures received the following treatments: ethanol, triiodothyronine, triiodothyronine plus 4-hydroxytamoxifen, 4-hydroxytamoxifen, estrogen, or estrogen plus 4-hydroxytamoxifen. Genes regulated by estrogen (TGFA, TGFB1, and PGR) and by triiodothyronine (TNFRSF9, BMP-6, and THRA) in vitro were evaluated. TSH levels in BrC patients did not differ from those of the control group (1.34 ± 0.60 versus 2.41 ± 1.10  μ U/mL), but FT4 levels of BrC patients were statistically higher than controls (1.78 ± 0.20 versus 0.95 ± 0.16 ng/dL). TGFA was upregulated and downregulated after estrogen and triiodothyronine treatment, respectively. Triiodothyronine increased PGR expression; however 4-hydroxytamoxifen did not block triiodothyronine action on PGR expression. 4-Hydroxytamoxifen, alone or associated with triiodothyronine, modulated gene expression of TNFRSF9, BMP-6, and THRA, similar to triiodothyronine treatment. Thus, our work highlights the importance of thyroid hormone status evaluation and its ability to interfere with estrogen target gene expression in BrC samples in menopausal women.

Bone morphogenetic proteins: promising molecules for bone healing, bioengineering, and regenerative medicine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hemodynamic response in one session of strength exercise with and without electrostimulation in heart failure patients: a randomized controlled trial

Background: Studies have investigated the influence of neuromuscular electrostimulation on the exercise/muscle capacity of patients with heart failure (HF), but the hemodynamic overload has never been investigated. The aim of our study was to evaluate the heart rate (HR), systolic and diastolic blood pressures in one session of strength exercises with and without neuromuscular electrostimulation (quadriceps) in HF patients and in healthy subjects. Methods: Ten (50% male) HF patients and healthy subjects performed three sets of eight repetitions with and without neuromuscular electrostimulation randomly, with one week between sessions. Throughout, electromyography was performed to guarantee the electrostimulation was effective. The hemodynamic variables were measured at rest, again immediately after the end of each set of exercises, and during the recovery period. Results: Systolic and diastolic blood pressures did not change during each set of exercises among either the HF patients or the controls. Without electrostimulation: among the controls, the HR corresponding to the first (85 ± 13 bpm, p = 0.002), second (84 ± 10 bpm, p < 0.001), third (89 ± 17, p < 0.001) sets and recuperation (83 ± 16 bpm, p = 0.012) were different compared to the resting HR (77 bpm). Moreover, the recuperation was different to the third set (0.018). Among HF patients, the HR corresponding to the first (84 ± 9 bpm, p = 0.041) and third (84 ± 10 bpm, p = 0.036) sets were different compared to the resting HR (80 ± 7 bpm), but this increase of 4 bpm is clinically irrelevant to HF. With electrostimulation: among the controls, the HR corresponding to the third set (84 ± 9 bpm) was different compared to the resting HR (80 ± 7 bmp, p = 0.016). Among HF patients, there were no statistical differences between the sets. The procedure was well tolerated and no subjects reported muscle pain after 24 hours. Conclusions: One session of strength exercises with and without neuromuscular electrostimulation does not promote a hemodynamic overload in HF patients. (Cardiol J 2011; 18, 1: 39-46)

Aedes aegypti proliferation in stormwater retained in Best Management Practices

The present study aimed to evaluate the risk of Aedes aegypti proliferation in structures used in compensatory techniques for urban drainage (Best Management PracticesBMPs). These drainage structures are utilised to reduce flood peaks due to surface runoff, and they have been used in many countries. However, many of these structures have been designed to keep water surfaces exposed for a certain period of time, depending on the type of project. Exposed water surfaces may become an ideal environment for A. aegypti proliferation in tropical and subtropical areas where the rainy season occurs during the summer. Thus, data regarding the mosquito life cycle, consecutive rainfall pattern and emptying time of these structures were collected. A comparison of these data led to the evaluation of the associated risk of A. aegypti proliferation in BMP structures. The risk of mosquito proliferation ranged from 1.1% to 3.3%, depending on the rainfall pattern, A. aegypti life cycle phase and BMP activity.

Monitoramento clínico do processo inflamatório periapical antes e após o uso de diferentes medicações intracanal através dos níveis de biomarcadores inflamatórios

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Monitoramento clínico do processo inflamatório periapical antes e após o uso de diferentes medicações intracanal através dos níveis de biomarcadores inflamatórios

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Human Bone Morphogenetic Protein-2 Use for Maxillary Reconstruction in Cleft Lip and Palate Patients

Background: The conventional methods of maxillary alveolar reconstruction in patient with cleft are the periosteoplasty and autologous bone grafting. As an important alternative of bone substitution, there is the recombinant human bone morphogenetic protein-2 (rhBMP-2). This study compares the rhBMP-2 with periosteoplasty and autologous bone grafting. Methods: Patients with cleft and alveolar defect were divided into 3 groups of 6 patients who underwent to autologous iliac crest bone grafting, resorbable collagen sponge with rhBMP2, and periosteoplasty, respectively. The analysis was performed through computed tomographic scan preoperatively and at months 3, 6, and 12 postoperatively. The variables analyzed were the alveolar defect volume, formed bone volume, bone formation rate, maxillary height repair rate, and the formed bone density mean. Results: The formed bone volume was similar comparing the bone graft and BMP groups at 1-year postoperative analysis (P = 0.58). Both of them had the formed bone volume significantly larger than the periosteoplasty group at 3 and 6 months postoperatively. In this last group, the 1-year follow-up was canceled because the bone formation was insufficient. The bone formation rate, the maxillary height repair rate, and the mean of density of the formed bone were similar in the bone graft and BMP groups at 1-year follow-up with P values of 0.93, 0.90, and 0.81, respectively. Conclusions: The amount of formed bone in the periosteoplasty group was insufficient. There was no difference among the bone graft and rhBMP-2 therapy considering the parameters analyzed.

Immunolocalization of bone morphogenetic protein 2 during the early healing events after guided bone regeneration

Objective. The objective of this study was to evaluate the immunolocalization of bone morphogenetic protein 2 (BMP-2) after autogenous block grafting covered or not with an e-PTFE membrane. Study Design. Forty-eight rats were divided into 2 groups, autogenous block graft (B) and autogenous block graft + e-PTFE membrane (MB), and were evaluated by immunohistochemistry at baseline and 3, 7, 14, 21, and 45 days. Results. The largest number of positive cells in the recipient bed was observed after 3 days in both groups. At the graft border, the largest number of positive cells was seen after 7 days in group B and after 14 days in group MB. The highest proportion of staining in the graft was observed after 3 days in group B and after 21 days in group MB. Conclusions. High proportions of stain were related to intense revascularization and osteogenesis. Except for the interface, BMP-2 staining occurred later in group MB than in group B in all structures analyzed. (Oral Surg Oral Med Oral Pathol Oral Radiol 2012;113:533-541)

Pore size regulates cell and tissue interactions with PLGA-CaP scaffolds used for bone engineering

A common subject in bone tissue engineering is the need for porous scaffolds to support cell and tissue interactions aiming at repairing bone tissue. As poly(lactide-co-glycolide)calcium phosphate (PLGACaP) scaffolds can be manufactured with different pore sizes, the aim of this study was to evaluate the effect of pore diameter on osteoblastic cell responses and bone tissue formation. Scaffolds were prepared with 85% porosity, with pore diameters in the ranges 470590, 590850 and 8501200 mu m. Rat bone marrow stem cells differentiated into osteoblasts were cultured on the scaffolds for up to 10 days to evaluate cell growth, alkaline phosphatase (ALP) activity and the gene expression of the osteoblast markers RUNX2, OSX, COL, MSX2, ALP, OC and BSP by real-time PCR. Scaffolds were implanted in critical size rat calvarial defects for 2, 4, and 8 weeks for histomorphometric analysis. Cell growth and ALP activity were not affected by the pore size; however, there was an increase in the gene expression of osteoblastic markers with the increase in the pore sizes. At 2 weeks all scaffolds displayed a similar amount of bone and blood vessels formation. At 4 and 8 weeks much more bone formation and an increased number of blood vessels were observed in scaffolds with pores of 470590 mu m. These results show that PLGACaP is a promising biomaterial for bone engineering. However, ideally, combinations of larger (similar to 1000 mu m) and smaller (similar to 500 mu m) pores in a single scaffold would optimize cellular and tissue responses during bone healing. Copyright (C) 2011 John Wiley & Sons, Ltd.

New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells

Abstract Background Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. Results From 150 μg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Conclusions Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells.

Studio del pathway della Displasia Ectodermica Anidrotica (EDA)

Anhidrotic Ectodermal Dysplasia (EDA), is the most frequent form among Ectodermal Dysplasias, hereditary genetic disorders causing ectodermal appendages defective development. Indeed, EDA is characterized by defective formation of hair follicles, sweat glands and teeth both in human patients and animals. EDA, the gene mutated in Anhidrotic Ectodermal Dysplasia, encodes Ectodysplasin, a TNF family member that activates NF-kB mediated transcription. This disease can occur with mutations in other EDA-NF-kB pathway members, as EDA receptor, EDAR and its adapter, EDARADD. Moreover, mutations in TRAF6, NEMO, IKB and NF-kBs genes are responsible for Immunodeficiency associated EDA (EDA-ID). Several molecules, as SHH, WNT/DKK, BMP and LTβ, have already been reported to be EDA pathway regulators or effectors although the knowledge of the full spectrum of EDA targets remains incomplete. During the first part of the research project a gene expression analysis was performed in primary keratinocytes from Wild-type and Tabby (EDA model mouse) mice to identify novel EDA target genes. Earlier expression profiling at various developmental time points in Tabby and Wild-type mouse skin reported genes differentially expressed in the two samples and, to increase the resolution to find genes whose expression may be restricted to epidermal cells, the study was extended to primary keratinocyte cultures established from E19 Wild-type and Tabby skin. Using microarrays bearing 44,000 gene probes, we found 385 “preliminary candidate” genes whose expression was significantly affected by Eda defect. By comparing expression profiles to those from Eda-A1 (where Eda-A1 is highly expressed) transgenic skin, we restricted the list to 38 “candidate EDA targets”, 14 of which were already known to be expressed in hair follicles or epidermis. This work confirmed expression changes for 3 selected genes, Tbx1, Bmp7, and Jag1, both in primary keratinocytes and in Wild-type and Tabby whole skin, by Q-PCR and Western blotting analyses. Thus, this study detected novel candidate pathways downstream of EDA. In the second part of the research project, plasmid constructs were produced and analyzed to create a transgenic mouse model for Immunodeficiency associated EDA disease (XL-EDA-ID). In particular, plasmids containing mouse Wild-type and mutated Nemo cDNA under K-17 epidermis-specific promoter control and a Flag tag, were prepared, on the way to confine transgene expression to mice epidermis and to determine EDA phenotype without immunodeficiency for a comparison to Tabby model phenotype. EDA-ID mutations reported in patients and selected for this study are: C417R (C409R in mouse), causing Zinc Finger protein domain destabilization and A288G (A282G in mouse) affecting oligomerization of the protein. Moreover, the ex-novo mutation, ZnF, C-terminal Zinc Finger domain deletion, was tested. Thus, the constructs were analyzed by transient transfection, Western blotting and luciferase assays techniques, detecting Nemo Wild-type and mutant protein products and residue NF-kB activity in presence of mutants, after TNF stimulation. In particular, MEF_Nemo-/- cell line was used to monitor NF-kB activity without endogenous Nemo gene. Results show reduced NF-kB activity in presence of mutated Nemo forms compared to Wild-type: 81% for A282G (A288G in human); 24% for C409R (C417R in human); 15% for ZnF. C409R mutation (C417R in human), reported in 6 EDA-ID human patients, was selected to prepare transgenic model mouse. Mice (white, FVP) born following K17-promoter-Flag-Nemo_C409R plasmid region pronuclear injection, were analyzed for the transgene presence in the genotype and a preliminar examination of their phenotype was performed. In particular, one mouse showed considerable coat defects if compared to Wild-type mice. This preliminar analysis suggests a possible influence of Nemo mutant over-expression in epidermis without immunodeficiency. Still, more microscopic studies to analyze hair subtypes, Guard, Awl and Zigzag (usually alterated inTabby mouse model), Immunohistochemistry experiments to detect epidermis restricted Nemo expression and sweat glands analysis, will follow. This and other transgene positive mice will be crossed with black mice C57BL6 to obtain at least two indipendent agouti lines to analyze. Theses mice will be used in EDA target genes detection through microarrays. Following, plasmid constructs containing other Nemo mutant forms (A282G and ZnF) might be studied by the same experimental approaches to prepare more transgenic model mice to compare to Nemo_C409R and Tabby mouse models.

3D nanostructured microcarriers for cell therapy in regenerative medicine

Supercritical Emulsion Extraction technology (SEE-C) was proposed for the production of poly-lactic-co-glycolic acid microcarriers. SEE-C operating parameters as pressure, temperature and flow rate ratios were analyzed and the process performance was optimized in terms of size distribution and encapsulation efficiency. Microdevices loaded with bovine serum insulin were produced with different sizes (2 and 3 µm) or insulin charges (3 and 6 mg/g) and with an encapsulation efficiency of 60%. The microcarriers were characterized in terms of insulin release profile in two different media (PBS and DMEM) and the diffusion and degradation constants were also estimated by using a mathematical model. PLGA microdevices were also used in a cultivation of embryonic ventricular myoblasts (cell line H9c2 obtained from rat) in a FBS serum free medium to monitor cell viability and growth in dependence of insulin released. Good cell viability and growth were observed on 3 µm microdevices loaded with 3 mg/g of insulin. PLGA microspheres loaded with growth factors (GFs) were charged into alginate scaffold with human Mesenchimal Steam Cells (hMSC) for bone tissue engineering with the aim of monitoring the effect of the local release of these signals on cells differentiation. These “living” 3D scaffolds were incubated in a direct perfusion tubular bioreactor to enhance nutrient transport and exposing the cells to a given shear stress. Different GFs such as, h-VEGF, h-BMP2 and a mix of two (ratio 1:1) were loaded and alginate beads were recovered from dynamic (tubular perfusion system bioreactor) and static culture at different time points (1st, 7th, 21st days) for the analytical assays such as, live/dead; alkaline phosphatase; osteocalcin; osteopontin and Van Kossa Immunoassay. The immunoassay confirmed always a better cells differentiation in the bioreactor with respect to the static culture and revealed a great influence of the BMP-2 released in the scaffold on cell differentiation.

Effects of low-density lipoprotein receptor-related protein 4 and apolipoprotein E isoforms on bone metabolism in vivo

LRP4, member of the LDLR family, is a multifunctional membrane-bound receptor that is expressed in various tissues. The expression of LRP4 by osteoblasts, its novel interaction with Wnt-signaling inhibitors Dkk1 and SOST, and the lower levels of activated beta-catenin in different bone locations described here, adds another player to the long list of established factors that modulate canonical Wnt-signaling in bone. By demonstrating that in addition to Wise, LRP4 is able to interact with two additional important modulators of Wnt- and BMP-signaling, our perspective of the complexity of the integration of BMP and Wnt-signaling pathways on the osteoblast surface has expanded further. Nevertheless the recently described association of both the SOST and LRP4 genes with BMD in humans, together with our findings suggest that LRP4 plays a physiologically important role in the skeletal development and bone metabolism not only in rodents, but in humans as well. The efficiency with which LRP4 binds both SOST and Dkk1, presumably at the osteoblastic surface, LRP4 may act as a sink and competes with LRP5/6 for the binding of these Wnt antagonists, which then are no longer available for suppression of the signal through the LRP5/6 axis. rnApoE, a 299 amino acid glycoprotein, is a crucial regulator in the uptake of triglyceride, phospholipids, cholesteryl esters, and cholesterol into cells. ApoE has been linked to osteoporosis, and such a role is further strengthened by the present of a high bone mass phenotype in ApoE null mice. Until recently, the effects of respective ApoE isoforms E2, E3, and E4, and their impact on bone metabolism, have been unclear. Here we report that respective human ApoE knockin mice display diverse effects on bone metabolism. ApoE2 mice show decreased trabecular bone volume per total volume in femoral bone and lumbar spine in comparison to ApoE3 and E4 animals. In this context, urinary bone resorption marker DPD is increased in these animals, which is accompanied by a low ratio of osteoclastogenesis markers OPG/RANKL. Interestingly, serum bone formation markers ALP and OCN are diminished in ApoE4 mice. In contrast to this finding, ApoE2 mice show the lowest bone formation of all groups in vivo. These findings cannot be explained by the low receptor-affinity of ApoE2 and subsequent decreased uptake of triglyceride-rich lipoproteins by osteoblasts, resulting in elevated levels of undercarboxylated osteocalcin. Thus, other crucial pathways relevant for bone metabolism, e. g. Wnt/beta-catenin-signaling pathways, must be, compared to the ApoE3/4 isoforms, more affected by the ApoE2 isoform.

Role of the Transcription Factor Sox2 in the Osteogenic Lineage

The Sox2 transcription factor is modified by sumoylation at the K247 position although the addition of SUMO1 and Pias1 promotes the sumoylation of Sox2 at the additional K123 site. The role of sumoylation on Sox2 biological functions was analyzed by comparing the activity of WT and sumoylation mutants on the transcription of the FGF4 gene in HeLa cells and on the downregulation of the Wnt pathwayvin 293T cells. When SUMO1 and PIAS1 promote the sumoylation of WT Sox2, the transcriptional activity of the FGF4 promoter is inhibited showing that Sox2 sumoylation is necessary for the repression function. However, there is no effect of Sox2 sumoylation on β-Catenin activity. Since we were interested in osteoblast differentiation we set up an inducible system for Sox2 in primary osteoblasts. Following Sox2 doxycycline induction, 158 genes were differentially expressed: 120 up-regulated and 38 down-regulated. We annotated as direct Sox2 targets a number of genes involved in osteoblast biology and we further analyzed 3 of them involved in the BMP pathway. The results show that Sox2 regulates the BMP pathway without affecting SMAD phosphorylation, and that Sox2 sumoylation is not necessary for this function. We also found that genes involved in the Hippo pathway were direct Sox2 targets. As the Hippo pathway is activated by Sox2 and Sox2 interacts with the NF2 promoter, we checked the effect of Sox2 on the expression of NF2. We showed that Sox2 down-regulates the transcriptional activity of the NF2 promoter, allowing the transcription of the YAP/TEAD genes in osteoblasts, thus acting as an upstream regulator of the Hippo pathway. We conclude that Sox2 induction in osteoblasts triggers FGF dependent inhibition of the BMP, Wnt and Hippo pathways.

Buone pratiche per la gestione delle acque meteoriche. Il caso studio dei tetti verdi della Scuola di Ingegneria e Architettura e la progettazione di un sistema integrato di raccolta e infiltrazione delle acque piovane

La tesi analizza i principi di gestione sostenibile della risorsa meteorica attraverso la progettazione di una pavimentazione drenante, di un Rain Garden ed un impianto di raccolta e riutilizzo dell’acqua piovana, con lo scopo di costituire un sistema integrato con il verde pensile già esistente. Nella prima fase di stesura della tesi si è dato importanza allo studio ed all'elaborazione dei dati di runoff provenienti dal sistema di monitoraggio dei tetti verdi installati sul laboratorio LAGIRN. In seguito si è dato risalto alla gestione sostenibile della risorsa idrica, dimensionando un sistema di riutilizzo dell’acqua piovana al fine di irrigare il verde pensile e le altre zone verdi posizionate in prossimità del laboratorio. Si è dedotto che l’efficienza nel riutilizzo dell’acqua piovana dipende essenzialmente dal rapporto tra la disponibilità di superficie di raccolta e conseguente domanda del riuso da soddisfare, che potrebbe portare ad un dimensionamento errato del serbatoio di accumulo con il risultato di influenzare il costo complessivo del sistema. Sono state progettate due tipologie di BMP finalizzate alla riduzione dell’impermeabilizzazione del suolo per migliorare quantitativamente e qualitativamente la permeabilità. Il Rain Garden risulta un metodo molto economico per riqualificare il tessuto urbano tenendo conto anche dei cambiamenti climatici in continua evoluzione. Le BMP appaiono quindi una soluzione economica e facilmente attuabile sia dai singoli cittadini che da enti comunali al fine di migliorare le qualità delle acque, diminuire il rischio idraulico e smaltire all'interno dei corpi idrici acque di buona qualità.