When the writer took an introductory course in Microtechnique in 1954-55, he was impressed by the inadequacy of some microtechnical procedures recommended in triple staining, and he felt disappointed and discouraged by the consistently inferior results. He resolved to attempt modifications or to develop a new technique which could be relied upon to achieve anticipated results. This is a report on two years of efforts to develop a simple dependable technique in polychrome staining for histological studies. The writer feels that the results are of importance to students and teachers of biological science and to microtomists. The herein proposed technique enables the observer readily to recognize fine details of microscopic structures, and it presents a more attractive picture than does the common Harris’ hematoxylin counterstained with eosin. This report also includes a list of polychrome procedures, divided into four groups: (1) hematoxylin and combinations, (2) safranin and combinations, (3) acid fuchsin and combinations, and (4) miscellaneous.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

The three-dimensional (3-D) structure of gp32*I, a major proteolytic fragment of the DNA helix-destabilizing protein from bacteriophage T4, has been determined at 18 A resolution by electron microscopy of negatively stained crystals and computer image analysis. The crystalline areas processed in 3-D have the symmetry of the space group P2₁, with a = 47 Å, b = 63 Å, c = 65 Å, and α = β = γ = 90°. This P2₁ unit cell contains one gp32*I molecule per asymmetric unit. The molecule is roughly V-shaped, containing two large domains linked by a smaller domain occupying the base of the V. The total length of the molecule is about 110 Å with an average diameter of about 25 Å. Systematic analysis of the symmetry in images of untilted crystals determined that the crystal could display several types of projection symmetry, pgg, pg corresponding to P2₁ symmetry with the screw axis along the a axis of the crystal, and pg corresponding to P2₁ symmetry with the screw axis along the b axis. Among images displaying pg symmetry along the b axis, two types of images with noticeably different appearances were obtained. A hypothesis was formed that explained the different types of symmetry as the result of the growth of the gp32*I crystal in the space group P2₁ 2₁ 2₁, in steps of 1/2 of a unit cell along the thin direction of the crystal. Two different types of 1/2 unit cell thick steps were postulated. Computer simulations were used to generate synthetic images of untilted crystals containing either one, two or three steps of each kind. The results of the simulations prove that the space group of the gp32*I crystal is P2₁ 2₁ 2₁. They suggest that careful analysis of the symmetry in images of untilted gp32*I crystals can provide information about the thickness of the crystals. A strategy is presented for determining the structure of the gp32*I crystal at higher resolution by electron microscopy of frozen, hydrated crystals. This strategy includes the use of symmetry analysis as a tool for determining the thickness of the crystals so that data from crystals of the same thickness can be combined in 3-D. A similar approach may prove useful in the 3-D electron microscopic analysis of other thin, multi-layered crystals.

No description available.

Thesis (MMed (Biomedical Sciences. Anatomy and Histology))--University of Stellenbosch, 1993. / Glial fibrillary acidic protein (GFAP) is found in varying amounts in the cytoplasm of most normal and neoplastic cells of astroglial origin. Though not glial specific, immunoelectron microscopy has shown that vimentin and GFAP are coexpressed as monomers of glial intermediate filaments. These structures display irreversible assembly and a slow metabolic turnover. Although currently applied as astroglial markers, these intermediate filament proteins may reflect the functional and developmental differentiation status of the cells in which they are expressed. Some authors have tried to apply these aspects as diagnostic parameters for grades of malignancy and anaplasia whilst other workers have indicated variable concentrations of GFAP in different astroglial cell types and entities. Different processing protocols, including the use of epoxy and acrylic resins, omission of osmium tetroxide and variations in concentration and incubation time of primary fixatives, were evaluated to find a compromise between antigen availability and acceptable ultrastructure. Thin sections were labelled on grid for GFAP (Dako A561) and vimentin (Dako M725) by means of the indirect immunogold method. For semi- quantification of relative antigen concentrations, a novel method was devised to calculate the labelling density, percentage heterogeneity of the particle distribution and the surface area investigated. This allowed expression of labelling results as a three figure unit. Standardized post-embedding immunoelectron microscopy was performed on 11 normal and neoplastic human tissue specimens. The tissue was exposed to conventional immersion fixation in glutaraldehyde and osmium tetroxide prior to modified embedding in LR White resin. The validity of these results was verified by correlation with conventional histopathological, immunohistochemical and clinical data obtained for each specimen. The presence of epoxy resin in thin sections was shown to reduce antigen availability to such an extent that very low to negative labelling was encountered. Acrylic LR White resin allowed more acceptable immunodetection, but at the cost of inferior ultrastructure and greater instability of thin sections in the electron beam. This masked the effects of glutaraldehyde fixation on the density of the tissuefixative matrix which included destruction of the vimentin and some GFAP associated epitopes. Although osmium tetroxide was required for acceptable ultrastructure, it reduced the labelling sensitivity by 20% and was responsible for premature curing of acrylic resin during impregnation of tissue. Despite superior resolution gained by electron microscopy and the advantage of semi-quantification of labeling results, the labelling sensitivity of this technique is lesser than that of light microscopical immunohistochemistry. Immunoelectron microscopy confirmed the association between GFAP and glial intermediate filaments in almost all the glial tumours studied, correlating well with GFAP expression in matching specimens demonstrated at light microscopical level. In the absence of intermediate filaments, no positivity for GFAP or vimentin was found in oligodendroglial components of mixed tumours. GFAP positivity in astrocytomas was demonstrated by between 17 and 126 particles / µm2, whilst lower figures were obtained for the glioblastoma (PD = 8) and some of the mixed gliomas (Pd = 6). Rosenthal fibres showed both peripheral and central positive labelling for GFAP, thus providing more evidence for their hypothetical degenerative, astroglial nature. The meningioma studied, was GFAP negative, but produced low density positivity for vimentin. Coexpression of GFAP and vimentin was demonstrated in an astroblastoma and degenerative infant brain tissue, thus supporting the presence of both these proteins development of glial structures. Although sites of likely glial intermediate filament synthesis were found, the antigen availability for vimentin was too low to allow a reliable assessment of specific vimentin localization and determination of the GFAP : vimentin ratio in individual intermediate filaments and/or astroglial fibres. Variations in particle densities (PD) which demonstrated GFAP in the various astroglial entities studied, were considered to be a result of variable technical and tissue processing factors rather than truly significant differences in expression of GFAP in individual intermediate filaments. This lead to the conclusion that the GFAP concentration / glial intermediate filament area is likely to be constant for mature glial intermediate filaments and therefore cannot be used to distinguish between different astroglial cells or entities. Whether each cell has a different number of glial intermediate filaments, has not been established satisfactorily. Following complementary conventional immunohistochemistry and careful orientation of biopsy material, the procedure can be applied to suitable specimens for the electron microscopical localization of high concentrations of aldehyde resistant, cytoplasmic antigens.

Dissertação composta por 5 artigos. / CAPES / A quitosana é um aminopolissacarídeo natural, biodegradável, hidrofílico, atóxico e biocompatível que pode ser encontrada na parede de micro-organismos, especialmente nas espécies Mucor, mas principalmente nas cascas de crustáceos oriundos dos resíduos da indústria pesqueira. Por possuir diversas características funcionais a quitosana tem se destacado como um excelente agente encapsulante, seja através da formação de microcápsulas ou de microesferas. Diversos estudos têm sido realizados a fim de demonstrar a eficiência das microesferas de quitosana em adsorver, proteger e liberar compostos ativos e resíduos, beneficiando assim, diversos segmentos como: farmacêutico, alimentar, agroindustrial, químico, biomédico e de cosméticos. / Chitosan is a natural amino polysaccharide, biodegradable, hydrophilic, biocompatible and low toxicity and can be found in the wall of microorganisms, especially in Mucor species, but especially in crustacean shells waste from the fishing industry. By owning several functional characteristics chitosan has emerged as an excellent encapsulating agent, either through the formation of microcapsules or microspheres. Several studies have been conducted to demonstrate the efficiency of chitosan microspheres in adsorption, protection and release of active compounds, thus benefiting, several segments, including pharmaceutical, food, agribusiness, chemical, biomedical and cosmetics. / 5000

Dissertação composta por 5 artigos. / CAPES / A quitosana é um aminopolissacarídeo natural, biodegradável, hidrofílico, atóxico e biocompatível que pode ser encontrada na parede de micro-organismos, especialmente nas espécies Mucor, mas principalmente nas cascas de crustáceos oriundos dos resíduos da indústria pesqueira. Por possuir diversas características funcionais a quitosana tem se destacado como um excelente agente encapsulante, seja através da formação de microcápsulas ou de microesferas. Diversos estudos têm sido realizados a fim de demonstrar a eficiência das microesferas de quitosana em adsorver, proteger e liberar compostos ativos e resíduos, beneficiando assim, diversos segmentos como: farmacêutico, alimentar, agroindustrial, químico, biomédico e de cosméticos. / Chitosan is a natural amino polysaccharide, biodegradable, hydrophilic, biocompatible and low toxicity and can be found in the wall of microorganisms, especially in Mucor species, but especially in crustacean shells waste from the fishing industry. By owning several functional characteristics chitosan has emerged as an excellent encapsulating agent, either through the formation of microcapsules or microspheres. Several studies have been conducted to demonstrate the efficiency of chitosan microspheres in adsorption, protection and release of active compounds, thus benefiting, several segments, including pharmaceutical, food, agribusiness, chemical, biomedical and cosmetics. / 5000

Ce travail de thèse s’inscrit dans la partie caractérisation d’un projet collaboratif ayant pour objectif d’élaborer des textiles antimicrobiens pour différents domaines d’application, en particulier les domaines de la santé et de l’agroalimentaire. La démarche analytique a consisté à combiner différentes techniques d’analyse de surface (techniques microscopiques (SEM, AFM, TEM) et spectroscopiques (XPS, ToF-SIMS, SIMS, EELS)) avec des analyses microbiologiques pour aider à la compréhension des mécanismes de protection antimicrobienne des textiles traités. Les agents antimicrobiens, l’argent et le Poly HexaMéthylène Biguanide (PHMB), ont été déposés respectivement par plasma (PVD / PECVD) et par foulardage. Les contraintes liées aux domaines d’application des textiles étudiés (implants herniaires et vêtements professionnels) ont été prises en compte (respectivement, quantité minimale de l’agent antimicrobien et résistance au lavage industriel). Malgré certaines contaminations inhérentes à des procédés industriels, les analyses de surface se sont révélées être un ensemble d’outils essentiel au développement des procédés (qualité du dépôt, influence des conditions de dépôt, influence du lavage). Selon les domaines d’application, l’analyse à très haute sensibilité en extrême surface et l’étude de la distribution en profondeur de l’agent antimicrobien ont été des étapes clés pour la compréhension des propriétés antimicrobiennes observées pour les dépôts, démontrant la pertinence de l’approche multi-analytique choisie dans ce travail de thèse / This thesis work concerns the characterization effort within a cooperation project aiming at developing antimicrobial textiles for various application fields, particularly health applications and food-processing industry. The analytical approach combined different surface analysis techniques (microscopy techniques (SEM, AFM, TEM) and spectroscopy techniques (XPS, ToF-SIMS, SIMS, EELS)) to microbiological tests in order to understand the antimicrobial activity of deposits at the surface of textiles. Silver and Poly Hexamethylene Biguanide (PHMB) antimicrobial agents were deposited by plasma (PVD / PECVD) and padding, respectively. Specific constraints related to the application fields (hernia implants and clothing) were considered (minimum concentration in antimicrobial agent and resistance to industrial washing, respectively). Despite some ubiquitous contamination related to industrial processes, surface analysis techniques proved to be an essential help to develop these processes (deposit quality, influence of deposition conditions, influence of washing). Depending on the application fields, high sensitivity surface analysis at the extreme surface and in-depth distribution of the antimicrobial agent were essential to understand the antimicrobial properties of the deposits, which confirms the relevance of the multi-analytical approach used in this thesis work

Orientador: Adilma Regina Pippa Scamparini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-08T02:50:35Z (GMT). No. of bitstreams: 1 Luvielmo_MarciadeMello_D.pdf: 3297730 bytes, checksum: dc80c8868bb6f8fa43b93b87d5e0bae3 (MD5) Previous issue date: 2007 / Resumo: O presente estudo teve como objetivo fazer uma seleção de linhagens de X. campestris para a produção de goma xantana e verificar o efeito do processo de ¿stress¿ alcalino em diferentes condições de produção deste biopolímero, através da avaliação da produção de goma e da viscosidade aparente, que é um fator de elevada relevância para designar em quais processos e setores da indústria a goma xantana será aplicada. A pesquisa também teve como objetivo investigar as mudanças na estrutura da goma xantana causadas pelo ¿stress¿ alcalino, e as mudanças na ultraestrutura das células, assim como a disposição da goma em diferentes etapas da sua produção e em diferentes tempos de ¿stress¿ alcalino, a fim de contribuir com uma maior compreensão desse complexo processo. Foi selecionada a linhagem X. campestris pv. manihotis 280-95, como a de melhor desempenho em produção e qualidade, com uma produção de goma xantana de 10,8 g/L. A partir destes estudos, X. campestris pv. manihotis 280-95 passou a ser a bactéria utilizada para os estudos posteriores. O maior valor de produção de goma xantana foi atingido em ¿stress¿ alcalino com pH 12 (EA12), seguido do ¿stress¿ alcalino em pH 11 (EA11) e ¿stress¿ alcalino em pH 8 (EA08). Porém a qualidade da goma obtida após o processo de EA12 é menor se comparada à obtida sem o processo de ¿stress¿ alcalino. O hidróxido de sódio (NaOH) foi o álcali que apresentou melhor desempenho para o processo de ¿stress¿ alcalino. A produção de goma xantana (g.L-1) e as viscosidades aparentes das gomas não foram afetadas nos diferentes tempos de ¿stress¿ alcalino (EA12) testados nesse estudo (1h, 2h, 3h e 4h), na faixa de taxa de deformação testada (0 ¿ 60 s-1). A produção de goma xantana obtida da fermentação realizada em bioreator de 2 L utilizando X. campestris pv. manihotis 280-95 foi maior após o processo de ¿stress¿ alcalino por 24 horas (EA24h). Ao final de 72 horas de fermentação 9,43 g.L-1 de goma xantana foram obtidos e após 24 horas de ¿stress¿ alcalino, a produção foi 74,8% maior (16,48 g.L-1). No estudo das ultraestruturas (capítulo IV) foi possível visualizar cada passo dos processos e verificar que, mesmo no inóculo puro, observa-se também uma pequena produção de goma xantana próximo a algumas células. No final da fermentação (FF), observou-se o início de mudanças estruturais nas bactérias, como a vacuolização citoplasmática e a descontinuidade da membrana, podendo sugerir um início no processo de lise bacteriana. Após o ¿stress¿ alcalino (EA12-1h), foram observadas acentuadas diferenças estruturais nas células bacterianas. O conteúdo citoplasmático das bactérias tornou-se mais vacuolizado e verificou-se descontinuidade nas membranas das células bacterianas, indicando processo de lise bacteriana. A goma xantana que se apresentou agrupada em grumos, adquiriu uma conformação organizada em círculos concêntricos. Finalmente, pode ser mencionado que novos estudos com diferentes linhagens e condições de processo devem ser realizados procurando-se sempre melhores resultados de produção e qualidade desse biopolímero / Abstract: The present study had as objective to make a selection of strains of X. campestris for the production of xanthan gum and to verify the effect of the process of alkaline "stress" at different conditions of production of this biopolymer, through the evaluation of the gum production and of the apparent viscosity, which is a factor of high relevance, in order to allocate which processes and sections of the industry the xanthan gum will be applied in. The research also aimed to investigate changes in the structure of the xanthan gum caused by the alkaline "stress", changes in the ultrastructure of the cells, as well as the disposition of the gum at different stages of its production and at different times of alkaline "stress", so as to contribute with a better understanding of such compound process. The strain was selected X. campestris pv. manihotis 280-95, as better acting in production and quality, with a production of xanthan gum of 10,8 g.L-1 starting from these studies, X. campestris pv. manihotis 280-95 started to be the bacterium used for the subsequent studies. The largest value of production of gum xantana was reached in alkaline "stress" with pH 12 (EA12), followiedby the alkaline "stress" in pH 11 (EA11) and alkaline "stress" in pH 8 (EA08). However, the quality of the gum obtained after the process of EA12 is worse compared to what was obtained without the process of alkaline "stress." The hydroxide of sodium (NaOH) was the alkali that presented better results in obtaining the process of alkaline "stress." The production of xanthan gum (g.L-1) and the apparent viscosities of the gums were not affected in the different times of alkaline "stress" (EA12) tested in that study (1:00, 2:00, 3:00 and 4:00), at the shear rate range tested (0 - 60 s-1). The production of xanthan gum obtained from the fermentation accomplished in bioreactor of 2 L using X. campestris pv. manihotis 280-95 was larger after the process of alkaline "stress" within 24 hours (EA24h). At the end of 72 hours of fermentation 9,43 g.L-1 of xanthan gum were obtained and after 24 hours of alkaline "stress", the production was 74,8% larger (16,48 g.L-1). In the study of the ultrastructures (chapter IV) it was possible to observe each step of the processes and to verify that, even in the pure inoculum, it is also observed a small production of gum close to some cells. Atthe end of the fermentation (FF), the beginning of structural changes was observed in the bacteria, as the vacuum cytoplasm and the discontinuity of the membrane, could suggest a beginning in the process of bacterial lise. After the alkaline "stress" (EA12-1h), accentuated structural differences were observed in the bacterial cells. The content cytoplasm of the bacteria became more vacuuming and discontinuity was verified in the membranes of the bacterial cells, indicating process of bacterial lysis. The resulting xanthan gum presented in clots, acquired an organized conformationin concentric circles. Finally, it can be mentioned that new studies with different strains and process conditions should be accomplished being always sought better production results and quality of that biopolymer / Doutorado / Doutor em Ciência de Alimentos

Indiana University-Purdue University Indianapolis (IUPUI) / The details of Polarization Modulation Near-Field Scanning Optical Microscopy (PM-NSOM) are presented. How to properly calibrate and align the system is also introduced. A measurement of Muscovite crystal is used to display the capabilities of the setup. Measurements of supported Lβʹ 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayers are presented, emphasizing how it was tooled in exploiting the anisotropic nature of the acyl chains. A discussion of how the effective retardance (ΔS = 2π( n_e-n_o )t/λ) and the direction of the projection of the acyl chains (θ) are measured simultaneously is given, (where t is the thickness of the bilayer and λ is the wavelength of light used). It is shown from ΔS the birefringence (ne-no) of the bilayer is determined, by assuming the acyl chain tilt with respect to the membrane's normal to be ϕ ≈ 32. Time varying experiments show lateral diffusions of ~ 2 x 10-12 cm2/s. Temperature controlled PM-NSOM is shown to be a viable way to determine the main phase transition temperature (Tm) for going from the gel Lβʹ to liquid disorder Lα state of supported DPPC bilayers. A change of ΔS ~ (3.8 +/- 0.3 mrad) at the main phase transition temperature Tm (≈41^o C) is observed. This agrees well with previous values of (ne-no) and translates to an assumed <ϕ> ~ 32^o when T < Tm and 0^o when T > Tm. Evidence of supper heating and supper cooling will be presented, along with a discussion of the fluctuations that occur around Tm. Finally it is shown how physical parameters such as the polarizability are extracted from the data. Values of the transverse (αt) and longitudinal (αl) polarizabilites of the acyl chains are shown to be, αt = 44.2 Å3 and αl = 94.4 Å3, which correspond well with the theoretical values of a single palmitic acid (C16) αt = 25.14 Å3 and αl = 45.8 Å3.