Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

Use of fluorescent probes to follow membrane traffic in nerve terminals

Optical tracers in conjunction with fluorescence microscopy have become widely used to follow the movement of synaptic vesicles in nerve terminals. The present review discusses the use of these optical methods to understand the regulation of exocytosis and endocytosis of synaptic vesicles. The maintenance of neurotransmission depends on the constant recycling of synaptic vesicles and important insights have been gained by visualization of vesicles with the vital dye FM1-43. A number of questions related to the control of recycling of synaptic vesicles by prolonged stimulation and the role of calcium to control membrane internalization are now being addressed. It is expected that optical monitoring of presynaptic activity coupled to appropriate genetic models will contribute to the understanding of membrane traffic in synaptic terminals.

The requirements of a feasible plastic packaging material for cultured dairy products

The objective of this Master´s Thesis was to conduct a wide scale preliminary survey regarding the package requirements of a cultured dairy package, and to compare the currently used material polystyrene to other suitable packaging materials. Polystyrene has a long history of use in dairy cups, but in recent years its price has increased significantly compared to other common packaging materials. The overall environmental effects of a package and a package material are today a part of designing a sustainable product life cycle. In addition, in certain contexts there has been discussion of the risks posed by styrene polymer for the environment and for humans. These risks are also discussed in this thesis. Polystyrene (PS) is still the most widely used material in dairy cups. In recent years, polypropylene (PP) cups have appeared in increasing numbers on market shelves. This study focuses on the differences of the suitable polymers and examines the suitability of alternative “suitable” polymers with regards to dairy packaging. Aside from focusing on the cup manufacturer, this thesis also examines its subject matter from the viewpoint of the dairy customer, as well as observing the concrete implications of material changes in the overall value chain. It was known in advance that material permeability would be one of the determining factors and that gas transmission testing would be a significant part of the thesis. Mechanical tests were the second part of the testing process, providing information regarding package strength and protectiveness during the package’s life cycle. Production efficiency, along with uninterrupted stable production, was another important factor that was taken into consideration. These two issues are sometimes neglected in similar contexts due to their self-evident nature. In addition, materials used in production may have a surprising significance to the production and efficiency. Consistent high quality is also partly based on material selection. All of the aforementioned factors have been documented and the results have been analyzed by the development team at Coveris Rigid Finland. Coveris is now calculating the total finance effects and capacities should the material changes be implemented in practice. There are many factors in favor of switching to polypropylene at the moment. The overall production costs, as well as the environmental effects of resin production are the primary influences for said switch from the converters’ perspective.

Energy and raw material consumption analysis of powder bed fusion: case study: CNC machining and laser additive manufacturing

Laser additive manufacturing (LAM), known also as 3D printing, is a powder bed fusion (PBF) type of additive manufacturing (AM) technology used to manufacture metal parts layer by layer by assist of laser beam. The development of the technology from building just prototype parts to functional parts is due to design flexibility. And also possibility to manufacture tailored and optimised components in terms of performance and strength to weight ratio of final parts. The study of energy and raw material consumption in LAM is essential as it might facilitate the adoption and usage of the technique in manufacturing industries. The objective this thesis was find the impact of LAM on environmental and economic aspects and to conduct life cycle inventory of CNC machining and LAM in terms of energy and raw material consumption at production phases. Literature overview in this thesis include sustainability issues in manufacturing industries with focus on environmental and economic aspects. Also life cycle assessment and its applicability in manufacturing industry were studied. UPLCI-CO2PE! Initiative was identified as mostly applied exiting methodology to conduct LCI analysis in discrete manufacturing process like LAM. Many of the reviewed literature had focused to PBF of polymeric material and only few had considered metallic materials. The studies that had included metallic materials had only measured input and output energy or materials of the process and compared to different AM systems without comparing to any competitive process. Neither did any include effect of process variation when building metallic parts with LAM. Experimental testing were carried out to make dissimilar samples with CNC machining and LAM in this thesis. Test samples were designed to include part complexity and weight reductions. PUMA 2500Y lathe machine was used in the CNC machining whereas a modified research machine representing EOSINT M-series was used for the LAM. The raw material used for making the test pieces were stainless steel 316L bar (CNC machined parts) and stainless steel 316L powder (LAM built parts). An analysis of power, time, and the energy consumed in each of the manufacturing processes on production phase showed that LAM utilises more energy than CNC machining. The high energy consumption was as result of duration of production. Energy consumption profiles in CNC machining showed fluctuations with high and low power ranges. LAM energy usage within specific mode (standby, heating, process, sawing) remained relatively constant through the production. CNC machining was limited in terms of manufacturing freedom as it was not possible to manufacture all the designed sample by machining. And the one which was possible was aided with large amount of material removed as waste. Planning phase in LAM was shorter than in CNC machining as the latter required many preparation steps. Specific energy consumption (SEC) were estimated in LAM based on the practical results and assumed platform utilisation. The estimated platform utilisation showed SEC could reduce when more parts were placed in one build than it was in with the empirical results in this thesis (six parts).

Hygro-elasto-plastic behavior of planar orthotropic material

The mechanical and hygroscopic properties of paper and board are factors affecting the whole lifecycle of a product, including paper/board quality, production, converting, and material and energy savings. The progress of shrinkage profiles, loose edges of web, baggy web causing wrinkling and misregistration in printing are examples of factors affecting runnability and end product quality in the drying section and converting processes, where paper or board is treated as a moving web. The structural properties and internal stresses or plastic strain differences built up during production also cause the end-product defects related to distortion of the shape of the product such as sheet or box. The objective of this work was to construct a model capable of capturing the characteristic behavior of hygroscopic orthotropic material under moisture change, during different external in-plane stretch or stress conditions. Two independent experimental models were constructed: the elasto-plastic material model and the hygroexpansivity-shrinkage model. Both describe the structural properties of the sheet with a fiber orientation probability distribution, and both are functions of the dry solids content and fiber orientation anisotropy index. The anisotropy index, introduced in this work, simplifies the procedure of determining the constitutive parameters of the material model and the hygroexpansion coefficients in different in-plane directions of the orthotropic sheet. The mathematically consistent elasto-plastic material model and the dry solids content dependent hygroexpansivity have been constructed over the entire range from wet to dry. The presented elastoplastic and hygroexpansivity-shrinkage models can be used in an analytical approach to estimate the plastic strain and shrinkage in simple one-dimensional cases. For studies of the combined and more complicated effects of hygro-elasto-plastic behavior, both models were implemented in a finite element program for a numerical solution. The finite element approach also offered possibilities for studying different structural variations of orthotropic planar material, as well as local buckling behavior and internal stress situations of the sheet or web generated by local strain differences. A comparison of the simulation examples presented in this work to results published earlier confirms that the hygro-elasto-plastic model provides at least qualitatively reasonable estimates. The application potential of the hygro-elasto-plastic model is versatile, including several phenomena and defects appearing in the drying, converting and end-use conditions of the paper or board webs and products, or in other corresponding complex planar materials.

A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 µM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Optical spectroscopy of organic material based on C60

The structure and optical properties of thin films based on C60 materials are studied. Reproducible vacuum method of thin fullerene films production with Cd impurity on Si, glass and mica surfaces is developed. Surface morphology of the films are investigated by AFM and SEM methods. The ab initio quantum - chemical calculations of the geometry, total energy and excited energy states of complex fullerene- cadmium telluride supramolecules are performed. Photoluminescence spectra of composite thin films based on C60 before and after X-ray irradiation were measured. The intensity of additional peaks is defined as the charge composition due to the type of substrate. These results are interpreted as an appearance of the dipole-allowed transitions in the fullerene excited singlet states spectrum cause of an interference with cadmium telluride. X-ray irradiated films were investigated, and additional peaks in photoluminescence spectra were detected. These peaks appear as a result of molecular complexes formation from C60CdTe mixture and dimerization of the films. Density functional B3LYP quantum-chemical calculations for C60CdTe, molecular complexes, (C60)2 and C120O dimers were performed to elucidate some experimental results.

Fluorescent quantitative PCR of Mycobacterium tuberculosis for differentiating intestinal tuberculosis from Crohn's disease

Intestinal tuberculosis (ITB) and Crohn's disease (CD) are granulomatous disorders with similar clinical manifestations and pathological features that are often difficult to differentiate. This study evaluated the value of fluorescent quantitative polymerase chain reaction (FQ-PCR) for Mycobacterium tuberculosis (MTB) in fecal samples and biopsy specimens to differentiate ITB from CD. From June 2010 to March 2013, 86 consecutive patients (38 females and 48 males, median age 31.3 years) with provisional diagnoses of ITB and CD were recruited for the study. The patients' clinical, endoscopic, and histological features were monitored until the final definite diagnoses were made. DNA was extracted from 250 mg fecal samples and biopsy tissues from each patient. The extracted DNA was amplified using FQ-PCR for the specific MTB sequence. A total of 29 ITB cases and 36 CD cases were included in the analysis. Perianal disease and longitudinal ulcers were significantly more common in the CD patients (P<0.05), whereas night sweats, ascites, and circumferential ulcers were significantly more common in the ITB patients (P<0.05). Fecal FQ-PCR for MTB was positive in 24 (82.8%) ITB patients and 3 (8.3%) CD patients. Tissue PCR was positive for MTB in 16 (55.2%) ITB patients and 2 (5.6%) CD patients. Compared with tissue FQ-PCR, fecal FQ-PCR was more sensitive (X2=5.16, P=0.02). We conclude that FQ-PCR for MTB on fecal and tissue samples is a valuable assay for differentiating ITB from CD, and fecal FQ-PCR has greater sensitivity for ITB than tissue FQ-PCR.

Estudo das características de adsorção de água e da estabilidade das microcápsulas de óleo essencial de laranja na seleção de material de parede

Este trabalho consistiu no estudo e comparação das características de adsorção de água de três amostras de microcápsulas de óleo essencial de laranja, obtidas pela secagem por atomização de três diferentes emulsões preparadas pela adição de óleo essencial de laranja (oel), a uma solução aquosa de material de parede (mp) constituída de capsul (5,0, 0,0 e 10,0%), goma arábica (5,0, 10,0 e 0,0%) sendo constante para as três emulsões a maltodextrina (36,0%), água (44,0%) e óleo essencial (10,0%). A microencapsulação foi realizada a 220 e 110° C de ar de entrada e saída do secador usando um atomizador rotativo a 20.000rpm. Com base à determinação das isotermas de adsorção de água a 30, 40 e 50° C e usando o modelo de GAB para ajustar os pontos experimentais foram avaliadas as características das isotermas, a estabilidade e área superficial de adsorção de água das diferentes amostras de microcápsulas obtidas. Os resultados indicaram ser importante o estudo das características de adsorção de água para estimar a estabilidade das microcápsulas de oel e a comparação destas mostrou que as microcápsulas obtidas pela secagem por atomização da emulsão preparada com 5,0% de capsul e 5,0% de goma arábica apresentaram o melhor resultado.

Microencapsulação de óleo essencial de laranja: seleção de material de parede

No presente trabalho se procedeu à comparação de agentes microencapsulantes, material de parede (mp), na microencapsulação de óleo essencial de laranja (material ativo) através da secagem por atomização. Foram preparadas três amostras de emulsões pela adição de óleo essencial de laranja a uma solução aquosa do mp composta de capsul (5,0, 0,0 e 10,0%), goma arábica (5,0, 10,0 e 0,0%) sendo constante para as três amostras maltodextrina (36,0%), água (44,0%) e óleo essencial (10,0%). Foram avaliadas as curvas de secagem das emulsões frente à retenção do óleo essencial de laranja e a tendência de formação de dobras superficiais das partículas e verificou-se a hipótese "menor a tendência de formação de dobras na superfície das microcápsulas maior a retenção de material ativo". A microencapsulação foi obtida pela secagem por atomização, com temperaturas de 220ºC e 110ºC do ar de entrada e de saída da câmara de secagem, respectivamente, e com atomizador rotativo (20.000rpm). A comparação das microcápsulas obtidas a partir das três amostras de emulsões mostrou que aquela preparada com 10,0% de capsul e 0,0% de goma arábica apresentou o maior resultado. O período de taxa constante de secagem desta mistura é curto e com maior retenção de umidade após a secagem. As microcápsulas obtidas apresentaram maior retenção de óleo essencial e dobras superficiais menos pronunciadas decorrentes do menor entumescimento das gotículas durante a secagem.

Monolithic zirconium dioxide as a full contour restorative material: With special emphasis on the optical and mechanical properties

Full contour monolithic zirconia restorations have shown an increased popularity in the dental field over the recent years, owing to its mechanical and acceptable optical properties. However, many features of the restoration are yet to be researched and supported by clinical studies to confirm its place among the other indirect restorative materials This series of in vitro studies aimed at evaluating and comparing the optical and mechanical properties, light cure irradiance, and cement polymerization of multiple monolithic zirconia material at variable thicknesses, environments, treatments, and stabilization. Five different monolithic zirconia materials, four of which were partially stabilized and one fully stabilized were investigated. The optical properties in terms of surface gloss, translucency parameter, and contrast ratio were determined via a reflection spectrophotometer at variable thicknesses, coloring, sintering method, and after immersion in an acidic environment. Light cure irradiance and radiant exposure were quantified through the specimens at variable thicknesses and the degree of conversion of two dual-cure cements was determined via Fourier Transform Infrared spectroscopy. Bi-axial flexural strength was evaluated to compare between the partially and fully stabilized zirconia prepared using different coloring and sintering methods. Surface characterization was performed using a scanning electron microscope and a spinning disk confocal microscope. The surface gloss and translucency of the zirconia investigated were brand and thickness dependent with the translucency values decreasing as the thickness increased. Staining decreased the translucency of the zirconia and enhanced surface gloss as well as the flexural strength of the fully stabilized zirconia but had no effect on partially stabilized zirconia. Immersion in a corrosive acid increased surface gloss and decreased the translucency of some zirconia brands. Zirconia thickness was inversely related to the amount of light irradiance, radiant exposure, and degree of monomer conversion. Type of sintering furnace had no effect on the optical and mechanical properties of zirconia. Monolithic zirconia maybe classified as a semi-translucent material that is well influenced by the thickness, limiting its use in the esthetic zones. Conventional acid-base reaction, autopolymerizing and dual-cure cements are recommended for its cementation. Its desirable mechanical properties give it a high potential as a restoration for posterior teeth. However, close monitoring with controlled clinical studies must be determined before any definite clinical recommendations can be drawn.

Preparação de um material de referência certificado para controle de agrotóxicos em hortifrutigranjeiros: estudo da homogeneidade

Neste trabalho são apresentados os esforços para garantir a homogeneidade de um material de referência. Níveis residuais (mg.kg-1) de quatro agrotóxicos (γ-HCH, fenitrotiona, clorpirifós e procimidona) foram adicionados à polpa de tomate com o objetivo de se preparar um material de referência certificado. As propriedades mais importantes desses materiais são a homogeneidade e a estabilidade. Antes de serem enviados a outros laboratórios, os materiais de referência precisam ter sua homogeneidade verificada. Nas etapas prévias, amostras foram avaliadas de modo a prover dados sobre o tratamento mais adequado para minimizar a variabilidade analítica do lote preparado e garantir a qualidade da amostra candidata a material de referência certificado, tão bem como a estimativa da incerteza associada à homogeneidade. A análise de variância fornece informações sobre a variabilidade do lote preparado e o grau de invariabilidade da amostra fortificada. Depois da preparação, as amostras foram expostas à radiação gama na dose de 2 kGy e submetidas a um estudo interlaboratorial para certificação. As concentrações certificadas dos agrotóxicos após caracterização foram 0,191 ± 0,047 mg.kg-1; 0,192 ± 0,068 mg.kg-1; 0,225 ± 0,076 mg.kg-1e 0,177 ± 0,051 mg.kg-1 para o γ-HCH, a fenitrotiona, clorpirifós e procimidona respectivamente.

Preparação de um material de referência certificado para controle de agrotóxicos em hortifrutigranjeiros: estudo da estabilidade

Neste trabalho são apresentados os resultados dos estudos de estabilidade referentes à produção de um material de referência certificado. Foram avaliados níveis residuais de concentração dos agrotóxicos γ-HCH, fenitrotiona, clorpirifós e procimidona em polpa de tomate. A pasteurização e a irradiação gama foram empregadas à polpa de tomate, visando manter a integridade da amostra candidata a material de referência. A polpa foi preparada e dividida em duas partes. Cada parte foi fortificada com os referidos agrotóxicos na faixa de concentração de 0,1 a 0,2 mg.kg-1. Uma das partes foi submetida à pasteurização a 90 °C por 4 minutos e a outra parte foi irradiada com dose de 2,0 kGy depois de homogeneizada. A estabilidade e a incerteza da amostra, correspondente ao período de tempo avaliado, foram determinadas através da análise de regressão em conjunto com a ANOVA. Os resultados indicaram que ambos os procedimentos de preparo da amostra são adequados para a conservação da polpa de tomate fortificada com os quatro agrotóxicos, com vantagem para o tratamento por irradiação com a dose de 2,0 kGy.

Microparticles obtained by complex coacervation: influence of the type of reticulation and the drying process on the release of the core material

Microparticles obtained by complex coacervation were crosslinked with glutaraldehyde or with transglutaminase and dried using freeze drying or spray drying. Moist samples presented Encapsulation Efficiency (%EE) higher than 96%. The mean diameters ranged from 43.7 ± 3.4 to 96.4 ± 10.3 µm for moist samples, from 38.1 ± 5.36 to 65.2 ± 16.1 µm for dried samples, and from 62.5 ± 7.5 to 106.9 ± 26.1 µm for rehydrated microparticles. The integrity of the particles without crosslinking was maintained when freeze drying was used. After spray drying, only crosslinked samples were able to maintain the wall integrity. Microparticles had a round shape and in the case of dried samples rugged walls apparently without cracks were observed. Core distribution inside the particles was multinuclear and homogeneous and core release was evaluated using anhydrous ethanol. Moist particles crosslinked with glutaraldehyde at the concentration of 1.0 mM.g-1 protein (ptn), were more efficient with respect to the core retention compared to 0.1 mM.g-1 ptn or those crosslinked with transglutaminase (10 U.g-1 ptn). The drying processes had a strong influence on the core release profile reducing the amount released to all dry samples