Academic literature on the topic 'Ultrastructure analysis'

Cilia and flagella are dynamic organelles of the eukaryotic cell that undergo cycles of assembly/disassembly, in a manner that is coordinated in time with the cell cycle. Cilia are composed by more than 600 peptides and their turnover occurs at the plus distal end of the axoneme; since these organelles lack the machinery required for protein synthesis, a bidirectional transport process known as the IntraFlagellar Transport (IFT) is required to provide flagellar precursors and remove turnover products. IFT is carried on by macromolecular complexes (the IFT particles) which are arranged in polymers (the IFT trains) in the space between the microtubular doublets and the flagellar membrane. IFT trains operate as platforms for cargoes and are moved bidirectionally by specific molecular motors, kinesin-2 as the anterograde motor and dynein-1b as the retrograde motor. Anterograde and retrograde IFT trains possess distinct architectures but, up to now, a high-resolution 3D-model is available only for the anterograde trains, while much less is known on the ultrastructure of retrograde trains. At the distal tip of the organelle, the anterograde transport (from the cell body up to the ciliary distal end or tip) is converted into the retrograde transport (from the tip back to the cell body). Such a turnaround process is strictly required for the correct functioning of the IFT process. So far, however, very little is known about the morpho-functional organization of the tip district, where IFT turnaround takes place. In particular, nothing is known on the interactions that might occur between IFT proteins and the distal tip structures. This doctoral work has been aimed at contributing new information for the comprehension of the IFT turnaround process in the model organism Chlamydomonas reinhardtii. We started our studies from the observation that thin sections of flat-embedded flagella often show anterograde IFT trains that contact the distal end of the central pair microtubules (CP), suggesting the direct involvement of CP capping structures (terminal plates and the ring above) in the IFT turnaround process. We confirmed the interaction of anterograde trains with the CP distal end by electron-tomographic reconstruction of flat-embedded flagellar tips. This approach revealed that anterograde trains split into three components after having reached the end of the A tubule, with the outer part of the train that remains associated with the membrane, the inner part, closer to the microtubule surface, that continues its travel and bends to contact the CP plates, and an intermediate part that stops before reaching the tip. The latter region was interpreted as the part of the train consisting of inactive dynein-1b, which is known to dissociate from the anterograde train before its activation and recruitment for the retrograde transport. Then, we sought to obtain further information on the ultrastructural organization of the distal CP segment. We were able to identify a ladder-like structure (LLS) which is distinctive of this region, is intercalated between the two CP tubules, and is resistant to the cold treatments used to depolymerize tubulin. In order to confirm the association between IFT anterograde trains and the capping CP structures, whole cells were treated with inhibitors of Ca++-dependent protein kinases before flagellar demembranation and negative staining. These inhibitors block the release of kinesin-2 from the anterograde trains and, consequently, IFT turnaround at the tip. As expected, we observed a massive accumulation of IFT particles around the CP terminus. Successively, we analyzed by immunoelectronmicroscopy the specific distribution of the three protein complexes present within the IFT particles. At this purpose, we carried out a series of immunolabeling experiments on grid-absorbed demembranated cells or on sections of resin-embedded samples, using antibodies directed against subunits of the IFT-A complex (IFT139), and of the two IFT-B subcomplexes IFT-B1 (IFT74 and IFT81) and IFT-B2 (IFT172 and IFT57). Our findings suggest that at the tip the IFT-A complex is closely associated with the membrane. On the contrary, both IFT-B1 and IFT-B2 antibodies labelled the distal CP region, though, interestingly, with distinct spatial distributions. IFT-B2 labeling was restricted to approximately the distal 200 nm-segment of the CP, which contains the LLS, and gold particles were never found more distally, above the terminal plates, while IFT-B1 labeling extend also to the ring. The whole set of immunoelectronmicroscopy data indicates that the IFT-B1 and IFT-B2 subcomplexes differentially interact with the distal CP region and its capping structures, and suggests that the IFT-B1 subcomplex might be a main component of the CP capping structures. Accordingly, in our negatively stained samples the cap was shown to consist of thin elongated elements, frequently with a sort of small knob at their mid region; these elements fit quite well with the available IFT-B 3D model. The possibility that IFT-B1 proteins are involved in the formation of the CP cap was confirmed by the analysis of a series Chlamydomonas mutants with defective IFT, which related the presence of the CP cap to the establishment of a fully cycling IFT process. Our data sustain a model of IFT turnaround in which i) the IFT-A complex turns around quickly, remaining associated with the membrane, ii) IFT-B1 and IFT-B2 follow a more complex pathway, during which they separate and differentially interact with the CP distal segment, iii) IFT-B1 directly contribute to the formation of the CP cap. The LLS component, which is ectopically assembled also in mutant strains devoid of the CP tubules, is likely to act as an anchoring structure for IFT-B2 during IFT turnaround.

The crayfish, Procambarus clarkii, has a multitude of ideal sites in which synaptic transmission may be studied. Its opener muscle, being innervated by a single excitatory neuron is a good model for studying the structure/function of neuromuscular junctions since the preparation is identifiable from animal to animal and the nerve terminals are visible using a vital dye. This allows ease in finding a suitable site to record from in each preparation and offers the ability to relocate it anatomically. Marking a recorded site and rebuilding it through electron microscopy gives good detail of synaptic struture for assesment.In the first of these studies, low output sites known as stems (which lie between varicosities) were used to reduce n (number of release sites) in order to minimize synaptic complexity so individual quantal events could be analyzed by their unique parameters (area, peak, tau, rise time and latency). This was in attempt to uncover specific quantal signatures that could be traced back to the structure of the area recorded. It was found that even at stem regions synaptic structure is still complex having multiple synapses each of which could harbor a number of AZs. This gives insight as to how quantal analysis should be treated. Even low output synapses n must be treated at the AZ level.Synaptic depression was studied at the crayfish extensor muscle. By depressing the phasic neuron and recording from the muscle it appears thatdepression is a presynaptic phenomenon. The use of 5-HT gave insight to vesicular dynamics within the nerve terminal, by delaying depression and increasing maximum EPSP amplitude. TEM of phasic nerve terminals reveals no change in numbers of dock or RRP vesicles. Short term facilitation and vesicular dynamics were studied with the use of 5-HT and a neurotoxin TBOA, which blocks the glutamate transporter. In this study I showed differential mechanisms that control RRP and RP vesicles. By blocking glutamate reuptake, the RRP is depleted as shown by reduced EPSPs, but recovered with 5-HT application. The understanding of vesicle dynamics in any system has relevance for all chemical synapses.

Afin de réduire l’impact écologique des processus industriels, on assiste à un intérêt croissant dans l’industrie pour les fibres végétales. En effet, en plus d’être biodégradables, ces fibres possèdent des propriétés mécaniques capables de concurrencer celles des fibres synthétiques. Cependant, leur usage est limité notamment par la variabilité de leurs propriétés mécaniques. De plus, ces fibres présentent un comportement mécanique non-linéaire qu’il convient d’élucider. Dans ce contexte, cette thèse se propose dans une première partie d’étudier l’influence de la morphologie des faisceaux de fibres de lin sur leur comportement mécanique. Les résultats révèlent une forte hétérogénéité de la morphologie et une dépendance du comportement mécanique par rapport aux paramètres morphologiques comme le vrillage des échantillons, l’état des lamelles mitoyennes ou la section transversale. Dans une deuxième partie, il est proposé une caractérisation par diffraction des rayons X de l’ultrastructure du lin afin d’améliorer la compréhension du comportement mécanique non-linéaire des fibres de lin. Le traitement des mesures de diffraction par la technique d’analyse combinée &lt;&lt; structure/microstructure/texture &gt;&gt; a rendu possible, entre autres, la détermination de la distribution de l’Angle Micro-Fibrillaire et la forme des cristallites de cellulose. Cette méthode a ensuite permis de suivre l’évolution sous traction de l’ultrastructure de la fibre de lin. Nous avons pu ainsi proposer un scénario susceptible d’expliquer le comportement mécanique non-linéaire des fibres de lin<br>In order to reduce the ecological impact of industrial processes, there is a growing interest in the industry for plant fibres. Indeed, in addition to being biodegradable, these fibres have remarkable mechanical properties, making them very competitive with synthetic fibres. However, the use of plant fibres is currently limited, in particular by the variability observed in their mechanical properties. In addition, these fibres have a non-linear mechanical behaviour that needs to be elucidated. In this context, this work proposes in a first part to study the influence of the morphology of flax fibre bundles on their mechanical behaviour. The results reveal a strong heterogeneity in the morphology and a dependence of the tensile mechanical behaviour on morphological parameters such as the twisting of the samples, the state of the middle lamellae or the cross-section. In a second part, an X-ray diffraction characterisation of the flax ultrastructure is proposed to improve the understanding of the non-linear mechanical behaviour of flax fibres. The use of the so-called combined analysis &lt;&lt; structure/microstructure/texture &gt;&gt; approach for the X-ray diffraction data fit allows the determination of the Micro-Fibrillar Angle distribution and the shape of the cellulose crystallites. This method then made it possible to follow the evolution of the ultrastructure of flax fibres under tensile loading. Finally, this allowed us to propose a scenario that could explain the non-linear mechanical behaviour of flax fibres

Several lines of evidence indicate that neuromuscular junction (NMJ) destruction and disassembly is an early phenomenon in the amyotrophic lateral sclerosis (ALS) neurodegenerative disease. Here we analyzed by confocal and electron microscopy the NMJ structure in the diaphragm of Superoxide dismutase (SOD)1 G93A mice at symptom onset and we compared these observation with animals sacrificed at the pathological end stage. In young transgenic mice, which provide a model for familial ALS, the present findings showed marked denervation both in the diaphragm and in the gastrocnemius, which partially spares soleus muscle. At the clinical end stage even the soleus is slight denervated, but less severely than other muscles. In addition, the size of the synaptic vesicle (SV) pool was found reduced and alterations of mitochondria were observed in approximately 40% of the remaining presynaptic terminals. Treatment of SOD1 G93A mice with the anabolic steroid nandrolone during the presymptomatic stage preserved the diaphragm muscle mass and features indicative of synaptic activity, represented by the number of vesicles docked within 200 nm from the presynaptic membrane and area of acetylcholine receptor clusters. Furthermore, structural preservation of mitochondria was documented in presynaptic terminals, but innervation of diaphragm muscle fibers was only slightly increased in nandrolone-treated SOD1-mutant mice. Altogether the results point out and define fine structural alterations of diaphragm NMJs in the murine model of familial ALS at symptom onset, and indicate that nandrolone may prevent or delay structural alterations in NMJ mitochondria and stimulate presynaptic activity but does not prevent muscle denervation in the disease.

Orientador: Marcelo Brocchi<br>Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia<br>Made available in DSpace on 2018-08-23T15:34:06Z (GMT). No. of bitstreams: 1 Lima_BrunadeAraujo_D.pdf: 3794605 bytes, checksum: 178a4bf447e5292f789a4713d5500b38 (MD5) Previous issue date: 2013<br>Resumo: A violaceína é um pigmento violeta produzido por algumas espécies bacterianas de origem ambiental, tais como Chromobacterium violaceum e Janthinobacterium lividum. Esta molécula apresenta várias propriedades biológicas incluindo antibacteriana, antifúngica, antiviral, antiprotozoária e antitumoral, apesar de sua função exata na fisiologia dos micro-organismos que a produz, ainda é desconhecido. No presente trabalho, a atividade antimicrobiana da violaceína produzida comercialmente, o extrato semi purificado e nanopartículas de vanadato de prata foram avaliados contra espécies de bacterianas gram-positivas e gram-negativas. A violaceína exibiu efeito antimicrobiano contra Staphylococcus aureus resistente à meticilina (MRSA) e Enterococcus resistente à vancomicina (VRE), que são micro-organismos frequentemente relacionados com infecções adquiridas em hospitais. Os valores de MIC (concentração inibitória mínima) e MBC (concentração bactericida mínima) da violaceína produzida comercialmente foram de 0,625 ?M e 1,25 ?M respectivamente e, análise de curvas de crescimento e tempo-morte revelaram um efeito antibacteriano durante 12 horas contra MRSA. A microscopia eletrônica de transmissão mostrou os efeitos da violaceína com alterações morfológicas e ultra estruturais, incluindo alterações na parede celular e formação de septos de divisão anormais. Nos resultados obtidos das análises de proteômica e transcriptoma a violaceína afetou a expressão de várias classes funcionais de proteínas e genes em MRSA, incluindo processos biológicos em biossíntese da parede celular e divisão celular que corroboram as alterações ultra estruturais visualizadas. Em conclusão, a violaceína produzida comercialmente demonstrou atividade antimicrobiana para S. aureus MRSA e pela primeira vez, os efeitos da violaceína sobre o metabolismo de S. aureus foram descritos, indicando possíveis alvos e vias metabólicas afetadas por esta droga. No seu conjunto, estes dados indicam a violaceína como uma droga potencial para o tratamento de infecções provocadas por MRSA<br>Abstract: Violacein is a violet pigment produced by some bacterial species of environmental source, such as Chromobacterium violaceum and Janthinobacterium lividum. This molecule has numerous biological properties including antibacterial, antifungal, antiviral, antiprotozoal and antitumor activity, although the exact role in the physiology of producing microorganisms is still unknown. In this study, the antimicrobial activity of violacein produced commercially, semi purified extract and silver vanadate nanoparticles were evaluated against several species of gram-positive and gram-negative bacteria. Violacein exhibited antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), microorganisms that are often related to hospital-acquired infections. MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) values of violacein produced commercially were 1.25 ?M mM and 0.625 ?M respectively, and analysis of growth and time-kill curves showed an antibacterial effect against MRSA for 12 hours. The transmission electron microscopy showed the effects of violacein with morphological and ultra-structural changes, including changes in cell wall formation and abnormal division septum. The results obtained from the analysis of proteomic and transcriptomic revealed that violacein affects the expression of several functional classes of proteins and genes in MRSA, including biological processes in cell wall biosynthesis and cell division, supporting ultra-structural changes. In conclusion, violacein produced commercially demonstrated antimicrobial activity against S. aureus MRSA and the effects on the metabolism of S. aureus have been described, indicating possible targets and pathways affected by this drug. These data indicate violacein as a potential drug for the treatment of infections caused by MRSA<br>Doutorado<br>Microbiologia<br>Doutora em Genética e Biologia Molecular