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1.
Microbiome ; 11(1): 159, 2023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-37491398

RESUMO

BACKGROUND: Cervicovaginal inflammation has been linked to negative reproductive health outcomes including the acquisition of HIV, other sexually transmitted infections, and cervical carcinogenesis. While changes to the vaginal microbiome have been linked to genital inflammation, the molecular relationships between the functional components of the microbiome with cervical immunology in the reproductive tract are understudied, limiting our understanding of mucosal biology that may be important for reproductive health. RESULTS: In this study, we used a multi'-omics approach to profile cervicovaginal samples collected from 43 Canadian women to characterize host, immune, functional microbiome, and metabolome features of cervicovaginal inflammation. We demonstrate that inflammation is associated with lower amounts of L. crispatus and higher levels of cervical antigen-presenting cells (APCs). Proteomic analysis showed an upregulation of pathways related to neutrophil degranulation, complement, and leukocyte migration, with lower levels of cornified envelope and cell-cell adherens junctions. Functional microbiome analysis showed reductions in carbohydrate metabolism and lactic acid, with increases in xanthine and other metabolites. Bayesian network analysis linked L. crispatus with glycolytic and nucleotide metabolism, succinate and xanthine, and epithelial proteins SCEL and IVL as major molecular features associated with pro-inflammatory cytokines and increased APCs. CONCLUSIONS: This study identified key molecular and immunological relationships with cervicovaginal inflammation, including higher APCs, bacterial metabolism, and proteome alterations that underlie inflammation. As APCs are involved in HIV transmission, parturition, and cervical cancer progression, further studies are needed to explore the interactions between these cells, bacterial metabolism, mucosal immunity, and their relationship to reproductive health. Video Abstract.


Assuntos
Infecções por HIV , Humanos , Feminino , Infecções por HIV/microbiologia , Proteômica , Teorema de Bayes , Canadá , Vagina/microbiologia , Inflamação/metabolismo , Citocinas , Células Apresentadoras de Antígenos/metabolismo , Xantinas/metabolismo
2.
Microbiome ; 8(1): 61, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32370783

RESUMO

BACKGROUND: Gender reassignment surgery is a procedure some transgender women (TW) undergo for gender-affirming purposes. This often includes the construction of a neovagina using existing penile and scrotal tissue and/or a sigmoid colon graft. There are limited data regarding the composition and function of the neovaginal microbiome representing a major gap in knowledge in neovaginal health. RESULTS: Metaproteomics was performed on secretions collected from the neovaginas (n = 5) and rectums (n = 7) of TW surgically reassigned via penile inversion/scrotal graft with (n = 1) or without (n = 4) a sigmoid colon graft extension and compared with secretions from cis vaginas (n = 32). We identified 541 unique bacterial proteins from 38 taxa. The most abundant taxa in the neovaginas were Porphyromonas (30.2%), Peptostreptococcus (9.2%), Prevotella (9.0%), Mobiluncus (8.0%), and Jonquetella (7.2%), while cis vaginas were primarily Lactobacillus and Gardnerella. Rectal samples were mainly composed of Prevotella and Roseburia. Neovaginas (median Shannon's H index = 1.33) had higher alpha diversity compared to cis vaginas (Shannon's H = 0.35) (p = 7.2E-3, Mann-Whitney U test) and were more similar to the non-Lactobacillus dominant/polymicrobial cis vaginas based on beta diversity (perMANOVA, p = 0.001, r2 = 0.342). In comparison to cis vaginas, toll-like receptor response, amino acid, and short-chain fatty acid metabolic pathways were increased (p < 0.01), while keratinization and cornification proteins were decreased (p < 0.001) in the neovaginal proteome. CONCLUSIONS: Penile skin-lined neovaginas have diverse, polymicrobial communities that show similarities in composition to uncircumcised penises and host responses to cis vaginas with bacterial vaginosis (BV) including increased immune activation pathways and decreased epithelial barrier function. Developing a better understanding of microbiome-associated inflammation in the neovaginal environment will be important for improving our knowledge of neovaginal health. Video Abstract.


Assuntos
Bactérias , Microbiota , Cirurgia de Readequação Sexual , Vagina/microbiologia , Adulto , Bactérias/classificação , Bactérias/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pessoas Transgênero
3.
Am J Reprod Immunol ; 83(6): e13235, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32196803

RESUMO

PROBLEM: Pregnant women are at increased risk of HIV acquisition, but the biological mechanisms contributing to this observation are not well understood. METHOD OF STUDY: Here, we assessed host immune and microbiome differences in the vaginal mucosa of healthy pregnant and non-pregnant women using a metaproteomics approach. Cervicovaginal lavage (CVL) samples were collected from 23 pregnant and 25 non-pregnant women. RESULTS: Mass spectrometry analysis of CVL identified 550 human proteins and 376 bacterial proteins from 11 genera. Host proteome analysis indicated 56 human proteins (10%) were differentially abundant (P < .05) between pregnant and non-pregnant women, including proteins involved in angiogenesis (P = 3.36E-3), cell movement of phagocytes (P = 1.34E-6), and permeability of blood vessels (P = 1.27E-4). The major bacterial genera identified were Lactobacillus, Gardnerella, Prevotella, Megasphaera, and Atopobium. Pregnant women had higher levels of Lactobacillus species (P = .017) compared with non-pregnant women. Functional pathway analysis indicated that pregnancy associated with changes to bacterial metabolic pathway involved in energy metabolism, which were increased in pregnant women (P = .035). CONCLUSION: Overall, pregnant women showed differences in the cervicovaginal proteome and microbiome that may be important for HIV infection risk.


Assuntos
Lactobacillus/fisiologia , Microbiota/imunologia , Mucosa/microbiologia , Gravidez , Vagina/imunologia , Adolescente , Adulto , Metabolismo Energético , Feminino , Humanos , Espectrometria de Massas , Pessoa de Meia-Idade , Proteoma , Vagina/microbiologia , Adulto Jovem
4.
J Int AIDS Soc ; 21(7): e25150, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30047573

RESUMO

INTRODUCTION: At its basic level, HIV infection requires a replication-competent virus and a susceptible target cell. Elevated levels of vaginal inflammation has been associated with the increased risk of HIV infection as it brings highly activated HIV target cells (CCR5+CD4+ T cells; CCR5+CD4+CD161+ Th17 T cells) to the female genital tract (FGT) where they interact with HIV. Decreased HIV risk has been associated with a phenotype of decreased immune activation, called immune quiescence, described among Kenyan female sex workers who were intensely exposed to HIV yet remain uninfected. Current prevention approaches focus on limiting viral access. We took the novel HIV prevention approach of trying to limit the number of HIV target cells in the genital tract by reducing inflammation using safe, affordable and globally accessible anti-inflammatory drugs. METHODS: We hypothesized that the daily administration of low doses of acetylsalicylic acid (ASA 81 mg) or hydroxychloroquine (HCQ 200 mg) would reduce inflammation thereby decreasing HIV target cells at the FGT. Low-risk HIV seronegative women from Nairobi, Kenya were randomized for six weeks therapy of ASA (n = 37) or HCQ (n = 39) and tested to determine the impact on their systemic and mucosal immune environment. RESULTS: The results showed that HCQ use was associated with a significant reduction in the proportion of systemic T cells that were CCR5+CD4+ (p = 0.01) and Th17 (p = 0.01). In the ASA arm, there was a 35% and 28% decrease in the proportion of genital T cells that were CD4+CCR5+ (p = 0.017) and Th17 (p = 0.04) respectively. Proteomic analyses of the cervical lavage showed ASA use was associated with significantly reduced amount of proteins involved in the inflammatory response and cell recruitment at the mucosa, although none of the individual proteins passed multiple comparison correction. These changes were more apparent in women with Lactobacillus dominant microbiomes. CONCLUSION: Together, these data indicate that taking low-dose ASA daily was associated with significant reduction in HIV target cells at the FGT. This study provides proof-of-concept for a novel HIV-prevention approach that reducing inflammation using safe, affordable and globally accessible non-steroidal anti-inflammatory agents is associated with significant reduction in the proportion of HIV-target cells at the FGT.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Aspirina/uso terapêutico , Genitália Feminina/efeitos dos fármacos , Infecções por HIV/prevenção & controle , Hidroxicloroquina/uso terapêutico , Adulto , Feminino , Genitália Feminina/citologia , Genitália Feminina/imunologia , Infecções por HIV/patologia , Humanos , Quênia , Mucosa/virologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Projetos Piloto , Proteômica , Profissionais do Sexo , Linfócitos T
5.
Am J Reprod Immunol ; 80(2): e12977, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29790240

RESUMO

The mucosal surface of the female genital tract contains physiological, immunological, and microbial components that collectively comprise a functioning "mucosal system" that is critical for reproductive health. Alterations or imbalances to any of these components can have significant consequences for susceptibility to sexually transmitted infections, such as HIV. In recent years the advent of advanced systems biology technologies, such as metaproteomics, has provided new toolsets to studying mucosal systems. Studies have linked an altered mucosal proteome to many HIV risk factors including mucosal inflammation, bacterial vaginosis, hormonal contraceptives, and reduced efficacy of antiretroviral drugs for HIV prevention. Herein we will discuss how metaproteomics has been used to study mucosal system components, including epithelial barriers, inflammation, and the microbiome, with a focus on what alterations may contribute to increased HIV transmission risk in women.


Assuntos
Infecções por HIV/transmissão , Microbiota/fisiologia , Mucosa/microbiologia , Proteoma/metabolismo , Vagina/microbiologia , Suscetibilidade a Doenças , Feminino , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Imunidade Inata/imunologia , Mucosa/imunologia , Vagina/imunologia
6.
Nat Protoc ; 6(12): 1953-68, 2011 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-22094731

RESUMO

This protocol describes a method for determining both the average number and variance of proteins, in the few to tens of copies, in isolated cellular compartments such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number, but lack information on the variance, or they are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling of the cellular compartment with fluorescent primary-secondary antibody complexes, total internal reflection fluorescence microscopic imaging of the cellular compartment, digital image analysis and deconvolution of the fluorescence intensity data. A minimum of 2.5 d is required to complete the labeling, imaging and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes.


Assuntos
Imunofluorescência , Proteínas/análise , Algoritmos , Compartimento Celular , Estruturas Citoplasmáticas/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microfluídica/métodos , Microscopia de Fluorescência/métodos , Proteínas/química , Software
7.
J Neurosci ; 31(4): 1461-70, 2011 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-21273430

RESUMO

Protein sorting represents a potential point of regulation in neurotransmission because it dictates the protein composition of synaptic vesicles, the organelle that mediates transmitter release. Although the average number of most vesicle proteins has been estimated using bulk biochemical approaches (Takamori et al., 2006), no information exists on the intervesicle variability of protein number, and thus on the precision with which proteins are sorted to vesicles. To address this, we adapted a single molecule quantification approach (Mutch et al., 2007) and used it to quantify both the average number and variance of seven integral membrane proteins in brain synaptic vesicles. We report that four vesicle proteins, SV2, the proton ATPase, Vglut1, and synaptotagmin 1, showed little intervesicle variation in number, indicating they are sorted to vesicles with high precision. In contrast, the apparent number of VAMP2/synaptobrevin 2, synaptophysin, and synaptogyrin demonstrated significant intervesicle variability. These findings place constraints on models of protein function at the synapse and raise the possibility that changes in vesicle protein expression affect vesicle composition and functioning.


Assuntos
Proteínas de Membrana/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Encéfalo/metabolismo , Técnicas In Vitro , Transporte Proteico , Ratos , Ratos Sprague-Dawley
8.
Thorax ; 65(7): 606-11, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20627917

RESUMO

BACKGROUND AND AIMS: Manual laboratory continuous positive airway pressure (CPAP) titration for obstructive sleep apnoea (OSA) is costly, time intensive and delays access to treatment. Automatic positive airway pressure (APAP) titration has the potential to reduce cost and improve access to treatment. The aim of this study was to assess the clinical efficacy and costs of APAP titration compared with manual titration in moderate-severe OSA. METHODS: Patients with moderate-severe OSA (apnoea/hypopnoea index >15 and Epworth Sleepiness Score > or = 8) who were free of co-morbidities that could impair APAP titration were eligible. 249 participants were randomised to manual titration, home APAP or laboratory APAP titration to determine a fixed pressure for CPAP. Clinical and direct cost outcomes were assessed after 4 weeks of treatment. RESULTS: Average nightly CPAP use, subjective sleepiness, SF36 quality of life, Trails A and B cognitive function and polysomnographic outcomes were similar among the per-protocol groups. Non-hypertensive patients had a lower resting heart rate (and greater reduction in heart rate) at 4 weeks after laboratory APAP titration compared with home APAP titration. Costs per patient were highest in manual (AU$817.84), followed by laboratory (AU$647.56) and home (AU$132.09) APAP titration. An intention-to-treat analysis confirmed the effectiveness of APAP titration compared with manual titration in the standard clinical setting. CONCLUSIONS: Among patients with moderate-severe OSA without serious co-morbidities, outcomes at 1 month indicate that APAP titration is more cost-effective than manual laboratory titration to determine an appropriate pressure for CPAP for long-term use; with the largest savings occurring in the home APAP patients.


Assuntos
Pressão Positiva Contínua nas Vias Aéreas/métodos , Apneia Obstrutiva do Sono/terapia , Adulto , Pressão Sanguínea/fisiologia , Pressão Positiva Contínua nas Vias Aéreas/economia , Feminino , Custos de Cuidados de Saúde/estatística & dados numéricos , Frequência Cardíaca/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Cooperação do Paciente , Polissonografia , Índice de Gravidade de Doença , Apneia Obstrutiva do Sono/economia , Apneia Obstrutiva do Sono/fisiopatologia , Resultado do Tratamento
9.
Lab Chip ; 7(7): 923-6, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17594014

RESUMO

Thermoset polyester (TPE) microfluidic devices were previously developed as an alternative to poly(dimethylsiloxane) (PDMS) devices, fabricated similarly by replica molding, yet offering stable surface properties and good chemical compatibility with some organics that are incompatible with PDMS. This paper describes a number of improvements in the fabrication of TPE chips. Specifically, we describe methods to form TPE devices with a thin bottom layer for use with high numerical aperture (NA) objectives for sensitive fluorescence detection and optical manipulation. We also describe plasma-bonding of TPE to glass to create hybrid TPE-glass devices. We further present a simple master-pretreatment method to replace our original technique that required the use of specialized equipment.


Assuntos
Dimetilpolisiloxanos/química , Técnicas Analíticas Microfluídicas/instrumentação , Microfluídica , Poliésteres/química , Silicones/química , Catálise , Eletroquímica , Desenho de Equipamento , Vidro , Microscopia de Fluorescência , Modelos Teóricos , Osmose , Silanos/química , Propriedades de Superfície
10.
Biophys J ; 92(8): 2926-43, 2007 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17259276

RESUMO

In fluorescence microscopy, images often contain puncta in which the fluorescent molecules are spatially clustered. This article describes a method that uses single-molecule intensity distributions to deconvolve the number of fluorophores present in fluorescent puncta as a way to "count" protein number. This method requires a determination of the correct statistical relationship between the single-molecule and single-puncta intensity distributions. Once the correct relationship has been determined, basis histograms can be generated from the single-molecule intensity distribution to fit the puncta distribution. Simulated data were used to demonstrate procedures to determine this relationship, and to test the methodology. This method has the advantages of single-molecule measurements, providing both the mean and variation in molecules per puncta. This methodology has been tested with the avidin-biocytin binding system for which the best-fit distribution of biocytins in the sample puncta was in good agreement with a bulk determination of the avidin-biocytin binding ratio.


Assuntos
Avidina/metabolismo , Lisina/análogos & derivados , Modelos Biológicos , Vesículas Sinápticas/metabolismo , Algoritmos , Animais , Encéfalo , Células Cultivadas , Simulação por Computador , Interpretação de Imagem Assistida por Computador/métodos , Lisina/metabolismo , Modelos Estatísticos , Ratos , Distribuições Estatísticas , Vesículas Sinápticas/ultraestrutura
11.
J Am Chem Soc ; 128(10): 3233-40, 2006 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-16522104

RESUMO

This article describes the first single-vesicle study of proton permeability across the lipid membrane of small (approximately 100 nm) uni- and multilamellar vesicles, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). To follow proton permeation into the internal volume of each vesicle, we encapsulated carboxyfluorescein, a pH-sensitive dye whose fluorescence was quenched in the presence of excess protons. A microfluidic platform was used for easy exchange of high- and low-pH solutions, and fluorescence quenching of single vesicles was detected with single-molecule total internal reflection fluorescence (TIRF) microscopy. Upon solution exchange and acidification of the extravesicular solution (from pH 9 to 3.5), we observed for each vesicle a biphasic decay in fluorescence. Through single-vesicle analysis, we found that rate constants for the first decay followed a Poisson distribution, whereas rate constants for the second decay followed a normal distribution. We propose that proton permeation into each vesicle first arose from formation of transient pores and then transitioned into the second decay phase, which occurred by the solubility-diffusion mechanism. Furthermore, for the bulk population of vesicles, the decay rate constant and vesicle intensity (dependent on size) correlated to give an average permeability coefficient; however, for individual vesicles, we found little correlation, which suggested that proton permeability among single vesicles was heterogeneous in our experiments.


Assuntos
Fluoresceínas/química , Corantes Fluorescentes/química , Lipossomos/química , Fosfatidilcolinas/química , Permeabilidade da Membrana Celular , Cinética , Técnicas Analíticas Microfluídicas/métodos , Microscopia de Fluorescência , Prótons
12.
Pain ; 120(1-2): 182-187, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16360266

RESUMO

Transcutaneous electrical nerve stimulation (TENS) reduces pain through central mechanisms involving spinal cord and brainstem sites. Since TENS acts through central mechanisms, we hypothesized that TENS will reduce chronic bilateral hyperalgesia produced by unilateral inflammation when applied either ipsilateral or contralateral to the site of muscle inflammation. Sprague-Dawley rats were injected with carrageenan in the left gastrocnemius muscle belly. Mechanical withdrawal threshold was tested bilaterally before and 2 weeks after carrageenan injection. After testing withdrawal thresholds at 2 weeks, rats received TENS treatment either ipsilateral or contralateral to the site of inflammation. In each of these groups, rats were randomized to control (no TENS), low frequency (4 Hz), or high frequency (100 Hz) TENS treatment. TENS was applied for 20 min at sensory intensity under light halothane anesthesia. Mechanical withdrawal thresholds were re-assessed after TENS or 'no TENS' treatment. Unilateral injection of carrageenan to the gastrocnemius muscle significantly reduced the mechanical withdrawal threshold (mechanical hyperalgesia) bilaterally 2 weeks later. Either low or high frequency TENS applied to the gastrocnemius muscle ipsilateral to the site of inflammation significantly reversed mechanical hyperalgesia, both ipsilateral and contralateral to the site of inflammation. Low or high frequency TENS applied to the gastrocnemius muscle contralateral to the site of inflammation also significantly reduced mechanical hyperalgesia, both ipsilateral and contralateral to the site of inflammation. Since ipsilateral or contralateral TENS treatments were effective in reducing chronic bilateral hyperalgesia in this animal model, we suggest that TENS act through modulating descending influences from supraspinal sites such as rostral ventromedial medulla (RVM).


Assuntos
Hiperalgesia/etiologia , Hiperalgesia/terapia , Miosite/complicações , Estimulação Elétrica Nervosa Transcutânea/métodos , Animais , Carragenina , Doença Crônica , Modelos Animais de Doenças , Hiperalgesia/diagnóstico , Hiperalgesia/fisiopatologia , Masculino , Miosite/induzido quimicamente , Miosite/diagnóstico , Miosite/fisiopatologia , Limiar da Dor , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento
13.
Langmuir ; 21(23): 10763-9, 2005 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-16262349

RESUMO

This paper demonstrates the ability to grow silica directly on a deposited surface of polyelectrolyte. Using this strategy, we describe the deposition of layered polyelectrolyte-silica coating on negatively charged surfaces of polystyrene particles and latex nanocapsules, which could not be coated directly with silica alone. By etching away the underlying polystyrene bead, we were able to form polyelectrolyte-silica capsules that were mechanically robust. Using scanning and transmission electron microscopy, we imaged and studied the coating after the deposition of each layer of polyelectrolyte and silica. We then applied this new coating to latex nanocapsules that were loaded with fluorescein molecules. We found that the coating procedure did not cause the loaded molecules to leak out from the capsules, and we determined that the variation in the number of loaded molecules among capsules arose from differences in the volume of the nanocavities and was not caused by the loading and coating of the capsules. This layered architecture permits the thickness of the coating to be controlled in principle over a wide dynamic range, but more importantly, this coating could act as an effective seal to prevent undesired leakage from nanocapsules and thus increase the long-term storability of loaded capsules. Over a 30-day period, we determined that leakage from uncoated capsules was significant but negligible for ones that were coated with two layers of polyelectrolyte-silica. Using single-pulse UV photolysis of individual nanocapsules, we demonstrate that the molecules contained within coated capsules could be released effectively and on demand with a single laser pulse.

14.
Anal Chem ; 76(9): 2492-7, 2004 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-15117188

RESUMO

This paper describes a method for manipulating and monitoring the rotational motion of single, optically trapped microparticles and living cells in a microvortex. To induce rotation, we placed the microparticle at the center of rotation of the vortex and used the recirculating fluid flow to drive rotation. We have monitored the rotation of single beads (which ranged in diameter from a few micrometers to tens of micrometers) and living cells in a microvortex. To follow the rotation of a smooth and symmetrically shaped bead, we first ablated a small region ( approximately 1 microm) on the bead. An Ar(+) laser was then tightly focused ( approximately 0.5-microm spot size) onto the bead, and rotation was tracked by recording changes in the level of backscattered laser light as the ablated region repeatedly transited the laser focus. Using this method, we have followed bead rotation that varied in frequency from 0.15 to 100 Hz and have studied the effect of bead diameter on the rate of rotation at a given fluid flow rate. To monitor the rotation of single living cells, we selectively stained portions of B-lymphocytes with the fluorescent dye DiOC(6). We observed rotation by following changes in the fluorescence signal as the dye-stained region transited the laser focal volume. This technique provides a simple and sensitive method for controlling and monitoring the rotational motion of microparticles in a microfluidic environment.


Assuntos
Linfócitos B/química , Técnicas Analíticas Microfluídicas/métodos , Micromanipulação , Microesferas , Óptica e Fotônica , Animais , Linfócitos B/citologia , Carbocianinas/química , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Micromanipulação/instrumentação , Micromanipulação/métodos , Movimento (Física) , Observação , Tamanho da Partícula , Rotação , Sensibilidade e Especificidade
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