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A study of proteinases of invasive cells using cryoultramicrotomy and immunogold labelling.Elliott, Edith. January 1993 (has links)
This study forms part of an investigation into the possible relevance of the
lysosomal proteinases, cathepsins B, H, Land D, in cancer cell invasion. In this
study, the main technique adopted was the Tokuyasu "cryo" method, in which the
tissues were fixed, frozen and sectioned and labelled using the relevant antibodies,
which were detected with protein A gold probes.
In order to implement the Tokuyasu technique, it was necessary to rebuild a knife
maker, for the production of adequately sharp glass knives, and to modify a sputter-coater
into a glow-discharger, for rendering carbon-coated grids hydrophilic, to
promote adhesion of hydrated sections.
This study was directed towards human tissues and peptide antibodies were
investigated as a means of avoiding isolation of proteins from scarce human tissue,
and as a means of obtaining antibodies that will target specific regions of proteins of
interest. Peptide antibodies were also considered promising for studies of
proteinase trafficking and as immunoinhibiting agents, potentially useful in cancer
therapy. Various prediction programmes were investigated for their effectiveness
in predicting whether a given peptide sequence will elicit antibodies that will react
with the native protein. Successful prediction would increase the success rate of
peptide antibody production and thus lower the cost.
Leucocytes were studied as a model of an invasive cell, since they are more readily
available than tumour cells and serve the purpose during the development of
methods. In the course of these studies, an optimal protocol for the fixation of
PMNs was developed, involving lateral fixation of cut sections, that should be
useful for future studies on these cells. Elastase and cathepsins D and G were found
on the surface of activated PMNs and could thus play a role in the invasive
properties of these cells.
Studies on MCF-10A "normal" breast epithelial cells and their ras-transformed
Neo-T counterparts revealed that upon transformation, lysosomes shift from a
perinuclear position, to a more peripheral position. None of the cathepsins studied
was found on the cell surface of either the normal or ras-transfected cells,
suggesting that surface distribution of these enzymes may not be a requirement for
invasiveness. These studies suggest that immunocytochemical investigation of
cells, in the process of invading through a barrier membrane, might be profitable in
elucidating the role of proteinases in invasive cancer. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1993.
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Morphological and physiological studies of the carbon concentrating mechanism in Chlamydomonas reinhardtiiChan, Kher Xing January 2019 (has links)
Chlamydomonas reinhardtii possesses a single-cell-based CO2-concentrating mechanism (CCM). The CCM is an important element of algal photosynthesis, metabolism, growth and biomass production, which works by increasing the concentration of inorganic carbon (Ci) in the pyrenoid, a dense RuBisCO-packed structure within the chloroplast. This suppresses RuBisCO oxygenase activity and associated photorespiration. The enhanced efficiency of CO2 assimilation in the pyrenoid via CCM had been modelled theoretically as a requirement for successful CCM in higher plant systems. The ultimate aim of my research is to understand the biogenesis of the pyrenoid using a set of CCM mutants with pyrenoidal defects. Immunofluorescence methods and spot growth tests under different CO2 concentrations were performed on mutants with CCM defects generated by an insertional mutagenesis screen. Morphological and physiological characterisation of these mutants revealed differences in the pyrenoid morphology, the ability for RuBisCO to aggregate into the pyrenoid and the formation of thylakoidal tubule network associated with the pyrenoid. The thylakoid tubule network may be linked to the transport of inorganic carbon into the pyrenoid as part of the CCM. Further characterisation of one of the mutants gave rise to the hypothesis that the gene of interest, Cre11.g467712 (SAGA), is a multi-functional anchor protein related to the structural formation of the pyrenoid and may be another essential component of the pyrenoid.
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Caractérisation, quantification et isolation de vésicules extracellulaires du plasma sanguin à l’aide de nanoparticules d’or ou magnétiques conjuguées à des protéines / Phenotyping, quantification and isolation of extracellular vesicles from blood plasma using gold or magnetic nanoparticles conjugated to proteinsLinares, Romain 02 December 2016 (has links)
Les vésicules extracellulaires (VEs) sont des vésicules membranaires de taille majoritairement submicrométrique présentes dans les fluides biologiques et émises par les cellules en réponse à divers stimuli. Les VEs sont impliquées dans de nombreux phénomènes physiologiques mais également dans des pathologies telles que cancers ou maladies cardiovasculaires. Elles pourraient donc être utilisées comme biomarqueurs de ces pathologies. Bien que les VEs soient aujourd’hui largement étudiées, nos connaissances sur le sujet demeurent limitées. Ceci est principalement dû aux difficultés de caractérisation des VEs et à l’absence de standardisation de leurs méthodes d’étude et d’isolation. La première partie de mon travail de thèse a porté sur le développement d’une méthode de thiolation de protéines. Des anticorps ont été modifiés pour exposer des thiols et ont été conjugués à des nanoparticules d’or fonctionnalisées par des maléimides. Le couplage des anticorps thiolés aux nanoparticules d’or a été étudié de manière quantitative et des conditions de conjugaison optimales ont été déterminées par des approches biochimiques. La seconde partie de ce travail a concerné la caractérisation des VEs du plasma sanguin de sujets sains par microscopie électronique à transmission (MET). La morphologie, la taille et le phénotype des VEs ont été déterminés par cryo- MET combinée au marquage par des nanoparticules d’or conjuguées à des protéines. La quantification objective des VEs du plasma sanguin a été réalisée à l’aide d’une méthode originale de MET basée sur la sédimentation de VEs sur grille de MET. La troisième partie de cette étude a consisté à mettre au point une méthode d’isolation de VEs à l’aide de particules magnétiques conjuguées à de l’AnxA5. Des conditions permettant d’extraire la totalité des VEs exposant la phosphatidylsérine contenues dans un plasma sanguin ont été déterminées par cytométrie en flux (CF). Ce travail a permis d’apporter une caractérisation détaillée des VEs du plasma sanguin du sujet sain et peut servir de référence pour des études ultérieures concernant les VEs contenues dans des plasmas ou autres liquides biologiques pathologiques. / Extracellular vesicles (EVs) are submicrometric membrane vesicles found in body fluids and produced by cells in response to various stimuli. EVs are involved in numerous physiological processes but also in pathologies as cancers or cardiovascular diseases. Even if EVs are largely studied, our knowledge about them remains limited. This is mainly caused by the difficulties to characterize EVs and by the lack of standardized methods allowing their characterization. The first part of my PhD work focused on the development and optimization of a protein thiolation method. Antibodies modified to expose few thiols were conjugated to gold nanoparticles functionalized with maleimides. The binding of thiolated antibodies to gold nanoparticles was quantitatively studied and optimal conjugation conditions were determined using biochemical methods. The second part of my PhD work concerned the characterization of blood plasma EVs from healthy subjects using transmission electron microscopy (TEM). EVs morphology, size and phenotype were determined by cryo-TEM combined with labelling with protein-conjugated gold nanoparticles. The near-absolute quantification of blood plasma EVs was achieved using an original TEM method based on the direct sedimentation of EVs onto TEM grids. The third part of this study consisted in developing an EV isolation method using AnxA5-conjugated magnetic particles. Conditions allowing total extraction of blood plasma phosphatidylserine-exposing EVs were determined using flow cytometry (FC). This study presents a detailed characterization of blood plasma EVs from healthy subjects and can serve as a reference for future studies on EVs contained in pathological plasmas or other body fluids.
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