RESUMEN

Papillary carcinomas of thyroid type rarely arise within struma ovarii. There are limited data on the immunohistochemical and molecular features of these tumors. Three cases of papillary carcinoma arising in struma ovarii (PCSO) were identified. The clinicopathological features were reviewed and immunohistochemical staining for HBME-1, cytokeratin (CK) 19, and CD56 was performed. Tumor DNA was sequenced for somatic mutations using a panel of 26 oncogenes, with a particular focus on BRAF and KRAS mutations. The patients were aged 22, 48, and 55 years. All cases were FIGO stage IA. Two tumors were of classical histological type, and one was a follicular variant papillary carcinoma. All tumors expressed HBME-1 and two were positive for CK19. CD56 was negative in all three cases. One tumor demonstrated a BRAF G469A mutation in exon 11, and in a second case, a KRAS Q61K double base mutation in exon 3 was detected. These mutations have not been described previously in PCSO. No mutations were detected in the benign follicular components of the tumors adjacent to the malignant papillary tissue. None of the patients had tumor recurrence on clinical follow-up (range 11 months to 8½ years). HBME-1, CK19, and CD56 are useful immunohistochemical markers of PCSO. Novel BRAF and KRAS mutations were identified in two of three tumors suggesting that mutations in PCSO may differ from those commonly identified in papillary carcinoma of the eutopic thyroid. The clinical significance of these mutations is uncertain but follow-up data in this small series support the generally good prognosis of PCSO.

Asunto(s)

Carcinoma/patología , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Estruma Ovárico/patología , Neoplasias de la Tiroides/patología , Adulto , Biomarcadores de Tumor , Carcinoma/genética , Carcinoma Papilar , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Mutación , Neoplasias Ováricas/genética , Estruma Ovárico/genética , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides/genética , Adulto Joven

RESUMEN

BACKGROUND: We have previously demonstrated the in vivo uptake of oligonucleotides in the rat eye and have continued with experiments to look at the effectiveness of targeted oligonucleotide sequences. Vascular endothelial growth factor (VEGF) is correlated with new blood vessel formation and has been implicated in numerous eye diseases characterised by abnormal blood vessel proliferation. An oligonucleotide targeted to the VEGF sequence was examined for its effect on VEGF production in vitro and the development of choroidal neovascularisation in vivo in the eye. METHODS: A series of sequences were assessed in an in vitro screening system using retinal pigment epithelial (RPE) cells to demonstrate a reduction in VEGF. A targeted sequence was further investigated using an animal model of choroidal neovascularisation where a krypton laser was used to produce a wound healing response in the choroid and retina. The oligonucleotide was injected into the vitreous and the development of choroidal neovascularisation assessed using fluorescein angiography. RESULTS: The targeted sequence was shown in vitro to downregulate the VEGF produced by RPE cells grown under hypoxic conditions and when injected into laser treated eyes was shown to be preferentially taken up in the laser lesion. Fluorescein angiography demonstrated that the test oligonucleotide was successful in reducing laser-mediated choroidal neovascularisation. CONCLUSIONS: A sequence corresponding to the 5'UTR of the VEGF gene has provided encouraging results for the treatment of neovascularisation.

Asunto(s)

Coroides/irrigación sanguínea , Factores de Crecimiento Endotelial/genética , Linfocinas/genética , Neovascularización Patológica/prevención & control , Oligodesoxirribonucleótidos Antisentido/uso terapéutico , Epitelio Pigmentado Ocular/irrigación sanguínea , Animales , Secuencia de Bases , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Coagulación con Láser , Linfocinas/antagonistas & inhibidores , Oligodesoxirribonucleótidos Antisentido/química , Oligodesoxirribonucleótidos Antisentido/farmacocinética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Relación Estructura-Actividad , Distribución Tisular , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

A major challenge of the antisense therapeutic strategies is the development of improved systems for the delivery of antisense oligodeoxynucleotides (AS ODNs) in order to enhance the cellular uptake, to assure a better efficiency in reaching the target tissue, and to provide sustained delivery over longer periods of time. Because the current methods for delivery (liposomes and cationic polymers) present some disadvantages, the attention was directed toward the use of neutral polymers as carriers for the AS ODNs. Based on our previous work on synthetic hydrogels for vitreous substitution, we developed a poly[1-vinyl-2-pyrrolidinone-co-(2-hydroxyethyl methacrylate)] hydrogel as a potential carrier for AS ODNs. We have previously demonstrated that such hydrogels are not cytotoxic, and they may have growth-promoting effects on cultured fibroblasts. This copolymer also has the advantage of being injectable. In this study, a specific AS ODN was synthesized and then covalently bound to the copolymer via carbodiimide coupling method. The resulting conjugate was subjected to in vitro release experiments over 46 days in the presence of bovine vitreous humor. Compared with the control (no enzyme present), a significant amount of covalently bound ODN was released from the ODN-hydrogel conjugate, suggesting the possibility of using such systems for the sustained delivery of AS ODNs.

Asunto(s)

Portadores de Fármacos , Hidrogeles , Metacrilatos/química , Oligonucleótidos Antisentido/administración & dosificación , Pirrolidinas/química , Secuencia de Bases , Cartilla de ADN , Oligonucleótidos Antisentido/química , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier

RESUMEN

Vascular endothelial growth factor (VEGF) has been strongly implicated in the development of choroidal neovascularization found in age-related macular degeneration. Normally expressed in low levels, this study investigates whether the overexpression of VEGF in the retinal pigment epithelium is sufficient to cause choroidal neovascularization in the rat retina. A recombinant adenovirus vector expressing the rat VEGF(164) cDNA (AdCMV.VEGF) was constructed and injected into the subretinal space. The development of neovascularization was followed by fluorescein angiography, which indicates microvascular hyperpermeability of existing and/or newly forming blood vessels, and histology. VEGF mRNA was found to be overexpressed by retinal pigment epithelial cells and resulted in leaky blood vessels at 10 days postinjection, which was maintained for up to 31 days postinjection. By 80 days postinjection, new blood vessels had originated from the choriocapillaris, grown through the Bruch's membrane to the subretinal space, and disrupted the retinal pigment epithelium. This ultimately led to the formation of choroidal neovascular membranes and the death of overlying photoreceptor cells. By controlling the amount of virus delivered to the subretinal space, we were able to influence the severity and extent of the resulting choroidal neovascularization. These results show that even temporary overexpression of VEGF in retinal pigment epithelial cells is sufficient to induce choroidal neovascularization in the rat eye.

Asunto(s)

Neovascularización Coroidal/genética , Factores de Crecimiento Endotelial/genética , Linfocinas/genética , Epitelio Pigmentado Ocular/metabolismo , Adenoviridae/genética , Animales , Permeabilidad Capilar , División Celular , Línea Celular , Células Cultivadas , Neovascularización Coroidal/etiología , Citomegalovirus/genética , Factores de Crecimiento Endotelial/metabolismo , Ojo/irrigación sanguínea , Ojo/metabolismo , Angiografía con Fluoresceína , Regulación de la Expresión Génica , Vectores Genéticos , Humanos , Linfocinas/metabolismo , Persona de Mediana Edad , Epitelio Pigmentado Ocular/citología , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Long-Evans , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Thymic myoid cells share structural and behavioural features with cells of the skeletal muscle lineage: they express regulatory genes and contractile proteins, and they can form myofibers in culture. Historically, those features suggested that myoid cells could be precursors for muscle repair in addition to the satellite cells in muscle that are typically designated as the only muscle precursors. Muscles of the mutant mdx dystrophic mouse strain have a large demand for precursors, which is greatest at a young age. In the present study, immunostaining for troponin T was used to localize myoid cells. We tested the hypothesis that the myoid cell population changes when there is a demand for muscle precursors and that these changes would be anticipated if myoid cells have a role as myogenic precursors or stem cells in muscle. Chronic demands for muscle precursors in mdx dystrophic mice were accompanied by lower myoid cell density in comparison with density in two normal strains (C57BL10/ScSn and Swiss Webster). Acute demand for precursors was accompanied by a sharp decline in thymic myoid cell density within 2 days after a crush injury to one tibialis anterior muscle in normal but not dystrophic animals. To standardize the developmental age of the thymus, density was determined in all animals at 28 days of age. Given the current interest in nonmuscle sources of myogenic stem cells, these data suggest that changes in the density of thymic myoid cells may accompany acute and chronic demands for muscle precursors. Further experiments are required to determine whether thymic myoid cells are participants in distant muscle cell proliferation, new fiber formation, or the establishment of new stem cells in regenerated muscle.

Asunto(s)

Músculo Esquelético/lesiones , Distrofia Muscular Animal/patología , Timo/patología , Animales , Autorradiografía , Recuento de Células , Núcleo Celular/ultraestructura , Citoplasma/química , Femenino , Técnica del Anticuerpo Fluorescente , Masculino , Ratones , Ratones Endogámicos , Microscopía Electrónica , Microscopía Fluorescente , Microscopía de Contraste de Fase , Músculo Esquelético/diagnóstico por imagen , Músculo Esquelético/ultraestructura , Músculo Liso Vascular/ultraestructura , Tamaño de los Órganos , Radiografía , Timo/anatomía & histología , Factores de Tiempo , Troponina T/análisis

RESUMEN

AIMS: To investigate the distribution, persistence, and stability of fluorescently labelled phosphorothioate oligonucleotides (PS-ODNs) in normal and laser photocoagulated retina following intravitreal injection in the rat. METHODS: Fluorescently labelled PS-ODNs were injected intravitreally into pigmented eyes at doses of 0.5-10.0 nmol in 2.0 microl solution. The dynamics of PS-ODNs was evaluated by fluorescent microscopy of cryosections and flat mounted retinal pigment epithelium (RPE)-choroid-sclera. Genescan analysis was used to assess the integrity of PS-ODNs in the retina after injection. The dynamics of PS-ODNs was also evaluated in the retina following krypton laser photocoagulation with a protocol producing choroidal neovascularisation (CNV). RESULTS: Following intravitreal injection the PS-ODNs demonstrated dose and time dependent distribution and persistence in the retina, where they accessed all neural layers. However, they preferentially accumulated in the RPE layer, demonstrated as bright granules in the cytoplasm of the cells. Injections of 5.0 and 7.5 nmol of PS-ODNs exhibited strong fluorescence in the retina for 6 weeks after injection. Genescan analysis demonstrated that the PS-ODNs remained almost completely intact for at least 12 weeks. Following laser treatment, the PS-ODNs were concentrated in the regions of laser photocoagulation and retained high intensity for at least 8 weeks after injection, particularly localised to macrophages, RPE, and the local choroidal tissue. CONCLUSIONS: These results indicate that PS-ODNs are stable and accessible to most neural layers of the retina, and they preferentially accumulate in the RPE layer following intravitreal injection. The successful delivery of PS-ODNs into normal and laser photocoagulated retina suggests that PS-ODNs may have potential in the development of therapy for attenuating retinal degenerations and CNV.

Asunto(s)

Coagulación con Láser , Oligonucleótidos/farmacocinética , Retina/metabolismo , Tionucleótidos/farmacocinética , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Factores de Crecimiento Endotelial/metabolismo , Técnica del Anticuerpo Fluorescente , Inyecciones , Linfocinas/metabolismo , Microscopía Confocal/métodos , Epitelio Pigmentado Ocular/metabolismo , Ratas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

This study coupled proton magnetic resonance spectroscopy (1H-NMR) and in situ hybridization plus autoradiography in a novel examination of different phenotypes of adult myogenesis that arise from genetic disruptions in mice. Study of muscle extracts from normal and dystrophin-deficient mdx limb and diaphragm muscles confirmed our previous findings linking taurine and muscle regeneration at the peak of damage and repair. 1H-NMR distinguished biochemical differences in regenerating muscles that were consistent with the extent of repair in three strains: mdx dystrophic mice; MyoD(-/-) mice that lack expression of the early myogenic regulatory gene MyoD; and a double-mutant mdx:MyoD(-/-) strain lacking expression of both MyoD and dystrophin. We tested the hypothesis that differences in spectra according to genotype and the regeneration phenotype are related specifically to proliferation by committed myogenic precursor cells. 1H-NMR distinguished the three mutant strains: Taurine was highest in mdx muscles, with the phenotype of most effective regeneration; lowest in MyoD(-/-) muscles, with the least effective formation of new muscle in repair, as reported previously; and intermediate in double-mutant muscles, now reported to show an intermediate repair phenotype. The early and late muscle precursors (mpcs) expressing myf5 and myogenin were examined for proliferation. Eighteen percent of mdx myf5-positive mpcs were proliferative, whereas myf5-positive mpcs did not proliferate in regenerating muscles that lacked MyoD expression. By contrast, whereas 30% of myogenin-positive mpcs were proliferative in mdx muscles, almost none were proliferative in MyoD(-/-) muscles, and 12% were proliferative in double-mutant muscles. Therefore, the extent of accumulated structural regeneration, taurine levels, and proliferation of late mpc (expressing myogenin) were congruent across genotypes. Proliferation by early mpc (expressing myf5) was inhibited by the lack of MyoD expression during muscle regeneration. These studies indicate the potential for 1H-NMR monitoring of muscle status in disease, regeneration, and treatment.

Asunto(s)

Distrofina/genética , Músculo Esquelético/metabolismo , Proteína MioD/genética , Regeneración/fisiología , Taurina/metabolismo , Animales , Autorradiografía , Recuento de Células , División Celular/fisiología , Síndrome de Aplastamiento/metabolismo , Síndrome de Aplastamiento/patología , Distrofina/deficiencia , Eliminación de Gen , Hibridación in Situ , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos mdx , Ratones Mutantes , Músculo Esquelético/lesiones , Músculo Esquelético/patología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Miocardio/patología

RESUMEN

The application of nonradioactive RNA probes for Northern blotting offers the advantage of a rapid turn-around time for results without the loss of sensitivity for target mRNA detection. However, a problem that has impeded the widespread use of nonradioactive RNA probes for use in Northern blotting is the difficulty in stripping these probes from nylon membranes after hybridization. In this report we describe two protocols for stripping digoxigenin (Dig)-labeled RNA probes from nylon membranes. One protocol utilizes a phosphate-buffered formamide stripping solution to remove nonchemically modified (regular) RNA probes while the other method utilizes strippable probes that were produced with a chemically modified nucleotide (CTP) and removed by a specific stripping solution. This latter method was developed by Ambion Inc. and is called Strip-EZ. We also describe a protocol for the detection of two separate rat mRNAs using both biotin and digoxigenin-labeled RNA probes that does not require stripping the membrane after hybridization. Finally, we describe the use of another new labeling technology, called Chem-Link, that quickly and conveniently labels RNA for use in Northern blotting.

Asunto(s)

Biotina , Northern Blotting/métodos , Digoxigenina , Indicadores y Reactivos , Sondas ARN , Animales , Masculino , Ratas , Ratas Sprague-Dawley

RESUMEN

Tissue culture studies using muscle cell lines suggest that in addition to mitogenic effects, fibroblast growth factors (FGF) inhibit skeletal muscle differentiation and the expression of members of a family of muscle-specific regulatory genes including MyoD and myogenin. We examined the possible coexpression of bFGF and myogenin by tandem in situ hybridization (detecting mRNA) and immunocytochemistry studies (detecting protein) to determine whether myogenic cells in vivo endogenously produce bFGF. Mdx mouse muscle, which shows characteristic dystrophic damage and regeneration, demonstrated mononuclear cells containing myogenin and bFGF transcripts in similar regions of adjacent sections of focal degeneration and repair, particularly near recent segmental fiber damage. Using immunocytochemistry and in situ hybridization concurrently on the same sections, bFGF protein and myogenin mRNA were colocalized in both muscle precursors and new myotubes. The in vivo results were confirmed in vitro using primary explant cultures of mdx muscle. Approximately one-half of mononuclear cells in vivo were myogenic by the criterion of myogenin mRNA expression. Both myogenin and bFGF mRNAs were also colocalized with bFGF protein, indicating endogenous expression of bFGF in a subpopulation of myogenic cells. Small numbers of myogenic mononuclear cells were differentiated, as determined by the presence of developmental myosin heavy chain protein (DevMHC). These cells and new myotubes also colocalized myogenin, DevMHC, and bFGF. Since bFGF and myogenin are colocalized in mpec and myotubes in vivo and in vitro, endogenous expression of bFGF is not mutually exclusive of myogenic regulatory gene expression, either before or after differentiation of the skeletal muscle phenotype. Such features of coexpression suggest an important and complex role for bFGF in muscle regeneration in vivo.

Asunto(s)

Factor 2 de Crecimiento de Fibroblastos/metabolismo , Músculos/metabolismo , Distrofia Muscular Animal/metabolismo , Miogenina/genética , Animales , Diferenciación Celular , Técnicas de Cultivo , Factor 2 de Crecimiento de Fibroblastos/genética , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Desarrollo de Músculos , Distrofia Muscular Animal/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

Nonspecific binding of a number of unrelated nucleic acid probes to cells in the crypts of Lieberkuhn was observed in the small intestine of mice with the in situ hybridization technique. Hybridization signal was localized to cells which, by virtue of their histological position, represented Paneth cells. This signal could not be removed by RNAse, DNAse, or proteinase K treatment, and was not removed after high-stringency washing conditions. This report indicates that caution must be exercised in the interpretation of in situ hybridization data when looking for nucleic acid sequences in the gastrointestinal tract.

Asunto(s)

Intestino Delgado/metabolismo , Sondas de Ácido Nucleico/metabolismo , Animales , Intestino Delgado/citología , Masculino , Ratones , Ratones Endogámicos BALB C , Hibridación de Ácido Nucleico , Ratas , Ratas Endogámicas

RESUMEN

Site-directed in vitro mutagenesis followed by in vitro transcription and translation has been used to study structure/function relationships for murine interferon-alpha 1 (MuIFN-alpha 1). The mature form of the MuIFN-alpha 1 protein was expressed as well as analogue forms with amino acid substitutions at positions 33, 71, 72, 123 and 133. These positions were chosen on the basis of known human interferon-alpha structure/function relationships. Biological assays for antiviral activity on murine cells and natural killer cell activation have been performed for each of the proteins produced. The data obtained have been interpreted in the light of previous human and murine interferon-alpha structure/function work and the recently published three-dimensional structure of murine type I interferon.

Asunto(s)

Virus de la Encefalomiocarditis/fisiología , Interferón-alfa/farmacología , Células Asesinas Naturales/inmunología , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Interferón-alfa/biosíntesis , Interferón-alfa/química , Interferón-alfa/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Relación Estructura-Actividad

RESUMEN

The skeletal muscle specific genes MyoD1 and myogenin are closely associated with commitment of cells to the myogenic lineage and differentiation of skeletal muscle precursor cells. The transcription of these genes was studied in the thymus where mononuclear cells termed myoid cells appear to closely resemble skeletal muscle precursors. In thymus from adult SJL/J and BALB/c mice, in situ hybridization with either MyoD1 or myogenin riboprobes showed probe-positive cells concentrated in the medullary region. In neonatal thymus, mRNA for these genes was not detected. These data are the first demonstration in a higher vertebrate of MyoD1 and myogenin expression in a tissue other than skeletal muscle. The sustained expression of MyoD1 and myogenin genes in thymi of adult mice shows that myoid cells are not equivalent to quiescent stem cells of mature skeletal muscle. In addition, studies with antistriational antibodies indicate that myoid cells do not continue to differentiate within the normal murine thymic environment. This arrested differentiation process presents an unusual model for investigating conditions regulating myogenesis in vivo.

Asunto(s)

Proteínas Musculares/genética , Proteína MioD , Proteínas Nucleares/genética , Fosfoproteínas/genética , Timo/citología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Expresión Génica/genética , Ratones , Ratones Endogámicos , Microscopía Fluorescente , Familia de Multigenes/genética , Miogenina , Hibridación de Ácido Nucleico , Timo/metabolismo , Transcripción Genética/genética

RESUMEN

The activation of mononuclear muscle precursor cells after crush injury to mouse tibialis anterior muscles was monitored in vivo by in situ hybridization with MyoD1 and myogenin probes. These genes are early markers of skeletal muscle differentiation and have been extensively studied in vitro. The role in vivo of these regulatory proteins during myogenesis of mature muscle has not been studied previously. MyoD1 and myogenin mRNA were present in occasional mononuclear cells of uninjured muscle. Increased MyoD1 and myogenin mRNA sequences in mononuclear cells were detected as early as 6 h after injury, peaked between 24 and 48 h, and thereafter declined to pre-injury levels at about 8 days. The mRNAs were detected in mononuclear cells throughout the muscle, with the majority of cells located some distance from the site of crush injury. The presence of MyoD1 and myogenin mRNA at 6 to 48 h indicates that transcription of these genes is occurring at the same time as replication of muscle precursor cells in vivo. At no time were significant levels of mRNA for these genes detected in myotubes. MyoD1 and myogenin provide precise markers for the very early identification and study of mononuclear skeletal muscle precursor cells in muscle regenerating in vivo.

Asunto(s)

Músculos/citología , Proteína MioD , Células Madre/citología , Animales , Biomarcadores , Masculino , Ratones , Sondas Moleculares , Proteínas Musculares/metabolismo , Músculos/lesiones , Músculos/metabolismo , Miogenina , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , ARN Mensajero/metabolismo , Regeneración , Células Madre/metabolismo

RESUMEN

The regeneration of skeletal muscle is dependent upon proliferation and fusion of activated mononuclear muscle precursor cells. Early and specific markers of this population of activated cells are the transcription factors MyoD and myogenin. Northern analysis was used to determine levels of MyoD and myogenin mRNA in (i) muscles regenerating after experimental crush injury and (ii) in limb muscles of dystrophic mdx mice at various ages in comparison to controls. In crush-injured muscle, MyoD and myogenin mRNA increased at 24 h, peaked between 2 to 6 days, and returned to uninjured control levels by 15 days after injury. In both mdx and control mice, MyoD and myogenin mRNA levels were high in fetal muscles and decreased rapidly during the 2 weeks after birth. In mdx muscles, the mRNA levels increased significantly from about 21 days, remained high until around 40 days, and then decreased to a relatively constant yet elevated level when compared to control muscles. The elevated levels persisted to 420 days of age. The results show that this technique can be used to provide sensitive quantitative information on the size of the population of activated precursor cells in skeletal muscle. As such, it represents a novel and convenient means of measuring regenerative activity in vivo in whole muscles.

RESUMEN

The purpose of this study was to determine the amount of variation for several vocal parameters across three times of the day (morning, noon, and afternoon). Connected speech samples from normal adult males (N = 10) and females (N = 10) were recorded during morning, early afternoon, and late afternoon. Results showed that males produced a statistically significant increase in speaking fundamental frequency (SFF) from morning to afternoon. Females did not demonstrate a statistically significant change in SFF across the three time periods. Vocal amplitude did not change significantly for either group. The SFF variability was higher for the females than for the males. Analysis of individual data revealed that the patterns of vocal change across the three times of day were not consistent among the subjects.

Asunto(s)

Ritmo Circadiano , Acústica del Lenguaje , Habla , Adolescente , Adulto , Femenino , Humanos , Masculino , Valores de Referencia