Orientador: Regina Helena Garcia Martins / Resumo: Introdução: denomina-se presbifonia o conjunto de alterações nos padrões vocais consequentes ao envelhecimento da laringe, podendo cursar com sintomas de disfonia, voz fraca, trêmula e baixa. Estudos histológicos e imunohistoquímicos da presbilaringe demonstram atrofia do epitélio, da lâmina própria e do músculo vocal, além de aumento de fibras colágenas e diminuição de fibras elásticas e das proteínas não fibrosas da matriz extracelular. Os estudos de microscopia eletrônica da presbilaringe são escassos e podem acrescentar detalhes ultraestruturais importantes e auxiliar na compreensão da fisiopatologia da presbifonia. Objetivos: descrever os achados de microscopia eletrônica de varredura e transmissão da prega vocal senil. Casuística e métodos: Foram removidas 16 laringes humanas durante necrópsia e distribuídas em dois grupos: controle (n-8; idade 30 - 50 anos; 6F e 2M) e idoso (n-8; idade 75- 92 anos; 6F e 2M). As porções medianas de ambas as pregas vocais foram dissecadas, fixadas em glutaraldeído 2,5% e preparadas para exames de microscopia eletrônica de varredura e transmissão. A espessura do epitélio foi medida nas fotografias de microscopia eletrônica de varredura, com aumentos semelhantes, utilizando-se o programa de morfometria digital Scandium. Resultados: Microscopia eletrônica de varredura: Grupo controle: epitélio composto por 5 a 7 camadas de células sobrepostas, raras células em descamação, e discreta ondulação. Lâmina própria com rede uniforme de fibras col... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Presbyphonia is called the set of changes in vocal patterns consequent aging of the larynx, which can present with symptoms of dysphonia, weak voice, trembling and low. Histological and immunohistochemical studies of presbylarynx show atrophy of the epithelium, lamina propria and the vocal muscle, and increase of collagen fibers and diminution of elastic fibers and non-fibrous proteins of the extracellular matrix. The studies of electron microscopy of presbylarynx are scarce and can add significant ultrastructural details contribute to further understanding of the pathophysiology of presbyphonia. Objectives: To describe the findings of scanning and transmission electron microscopy of senile vocal folds. Methods: 16 human larynx were removed during autopsy and distributed into two groups: control (n-8; age 30-50 years; 6F and 2M) and elderly (n-8, age 75- 92 years; 6F and 2M). The median portions of the right and left vocal folds were dissected, fixed in 2.5% glutaraldehyde and prepared for examination using scanning and transmission electron microscopy respectively. The thickness of the epithelium was analyzed in the pictures of the scanning electron microscopy with similar magnification, using the Scandium morphometric digital program. Results: Scanning electron microscopy: Control Group: epithelium composed of 5-7 layers of overlapping cells, rare cells in flaking and slight ripple. Lamina propria with uniform network of collagen and elastic fibers running pa... (Complete abstract click electronic access below) / Mestre

<p>Flaviviruses are enveloped, positive-strand RNA viruses that are spread by mosquitoes and ticks and can cause serious disease in humans. Flavivirus virions undergo extensive structural changes during their life cycle, including during maturation and fusion. Flaviviruses are initially assembled at the endoplasmic reticulum in a non-infectious, immature state, and then traffic to the trans-Golgi network, where a pH drop triggers a structural rearrangement of glycoproteins prM and E on the virus surface from 60 trimers to 90 dimers. A host protease, furin, then cleaves prM which makes the transition irreversible. Upon exiting the host cell, pr disassociates from the virus and the infectious, mature virus is able to enter a new cell. <br></p><p><br></p> <p> </p> <p>In Chapter 1, an overview of flaviviruses is presented, including a brief history of their discovery and interaction with humans, followed by what is known about their life cycle and the maturation process. The structure of a mature flavivirus is then described, including the symmetrical arrangement of glycoproteins on the virion surface, the lipid membrane, and the nucleocapsid core, followed by an introduction of the structural proteins that assemble into the virion. The structure of the immature flavivirus is then described. The chapter concludes with a description of the dynamics and heterogeneity observed for flaviviruses.</p><p><br></p> <p> </p> <p>The conformational rearrangements that occur during flavivirus maturation remain unclear. The structures of immature and mature flaviviruses determined with cryo-electron microscopy (cryo-EM) demonstrated that flaviviruses are icosahedral particles with 180 copies of glycoproteins on their surface. Icosahedral viruses typically have a quasi-equivalent arrangement of glycoproteins, but flaviviruses lack quasi-equivalence and instead the three subunits within an asymmetric unit occupy different chemical environments. Although the subunits are the same proteins, the unique environment of each subunit can be exploited for tracking subunits during conformational rearrangements. For example, the unique labeling of a subunit can be used to identify it in the immature and mature virion.</p><p><br></p> <p> </p> <p>In Chapter 2, the maturation process was studied by developing tools to differentially label protein subunits and trap potential intermediates of maturation. The tools included heavy-atom compounds and antibody Fabs, which were used to probe Kunjin virus (KUNV), an Australian subtype of West Nile virus (WNV). One heavy-atom compound, potassium tetranitroplatinate(II), was found to derivatize immature KUNV, likely at sites on both E and prM. Higher-resolution studies will be required to determine if the compound differentially labeled the three subunits. The other tool developed was the E16 Fab. E16 Fab, originally isolated from a mouse immunized with WNV E and found to bind to two out of three subunits on mature WNV, was used to differentially label subunits in immature KUNV. Based on poor epitope accessibility on immature KUNV, E16 Fab was hypothesized to trap an intermediate state of maturation. In the cryo-EM reconstruction of E16 Fab bound to immature KUNV it was found that the virion had localized distorted density and apparent non-uniform binding of the E16 Fab. Based on this result it was proposed that flaviviruses had imperfect icosahedral symmetry. <br></p><p><br></p> <p> </p> <p>The structural asymmetry of immature and mature flaviviruses was investigated in Chapter 3. Icosahedral symmetry has always been imposed during cryo-EM reconstructions of flaviviruses, as it led to stable convergence of orientations. When reconstructions of immature KUNV and ZIKV were performed without imposing symmetry, the reconstructions showed that the flaviviruses had an eccentric nucleocapsid core, which was positioned closer to the membrane at one pole. At the opposite pole, the glycoprotein and inner leaflet densities were weak and distorted. Furthermore, there were protrusions from the core that contacted the transmembrane helices of the glycoproteins. In the asymmetric reconstruction of mature KUNV, the core was positioned concentric with the glycoprotein shell, in contrast to the immature virion, indicating that maturation alters the interactions between the core and the glycoproteins. The asymmetric reconstructions suggested that there is variable contact between the core and glycoproteins during assembly, which may be due to membrane curvature restrictions in the budding process. </p> <p> </p> <p><br></p><p>In Chapter 4, extracellular vesicles (EVs) that were released during dengue virus (DENV) infection were characterized by mass spectrometry. EVs may play a significant role in flavivirus infection, as they have been shown to transport both viral proteins and infectious RNA. EVs likely represent alternative modes of virus transmission and aid in immune evasion. However, previous studies on EVs are controversial because EVs are potential contaminated during assays by co-purifying virions and other particulates. The identification of EV biomarkers would greatly reduce contamination because biomarkers would enable isolation of pure EVs by affinity purification. Therefore, a strategy was developed to isolate EVs and profile them with proteomics. The four proteins cystatin-A, filamin B, fibrinogen beta chain, and endothelin converting enzyme 1 were found to be statistically enriched in the DENV sample and represent potential EV biomarkers. </p> <p> </p>

Vitamin A is an essential nutrient; it is not synthesized by mammals and therefore must be obtained through the diet. During times of fasting or dietary vitamin A insufficiency, retinol, the alcohol form of the vitamin is released from the liver, its main storage tissue, for circulation in complex with retinol-binding protein 4 (RBP) to provide an adequate supply to peripheral tissues. Stimulated by Retinoic Acid 6 (STRA6), the transmembrane RBP receptor, mediates retinol uptake across blood-tissue barriers such as the retinal pigment epithelium of the eye, the placenta and the choroid plexus of the brain. Our understanding as to how this protein functions has been greatly enhanced by the high-resolution 3D structure of zebrafish STRA6 in complex with calmodulin (CaM) solved by single-particle cryogenic-electron microscopy. However, the nature of the interaction of STRA6 with retinol remains unclear. Here, I present the high-resolution structures of zebrafish and sheep STRA6 reconstituted in nanodisc lipid bilayers in the presence and absence of retinol. The nanodisc reconstitution system has allowed us to study this protein in a close to physiological environment and examine its interaction with the cell membrane and relationship with its ligand, retinol. We also present the structure of sheep STRA6 in complex with human RBP. The structure of the STRA6-RBP complex confirms predictions in the literature as to the nature of the protein-protein interaction needed for retinol transport. Calcium-bound CaM is bound to STRA6 in the RBP-STRA6 structure, consistent with a regulatory role of this calcium binding protein in STRA6-RBP interaction. The analysis of the three states of STRA6 – pre, post and during interaction with retinol – provide unique insights into the mechanism of STRA6-mediated cellular retinol uptake.

Předložená práce se zabývá problematikou slepé filtrace obrazů z transmisního elektronového mikroskopu. V úvodu práce je uveden popis transmisního elektronového mikroskopu. Navazující část popisuje mechanismy interakce elektronů se zkoumaným vzorkem a z toho vyplývající zobrazovací techniky elektronové mikroskopie. Poslední kapitola teoretické části práce zahrnuje popis vybraných metod slepé filtrace obrazu zejména s využitím dekompozice obrazu na charakteristické složky. Taktéž je zde uveden výčet metod pro zhodnocení úspěšnosti filtrace. V praktické části jsou popsány aplikované metody slepé filtrace obrazů a výsledky filtrování. Jednotlivé metody jsou mezi sebou porovnány. Získané výsledky a využitelnost aplikovaných metod jsou zhodnoceny v diskuzi.

abstract: Interconnection methods for IBC photovoltaic (PV) module integration have widely been explored yet a concrete and cost-effective solution has yet to be found. Traditional methods of tabbing and stringing which are still being used today impart increased stress on the cells, not to mention the high temperatures induced during the soldering process as well. In this work and effective and economical interconnection method is demonstrated, by laser welding an embossed aluminum (Al) electrode layer to screen-printed silver (Ag) on the solar cell. Contact resistivity below 1mΩ.cm2 is measured with the proposed design. Cross-sectional analysis of interfaces is conducted via Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDXS) methods. Typical laser weld phenomenon observed involves Al ejection at the entrance of the weld, followed by Al and Ag fusing together mid-way through the weld spot, as revealed by cross-sectional depth analysis. The effects of voltage and lamp intensity are also tested on the welding process. With the range of voltages tested, 240V seems to show the least process variability and the most uniform contact between Al and Ag layers, upon using an Ethylene-Vinyl Acetate (EVA) encapsulant. Two lamp intensities were also explored with a Polyolefin (POE) encapsulant with Al and Ag layers seen welded together as well. Smaller effect sizes at lamp 2 intensity showed better contact. A process variability analysis was conducted to understand the effects of the two different lamps on welds being formed. Lamp 2 showed a bi-modal size distribution with a higher peak intensity, with more pulses coupling into the sample, as compared to lamp 1. / Dissertation/Thesis / Masters Thesis Materials Science and Engineering 2019

abstract: Photocatalytic water splitting over suspended nanoparticles represents a potential solution for achieving CO2-neutral energy generation and storage. To design efficient photocatalysts, a fundamental understanding of the material’s structure, electronic properties, defects, and how these are controlled via synthesis is essential. Both bulk and nanoscale materials characterization, in addition to various performance metrics, can be combined to elucidate functionality at multiple length scales. In this work, two promising visible light harvesting systems are studied in detail: Pt-functionalized graphitic carbon nitrides (g-CNxHys) and TiO2-supported CeO2-x composites. Electron energy-loss spectroscopy (EELS) is used to sense variations in the local concentration of amine moieties (defects believed to facilitate interfacial charge transfer) at the surface of a g-CNxHy flake. Using an aloof-beam configuration, spatial resolution is maximized while minimizing damage thus providing nanoscale vibrational fingerprints similar to infrared absorption spectra. Structural disorder in g-CNxHys is further studied using transmission electron microscopy at low electron fluence rates. In-plane structural fluctuations revealed variations in the local azimuthal orientation of the heptazine building blocks, allowing planar domain sizes to be related to the average polymer chain length. Furthermore, competing factors regulating photocatalytic performance in a series of Pt/g-CNxHys is elucidated. Increased polymer condensation in the g-CNxHy support enhances the rate of charge transfer to reactants owing to higher electronic mobility. However, active site densities are over 3x lower on the most condensed g-CNxHy which ultimately limits its H2 evolution rate (HER). Based on these findings, strategies to improve the cocatalyst configuration on intrinsically active supports are given. In TiO2/CeO2-x photocatalysts, the effect of the support particle size on the bulk/nanoscale properties and photocatalytic performance is investigated. Small anatase supports facilitate highly dispersed CeO2-x species, leading to increased visible light absorption and HERs resulting from a higher density of mixed metal oxide (MMO) interfaces with Ce3+ species. Using monochromated EELS, bandgap states associated with MMO interfaces are detected, revealing electronic transitions from 0.5 eV up to the bulk bandgap onset of anatase. Overall, the electron microscopy/spectroscopy techniques developed and applied herein sheds light onto the relevant defects and limiting processes operating within these photocatalyst systems thus suggesting rational design strategies. / Dissertation/Thesis / Doctoral Dissertation Materials Science and Engineering 2019

abstract: Extended crystal defects often play a critical role in determining semiconductor device performance. This dissertation describes the application of transmission electron microscopy (TEM) and aberration-corrected scanning TEM (AC-STEM) to study defect clusters and the atomic-scale structure of defects in compound semiconductors. An extensive effort was made to identify specific locations of crystal defects in epitaxial CdTe that might contribute to degraded light-conversion efficiency. Electroluminescence (EL) mapping and the creation of surface etch pits through chemical treatment were combined in attempts to identify specific structural defects for subsequent TEM examination. Observations of these specimens revealed only surface etch pits, without any visible indication of extended defects near their base. While chemical etch pits could be helpful for precisely locating extended defects that intersect with the treated surface, this study concluded that surface roughness surrounding etch pits would likely mitigate against their usefulness. Defect locations in GaAs solar-cell devices were identified using combinations of EL, photoluminescence, and Raman scattering, and then studied more closely using TEM. Observations showed that device degradation was invariably associated with a cluster of extended defects, rather than a single defect, as previously assumed. AC-STEM observations revealed that individual defects within each cluster consisted primarily of intrinsic stacking faults terminated by 30° and 90° partial dislocations, although other defect structures were also identified. Lomer dislocations were identified near locations where two lines of strain contrast intersected in a large cluster, and a comparatively shallow cluster, largely constrained to the GaAs emitter layer, contained 60° perfect dislocations associated with localized strain contrast. In another study, misfit dislocations at II-VI/III-V heterovalent interfaces were investigated and characterized using AC-STEM. Misfit strain at ZnTe/GaAs interfaces, which have relatively high lattice mismatch (7.38%), was relieved primarily through Lomer dislocations, while ZnTe/InP interfaces, with only 3.85% lattice mismatch, were relaxed by a mixture of 60° perfect dislocations, 30° partial dislocations, and Lomer dislocations. These results were consistent with the previous findings that misfit strain was relaxed primarily through 60° perfect dislocations that had either dissociated into partial dislocations or interacted to form Lomer dislocations as the amount of misfit strain increased. / Dissertation/Thesis / Doctoral Dissertation Physics 2020

Biofilms of Actinobacillus succinogenes, grown in a biofilm reactor system, were investigated for structure and cell viability, through microscopic visualisation with a confocal scanning laser microscope (CSLM) and a scanning electron microscope (SEM). Biofilms were sampled and visualised at steady state conditions with the broth containing succinic acid titres between 15 and 21 g/L. All sampled biofilm was 6 days old. Six-day-old biofilms of A. succinogenes showed a heterogeneous biofilm architecture composed of cell micro-colony pillars which varied considerably in thickness, area and shape. Microcolony pillars consisted of a densely packed entanglement of sessile cells. Quantitative analysis revealed that the pillars were mostly large, with a mean pillar diameter of 170 m and a mean thickness of 92 m, although pillar diameter and thickness were variable as they ranged from 25 – 500 m and 30 – 300 m, respectively. In the regions close to the substratum surface, pillars were characterised by having defined borders with a network of channels ranging from 40 – 200 m in width separating them. However, towards the middle of the biofilm depth some of the pillars coalesced. For this reason low cross sectional area coverage of biofilm consistently occurred at the bottom portion of the biofilm whilst the highest coverage was in the middle portion of the biofilm. Regarding cell morphology, very large differences were observed. Planktonic cells were rod-shaped, whereas sessile cells expressed an elongated rod morphology and thus were much longer and thinner compared with planktonic cells. Planktonic cells were 1 – 2 m thick and 4 – 5 m long, while sessile cells were 0.5 – 1 m thick and 5 – 100 m long. Long sessile cells resulted in extensive tangling in microcolony pillars, which may have contributed to the structural stability of the pillars. Fibre-like connections of constant diameter were observed between cells, and between the cells and surface. The diameter of these connections was approximately 20 – 30 nm. Viability stains showed that in the bottom portion (from 0 - 20 m above the substratum surface) of the biofilm, most of the cells were dead. However, the portion of covered area attributed to living cells increased past the middle of the biofilm towards the top part of the biofilm. A high percentage of living cells was thus found towards the top part of the biofilm. Overall, 65% (with 2% standard deviation) of the entire biofilm was composed of dead cells. In this way, the results show that operation at high acid conditions comes at a cost of low overall biomass productivity due to decreased active biomass. / Dissertation (MEng)--University of Pretoria, 2017. / Chemical Engineering / MEng / Unrestricted

Dichapetalum cymosum (poison leaf) is a very common problem plant in southern Africa. Fluoroacetic acid, believed to be the poisonous entity in the plant, is produced by the plant, but the micro-organisms associated with this plant may also play a role in the production thereof. A previous study on Burolderia cepacia, an endophyte of D. cymosum showed active metabolism of fluoroacetate by this endophyte. The isolated endophytes from D. cymosum were studied to determine whether they synthesise any fluorinated compounds. It seemed from preliminary results that symbionts might play a role in the synthesis of the poisonous entities in D. cymosum, but further investigation is required. The detection of glandular lesions on the abaxial side of the leaf led to closer examination and the cross sections revealed unusually deformed epidermis cells with adjacent cells containing vacuoles filled with phenolic-like crystals. Transmission electron microscopy (TEM) of the spongy parenchyma cells directly above the glandular lesions indicated the presence of clusters of small, virus-like particles (VLPs) in the chloroplasts. Observations by TEM showed that these VLPs have analogous structures to phytoferritin. Tapura fischeri (leafberry tree) is a tree member of the same family, and it was found to also contain a fluorinated compound. Endophytes were also found in the plant and similar glandular lesions with analogous VLPs were observed at these sites. This might indicate that endophytes have a share in the biosynthesis of the fluorinated compounds found in Dichapetalaceae. Numerous factors ought to be considered in order to fully understand the chemical ecology of the intricate system regarding the endophytes and the possible toxicity of the family Dichapetalaceae. / Dissertation (MSc)--University of Pretoria, 2012. / Plant Science / unrestricted

No description available.