1,721,015 research outputs found
STUDY OF α-SYNUCLEIN AGGREGATION IN A HUMANIZED YEAST MODEL
Get PDFParkinson’s Disorder (PD) is one of the major age-related neurodegenerative disorders that mainly affects the motor system, leading to the loss of dopaminergic neurons. Although PD is a progressive neurodegenerative disorder that primarily affects individuals over the age of 60; mutations of genes associated with hereditary forms of early-onset PD have been identified. This project focuses on the SNCA gene that encodes α-synuclein (α-syn), the main component of Lewy bodies which are found in the substantia nigra pars compacta of PD patients. Missense mutations in SNCA (also known as PARK1) were the first to be identified in familial cases of PD. Furthermore, duplications and triplications of SNCA have a gene dosage effect on the severity of the disorder. Currently, there is no cure for PD, but treatments that reduce the symptoms and improve the patient’s quality of life are available. Levodopa is the first-line drug in the treatment of the disorder and the more recent therapy of deep brain stimulation (DBS) effectively improves the patient’s movement. This doctoral project aimed to investigate the mechanisms underlying a-syn toxicity by studying the protein in yeast, a simple and comprehensive model organism. The resulting findings define molecular pathways that in the future may be targeted for the treatment of synucleinopathies.
This work started with the observation that cobalt, nickel and deferoxamine can rescue the toxicity induced by α-syn aggregation in yeast cells. These compounds were identified as promising candidates in a phenotypic high-throughput screening of an FDA-approved drug library on yeast engineered to overexpress human α-syn fused to GFP. After confirming that the three highlighted compounds do not directly decrease the expression level of the human protein, we concluded that the observed protective effect must involve a more complex molecular mechanism. To uncover this mechanism, we performed different analyses. Fluorescence microscopy revealed that treatment with Cobalt, Nickel, and Deferoxamine significantly reduced the number of α-syn aggregates in yeast cells. The formation and clearance of the inclusions were quantitatively monitored by time-lapse imagining in cells grown in a microfluidic system. The three treatments reduced the number and size of aggregates through different kinetics. Cobalt, in particular, eliminated all aggregates within the first 6 hours and prevented the formation of new ones.
We also observed a rescue effect by increasing the oxygenation level of the media. To assess the importance of oxygen, we cultivated the cells in different formats with decreased oxygenation until we reached complete anoxia. We were able to observe the toxic phenotype only when the cells were grown under hypoxia conditions.
To identify the key mechanism involved, we performed transcriptomic profiling coupled with pathway enrichment analysis on the strains without and with Cobalt and Deferoxamine treatment. Our analyses showed significant changes in fatty acid metabolism, ergosterol homeostasis, iron metabolism, and, more unexpectedly, in the thiamine biosynthesis pathway. The importance of the thiamine biosynthetic process was further investigated by generating a-syn strains overexpressing the enzymes THI4, THI5, THI11, THI12 and THI13. A moderate growth rescue effect was observed for some of these overexpression strains.
Following a multi-omics approach, we conducted metabolomic and lipidomic profiling to assess the impact of a-syn expression on the cell metabolic processes and on the membrane composition with a focus on phospholipids, which were also quantified through 31P Nuclear Magnetic Resonance (31P-NMR). We found that α-syn expression decreases the levels of TCA cycle and one-carbon metabolism intermediates and alters the phospholipid profile. In particular, we noticed an increased level of intermediates involved in phosphatidylinositol biosynthesis in the strain that overexpresses α-syn. This result supports the recent literature on the initiator role of Phosphatidylinositol 3-phosphate (PIP3) in the aggregation of α-syn. Interestingly, Cobalt treatment inverted the observed trend, reducing the levels of phosphoinositide.
Our study highlights that hypoxia triggers a-syn aggregation and toxicity, suggesting that oxygen availability plays an important role in neurodegenerative processes. Furthermore, pathological α-syn expression compromises central metabolic processes, potentially altering membrane lipid composition and further promoting protein aggregation in a self-perpetuating cycle. Small molecules able to modulate the hypoxia response, most likely at the level of lipid and iron metabolism, can protect against α-syn toxicity. The results offer important insights into the mechanisms of α-syn toxicity and potential therapeutic interventions for neurodegenerative diseases involving protein aggregation.STUDYOF ¸-SYNUCLEIN AGGREGATIONINAHUMANIZED YEAST MODE
Generation and phenotyping of NAD(P)HX repair deficient zebrafish and iPSC-derived microglia-like cells
No full textPEBEL (progressive early onset encephalopathy with brain edema and/or leukoencephalopathy) disorder
is a severe infantile neurometabolic disorder that falls within the broader spectrum of rare inherited
diseases known as Inborn Errors of Metabolism. The underlying cause of PEBEL is a genetic deficiency in
the NAD(P)HX metabolite repair system. This system is responsible for correcting hydration damage of
the central cofactors NADH and NADPH, which leads to the formation of non-canonical and inactive
metabolites called NAD(P)HX.
The NAD(P)HX repair system comprises two key enzymes: S-NAD(P)HX dehydratase, also referred to as
NAXD, and NAD(P)HX epimerase, known as NAXE. Mutations in either NAXD or NAXE result in the onset
of PEBEL during the early years of life, and in most documented cases, it has so far ledto premature death.
The appearance of disease symptoms is typically triggered by febrile incidents and/or viral/bacterial
infections. In a few recently published trials, the treatment of PEBEL patients with high doses of Vitamin
B3, a precursor of NAD+, was very efficient in treating the severe skin lesions and exerted a stabilizing
effect of the neurological condition of patients. However, the exact molecular mechanisms underlying the
disorder remain unclear. It remains yet to be establishedwhether the depletion of normal cofactors, the
accumulation of non-canonical metabolites, or a combination of both factors or even additional
perturbations play most critical roles in disease development. Similarly, the reason for the immunerelated trigger-dependent onset of the disease symptoms remains fully unclear.
To shed light on these aspects, in this study, we aimed to establish zebrafish models of NAXD and NAXE
deficiency using the CRISPR/Cas9 technology. Through gross and molecular phenotyping of these
zebrafish models, we observed that naxe mutants exhibit a mild immune deficiency that does not
apparently impact their behavior or survival. On the other hand, naxd mutants display a severe
phenotype, with readily detectable differences in locomotion behavior and a significant decrease in
survival rate.
To complement the zebrafish model work and study specifically the impact of NAXD deficiency on the
immune system, we used patient-derived and genetically modified induced pluripotent stem cells (iPSCs)
to generate microglia-like cells(iMGLs) lacking functional NAXD. Patient-derived microglia-like cells with
NAXD deficiency demonstrated main hallmarks of this cell type showing that the methodology can be
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used to generate a suitable disease model. NADHX accumulation, the main molecular characteristic of
NAXD disease, could also be shown to occur in the NAXD deficient iMGLs. Significant differences between
NAXD mutant and isogenic control iMGLs could be measured for several microglia specific functions such
as phagocytic activity, ramification morphology, and expression of inflammation responsive genes. For
some of these parameters, conflicting results were obtained in NAXD patient-derived and knockout
iMGLs, which calls for repeating this type of phenotyping in a larger collection of cell lines with different
genetic backgrounds, to identify the traits specifically affected by mutations in the NAXD gene.
The original findings in this thesis provide initial insights into the effect of NAXD and NAXE deficiency in
developmental processes and their connection with the immune system in the context of PEBEL disorder.
Longitudinal studies in our cell and whole-organism models, guided by our findings here, will be required
to understand the causal chain of events leading from a failure to repair damaged NAD(P)H to an impaired
immune function and an eventually fatal neurological decline
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Millifluidic culture improves human midbrain organoid vitality and differentiation
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Mechanism and regulation of the formation of D-glucuronate, the precursor of vitamin C, in rat liver
No full textLe L-ascorbate, un antioxydant et cofacteur enzymatique important chez les animaux et les plantes, est synthétisé par une grande majorité de vertébrés et par toutes les espèces de plantes analysées jusqu'à ce jour. L'enzyme catalysant l'étape finale de la voie de biosynthèse de l'ascorbate est toutefois absente chez les primates (y compris les humains) et le cobaye, et pour eux ce composé est donc devenu une vitamine (vitamine C). Une des premières étapes de la voie de biosynthèse consiste en la réduction du D-glucuronate, le précurseur de la vitamine C chez les animaux, en L-gulonate. Ce dernier peut être converti en ascorbate par lactonisation et oxydation, mais peut également entrer dans la voie des pentoses dans laquelle il est successivement oxydé et décarboxylé en L-xylulose. La voie des pentoses est opérationnelle chez tous les mammifères et son fonctionnement chez l'homme est démontré par le fait qu'une déficience en L-xylulose réductase mène à une excrétion urinaire anormalement élevée de L-xylulose (pentosurie). Il est connu depuis longtemps que l'administration d'une série de médicaments tels que l'aminopyrine, le barbital et la chlorétone, augmente la formation de vitamine C chez les animaux et l'excrétion de L-xylulose chez les sujets pentosuriques. L'objectif de cette thèse était d'élucider le mécanisme à la base de ces effets stimulateurs. L'observation que certains xénobiotiques stimulent à la fois l'excrétion de vitamine C et de L-xylulose indique qu'ils augmentent la formation d'un précurseur commun des voies métaboliques impliquées. Ceci nous a amenés à mettre au point une méthode de dosage spectrophotométrique pour le D-glucuronate basée sur l'utilisation de deux enzymes bactériennes, la mannonate déshydrogénase et l'uronate isomérase. Ces enzymes ont été surepxrimées chez E. coli, purifiées à quasi-homogénéité et partiellement caractérisées. Nous avons observé que l'uronate isomérase est inhibée par l'EDTA et stimulée par le Zn2+, le Co2+ et le Mn2+. La méthode de dosage enzymatique développée est spécifique et sensible, et peut également être utilisée pour mesurer les concentrations de ?-glucuronides et de glucuronate 1-phosphate. A l'aide de ce dosage, nous avons pu montrer que certains agents (par exemple l'aminopyrine, l'antipyrine et la chlorétone) connus pour stimuler la formation de vitamine C in vivo, mais également d'autres composés (métyrapone, proadifen, clotrimazole) augmentent de plusieurs fois, et en moins de 10 minutes, la formation de glucuronate dans des hépatocytes isolés de rat. Cet effet était considérablement amplifié en présence de sorbinil, un inhibiteur de la glucuronate réductase. L'augmentation de la formation de glucuronate était accompagnée par une augmentation de la formation de vitamine C et par une diminution d'environ deux fois de la concentration en UDP-glucuronate. Les agents stimulateurs ne donnaient généralement pas lieu à la formation de quantités détectables de ?-glucuronides et n'avaient pas d'effet sur la concentration de glutathion. D'un autre côté, le résorcinol, qui est un bon substrat de la glucuronoconjugaison, et des agents diminuant la concentration de glutathion (diamide, L-buthionine sulfoximine) ne stimulent pas la formation de glucuronate. Ces résultats indiquent que l'effet stimulateur des xénobiotiques sur la formation de vitamine C est médié par une augmentation rapide de la conversion d'UDP-glucuronate en glucuronate libre, n'impliquant apparemment pas un cycle de glucuronoconjugaison-déconjugaison ou une déplétion en glutathion. Nous avons ensuite voulu reproduire les effets de l'aminopyrine, de la métyrapone et de la chlorétone sur la formation de glucuronate dans un système acellulaire. Les trois composés stimulent la formation de glucuronate à partir d'UDP-glucuronate dans des extraits de foie à condition que ceux-ci soient enrichis en ATP-Mg. La formation et l'hydrolyse du glucuronate 1-phosphate ne sont cependant pas affectées par la métyrapone et les autres agents. Ceci indique que, contrairement à ce qui est généralement admis, le glucuronate 1-phosphate n'est pas un intermédiaire dans la formation de glucuronate à partir d'UDP-glucuronate et suggère que cette réaction consiste en une hydrolyse directe de l'UDP-glucuronate en UDP et glucuronate par une "UDP-glucuronidase". Cette activité enzymatique possède plusieurs propriétés similaires à celle catalysée par les UDP-glucuronosyltransférases (association avec la fraction microsomiale, stimulation par l'UDP-N-acétylglucosamine, sensibilité aux détergents) et l'observation qu'elle est inhibée par des substrats de la glucuronoconjugaison ...L-Ascorbate, an important antioxidant and enzyme cofactor in animals and plants, is synthesized by a great majority of vertebrates and by all plant species examined so far. Primates, including humans, and guinea pigs do not form ascorbate because they lack the enzyme catalyzing the final step of the ascorbate biosynthesis pathway, and for them ascorbate has thus become a vitamin (vitamin C). An early step of the biosynthetic pathway consists of the reduction of D-glucuronate, the precursor of vitamin C in animals, to L-gulonate. The latter can be converted to ascorbate through lactonization and oxidation, but can also enter the pentose pathway by which it is successively oxidized and decarboxylated to L-xylulose. The pentose pathway is operational in all mammals and its occurrence in man is demonstrated by the fact that a deficiency in L-xylulose reductase leads to urinary excretion of abnormal amounts of L-xylulose (pentosuria). It has been known for a long time that the administration of a series of drugs including aminopyrine, barbital and chloretone, increases the formation of vitamin C in animals and the excretion of L-xylulose in pentosuric subjects. The aim of this thesis was to elucidate the mechanism underlying the stimulatory effects observed. The finding that some xenobiotics stimulate the excretion of both vitamin C and L-xylulose indicates that they increase the formation of a common precursor of the metabolic pathways involved. This prompted us to set up a spectrophotometric assay for D-glucuronate based on the use of two bacterial enzymes, mannonate dehydrogenase and uronate isomerase. These enzymes have been overexpressed in E. coli, purified to near-homogeneity and partially characterized. Uronate isomerase was found to be inhibited by EDTA and stimulated by Zn2+, Co2+ and Mn2+. The enzymatic assay developed is specific and sensitive, and can also be used to determine the concentration of ?-glucuronides and glucuronate 1-phosphate. Using this assay, we showed that agents (e.g. aminopyrine, antipyrine and chloretone) known to stimulate vitamin C formation in vivo, but also some other compounds (metyrapone, proadifen, clotrimazole) induced in less than 10 minutes a several-fold increase in the formation of glucuronate in isolated rat hepatocytes. This effect was considerably amplified by the presence of sorbinil, an inhibitor of glucuronate reductase. The increase in glucuronate formation was accompanied by an increase in the formation of vitamin C and an approximate 2-fold decrease in the concentration of UDP-glucuronate. The stimulating compounds did not generally give rise to the formation of detectable amounts of ?-glucuronides and exerted no effect on glutathione levels. On the other hand, resorcinol, a good substrate of glucuronidation, and glutathione-depleting agents (diamide, L-buthionine sulfoximine) did not stimulate the formation of glucuronate. These observations indicated that the stimulatory effect of some xenobiotics on vitamin C formation is mediated by a rapid increase in the conversion of UDP-glucuronate to free glucuronate, which does apparently not involve a glucuronidation-deglucuronidation cycle or glutathione depletion. We then wanted to reproduce the effects of aminopyrine, metyrapone and chloretone on the formation of glucuronate in a cell-free system. The three compounds stimulated glucuronate formation from UDP-glucuronate in liver extracts provided these were enriched with ATP-Mg. The formation and hydrolysis of glucuronate 1-phosphate were, however, unaffected by metyrapone and the other agents. This indicated that, unlike as generally believed, glucuronate 1-phosphate is not an intermediate in the formation of glucuronate from UDP-glucuronate and suggested that this reaction consists of direct hydrolysis of UDP-glucuronate to UDP and glucuronate by a UDP-glucuronidase. This enzymatic activity shared several properties with that catalyzed by UDP-glucuronosyltransferases (association with the microsomal fraction, stimulation by UDP-N-acetylglucosamine, sensitivity to detergents) and the observation that it was inhibited by substrates of glucuronidation indicated that it could in fact be contributed by one or several isoform(s) of the UDP-glucuronosyltransferase superfamily. Inhibitors of ?-glucuronidase and esterases had no effect on glucuronate formation, further arguing against involvement of a glucuronidation-deglucuronidation cycle. The stimulatory effect of metyrapone and other agents was lost in washed microsomes, but could be restored by adding a heated liver high-speed supernatant or CoASH. In conclusion, our results indicate that the increase in vitamin C formation in animals and of L-xylulose excretion in pentosuric subjects induced by several xenobiotics is mediated by a rapid stimulatory effect of these agents on UDP-glucuronidase, an enzyme closely related to UDP-glucuronosyltransferases. The presence of ATP-Mg and CoASH, which together inhibit UDP-glucuronidase activity, is necessary to observe the stimulatory effect of xenobiotics in liver microsomes.Thèse de doctorat en sciences biomédicales (biochimie et biologie cellulaire) (BCHM)-- UCL, 200
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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