The presence of a vocal ligament in fetuses: a histochemical and ultrastructural study

Although it is currently believed that the vocal ligament of humans undergoes considerable development postnatally, there is no consensus as to the age at which it first emerges. In the newborn infant, the lamina propria has been described as containing a sparse collection of relatively unorganized fibres. In this study we obtained larynges from autopsy of human fetuses aged 7-9 months and used light and electron microscopy to study the collagenous and elastic system fibres in the lamina propria of the vocal fold. Collagen fibres were viewed using the Picrosirius polarization method and elastic system fibres were stained using Weigert`s resorcin-fuchsin after oxidation with oxone. The histochemical and electron microscopic observations were consistent, showing collagen populations with an asymmetric distribution across different compartments of the lamina propria. In the central region, the collagen appeared as thin, weakly birefringent, greenish fibres when viewed using the Picrosirius polarization method, whereas the superficial and deep regions contained thick collagen fibres that displayed a strong red or yellow birefringence. These findings suggest that the thin fibres in the central region consist mainly of type III collagen, whereas type I collagen predominates in the superficial and deep regions, as has been reported in studies of adult vocal folds. Similarly, elastic system fibres showed a differential distribution throughout the lamina propria. Their distribution pattern was complementary to that of collagen fibres, with a much greater density of elastic fibres apparent in the central region than in the superficial and deep regions. This distribution of collagen and elastic fibres in the fetal vocal fold mirrors that classically described for the adult vocal ligament, suggesting that a vocal ligament has already begun to develop by the time of birth. The apparently high level of organization of connective tissue components in the newborn is in contrast to current hypotheses that argue that the mechanical stimuli of phonation are essential to the determination of the layered structure of the lamina propria and suggests that genetic factors may play a more significant role in the development of the vocal ligament than previously believed.

Oral lesions in lupus erythematosus-cytokines profiles of inflammatory infiltrate

Background: Lupus erythematosus (LE) is a chronic inflammatory disease. Presence of type 1 cytokines in cutaneous discoid lesions suggests that they may be critical for induction, development and maintenance of these manifestations. Type 2 cytokines in combination with local interferon gamma (INF-gamma) are thought to be related to the physiopathology of cutaneous LE. Cytokines profiles are still unknown in oral LE lesions. Materials and Methods: Expression of Th1 and Th2 cytokines (including IL-4, IL-5, IL-6, IL-10, IL-12, tumor necrosis factor alpha (TNF-alpha) and INF-gamma was investigated and compared in 29 biopsies of intra-oral (sun-protected) and labial lesions (sun-exposed) of LE using immunohistochemistry. Results: Inflammatory infiltrate of LE lesions was strongly positive for IFN-gamma (97%) and TNF-alpha (90%), both Th1 type cytokines. Interleukin-10, a Th2 cytokine was also strongly expressed. Other cytokines were only mildly positive. Cytokines patterns were similar in intra-oral (sun-covered) and labial (sun-exposed) LE lesions. Conclusions: Oral LE lesions are associated with both type 1 and type 2 cytokines, characterized by stronger expression of INF-gamma, TNF-alpha and IL-10. These findings suggest that although ultraviolet (UV) light is involved in the induction of LE lesions, mechanisms of lesions formation may be similar in sun-exposed as well as sun-covered areas.

In Vitro Phototoxicity of Liposomes and Nanocapsules Containing Chloroaluminum Phthalocyanine on Human Melanoma Cell Line

In this study, the photodynamic action of liposomes (LP) and nanocapsules (NC) containing Chloroaluminum phthalocyanine (CIAIPc), on the human melanoma cell (WM 1552C), was assessed. The light source was setup at 672 nm, which corresponds to the maximum absorption wavelength of the CIAIPc. Both colloidal carriers presented size in nanometric scale as well as negative zeta potential. The cellular damage was light dose dependent ranging from 30% of cell death at 70 mJ.cm(-2) to 90% of death at 700 mJ.cm(-2). However, the photocytotoxic effect of LP at 70 mJ.cm(-2) was slightly more efficient to induce cellular death than NC formulation. At 140 mJ.cm(-2), and 700 mJ.cm(-2) both nanocarriers were equally efficient to induce cellular damage. Therefore, in the present work, the maximum phototoxic effect was obtained with 700 mJ.cm(-2) of light dose, in combination with 0.29 mu g.mL(-1) of CIAIPc encapsulated into LP and NC. The cells were also positive to annexin V, after the PDT treatment with LP and NC, showing that one of the mechanisms of cellular death involved is apoptosis. In summary, the potential of LP and NC as a drug delivery system, in Photodynamic Therapy (PDT) against melanoma, has been confirmed using a lower concentration of the photosensitizer and lower light doses than that applied in current protocols. This is an innovative proposal to treat melanoma cell lines that until now have not received the benefit of the PDT protocol for treatment.

trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3)center dot H(2)O encapsulated in PLGA microparticles for delivery of nitric oxide to B16-F10 cells: Cytotoxicity and phototoxicity

The NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (py = pyridine) was loaded into poly-lactic-co-glycolic acid (PLGA) microparticles using the double emulsification technique. Scanning electron microscopy (SEM) and dynamic light scattering revealed that the particles are spherical in shape, have a diameter of 1600 nm, and have low tendency to aggregate. The entrapment efficiency was 25%. SEM analysis of the melanoma cell B16-F10 in the presence of the microparticles containing the complex trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (pyMP) showed that the microparticles were adhered to the cell surface after 2 h of incubation. The complex with concentrations lower than 1 x 10(-4) M did not show toxicity in B16-F 10 murine cells. The complex in solution is toxic at higher concentrations (> 1 x 10(-3) M), with cell death attributed to NO release following the reduction of the complex. pyMP is not cytotoxic due to the lower bioavailability and availability of the entrapped complex to the medium and its reducing agents. However, pyMP is phototoxic upon light irradiation. The phototoxicity strongly suggests that cell death is due to NO release from trans-[Ru(NO)(NH(3))(4)(py)](3+). This work shows that pyMP can serve as a model for a drug delivery system carrying the NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O, which can release NO locally at the tumor cell by radiation with light only. (c) 2007 Elsevier Inc. All rights reserved.

Evaluation of the radiopacity of some commercial and experimental root-end filling materials

Objective. The aim of the present study was to evaluate the radiopacity of 5 root end filling materials (white MTA Angelus, MTA Bio, light-cured MTA, Sealepox RP, and Portland cement clinker with bismuth oxide and calcium sulfate). Method. Five specimens, 10 mm in diameter and 1 mm in thickness according to specification ISO 6876: 2001 were fabricated from each material and radiographed using Insigth occlusal films close to a graduated aluminum step-wedge (2 to 16 mm in thickness). Radiographs were digitized and compared to the aluminum step-wedge. The radiographic density data were converted into millimeters of aluminum (mm Al), using the Digora 1.51 software. Results were evaluated statistically using the analysis of variance (ANOVA) followed by Tukey test. The level of significance was set at 5% (P<.05%). Results. Radiopacity values ranged from 1.21 mm Al (light-cured MTA) to 6.45 mm Al (MTA Angelus). Comparison between materials showed significant difference (P<.05) between MTA Angelus and all other materials, between Sealepox RP and MTA Bio, and between light-cured MTA and Portland cement clinker. Light-cured MTA was significantly less radiopaque than all other materials. No significant difference (P>.05) was found between MTA Bio and Portland cement clinker. Conclusions. All retrograde filling materials evaluated showed greater radiopacity than dentin. All the materials, except light-cured MTA met the minimum radiopacity standards of 3 mm Al recognized by the ISO 6876: 2001 and ADA n.57. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108: e35-e38)

Bone repair in mandibular body osteotomy after using 2.0 miniplate system - histological and histometric analysis in dogs

The objective of this study was to evaluate the bone repair along a mandibular body osteotomy after using a 2.0 miniplate system. Nine adult mongrel dogs were subjected to unilateral continuous defect through an osteotomy between the mandibular 3rd and 4th premolars. Two four-hole miniplates were placed in accordance with the Arbeitgeimeinschaft fur Osteosynthesefragen Manual. Miniplates adapted to the alveolar processes were fixed monocortically with 6.0-mm-length titanium alloy self-tapping screws, whereas miniplates placed near the mandible bases were fixed bicortically. At 2, 6 and 12 weeks, three dogs were sacrificed per period, and the osteotomy sites were removed, divided into three thirds (Tension Third, TT; Intermediary Third, IT; Compression Third, CT) and prepared for conventional and polarized light microscopy. At 6 weeks, while the CT repaired faster and showed bone union by woven bone formation, the TT and IT exhibited a ligament-like fibrous connective tissue inserted in, and connecting, newly formed woven bone overlying the parent lamellar bone edges. At 12 weeks, bone repair took place at all thirds. Histometrically, proportions of newly formed bone did not alter at TT, IT and CT, whereas significantly enhanced bone formation was observed for the 12-week group, irrespective of the third. The results demonstrated that although the method used to stabilize the mandibular osteotomy allowed bone repair to occur, differences in the dynamics of bone healing may take place along the osteotomy site, depending on the action of tension and compression forces generated by masticatory muscles.

Variations in cone photoreceptor abundance and the visual ecology of birds

The relative abundance and topographical distribution of retinal cone photoreceptors was measured in 19 bird species to identify possible correlations between photoreceptor complement and visual ecology. In contrast to previous studies, all five types of cone photoreceptor were distinguished, using bright field and epifluorescent light microscopy, in four retinal quadrants. Land birds tended to show either posterior dorsal to anterior ventral or anterior dorsal to posterior ventral gradients in cone photoreceptor distribution, fundus coloration and oil droplet pigmentation across the retina. Marine birds tended to show dorsal to ventral gradients instead. Statistical analyses showed that the proportions of the different cone types varied significantly across the retinae of all species investigated. Cluster analysis was performed on the data to identify groups or clusters of species on the basis of their oil droplet complement. Using the absolute percentages of each oil droplet type in each quadrant for the analysis produced clusters that tended to reflect phylogenetic relatedness between species rather than similarities in their visual ecology. Repeating the analysis after subtracting the mean percentage of a given oil droplet type across the whole retina (the 'eye mean') from the percentage of that oil droplet type in each quadrant, i.e. to give a measure of the variation about the mean, resulted in clusters that reflected diet, feeding behaviour and habitat to a greater extent than phylogeny.

MC1R genotype modifies risk of melanoma in families segregating CDKN2A mutations

Mutations in the exons of the cyclin-dependent kinase inhibitor gene CDKN2A are melanoma-predisposition alleles which have high penetrance, although they have low population frequencies. In contrast, variants of the melanocortin-1 receptor gene, MC1R, confer much lower melanoma risk but are common in European populations. Fifteen Australian CDKN2A mutation-carrying melanoma pedigrees were assessed for MC1R genotype, to test for possible modifier effects on melanoma risk. A CDKN2A mutation in the presence of a homozygous consensus MC1R genotype had a raw penetrance of 50%, with a mean age at onset of 58.1 years. When an MC1R variant allele was also present, the raw penetrance of the CDKN2A mutation increased to 84%, with a mean age at onset of 37.8 years (P=0.1). The presence of a CDKN2A mutation gave a hazard ratio of 13.35, and the hazard ratio of 3.72 for MC1R variant alleles was also significant. The impact of MC1R variants on risk of melanoma was mediated largely through the action of three common alleles, Arg151Cys, Arg160Trp, and Asp294His, that have previously been associated with red hair, fair skin, and skin sensitivity to ultraviolet light.

Photosynthetic responses of the coral Montipora digitata to cold temperature stress

Coral bleaching events have become more frequent and widespread, largely due to elevated sea surface temperatures. Global climate change could lead to increased variability of sea surface temperatures, through influences on climate systems, e.g. El Nino Southern Oscillation (ENSO). Field observations in 1999, following a strong ENSO, revealed that corals bleached in winter after unusually cold weather. To explore the basis for these observations, the photosynthetic responses of the coral species Montipora digitata Studer were investigated in a series of temperature and light experiments. Small replicate coral colonies were exposed to ecologically relevant lower temperatures for varying durations and under light regimes that ranged from darkness to full sunlight. Photosynthetic efficiency was analyzed using a pulse amplitude modulated (PAM) fluorometer (F-0, F-m, F-v/F-m), and chlorophyll a (chl a) content and symbiotic dinoflagellate density were analyzed with spectrophotometry and microscopy, respectively. Cold temperature stress had a negative impact on M digitata colonies indicated by decreased photosynthetic efficiency (F-v/F-m), loss of symbiotic dinoflagellates and changes in photosynthetic pigment concentrations. Corals in higher light regimes were more susceptible to cold temperature stress, Moderate cold stress resulted in photoacclimatory responses, but severe cold stress resulted in photodamage, bleaching and increased mortality. Responses to cold temperature stress of M digitata appeared similar to that observed in corals exposed to warmer than normal temperatures, suggesting a common mechanism. The results of this study suggest that corals and coral reefs may also be impacted by exposure to cold as well as warm temperature extremes as climate change occurs.

Alternatively spliced products of the human kinesin light chain 1 (KNS2) gene

Conventional kinesin is a microtubule-based molecular motor involved in the transport of membranous and non-membranous cargoes. The kinesin holoenzyme exists as a heterotetramer, consisting of two heavy chain and two light chain subunits. It is thought that one function of the light chains is to interact with the cargo. Alternative splicing of kinesin light chain pre-mRNA has been observed in lower organisms, although evidence for alternative splicing of the human gene has not been reported. We have identified 19 variants of the human KNS2 gene (KLC1) that are generated by alternative splicing of downstream exons, but calculate that KNS2 has the potential to produce 285919 spliceforms. Corresponding spliceforms of the mouse KLC1 gene were also identified. The alternative exons are all located 3' of exon 12 and the novel spliceforms produce both alternative carboxy termini and alternative 3' untranslated regions. The observation of multiple light chain isoforms is consistent with their proposed role in specific cargo attachment.

Direct Color Sensor, Optical Amplifier and Demux Device Integrated on a Single Monolithic SiC Photodetector

A pi'n/pin a-SiC:H voltage and optical bias controlled device is presented and its behavior as image and color sensor, optical amplifier and demux device is discussed. The design and the light source properties are correlated with the sensor output characteristics. Different readout techniques are used. When a low power monochromatic scanner readout the generated carriers the transducer recognizes a color pattern projected on it acting as a direct color and image sensor. Scan speeds up to 10(4) lines per second are achieved without degradation in the resolution. If the photocurrent generated by different monochromatic pulsed channels is readout directly, the information is demultiplexed. Results show that it is possible to decode the information from three simultaneous color channels without bit errors at bit rates per channel higher than 4000 bps. Finally, when triggered by light of appropriated wavelength, it can amplify or suppress the generated photocurrent working as an optical amplifier (C) 2009 Published by Elsevier Ltd.

Particle agglomeration study in rf silane plasmas: In situ study by polarization-sensitive laser light scattering

To determine self-consistently the time evolution of particle size and their number density in situ multi-angle polarization-sensitive laser light scattering was used. Cross-polarization intensities (incident and scattered light intensities with opposite polarization) measured at 135 degrees and ex situ transmission electronic microscopy analysis demonstrate the existence of nonspherical agglomerates during the early phase of agglomeration. Later in the particle time development both techniques reveal spherical particles again. The presence of strong cross-polarization intensities is accompanied by low-frequency instabilities detected on the scattered light intensities and plasma emission. It is found that the particle radius and particle number density during the agglomeration phase can be well described by the Brownian free molecule coagulation model. Application of this neutral particle coagulation model is justified by calculation of the particle charge whereby it is shown that particles of a few tens of nanometer can be considered as neutral under our experimental conditions. The measured particle dispersion can be well described by a Brownian free molecule coagulation model including a log-normal particle size distribution. (C) 1996 American Institute of Physics.

Estudo electroquímico de compostos naturais e semicondutores com possível utilização em células foto voltaicas

As células foto voltaicas orgânicas ou células de Gräetzel (depois do seu descobridor) são aparelhos para a colecta de energia solar que utilizam um semicondutor inorgânico e uma molécula orgânica. Dita molécula orgânica é capaz de excitar-se na presença de radiação electromagnética e ceder esta energia através da doação de electrões a este semicondutor. Embora estas estruturas e o seu processo de fabrico sejam relativamente pouco onerosas, o aproveitamento da energia solar é ainda muito baixo. Para além desta deficiência, os corantes sintéticos sofrem de “bleaching” ou então são reduzidos ou oxidados facilmente quando não conseguem transferir a energia que foi absorvida ou quando é difícil voltar ao estado original por dificuldades no completamento de circulação de electrões. Neste trabalho pretende-se então estudar o comportamento de moléculas e misturas complexas de moléculas com capacidade para serem excitadas pela luz solar. Como a dita xcitação promove a transferência de um electrão, este processo será seguido pela técnica de Voltametria cíclica. Como substâncias absorventes de luz utilizaremos compostos naturais (principalmente flavonóides) puros, ou então na forma de complexos naturais extraídos de algumas plantas. Estas misturas de corantes serão extractos aquosos (infusões) de casca de laranja e limão assim como extractos de folhas de cerejeira, com o objectivo de proporcionar lternativas aos flavonóides utilizados neste estudo. A caracterização voltamétrica desta célula é feita em diferentes formas de iluminação. Sobre a célula assim formada faz-se incidir rimeiro luz de lâmpadas fluorescentes, depois luz ultra violeta e por fim sem qualquer tipo de luz incidente. Na base do fabrico da variante mais clássica destas células está o semicondutor óxido de itânio (TiO2), por ser uma substância muito comum e barata e com propriedades semicondutoras notáveis. Uma forma comum de melhorar a eficiência deste material é introduzir dopantes com o intuito de melhorar a eficiência do processo de transferência electrónica. Um segundo objectivo deste trabalho é o estudo de sistemas semicondutor/molécula foto activa. Semicondutores como ZnO, TiO2 e TiO2 dopado serão então estudados. O gels de TiO2 ou o TiO2 dopado serão depositados sobre lâminas de vidro comum, nas quais foi anteriormente depositado uma película de alumínio que serve de condutor (eléctrodo egativo). Uma outra variante será a utilização de óxido de zinco, um semicondutor de baixo custo que por sua vez vai ser depositado em lâminas de alumínio comercial. A nossa célula foto electroquímica será então formada por moléculas de corante, uma lâmina e um semicondutor (que funcionará como eléctrodo de trabalho), com ou sem electrólito/catalizador (solução de iodo/iodeto), e eléctrodos de referência de Ag/AgCl, e outro auxiliar de grafite. Um outro objectivo é fazer um pequeno estudo sobre influencia do catalisador I2/etilenodiamina no comportamento electroquímico da célula, de forma a poder utilizar o solvente (etilenodiamina) com menor volatilidade do que a água, que é empregada no par I2/I3.m A importância deste facto prende-se com a limitada vida destas células quando o electrólito/solvente é evaporado pelas altas temperaturas da radiação incidente.

Human ceruloplasmin and neurotransmitters: complex stabilization and crystallization

Dissertation to obtain a Master Degree in Molecular Genetics and Biomedicine at Faculty of Sciences and Technology,Universidade Nova de Lisboa

Design and development of a microfluidic platform for use with colorimetric gold nanoprobe assays

Due to the importance and wide applications of the DNA analysis, there is a need to make genetic analysis more available and more affordable. As such, the aim of this PhD thesis is to optimize a colorimetric DNA biosensor based on gold nanoprobes developed in CEMOP by reducing its price and the needed volume of solution without compromising the device sensitivity and reliability, towards the point of care use. Firstly, the price of the biosensor was decreased by replacing the silicon photodetector by a low cost, solution processed TiO2 photodetector. To further reduce the photodetector price, a novel fabrication method was developed: a cost-effective inkjet printing technology that enabled to increase TiO2 surface area. Secondly, the DNA biosensor was optimized by means of microfluidics that offer advantages of miniaturization, much lower sample/reagents consumption, enhanced system performance and functionality by integrating different components. In the developed microfluidic platform, the optical path length was extended by detecting along the channel and the light was transmitted by optical fibres enabling to guide the light very close to the analysed solution. Microfluidic chip of high aspect ratio (~13), smooth and nearly vertical sidewalls was fabricated in PDMS using a SU-8 mould for patterning. The platform coupled to the gold nanoprobe assay enabled detection of Mycobacterium tuberculosis using 3 8l on DNA solution, i.e. 20 times less than in the previous state-of-the-art. Subsequently, the bio-microfluidic platform was optimized in terms of cost, electrical signal processing and sensitivity to colour variation, yielding 160% improvement of colorimetric AuNPs analysis. Planar microlenses were incorporated to converge light into the sample and then to the output fibre core increasing 6 times the signal-to-losses ratio. The optimized platform enabled detection of single nucleotide polymorphism related with obesity risk (FTO) using target DNA concentration below the limit of detection of the conventionally used microplate reader (i.e. 15 ng/μl) with 10 times lower solution volume (3 μl). The combination of the unique optical properties of gold nanoprobes with microfluidic platform resulted in sensitive and accurate sensor for single nucleotide polymorphism detection operating using small volumes of solutions and without the need for substrate functionalization or sophisticated instrumentation. Simultaneously, to enable on chip reagents mixing, a PDMS micromixer was developed and optimized for the highest efficiency, low pressure drop and short mixing length. The optimized device shows 80% of mixing efficiency at Re = 0.1 in 2.5 mm long mixer with the pressure drop of 6 Pa, satisfying requirements for the application in the microfluidic platform for DNA analysis.