Mediation analysis of an effective sexual health promotion intervention for Spanish adolescents.

The objective of this study is to determinate the factors that mediate in the self-reported consistent condom use over the 24-months post-intervention period in adolescents who received COMPAS, a sexual health promotion intervention targeted to Spanish adolescents. Twelve high schools located in Spain were randomized to an intervention or a control group with baseline, immediate-post, 12 and 24-month post-intervention assessments. Self-reported consistent condom use by 24 months post-intervention was the primary outcome. Based on the theory of planned behavior, we identified which theory-based variables mediated the intervention's effect on consistent condom use. Serial multiple mediation analysis indicated that attitudes toward condom use, when there are obstacles to use it, and self-efficacy mediated the COMPAS's effect in increasing consistent condom use. This is the first study that identifies the theoretical constructs that mediate the efficacy of a school-based intervention to promote sexual health in adolescents from Spain.

Cryptomphalus aspersa mollusc eggs extract promotes migration and prevents cutaneous ageing in keratinocytes and dermal fibroblasts in vitro.

BACKGROUND: The search of substances that minimize cutaneous ageing has increased in the last few years. Previous studies have described the regenerative properties of the secretion of the mollusc Cryptomphalus aspersa (C. aspersa) when applied topically. OBJECTIVE: We evaluate the in vitro effects of a new product derived from the eggs of C. aspersa, IFC-CAF, on cell proliferation, migration, distribution of cytoskeletal proteins, production of extracellular components as well as its ability to prevent cutaneous ageing because of intrinsic or extrinsic factors (exposure to UVB) by determination of ageing markers. METHODS: We have used the human keratinocyte cell line (HaCaT cells), primary dermal fibroblasts (HDF) and senescent dermal fibroblasts (SHDF). The effects of the compound on cell proliferation and on the cell cycle were determined by the MTT colorimetric assay, estimation of total protein and/or trypan blue test and by flow cytometry, respectively. We also studied cell migration using the wound-healing migration assay, whereas ELISA assays, Western Blot and immunofluorescence microscopy were carried out to test the expression of proteins related to cytoskeleton, extracellular matrix and with ageing. RESULTS: We have found that IFC-CAF does not promote proliferation but induces migration of HaCaT, HDF and SHDF in a time- and dose-dependent manner; a better organization of cytoskeletal proteins (F-actin and vimentin) and promotes the production of extracellular components (fibronectin, collagen 1 and MMPs) and the adhesion to cell-substrate vinculin protein. IFC-CAF also prevents cutaneous ageing. The treatment decreases the expression of the ageing-related markers b-Gal, p53 and p16INK4 in SDDF cells, and improves cell survival after UVB irradiation and nuclear repair in HaCaT cells. CONCLUSION: IFC-CAF has regenerative properties and protects against ageing factors being, therefore, a potential therapeutic agent for treating or preventing skin ageing.

New experimental models of skin homeostasis and diseases.

Homeostasis, whose regulation at the molecular level is still poorly understood, is intimately related to the functions of epidermal stem cells. Five research groups have been brought together to work on new in vitro and in vivo skin models through the SkinModel-CM program, under the auspices of the Spanish Autonomous Community of Madrid. This project aims to analyze the functions of DNA methyltransferase 1, endoglin, and podoplanin in epidermal stem cell activity, homeostasis, and skin cancer. These new models include 3-dimensional organotypic cultures, immunodeficient skin-humanized mice, and genetically modified mice. Another aim of the program is to use skin-humanized mice to model dermatoses such as Gorlin syndrome and xeroderma pigmentosum in order to optimize new protocols for photodynamic therapy.

Spanish version of the Sleep Self-Report (SSR): factorial structure and psychometric properties.

BACKGROUND: The Sleep Self-Report (SSR) is a questionnaire initially created for use with a sample from the USA to assess sleep patterns and problems in school-aged children. The objective of this study was to validate the SSR among a Spanish sample. METHODS: Participants were 1228 Spanish children from 8 to 12 years of age who completed the questionnaires at school anonymously. RESULTS: Internal consistency was good (ω = 0.85). Convergent validity with anxiety (r = 0.54) and perceived welfare (r = -0.53) measures, and divergent validity with a measure of academic performance and positive influence of peers (r = -0.22) were acceptable. Exploratory analysis suggested a factorial structure composed by four subscales: sleep quality, sleep anxiety, bedtime refusal and sleep routines. Confirmatory analysis indicated a good fit for the model (RMSEA = 0.04; GFI = 0.95; AGFI = 0.93; χ(2)/gl = 2.48). CONCLUSIONS: The SSR has demonstrated to have good psychometric properties in the Spanish-speaking sample of children. The factorial structure supported by exploratory and confirmatory analysis examines the most relevant areas of sleep in children. The satisfactory psychometric properties support the use of the Spanish version of the SSR by researchers and clinicians.

H-Ras activation promotes cytoplasmic accumulation and phosphoinositide 3-OH kinase association of beta-catenin in epidermal keratinocytes.

The mechanisms underlying downregulation of the cadherin/catenin complexes and beta-catenin signaling during tumor progression are not fully understood. We have analyzed the effect of oncogenic H-Ras on E-cadherin/catenin complex formation/stabilization and beta-catenin distribution in epidermal keratinocytes. Microinjection or stable expression of V12Ras into keratinocytes promotes the loss of E-cadherin and alpha-catenin and relocalization of beta-catenin to the cytoplasm and nucleus. Moreover, these effects are dependent on PI3K (phosphoinositide 3-OH kinase) activity. Interestingly, a strong association of p85alpha and p110alpha subunits of PI3K with beta-catenin is induced in V12Ras-expressing keratinocytes, and in vitro binding assays show a direct interaction between beta-catenin and p85alpha. Overexpression of either V12Ras or constitutively active p110alpha induces metabolic stabilization of beta-catenin and promotes its accumulation in cytoplasmic and nuclear pools. In addition, the interaction of beta-catenin with the adenomatous polyposis coli protein is blocked in V12Ras and p110alpha transformants though no changes in glycogen synthase kinase 3 beta activity could be detected. Nevertheless, in V12Ras transformants the in vivo phosphorylation of beta-catenin in Ser residues is strongly decreased. These results indicate that H-Ras activation induces the relocalization and cytoplasmic stabilization of beta-catenin by a mechanism involving its interaction with PI3K.

Vitamin D(3) promotes the differentiation of colon carcinoma cells by the induction of E-cadherin and the inhibition of beta-catenin signaling.

The beta-catenin signaling pathway is deregulated in nearly all colon cancers. Nonhypercalcemic vitamin D3 (1alpha,25-dehydroxyvitamin D(3)) analogues are candidate drugs to treat this neoplasia. We show that these compounds promote the differentiation of human colon carcinoma SW480 cells expressing vitamin D receptors (VDRs) (SW480-ADH) but not that of a malignant subline (SW480-R) or metastasic derivative (SW620) cells lacking VDR. 1alpha,25(OH)2D(3) induced the expression of E-cadherin and other adhesion proteins (occludin, Zonula occludens [ZO]-1, ZO-2, vinculin) and promoted the translocation of beta-catenin, plakoglobin, and ZO-1 from the nucleus to the plasma membrane. Ligand-activated VDR competed with T cell transcription factor (TCF)-4 for beta-catenin binding. Accordingly, 1alpha,25(OH)2D(3) repressed beta-catenin-TCF-4 transcriptional activity. Moreover, VDR activity was enhanced by ectopic beta-catenin and reduced by TCF-4. Also, 1alpha,25(OH)2D(3) inhibited expression of beta-catenin-TCF-4-responsive genes, c-myc, peroxisome proliferator-activated receptor delta, Tcf-1, and CD44, whereas it induced expression of ZO-1. Our results show that 1alpha,25(OH)2D(3) induces E-cadherin and modulates beta-catenin-TCF-4 target genes in a manner opposite to that of beta-catenin, promoting the differentiation of colon carcinoma cells.

Epigenetic inactivation of the Wnt antagonist DICKKOPF-1 (DKK-1) gene in human colorectal cancer.

Colorectal cancer is a major cause of cancer death worldwide. A number of key oncogenes and tumor suppressor genes have been proposed to drive progression from healthy colonic epithelia to malignant tumors, including members of the Wnt/beta-catenin pathway. Recently, CpG island promoter hypermethylation was shown to cause inactivation of two extracellular Wnt inhibitors in colon cancer: secreted frizzled-related proteins (sFRPs) and Wnt inhibitory factor-1 (WIF-1). Here, we show for the first time that another extracellular Wnt inhibitor, the DICKKOPF-1 (DKK-1) gene, is transcriptionally silenced by CpG island promoter hypermethylation in colon cancer cell lines (n=9), whereas treatment with the DNA-demethylating agent 5-aza-2-deoxycytidine restored DKK-1 expression. Restoration of DKK-1 function in non-expressing cells bearing a truncated APC (Adenomatous Polyposis Coli) gene had no effect on beta-catenin/T-cell factor-dependent transcription, but induced tumor suppressor-like features such as reduced colony formation density and tumor growth inhibition in nude mice. These results suggest additional functions for DKK-1 other than inhibiting canonical Wnt signaling. In primary colorectal tumors, DKK-1 was found hypermethylated in 17% (nine of 54) of cases. Furthermore, while for both SFRP-1 and WIF-1 methylation-associated silencing occurred across the whole spectrum of colorectal tumorigenesis, DKK-1 promoter was selectively hypermethylated in advanced colorectal neoplasms (Duke's C and D tumors).

[Experiences with the Eurotest in neuropsychological examinations. A screening test]. / Experiencia con el Eurotest en la exploración neuropsicológica. Un test de cribado.

INTRODUCTION: The increasing number of foreign patients being attended in our health centres makes it necessary to have tests that are compatible among different countries, especially in Europe. The objective of this study is to compare the Eurotest with other screening tests and to determine whether there are any differences between European (other than Spanish) and Spanish patients. PATIENTS AND METHODS: We included patients who were referred because of complaints that were compatible with cognitive impairment and they were asked to carry out a protocol of tests that included the global deterioration scale (GDS), Folstein's MMSE (Mini-Mental State Examination), the Clock Test (CT) and the Eurotest. Epidemiological data were collected. The sample used in the study included 45 patients, 21 males and 24 females, with a mean age of 69.76 years; 34 were born in Spain and 11 abroad. RESULTS: Diagnoses were 17 with mild cognitive impairment, 8 with Alzheimer's disease, 6 with age-associated impairment, 5 with vascular dementia, 5 with a psychiatric disorder, 1 with mixed dementia and 1 with dementia with Lewy bodies. The distribution according to the GDS was as follows: 10 with a GDS of 2, 18 with a GDS of 3, and 17 with a GDS of 4. All the patients with a GDS 2 completed the tests correctly, those with a GDS 3 scored below the cut-off point 5 for the Eurotest, 4 for the MMSE and 3 for CT, and in the GDS 4 they were 9, 7 and 6, respectively. Results did not vary between Spaniards and foreigners, the kappa index between MMSE and the Eurotest was 0.39 and the Pearson coefficient was 0.67. CONCLUSION: The Eurotest was slightly more useful in patients with a greater degree of impairment.

An Update on Src Family of Nonreceptor Tyrosine Kinases Biology.

The members of the Src family of nonreceptor tyrosine kinases (SFKs) are implicated in multiple signaling processes that regulate key cellular functions, including proliferation, migration, differentiation, and survival. SFKs are activated by a large number of receptors for growth factors, cytokines, steroid hormones, G protein-coupled receptors, and also by adhesion proteins and other signaling partners. Through their common modular kinase an adapter protein domains, SFKs critically contribute to diversify different signal inputs, weaving a complex and dynamic network of cellular responses. Not surprisingly, SFKs are involved in embryo development and in the maintenance of different adult tissues and organs. Conversely, dysfunction of SFKs is associated with different pathologies, including cancer. Despite the continuous research in the field, several aspects of SFKs regulation and function are still not well defined and new roles for these proteins are steadily reported. The aim of this review is to provide an update on the major regulatory mechanisms of SFKs activity, including the emerging redox-dependent pathway. We have also focused on the functional implications of SFKs in Prolactin signaling and breast cancer development, two increasingly important aspects of SFKs biology. Finally, we briefly revisited the role of SFKs during embryo development and provide insights on the involvement of these proteins in the regulation of embryonic, somatic, and breast cancer stem cells.

Cytochemical application of tris (2,2'-bipyridine) ruthenium (II): fluorescence reaction with sulfated polyanions of mast cell granules.

We describe the use of tris (2,2'-bipyridine) ruthenium (II) (Rubipy) as a cationic fluorochrome for cytochemical and histochemical studies. After staining with Rubipy, mast cell granules (MCGs) and lymphocyte nuclei (LN) from mouse peritoneal cavity and human breast carcinoma showed intense orange fluorescence and no fading under blue or blue-violet exciting light. Staining at low pH (< 2) or pre-treatment with Al3+ ions strongly diminished the fluorescence of LN, whereas that of MCG was less affected. Ca2+ and Ba2+ ions only diminished MCG fluorescence. Blots of DNA, pectic acid, heparin, and other sulfated polysaccharides stained with Rubipy showed high emission, which was reduced in DNA and pectic acid staining at low pH. Studies with chemically modified heparins suggested that O-sulfates were more important than N-sulfates in Rubipy-heparin interactions. These results are in agreement with an ionic binding mode between Rubipy and heparin. A very suitable method for mast cell detection was found with Mayer's hematoxylin before Rubipy staining, which could be of great value for histopathological studies. This procedure allowed visualization of the mast cells by fluorescence microscopy, and nuclei and tissue morphology were easily visualized under brightfield illumination.

Immediate and follow-up findings after stent treatment for severe coarctation of aorta.

Experimental studies have shown that stents implanted at the aorta become incorporated within the aortic wall and can be further expanded in growing animals. Few clinical studies have shown that the stent repair of severe coarctation of aorta provides excellent initial results, and little is known on the follow-up of these patients. We assessed the immediate and follow-up results obtained in a series of 48 patients (mean age 14+/-12 years) with severe coarctation of the aorta who were treated by Palmaz stent implantation; 30 of them (63%) underwent angiographic follow-up studies at a mean of 25+/-11 months after treatment. Quantitative serial analysis of the aortogram (baseline, after treatment, and at follow-up) was performed. Significant relief (mean residual gradient 3+/-4 mm Hg) was always obtained after stent implantation. The isthmus, when hypoplastic (60%), was always expanded with the stent. One associated aneurysm became occluded after the implant. Complications included aortic disruption, stent migration, and decreased or absent femoral pulses. At angiographic follow-up, the stent remained always in place, without recoil. In 22 patients (73%), there were no detectable neointimal proliferation at late angiogram; however, 8 patients (27%) had some degree of intimal thickening (1 to 5 mm), causing mild restenosis in 3 patients treated at early age, and nonsignificant lumen reduction in 5. The serial aortogram analysis revealed a minor but significant increase in nonstented aortic diameters that seemed related to the normal growth of children. No need for stent reexpansion was observed at 2-year follow-up (mean). Two patients (7%) developed late small aneurysm formation at the stented wall; both were occluded by the insertion of coils through the stent orifices. We conclude that stent treatment for severe coarctation of aorta provides excellent immediate and long-term results in young adults and children. However, at early age, restenosis by intimal growth may develop.

Photodamage induced by Zinc(II)-phthalocyanine to microtubules, actin, alpha-actinin and keratin of HeLa cells.

We have studied the photosensitizing effects of zinc(II)-phthalocyanine (ZnPc) on the cytoskeleton of HeLa cells using sublethal (10(-7) M, followed by 1 or 3 min of red light to induce 20%, LD20, or 60%, LD60, cell death, respectively) or lethal (5 x 10(-6) M and 15 min of irradiation, LD100) experimental conditions. The immunofluorescent analysis of the cytoskeleton showed a variable photodamage to microtubules (MT), actin microfilaments (AF) and intermediate filaments of keratin (KF), as well as on alpha-actinin, which was dependent on treatment conditions. Both sublethal treatments induced deep alterations on interphase and mitotic MT. The mitotic index increased with time with the maximum at 18 h (12%) or 24 h (14%) after LD20 or LD60, respectively. The alterations on AF and alpha-actinin were much more severe than those observed on KF at any evaluated time. With the exception of the KF, which remained partially organized, the MT and AF network was severely damaged by the lethal treatment. Western blot analysis for alpha-tubulin, G-actin and alpha-actinin from soluble and insoluble fractions confirmed the results observed by immunofluorescence, thus indicating that these cytoskeletal components are involved in cell damage and death by ZnPc photosensitization.

Fluorescence of bisazo dye reaction products from the coupled tetrazonium method for proteins.

The coupled tetrazonium reaction is a well known histochemical method for proteins. This method, using Fast Blue B salt and 1-naphthol, has been applied on paraffin sections of grasshopper testis and rabbit trachea, as well as on horse blood smears. Microscopic observation under bright field illumination revealed the expected purple staining of protein-rich cell and tissue structures, which also showed a strong red fluorescence under ultraviolet, violet, violet-blue and green exciting light. Some weakly stained cell components (e.g., meiotic spindles) were easily visualized by fluorescence microscopy. Control preparations lacking either the tetrazonium or naphthol treatment, and spectroscopic studies on the bisazo dye produced in vitro (showing an emission peak at 660 nm) confirmed that the red fluorescence of stained structures arises from the protein-tetrazonium-naphthol reaction product formed in situ.

New cationic fluorochromes from diaryloxazole scintillators: fluorescence of chromatin DNA induced by N-quaternary POPOP derivatives.

N-quaternary derivatives of the diaryloxazole scintillators POPOP and dimethyl-POPOP (dmPOPOP) in chloroform solution were obtained by methylation with dimethylsulfate. After drying, aqueous solutions of the corresponding oxazolium compounds (Q-POPOP and Q-dmPOPOP) revealed strong fluorescence (peaks at 485 and 493 nm, respectively). Under 365 nm excitation, both N-quaternary derivatives induced a bright greenish blue fluorescence in nuclei of chicken erythrocytes and human buccal cells, as well as in the kinetoplast DNA of Trypanosoma cruzi epimastigotes; mouse mast cell granules showed a green-yellow metachromatic emission. Chromatin fluorescence was dependent on the presence of DNA; it was abolished by washing with a 10 mM solution of the bisguanidine compound Phenformin, whereas 1 M NaCl or MgCl2 had no effect. The oxazolium derivatives were hydrophilic (log P: -6.409 and -5.373 for Q-POPOP and Q-dmPOPOP, respectively). Molecular modelling studies revealed that these cationic and non-rigid (cis) scintillator derivatives are well suited to locate along the convex floor of the narrow DNA minor groove from adenine-thymine regions.

[Efficiency of investigators in recruitment of patients for clinical trials: apropos of a multinational study]. / Eficiencia de los investigadores en la selección de los enfermos para ensayos clínicos: a propósito de un estudio multinacional.

BACKGROUND: Performance and efficiency in patient selection are essential for conducting clinical trials. Data on these are presented from a multinational trial. PATIENTS AND METHODS: A randomized, double-blind, placebo-controlled, parallel-group study in asthma, with a screening phase followed after randomization by a treatment period, was selected. Number of patients screened and randomized by centre and country, centres achieving the minimum recruitment (> or = 10 patients randomized), and efficiency of investigators (randomized/screened x 100) were determined and compared. RESULTS: 564 patients, out of 836 screened, were randomized at 69 centres in 11 countries. Twenty-four centres (35%) randomized > or = 10 patients each, accounting for 70% (n = 395) of the total number recruited. Efficiency was significantly higher among these "high-performance" centres (81.4%; p < 0.001; OR, 4.7; CI 95%, 3.4-6.5) than in the remaining ones (48.1%). Five countries had > or = 2 "high-performance" centres. Efficiency was also significantly higher (p < 0.001) among those (370 randomized/455 screened, 82.1%) than in the remaining centres of the same countries (82/140; 58.6%; OR, 3.0; CI 95%, 2.7-4.7). A relevant number of centres (n = 17; 25%) randomized 0-1 patient (7 randomized/58 screened). CONCLUSION: The analysis of patient selection in clinical trials showed that a minority of centres accounted for most of the patients recruited. Those are not only the most productive (more patients randomized) but also the more efficient (better quality of screening process).

[Nutrition and bacterial translocation]. / Nutrición y translocación bacteriana.

The present work is part of a presentation given at the Scientific Meeting of the Association for Surgical Nutrition and Metabolism, during the XX National Congress for Surgery (Madrid, November 1994). The authors, prior to presenting their experiences, define and high light the importance of the phenomenon of "Bacterial Translocation" (BT). Afterwards, and based on several experimental studies performed by them, they attempt to answer two questions: 1) Is the term BT correct? 2) Is BT a physiological or a pathological state? Finally they review the relationship which exists between bacterial translocation and nutrition, both from a causative point of view as from the prevention and therapy of the same.

[Glossopharyngeal neuralgia associated with syncopes and secondary to neck carcinoma]. / Neuralgia glosofaringea asociada a sincopes y secundaria a carcinoma de cuello.

TITLE: Neuralgia glosofaringea asociada a sincopes y secundaria a carcinoma de cuello.

Novel group A rotavirus G8 P[1] as primary cause of an ovine diarrheic syndrome outbreak in weaned lambs.

Rotavirus is a worldwide major cause of diarrhea outbreaks in neonatal ruminants. An outbreak of ovine diarrheic syndrome (ODS) in 50-75 days-old lambs (weaned lambs) is described. Fecal immunochromatography and intestinal immunohistochemistry for rotavirus group A were performed. In addition, semi-nested multiplex RT-PCR for G and P rotavirus genotyping in combination with sequencing were performed, to support the diagnosis and identify the viral strain. A novel ovine rotavirus group A G8 P[1] strain was determined as the main cause of the ODS observed, whereas other pathogens were ruled out.

Optimización de cultivos primarios de células de carcinoma renal de células claras como modelo "in vitro" para estudios metabólicos y determinantes de progresión neoplásica / Optimization of primary cultures of clear cell renal carcinoma cells as an "in vitro" model for metabolic studies and determinants of neoplastic progression

El objetivo del presente estudio fue optimizar la implementación de cultivos primarios a partir de muestras de carcinoma renal de células claras (CRCC) para comprobar la conservación del fenotipo lipogénico contra cortes fijados del mismo origen. Se utilizaron muestras de pacientes con CRCC, evaluándose diversas metodologías y condiciones experimentales de digestión de muestras, adherencia y despegue celular, fenotipo lipogénico, potencial de clonación, proliferación y capacidad de migración. El mayor rendimiento y viabilidad celular se verificó mediante digestión con colagenasa. La adherencia inicial se logró a las 24 hs de incubación, utilizando placas plásticas de cultivo, recubiertas con colágeno comercial y gelatina 0,2% en la mayoría de las muestras analizadas (60% de los casos). Se obtuvieron monocapas, con potencial de migración, en un 40% de los casos, tras 5 ± 1 días de incubación. El promedio de subcultivos fue de 3 ± 1. Este estudio permitió estandarizar cultivos primarios de CRCC comprobándose la conservación de la fenotipia lipogénica, logrando de dicha manera una herramienta importante y útil para el estudio de la biología tumoral y el ensayo de nuevas terapéuticas

The aim of this study was to optimize the implementation of primary cultures from samples of renal clear cell carcinoma (CRCC) to check the conservation of the lipogenic phenotype. CRCC Patient samples were used, in order to evaluate different methodologies and the experimental conditions of sample digestion, cell adhesion and lipogenic phenotype, proliferation and migration ability. The highest yield in cell number and viability was assessed using collagenase digestion. The initial adhesion was achieved after 24 hours of incubation in plastic plates recoverd with commercial collagen or 0.2% gelatin (60% of cases). Monolayers, with migration potential, were obtained in 40% of all cases, after 5 ± 1 days of incubation. The subcultures average was 3 ± 1. This study allowed us to standardize primary cultures of CRCC and check the conservation of the lipogenic phenotyping, achieving in this way an important and useful tool to study the tumor biology.

O objetivo deste trabalho foi otimizar a implementação de culturas primárias de amostras de carcinoma de células claras renal (CRCC) para verificar conservação fenótipo lipogenic contra os cortes previstos a mesma origem. As amostras dos pacientes foram utilizados CRCC, avaliando diferentes metodologias e as condições experimentais da digestão de amostras, adesão celular e fenótipo clonagem potencial take-lipogenic, proliferação e capacidade de migração. O maior rendimento e a viabilidade celular foi avaliada por digestão com colagenase. A adesão inicial foi obtida após 24 horas de incubação com colagénio e gelatina comercial 0,2% em 60% dos casos. As monocamadas foram obtidos em 40% após 5 ± 1 dias de incubação com o potencial de migração. As subculturas média foi de 3 ± 1. Este estudo nos permitiu padronizar culturas primárias de CRCC são verificados quanto à conservação da fenotipagem lipogenic, conseguindo desta forma um importante e útil para o estudo da biologia do tumor e teste de nova ferramenta terapêutica