Investigación de defectos genéticos en el sistema de la fosforilasa hepática en pacientes argentinos. Definición nosológica mediante una estrategia de análisis molecular. Investigation of genetic defects in the hepatic phosphorylase system in Argentinean patients. Nosological definition by molecular analysis strategy.

Las Enfermedades de Atesoramiento de Glucógeno (EAGs) también llamadas Glucogenosis comprenden un grupo de entidades causadas por una deficiencia enzimática específica relacionada con la vía de síntesis o degradación de esta macromolécula. La heterogeneidad fenotípica de los pacientes afectados dificulta la identificación de las diferentes variantes de EAG y por ende la correcta definición nosológica. En el Centro de Estudio de las Metabolopatías Congénitas, CEMECO, se fueron definiendo los diferentes tipos de Glucogenosis a través de una estrategia multidisciplinaria que integra distintos niveles de investigación clínica y complementaria, laboratorio metabólico especializado, enzimático, histomorfológico y de análisis molecular. Sin embargo, en algunos enfermos, entre los que se encuentran aquellos con defectos en el sistema de la fosforilasa hepática (EAG-VI y EAG-IX), la exacta definición nosológica aún no está resulta. La EAG-VI se refiere a un defecto en la fosforilasa hepática, enzima codificada por el gen PYGL, mientras que la EAG-IX es causada por un defecto genético en una de las subunidades de la fosforilasa b quinasa hepática codicadas por los genes PHKA2, PHKB y PHKG2, respectivamente. El objetivo del presente trabajo es propender a la definición nosológica de pacientes con defectos en el sistema de la fosforilasa mediante una estrategia de análisis molecular investigando los genes PYGL, PHKA2, PHKB y PHKG2. Los pacientes incluidos en este estudio deberán ser compatibles de padecer una EAG-VI o EAG-IX sobre la base de síntomas clínicos y hallazgos bioquímicos. La metodología incluirá la determinación de la enzima fosforilasa b quinasa en glóbulos rojos y dentro del análisis molecular la extracción de DNA genómico a partir de sangre entera para la amplificación por PCR de los exones más las uniones exon/intron de los genes PHKG2 y PYGL y la extracción de RNA total y obtención de cDNA para posterior amplificación de los cDNA PHKA2 y PHKB. Todos los fragmentos amplificados serán sometidos a análisis de secuencia de nucleótidos. Resultados esperados. Este trabajo, primero en Argentina, permitirá establecer las bases moleculares de los defectos del sistema de la fosforilasa hepática (EAG-VI y EAG-IX). El poder lograr este nivel de investigación traerá aparejado, una oferta integrativa en el vasto capítulo de las glucogenosis hepáticas, con extraordinaria significación en la práctica asistencial para el manejo, pronóstico y correspondiente asesoramiento genético. Hepatic glycogen storage diseases (GSDs) are a group of disorders produced by a deficiency in a specific protein involved in the metabolism of glycogen causing different types of GSDs. Phenotypic heterogeneity of affected patients difficult to identify the different GSD variants and therefore the correct definition of the disease. In the “Centro de Estudio de las Metabolopatías Congénitas”, CEMECO, were defined the different GSD types by a protocol which included complex gradual levels of clinical, biochemical, enzymatic and morphological investigation. However, in some patients, like those one with defects in the hepatic phosphorylase system (GSD-VI and GSD-IX) the exact definition of the disease has not yet been resolved. The GSD-VI is produced by a defect in the PYGL gen that encode the liver phosphorylase, while the GSD-IX is caused by a genetic defect in one of the Phosphorylase b kinase subunits, encoded by the PHKA2, PHKB and PHKG2 genes, respectively. The aim of the present study is to define the phosphorylase system defects in argentinian patients through a molecular strategy that involve the investigation of PYGL, PHKA2, PHKB and PHKG2 genes. Patients included in the present study must be compatible with a GSD-VI or GSD-IX on the bases of clinical symptoms and biochemical findings. The phosphorylase b kinase activity will be assay on in blood red cells. The molecular study will include genomic DNA extraction for the amplification of PHKG2 and PYGL genes and the total RNA extraction for amplification of the PHKA2 and PHKB cDNA by PCR. All PCR-amplified fragments will be subjected to direct nucleotide sequencing. This work, first in Argentina, will make possible to establish the molecular basis of the defects on the hepatic phosphorylase system (GSD-VI and GSD IX). To achieve this level of research will entail advance in the study of the hepatic glycogen storage disease, with extraordinary significance in the treatment, prognosis and the genetic counselling.

Convex and toric geometry to analyze complex dynamics in chemical reaction systems

Convex cone, toric variety, graph theory, electrochemical catalysis, oxidation of formic acid, feedback-loopsbifurcations, enzymatic catalysis, Peroxidase reaction, Shil'nikov chaos

Regenerative action of the wall on the heat transfer for directly and indirectly heated rotary kilns

Rotary kilns, Regenerative wall, heat transfer, directly fired, indirectly fired

Analysis of complex microwave structures using suitable computational electromagnetic techniques

Complex Microwave Structures Wake Field Computatation PETRA III Generalized Multipole Technique Antenna Antennen Wakefelder Berechnung

Ricci-flat complex geometry and its applications

Magdeburg, Univ., Fak. für Mathematik, Habil.-Schr., 2010

Dissecando o GEN

In thee present paper the classical concept of the corpuscular gene is dissected out in order to show the inconsistency of some genetical and cytological explanations based on it. The author begins by asking how do the genes perform their specific functions. Genetists say that colour in plants is sometimes due to the presence in the cytoplam of epidermal cells of an organic complex belonging to the anthocyanins and that this complex is produced by genes. The author then asks how can a gene produce an anthocyanin ? In accordance to Haldane's view the first product of a gene may be a free copy of the gene itself which is abandoned to the nucleus and then to the cytoplasm where it enters into reaction with other gene products. If, thus, the different substances which react in the cell for preparing the characters of the organism are copies of the genes then the chromosome must be very extravagant a thing : chain of the most diverse and heterogeneous substances (the genes) like agglutinins, precipitins, antibodies, hormones, erzyms, coenzyms, proteins, hydrocarbons, acids, bases, salts, water soluble and insoluble substances ! It would be very extrange that so a lot of chemical genes should not react with each other. remaining on the contrary, indefinitely the same in spite of the possibility of approaching and touching due to the stato of extreme distension of the chromosomes mouving within the fluid medium of the resting nucleus. If a given medium becomes acid in virtue of the presence of a free copy of an acid gene, then gene and character must be essentially the same thing and the difference between genotype and phenotype disappears, epigenesis gives up its place to preformation, and genetics goes back to its most remote beginnings. The author discusses the complete lack of arguments in support of the view that genes are corpuscular entities. To show the emharracing situation of the genetist who defends the idea of corpuscular genes, Dobzhansky's (1944) assertions that "Discrete entities like genes may be integrated into systems, the chromosomes, functioning as such. The existence of organs and tissues does not preclude their cellular organization" are discussed. In the opinion of the present writer, affirmations as such abrogate one of the most important characteristics of the genes, that is, their functional independence. Indeed, if the genes are independent, each one being capable of passing through mutational alterations or separating from its neighbours without changing them as Dobzhansky says, then the chromosome, genetically speaking, does not constitute a system. If on the other hand, theh chromosome be really a system it will suffer, as such, the influence of the alteration or suppression of the elements integrating it, and in this case the genes cannot be independent. We have therefore to decide : either the chromosome is. a system and th genes are not independent, or the genes are independent and the chromosome is not a syntem. What cannot surely exist is a system (the chromosome) formed by independent organs (the genes), as Dobzhansky admits. The parallel made by Dobzhansky between chromosomes and tissues seems to the author to be inadequate because we cannot compare heterogeneous things like a chromosome considered as a system made up by different organs (the genes), with a tissue formed, as we know, by the same organs (the cells) represented many times. The writer considers the chromosome as a true system and therefore gives no credit to the genes as independent elements. Genetists explain position effects in the following way : The products elaborated by the genes react with each other or with substances previously formed in the cell by the action of other gene products. Supposing that of two neighbouring genes A and B, the former reacts with a certain substance of the cellular medium (X) giving a product C which will suffer the action, of the latter (B). it follows that if the gene changes its position to a place far apart from A, the product it elaborates will spend more time for entering into contact with the substance C resulting from the action of A upon X, whose concentration is greater in the proximities of A. In this condition another gene produtc may anticipate the product of B in reacting with C, the normal course of reactions being altered from this time up. Let we see how many incongruencies and contradictions exist in such an explanation. Firstly, it has been established by genetists that the reaction due.to gene activities are specific and develop in a definite order, so that, each reaction prepares the medium for the following. Therefore, if the medium C resulting from the action of A upon x is the specific medium for the activity of B, it follows that no other gene, in consequence of its specificity, can work in this medium. It is only after the interference of B, changing the medium, that a new gene may enter into action. Since the genotype has not been modified by the change of the place of the gene, it is evident that the unique result we have to attend is a little delay without seious consequence in the beginning of the reaction of the product of B With its specific substratum C. This delay would be largely compensated by a greater amount of the substance C which the product of B should found already prepared. Moreover, the explanation did not take into account the fact that the genes work in the resting nucleus and that in this stage the chromosomes, very long and thin, form a network plunged into the nuclear sap. in which they are surely not still, changing from cell to cell and In the same cell from time to time, the distance separating any two genes of the same chromosome or of different ones. The idea that the genes may react directly with each other and not by means of their products, would lead to the concept of Goidschmidt and Piza, in accordance to which the chromosomes function as wholes. Really, if a gene B, accustomed to work between A and C (as for instance in the chromosome ABCDEF), passes to function differently only because an inversion has transferred it to the neighbourhood of F (as in AEDOBF), the gene F must equally be changed since we cannot almH that, of two reacting genes, only one is modified The genes E and A will be altered in the same way due to the change of place-of the former. Assuming that any modification in a gene causes a compensatory modification in its neighbour in order to re-establich the equilibrium of the reactions, we conclude that all the genes are modified in consequence of an inversion. The same would happen by mutations. The transformation of B into B' would changeA and C into A' and C respectively. The latter, reacting withD would transform it into D' and soon the whole chromosome would be modified. A localized change would therefore transform a primitive whole T into a new one T', as Piza pretends. The attraction point-to-point by the chromosomes is denied by the nresent writer. Arguments and facts favouring the view that chromosomes attract one another as wholes are presented. A fact which in the opinion of the author compromises sereously the idea of specific attraction gene-to-gene is found inthe behavior of the mutated gene. As we know, in homozygosis, the spme gene is represented twice in corresponding loci of the chromosomes. A mutation in one of them, sometimes so strong that it is capable of changing one sex into the opposite one or even killing the individual, has, notwithstading that, no effect on the previously existing mutual attraction of the corresponding loci. It seems reasonable to conclude that, if the genes A and A attract one another specifically, the attraction will disappear in consequence of the mutation. But, as in heterozygosis the genes continue to attract in the same way as before, it follows that the attraction is not specific and therefore does not be a gene attribute. Since homologous genes attract one another whatever their constitution, how do we understand the lack cf attraction between non homologous genes or between the genes of the same chromosome ? Cnromosome pairing is considered as being submitted to the same principles which govern gametes copulation or conjugation of Ciliata. Modern researches on the mating types of Ciliata offer a solid ground for such an intepretation. Chromosomes conjugate like Ciliata of the same variety, but of different mating types. In a cell there are n different sorts of chromosomes comparable to the varieties of Ciliata of the same species which do not mate. Of each sort there are in the cell only two chromosomes belonging to different mating types (homologous chromosomes). The chromosomes which will conjugate (belonging to the same "variety" but to different "mating types") produce a gamone-like substance that promotes their union, being without action upon the other chromosomes. In this simple way a single substance brings forth the same result that in the case of point-to-point attraction would be reached through the cooperation of as many different substances as the genes present in the chromosome. The chromosomes like the Ciliata, divide many times before they conjugate. (Gonial chromosomes) Like the Ciliata, when they reach maturity, they copulate. (Cyte chromosomes). Again, like the Ciliata which aggregate into clumps before mating, the chrorrasrmes join together in one side of the nucleus before pairing. (.Synizesis). Like the Ciliata which come out from the clumps paired two by two, the chromosomes leave the synizesis knot also in pairs. (Pachytene) The chromosomes, like the Ciliata, begin pairing at any part of their body. After some time the latter adjust their mouths, the former their kinetochores. During conjugation the Ciliata as well as the chromosomes exchange parts. Finally, the ones as the others separate to initiate a new cycle of divisions. It seems to the author that the analogies are to many to be overlooked. When two chemical compounds react with one another, both are transformed and new products appear at the and of the reaction. In the reaction in which the protoplasm takes place, a sharp difference is to be noted. The protoplasm, contrarily to what happens with the chemical substances, does not enter directly into reaction, but by means of products of its physiological activities. More than that while the compounds with Wich it reacts are changed, it preserves indefinitely its constitution. Here is one of the most important differences in the behavior of living and lifeless matter. Genes, accordingly, do not alter their constitution when they enter into reaction. Genetists contradict themselves when they affirm, on the one hand, that genes are entities which maintain indefinitely their chemical composition, and on the other hand, that mutation is a change in the chemica composition of the genes. They are thus conferring to the genes properties of the living and the lifeless substances. The protoplasm, as we know, without changing its composition, can synthesize different kinds of compounds as enzyms, hormones, and the like. A mutation, in the opinion of the writer would then be a new property acquired by the protoplasm without altering its chemical composition. With regard to the activities of the enzyms In the cells, the author writes : Due to the specificity of the enzyms we have that what determines the order in which they will enter into play is the chemical composition of the substances appearing in the protoplasm. Suppose that a nucleoproteln comes in relation to a protoplasm in which the following enzyms are present: a protease which breaks the nucleoproteln into protein and nucleic acid; a polynucleotidase which fragments the nucleic acid into nucleotids; a nucleotidase which decomposes the nucleotids into nucleoids and phosphoric acid; and, finally, a nucleosidase which attacs the nucleosids with production of sugar and purin or pyramidin bases. Now, it is evident that none of the enzyms which act on the nucleic acid and its products can enter into activity before the decomposition of the nucleoproteln by the protease present in the medium takes place. Leikewise, the nucleosidase cannot works without the nucleotidase previously decomposing the nucleotids, neither the latter can act before the entering into activity of the polynucleotidase for liberating the nucleotids. The number of enzyms which may work at a time depends upon the substances present m the protoplasm. The start and the end of enzym activities, the direction of the reactions toward the decomposition or the synthesis of chemical compounds, the duration of the reactions, all are in the dependence respectively o fthe nature of the substances, of the end products being left in, or retired from the medium, and of the amount of material present. The velocity of the reaction is conditioned by different factors as temperature, pH of the medium, and others. Genetists fall again into contradiction when they say that genes act like enzyms, controlling the reactions in the cells. They do not remember that to cintroll a reaction means to mark its beginning, to determine its direction, to regulate its velocity, and to stop it Enzyms, as we have seen, enjoy none of these properties improperly attributed to them. If, therefore, genes work like enzyms, they do not controll reactions, being, on the contrary, controlled by substances and conditions present in the protoplasm. A gene, like en enzym, cannot go into play, in the absence of the substance to which it is specific. Tne genes are considered as having two roles in the organism one preparing the characters attributed to them and other, preparing the medium for the activities of other genes. At the first glance it seems that only the former is specific. But, if we consider that each gene acts only when the appropriated medium is prepared for it, it follows that the medium is as specific to the gene as the gene to the medium. The author concludes from the analysis of the manner in which genes perform their function, that all the genes work at the same time anywhere in the organism, and that every character results from the activities of all the genes. A gene does therefore not await for a given medium because it is always in the appropriated medium. If the substratum in which it opperates changes, its activity changes correspondingly. Genes are permanently at work. It is true that they attend for an adequate medium to develop a certain actvity. But this does not mean that it is resting while the required cellular environment is being prepared. It never rests. While attending for certain conditions, it opperates in the previous enes It passes from medium to medium, from activity to activity, without stopping anywhere. Genetists are acquainted with situations in which the attended results do not appear. To solve these situations they use to make appeal to the interference of other genes (modifiers, suppressors, activators, intensifiers, dilutors, a. s. o.), nothing else doing in this manner than displacing the problem. To make genetcal systems function genetists confer to their hypothetical entities truly miraculous faculties. To affirm as they do w'th so great a simplicity, that a gene produces an anthocyanin, an enzym, a hormone, or the like, is attribute to the gene activities that onlv very complex structures like cells or glands would be capable of producing Genetists try to avoid this difficulty advancing that the gene works in collaboration with all the other genes as well as with the cytoplasm. Of course, such an affirmation merely means that what works at each time is not the gene, but the whole cell. Consequently, if it is the whole cell which is at work in every situation, it follows that the complete set of genes are permanently in activity, their activity changing in accordance with the part of the organism in which they are working. Transplantation experiments carried out between creeper and normal fowl embryos are discussed in order to show that there is ro local gene action, at least in some cases in which genetists use to recognize such an action. The author thinks that the pleiotropism concept should be applied only to the effects and not to the causes. A pleiotropic gene would be one that in a single actuation upon a more primitive structure were capable of producing by means of secondary influences a multiple effect This definition, however, does not preclude localized gene action, only displacing it. But, if genetics goes back to the egg and puts in it the starting point for all events which in course of development finish by producing the visible characters of the organism, this will signify a great progress. From the analysis of the results of the study of the phenocopies the author concludes that agents other than genes being also capaole of determining the same characters as the genes, these entities lose much of their credit as the unique makers of the organism. Insisting about some points already discussed, the author lays once more stress upon the manner in which the genes exercise their activities, emphasizing that the complete set of genes works jointly in collaboration with the other elements of the cell, and that this work changes with development in the different parts of the organism. To defend this point of view the author starts fron the premiss that a nerve cell is different from a muscle cell. Taking this for granted the author continues saying that those cells have been differentiated as systems, that is all their parts have been changed during development. The nucleus of the nerve cell is therefore different from the nucleus of the muscle cell not only in shape, but also in function. Though fundamentally formed by th same parts, these cells differ integrally from one another by the specialization. Without losing anyone of its essenial properties the protoplasm differentiates itself into distinct kinds of cells, as the living beings differentiate into species. The modified cells within the organism are comparable to the modified organisms within the species. A nervo and a muscle cell of the same organism are therefore like two species originated from a common ancestor : integrally distinct. Like the cytoplasm, the nucleus of a nerve cell differs from the one of a muscle cell in all pecularities and accordingly, nerve cell chromosomes are different from muscle cell chromosomes. We cannot understand differentiation of a part only of a cell. The differentiation must be of the whole cell as a system. When a cell in the course of development becomes a nerve cell or a muscle cell , it undoubtedly acquires nerve cell or muscle cell cytoplasm and nucleus respectively. It is not admissible that the cytoplasm has been changed r.lone, the nucleus remaining the same in both kinds of cells. It is therefore legitimate to conclude that nerve ceil ha.s nerve cell chromosomes and muscle cell, muscle cell chromosomes. Consequently, the genes, representing as they do, specific functions of the chromossomes, are different in different sorts of cells. After having discussed the development of the Amphibian egg on the light of modern researches, the author says : We have seen till now that the development of the egg is almost finished and the larva about to become a free-swimming tadepole and, notwithstanding this, the genes have not yet entered with their specific work. If the haed and tail position is determined without the concourse of the genes; if dorso-ventrality and bilaterality of the embryo are not due to specific gene actions; if the unequal division of the blastula cells, the different speed with which the cells multiply in each hemisphere, and the differential repartition of the substances present in the cytoplasm, all this do not depend on genes; if gastrulation, neurulation. division of the embryo body into morphogenetic fields, definitive determination of primordia, and histological differentiation of the organism go on without the specific cooperation of the genes, it is the case of asking to what then the genes serve ? Based on the mechanism of plant galls formation by gall insects and on the manner in which organizers and their products exercise their activities in the developing organism, the author interprets gene action in the following way : The genes alter structures which have been formed without their specific intervention. Working in one substratum whose existence does not depend o nthem, the genes would be capable of modelling in it the particularities which make it characteristic for a given individual. Thus, the tegument of an animal, as a fundamental structure of the organism, is not due to gene action, but the presence or absence of hair, scales, tubercles, spines, the colour or any other particularities of the skin, may be decided by the genes. The organizer decides whether a primordium will be eye or gill. The details of these organs, however, are left to the genetic potentiality of the tissue which received the induction. For instance, Urodele mouth organizer induces Anura presumptive epidermis to develop into mouth. But, this mouth will be farhioned in the Anura manner. Finalizing the author presents his own concept of the genes. The genes are not independent material particles charged with specific activities, but specific functions of the whole chromosome. To say that a given chromosome has n genes means that this chromonome, in different circumstances, may exercise n distinct activities. Thus, under the influence of a leg evocator the chromosome, as whole, develops its "leg" activity, while wbitm the field of influence of an eye evocator it will develop its "eye" activity. Translocations, deficiencies and inversions will transform more or less deeply a whole into another one, This new whole may continue to produce the same activities it had formerly in addition to those wich may have been induced by the grafted fragment, may lose some functions or acquire entirely new properties, that is, properties that none of them had previously The theoretical possibility of the chromosomes acquiring new genetical properties in consequence of an exchange of parts postulated by the present writer has been experimentally confirmed by Dobzhansky, who verified that, when any two Drosophila pseudoobscura II - chromosomes exchange parts, the chossover chromosomes show new "synthetic" genetical effects.

Storing Data in the Trial of Complex Technical Products

The article considers the ways of organization of databases for the storage of the results obtained during testing. A new variant of the organization of the data to ensure the ability to write to the database different sets of parameters in the form of chronological series. The required set of parameters depends on the modification of the tested technical installation.

Selection theory of dendritic growth in complex systems

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2015

Serotonergic modulation of action monitoring and cognitive control

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2015

Model-based diagnosis system: application to constructed wetlands

Report for the scientific sojourn carried out at the Model-based Systems and Qualitative Reasoning Group (Technical University of Munich), from September until December 2005. Constructed wetlands (CWs), or modified natural wetlands, are used all over the world as wastewater treatment systems for small communities because they can provide high treatment efficiency with low energy consumption and low construction, operation and maintenance costs. Their treatment process is very complex because it includes physical, chemical and biological mechanisms like microorganism oxidation, microorganism reduction, filtration, sedimentation and chemical precipitation. Besides, these processes can be influenced by different factors. In order to guarantee the performance of CWs, an operation and maintenance program must be defined for each Wastewater Treatment Plant (WWTP). The main objective of this project is to provide a computer support to the definition of the most appropriate operation and maintenance protocols to guarantee the correct performance of CWs. To reach them, the definition of models which represent the knowledge about CW has been proposed: components involved in the sanitation process, relation among these units and processes to remove pollutants. Horizontal Subsurface Flow CWs are chosen as a case study and the filtration process is selected as first modelling-process application. However, the goal is to represent the process knowledge in such a way that it can be reused for other types of WWTP.

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.

The role of the hypothalamic kisspeptin/GPR54 system in the control of GnRH neurons

Summary : The hypothalamus represents less than 1 % of the total volume of the brain tissue, yet it plays a crucial role in endocrine regulations. Puberty is defined as a process leading to physical, sexual and psychosocial maturation. The hypothalamus is central to this process, via the activation of GnRH neurons. Pulsatile GnRH secretion, minimal during childhood, increases with the onset of puberty. The primary function of GnRH is to regulate the growth, development and function of testes in boys and ovaries in girls, by stimulating the pituitary gland secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Several factors contribute to the timing of puberty, including sex and ethnicity, genetics, dietary intake and energy expenditure. Kisspeptins constitute a family of small peptides arising from the proteolytic cleavage of metastin, a peptide with 54 amino acids initially purified from human placenta. These kisspeptins were the subject of much attention following their discovery because of their antimetastatic properties, but it was more recently that their determining role in the reproductive function was demonstrated. It was shown that kisspeptins are ligands of a receptor, GPR54, whose natural inactivating mutation in humans, or knockout in the mouse, lead to infertility. GnRH neurons play a pivotal role in the central regulation of fertility. Kisspeptin greatly increases GnRH release and GnRH neuron firing activity, but the neurobiological mechanisms for these actions are unknown. Gprotein-coupled receptor 54, the receptor for kisspeptin, is expressed by GnRH neurons as well as other hypothalamic neurons, suggesting that both direct and indirect effects are possible. In the first part of my thesis, we investigated a possible connection between the acceleration of sexual development induced by leptin and hypothalamic metastin neurons. However, the data generated by our preliminary experiments confirmed that the commercially available antibodies are non-specific. This finding constituted a major drawback for our studies, which relied heavily upon the neuroanatomical study of the hypothalamic metastinergic pathways to elucidate their sensitivity to exogenous leptin. Therefore, we decided to postpone any further in vivo experiment until a better antibody becomes available, and focused on in vitro studies to better understand the mechanisms of action of kisspeptins in the modulation of the activity of GnRH neurons. We used two GnRH-expressing neuronal cell lines to investigate the cellular and molecular mechanisms of action of metastin in GnRH neurons. We demonstrated that kisspeptin induces an early activation of the MAP kinase intracellular signaling pathway in both cell lines, whereas the SAP/JNK or the Akt pathways were unaffected. Moreover, we found an increase in GnRH mRNA levels after 6h of metastin stimulation. Thus, we can conclude that kisspeptin regulates GnRH neurons both at the secretion and the gene expression levels. The MAPK pathway is the major pathway activated by metastin in GnRH expressing neurons. Taken together, these data provide the first mechanism of action of kisspeptin on GnRH neurons. Résumé : L'hypothalamus est une zone située au centre du cerveau, dont il représente moins de 1 du volume total. La puberté est la période de transition entre l'enfance et l'age adulte, qui s'accompagne de transformations somatiques, psychologiques, métaboliques et hormonales conduisant à la possibilité de procréer. La fonction principale de la GnRH est la régulation de la croissance, du développement et de la fonction des testicules chez les hommes, et des ovaires chez les femmes en stimulant la sécrétion de l'hormone lutéinisante (LH) et de l'hormone folliculostimulante (FSH) par la glande hypophysaire. Plusieurs facteurs contribuent au déclanchement de la puberté, y compris le sexe et l'appartenance ethnique, la génétique, l'apport alimentaire et la dépense énergétique. Les Kisspeptines constituent une famille de peptides résultant de la dissociation proteolytique de la métastine, un peptide de 54 acides aminés initialement purifié à partir de placenta humain. Ces kisspeptines ont fait l'objet de beaucoup d'attention à la suite de leur découverte en raison de leurs propriétés anti-metastatiques, et c'est plus récemment que leur rôle déterminant dans la fonction reproductive a été démontré. Les kisspeptines sont des ligands du récepteur GPR54, dont la mutation inactivatrice chez l'homme, ou le knockout chez la souris, conduisent à l'infertilité par hypogonadisme hypogonadotrope. Les neurones à GnRH jouent un rôle central dans le règlement des fonctions reproductrices et la kisspeptine stimule l'activité des neurones à GnRH et la libération de GnRH par ces neurones. Toutefois, les mécanismes neurobiologiques de ces actions ne sont pas connus. Dans la première partie de ma thèse, nous avons étudié le lien potentiel entre l'accélération du développement sexuel induite par la leptine et les neurones hypothalamiques à metastine. Les données générées dans cette première série d'expériences ont malheureusement confirmé que les anticorps anti-metastine disponibles dans le commerce sont aspécifiques. Ceci a constitué un inconvénient majeur pour nos études, qui devaient fortement s'appuyer sur l' étude neuroanatomique des neurones hypothalamiques à metastine pour évaluer leur sensibilité à la leptine exogène. Nous avons donc décidé de focaliser nos travaux sur une étude in vitro des mécanismes d'action de la kisspeptine pour moduler l'activité des neurones à GnRH. Nous avons utilisé deux lignées de cellules neuronales exprimant la GnRH pour étudier les mécanismes d'action cellulaires et moléculaires de la metastine dans des neurones. Nous avons ainsi pu démontrer que la kisspeptine induit une activation précoce de la voie f de signalisation de la MAP kinase dans les deux lignées cellulaires, alors que nous n'avons observé aucune activation de la voie de signalisation de la P13 Kinase et de la SAP/JNK. Nous avons en outre démontré une augmentation de l'expression de la GnRH par la stimulation avec la Kisspeptine. L'ensemble de ces données contribue à élucider le mécanisme d'action avec lequel la kisspeptine agit dans les neurones à GnRH, en démontrant un effet sur l'expression génique de la GnRH. Nous pouvons également conclure que la voie de la MAPK est la voie principale activée par la metastine dans les neurones exprimant la GnRH.

Profiling of T-cell receptor signaling complex assembly in human CD4 T-lymphocytes using RP protein arrays.

The RP protein (RPP) array approach immobilizes minute amounts of cell lysates or tissue protein extracts as distinct microspots on NC-coated slide. Subsequent detection with specific antibodies allows multiplexed quantification of proteins and their modifications at a scale that is beyond what traditional techniques can achieve. Cellular functions are the result of the coordinated action of signaling proteins assembled in macromolecular complexes. These signaling complexes are highly dynamic structures that change their composition with time and space to adapt to cell environment. Their comprehensive analysis requires until now relatively large amounts of cells (>5 x 10(7)) due to their low abundance and breakdown during isolation procedure. In this study, we combined small scale affinity capture of the T-cell receptor (TCR) and RPP arrays to follow TCR signaling complex assembly in human ex vivo isolated CD4 T-cells. Using this strategy, we report specific recruitment of signaling components to the TCR complex upon T-cell activation in as few as 0.5 million of cells. Second- to fourth-order TCR interacting proteins were accurately quantified, making this strategy specially well-suited to the analysis of membrane-associated signaling complexes in limited amounts of cells or tissues, e.g., ex vivo isolated cells or clinical specimens.

Structure-function relationships of the alpha1b-adrenergic receptor.

The alpha1b-adrenergic receptor (AR) is a member of the large superfamily of seven transmembrane domain (TMD) G protein-coupled receptors (GPCR). Combining site-directed mutagenesis of the alpha1b-AR with computational simulations of receptor dynamics, we have explored the conformational changes underlying the process of receptor activation, i.e. the transition between the inactive and active states. Our findings suggest that the structural constraint stabilizing the alpha1b-AR in the inactive form is a network of H-bonding interactions amongst conserved residues forming a polar pocket and R143 of the DRY sequence at the end of TMDIII. We have recently reported that point mutations of D142, of the DRY sequence and of A293 in the distal portion of the third intracellular loop resulted in ligand-independent (constitutive) activation of the alpha1b-AR. These constitutively activating mutations could induce perturbations resulting in the shift of R143 out of the polar pocket. The main role of R143 may be to mediate receptor activation by triggering the exposure of several basic amino acids of the intracellular loops towards the G protein. Our investigation has been extended also to the biochemical events involved in the desensitization process of alpha1b-AR. Our results indicate that immediately following agonist-induced activation, the alpha1b-AR can undergo rapid agonist-induced phosphorylation and desensitization. Different members of the G protein coupled receptor kinase family can play a role in agonist-induced regulation of the alpha1b-AR. In addition, constitutively active alpha1b-AR mutants display different phosphorylation and internalization features. The future goal is to further elucidate the molecular mechanism underlying the complex equilibrium between activation and inactivation of the alpha1b-AR and its regulation by pharmacological substances. These findings can help to elucidate the mechanism of action of various agents displaying properties of agonists or inverse agonists at the adrenergic system.

Structure-function relationships for the interleukin 2 receptor system

Receptors for interleukin 2 (IL-2) esit in at least three forms which differ in their subunit compositio, their affinity for ligand and their ability to mediate a cellular reponse. Type I receptors occur following cellular acitivation and consist of the 55,000 m. w. glycoprotein Tac. These receptors bind IL-2 with a low affinity, do not internalize ligand and have not been definitively associated with any response. Type II receptors, on the other hand, conssit of one or more glycoproteins of 70,000 m. w. which have been termed "beta ([beta]) chains." They bind IL-2 with an intermediate affinity and rapidly internalize the ligand. [Beta] proteins mediate many cellular IL-2-dependent reponses, including the short-term activation of natural killer cells and the induction of Tac protein expression. Type III receptors consist of a ternary complex of the Tac protein, the [beta] chain(s) and IL-2. They are characterized by a paricularly high affinity for ligand association. Type III receptors also internalize ligand and mediate IL-2-dependent responses at low factor concentrations. The identification of two independent IL-2-binding molecules, Tac and [beta], thus provides the elusive molecular explanation for the differences in IL-2 receptor affinity and suggests the potential for selective therapeutic manipulation of IL-2 reponses.