A brief review of equipment for use in serological procedures and equipment for isolating and identifying bacteria is followed by a study of basic laboratory test procedures used in medical diagnostic bacteriology, an analysis of the problems of developing automatic methods in diagnostic bacteriology and a discussion of possible fields of further study. The conclusions are stated. Extensive investigations of some diagnostic procedures that lend themselves to mechanization and automation, and evaluations of the performance of definitive diagnostic test equipment are reported and discussed: the procedures investigated include a method for serially diluting antibiotic and serum, as required for the measurement of the minimal inhibitory concentration of antibiotic and the serum antibiotic level, and a method for distributing the reagents for the Wassermann reaction. Detailed investigations of some techniques that may lead to the development of further definitive diagnostic test equipment are reported and discussed. The techniques include automatically spreading a culture over an agar plate, and an r. 1% induotion heating method for sterilizing vessels with which infected material oomos into contact in situ, and in a reasonable time: our inability to sterilize vessels in situ has hitherto been a major obstacle to the further development of automatic methods in diagnostic bacteriology. The conclusions drawn from the investigations are stated and many suggestions for further work are made.

Les gènes Hox codent des facteurs de transcription à homéodomaines conservés qui coordonnent la spécification de l'identité régionale du corps pendant le développement des animaux bilatériens. Au nombre de 39 chez les vertébrés supérieurs, ils sont organisés en 4 complexes (A à D) sur les chromosomes. Treize groupes de gènes paralogues occupent des positions, des modes d'expression et des fonctions similaires. En plus de leurs fonctions spécifiques, des études récentes ont mis en évidence des fonctions "génériques", communes en dehors des groupes de paralogie. L'objectif de cette thèse était de rechercher de nouvelles fonctions génériques des protéines Hox vertébrées, en se concentrant sur le développement de la moelle épinière chez l’embryon de poulet et de souris. Nous avons identifié deux de ces fonctions, impliquant en particulier les gènes Hox du complexe B. La première concerne le contrôle de l’autophagie, qui a été précédemment établi dans le corps gras de Drosophile. Mon travail a établi la dynamique spatio-temporelle de l'autophagie et des protéines Hox au cours du développement de la moelle épinière. Ces dynamiques ont permis de suggérer un rôle générique pour les protéines Hox dans la répression de l'autophagie, qui a été confirmé par gain de fonction chez l’embryon de poulet. La deuxième fonction générique des protéines Hox concerne le contrôle de la neurogenèse. L'étude de l'expression des gènes Hox a mis en évidence une expression prédominante des gènes Hox du complexe B dans la zone intermédiaire du tube neural, où ils activent l'expression du gène Lzts1, dont le produit module la signalisation AKT pour finalement contrôler la différenciation neuronale. / Hox genes encode conserved homeodomain transcription factors that coordinate the specification of regional body identity during the development of bilaterian animals. There are 39 Hox genes in higher vertebrates, organized into four complexes (named A to D) on the chromosomes. Thirteen groups of paralogue genes occupy similar positions within the complexes and exhibit similar modes of expression and functions. In addition to their specific functions, for which these transcription factors are well known, some recent studies are suggestive of "generic" functions, i.e. functions common outside paralogy groups. The goal of this thesis was to look for generic functions of vertebrate Hox proteins, focusing on the development of the spinal cord in chick and mouse. We identified two such functions, implying B cluster Hox genes. The first regards the potential of Hox proteins to control autophagy, which was previously established for Drosophila Hox proteins in the fat body. My work established the spatio temporal dynamic of autophagy during spinal cord development in chick and mouse embryos. This dynamic identified complementary autophagy and Hox patterns, suggesting a generic role for Hox proteins in the repression of autophagy, which could be confirmed by gain of function in chick embryo. The second Hox generic function regards the control of spinal cord neurogenesis. The study of Hox expression highlighted a predominant expression of B cluster Hox genes in the neural tube Intermediate Zone (IZ), where they activate the expression of the Lzts1 gene, the product of which modulates AKT signaling to ultimately control neuronal differentiation.

Over 600 temperature-sensitive mutants of B. subtilis were isolated at random from Nitroso-guanidine-treated cells. Mutants defective in DNA synthesis (ts DNA mutants) were identified by determining the ratio of the amount of protein to DNA synthesized at 45°C. Thirty-eight mutants were specifically inhibited in DNA synthesis. Genetic analysis by transformation of 29 of these ts DNA mutants shows that they are distributed in 4 small linkage groups designated A, B, C and D. By transduction it is shown that these groups are located in different regions of the B. subtilis genetic map. The following physiological properties at 45°C of 27 ts DNA mutants were determined: residual DNA and protein synthesis, viability, PBSX induction and morphology. After cessation of DNA synthesis at 45°C mutant cells of groups A and C elongate but cannot divide, whereas U and D group mutant cells do divide and produce anucleate cells. Preliminary experiments suggest that at 45°C B and D group mutants complete all current rounds of replication but cannot initiate new ones.

Following the administration to humans of [4-14C] norethisterone and [4-14C] lynestrenol, 54% and 43% of the radioactivity was excreted in urine, mostly within two days. The plasma level of radioactivity was higher in the case of norethisterone than that of lynestrenol and remained in the circulation for a longer period. The urinary metabolites of the steroids were studied, and a considerable metabolism of the compounds occurred in the body. Most of the urinary metabolites carried the ethynyl side chain and were polar compounds, probably hydroxylated in more than two positions. Possible conversion of lynestrenol to norethisterone prior to further metabolism is discussed. Following the administration of [4-14C] norethisterone to rabbits, 45% of the radioactivity was excreted in urine and 2.7% in faeces during seven days. Tissue concentration of radioactivity was highest at 5 hr. following the injection and had decreased appreciably by 24 hr. Appreciable concentrations.of radioactivity were detected in liver, bile, intestine and kidney. Uterus seemed to retain the radioactivity, suggesting a possible binding of the steroid metabolites. Considerable metabolism of norethisterone occurred in rabbits, the metabolites being mostly polar compounds. Little metabolism of the ethynyl grouping occurred. The specificity of the method of Fotherby and Love (1960) for the estimation of pregnanetriol excretion in pregnancy is examined. The excretion of pregnanetriol during pregnancy was measured in 15 subjects. The pattern of excretion was different from that of pregnanediol and in one half of the subjects studied there was a rise in the excretion of pregnanetriol during the last trimester of pregnancy. Following progesterone administration, the pregnanetriol excretion was affected in only one of five subjects. Three steroids which contaminated the pregnanetriol fraction of urine during pregnancy were isolated and characterized as 3, 6a-dihydroxy-5β-pregnan-20-one, 3, 16a-dihydroxy-5β-pregnan-20-orie, and 5β-pregnane, 3, 6a, 20β triol. The latter two compounds have not previously been identified in urine.

The megakaryocytes are normal constituents of the bone marrow. These cells, or fragments of them, may be found in the peripheral blood of human beings and animals under physiological conditions. It has been reported that circulating megakaryocytes are more frequently seen in males than in females. In human beings, they are normally observed during the foetal stage, and less and less frequently in childhood and adult life. Megakaryocytosia, or the increased incidence of these cells in various tissues, has been observed in diseases such as myelosclerosis, myeloid leukaemia and inflammatory conditions, as well as after accidental or sudden death. Cirrhosis causes a reduction of megakaryocytes, even in the bone marrow. The incidence of circulating megakaryocytes is notably high in patients with cancer. Investigators in the field of cancer research have in the past frequently mistaken them for cancer cells. Then a more detailed study established a distinct morphological difference between the two cell types, and it was found that many so-called cancer cells were in fact circulating megakaryocytes. This, almost accidental, discovery drew attention to the biological importance of these cells in neoplastic disease, even if there are no bone metastases. It is not yet known why megakaryocytes are mobilized, and migrate from the bone marrow (myeloid tissue) to the blood, in malignant neoplasia. The present investigation was designed to determine whether megakaryocytaemia is in fact related to malignant disease and, if so, what factors cause and control it. Various causative factors have been suggested. They include stress, changes in the blood platelet level, anoxaemiai softening of the bone marrow, and a biological reaction to neoplastic tissue. It was therefore decided to study the incidence of circulating megakaryocytes in relation to the blood platelet level, stress, adrenalectomy, spenectomy and other experimental conditions. Hale Wistar rats were used in all experiments. For transplant tumours, Walker 256 carcinoma was injected subcutaneously in the right flank. Blood for the megakaryocyte counts was taken from the heart. The simple smear technique was considered inadequate for the purpose of this investigation, and a cytological technique was used instead.

Le syndrome de Prade-Willi(PWS)est une pathologie génétique neurodéveloppementale ,caractérisée principalement par des altérations du comportement alimentaire et social,que l’on retrouve dans les pathologies du spectre autistique(ASD).PWS est dû à la perte d’expression de plusieurs gènes contigus,parmi lesquels MAGEL2,alors que des mutations ponctuelles de MAGEL2 ont été identifiées dans patients avec Schaaf-Yang syndrome(SYS).Le modèle de souris Magel2-/- présente altérations du comportement alimentaire,communication sociale et comportement cognitif,constituant un modèle animal valide.L’injection postnatal d’ocytocine(OT)rétablit un comportement alimentaire normal et corrige les altérations du comportement social chez les souris Magel2-/-.Les objectifs de ma thèse étaient d’expliquer comment l’absence de Magel2 modifie,via le systèmeOT,le comportement social chez les souris Magel2-/- et les mécanismes sous-jacent le sauvetage de ces altérations après injection d’OT.Premièrement nous avons caractérisé des nouveaux modèles de souris mutantes pour Magel2 , présentant des mutations ponctuelles ou des délétions génomiques différentes du modèle Magel2-/-.Nous avons montré que,en fonction de la mutation génomique,le comportement social et les altérations cellulaires sont différents,reflétant les différences cliniques entre SYS et PWS. Deuxièmement nous avons étudié la cause des altérations comportementales dans le modèle Magel2-/-.Nous avons démontré une altération de la balance entre excitation(E)et inhibition(I)dans l’hippocampe.Nous avons aussi abordé la question de l’effet du traitement OT précoce et nous avons observé un effet à long terme de ce traitement sur la balance E/I. / Prader-Willi syndrome (PWS) is a neurodevelopmental genetic disease, characterized mainly as a feeding disorder with behavioral disturbances, overlapping with autism spectrum disorder (ASD). PWS results from the lack of expression of several contiguous genes, including MAGEL2, and specific point mutations in MAGEL2 have been identified in patients with Schaaf-Yang syndrome (SYS). Magel2-deficient (-/-) mice present alterations in early feeding behavior and in social communication and cognition, constituting a valuable translational animal model. A postnatal injection of oxytocin (OT) restores a normal feeding behavior and cures alterations in social behavior in the Magel2-/- mice. The aims of my thesis were to elucidate how the lack of Magel2, via the OT system, alters the social behavior in Magel2-/- mice and the mechanisms underlying rescue of these alterations by OT administration. Firstly we characterized new Magel2 mouse mutants with point mutations or different genomic deletions compared with the Magel2-/- model. Those models allowed us to show that depending on the genomic mutations the behavior and cellular defects are different, refletting the differences between SYS and PWS. Secondly, we combined behavioral, histological and electrophysiological approaches to unravel the cause of behavioral alterations in the Magel2-/- model. In this study, we were able to reveal an imbalance between excitatory (E) and inhibitory (I) pathways in hippocampus. Moreover, we addressed the issue of the effect of OT treatment in early life and we observed a long term effect of the treatment on this E/I balance.

Il y a exactement un siècle, D'Arcy Thompson a proposé dans son ouvrage "On Growth and Form" que certains principes mathématiques et physiques généraux régissent la diversité de l'organisation cellulaire et tissulaire. L'un de ces principes est l'existence d'une tension superficielle entre les contacts cellulaires, une grandeur physique qui détermine la forme des cellules. On sait aujourd'hui que la tension mécanique au niveau des contacts cellulaires dépend de deux systèmes biologiques: le cytosquelette, un réseau actif générant des forces contractiles, et les molécules d’adhésion, qui lient les cellules et les maintiennent en contact. Les molécules d'adhésion maintiennent les cellules ensemble, mais couplent également les membranes cellulaires à un réseau d'actomyosine contractile, ce qui limite l'expansion des contacts cellulaires. Le rôle des molécules d'adhésion et de la contractilité de l'actomyosine dans la morphogenèse tissulaire est bien établi, mais leur importance et leur coordination dans l'obtention de formes cellulaires et d'arrangements cellulaires demeurent floues. Pour aborder cette question in vivo, nous utilisons l'œil de drosophile comme système modèle. Nous montrons que les formes de cellules cônes dépendent peu des liaisons d'adhésion, et surtout des forces contractiles. Cependant, la N-cadhérine a un contrôle indirect sur la forme des cellules cônes. Aux contacts homotypiques, la N-cadhérine jonctionelle réduit la contractilité de la myosine-II. Aux contacts hétérotypiques avec l' E-cadhérine, la N-cadhérine non liée induit une accumulation asymétrique de Myosin-II aux jonctions, ce qui conduit à une interface cellulaire très contractile. / Exactly a century ago, D'Arcy Thompson proposed in his work "On Growth and Form" that some general mathematical and physical principles govern the diversity of cellular and tissue organization. One of his stated principles is the existence of a surface tension between cell contacts, a physical quantity that determines the shape of the cells. We know today that the mechanical tension at the cellular contacts depends on two biological systems: the cytoskeleton, an active network generating contractile forces and the adhesive molecules, which bind the cells and keep them in contact. Adhesion molecules hold cells together, but also couple cell membranes to a contractile actomyosin network, which limits the expansion of the cell contacts. The role of adhesion molecules and actomyosin contractility in tissue morphogenesis is well established, but their importance and co-ordination in achieving cell shapes and cell arrangements remain unclear. In order to tackle this question in vivo, we use the Drosophila eye as a model system. We show that cone cell shapes depend little on adhesion bonds and mostly on contractile forces. However, N-cadherin has an indirect control on cone cell shape. At homotypic contacts, junctional N-cadherin bonds downregulate Myosin-II contractility. At heterotypic contacts with E-cadherin, unbound N-cadherin induces an asymmetric accumulation of Myosin-II at junctions, which leads to a highly contractile cell interface. Such differential regulation of contractility by N-cadherins is essential for morphogenesis as loss of N-cadherin disrupts cell rearrangements.

Chez les vertébrés, les cellules progénitrices du second champ cardiaque (SHF) situées dans le mésoderme cardiopharyngé (CPM) s’ajoutent aux pôles artériel et veineux du tube cardiaque embryonnaire et contribuent à son élongation. La perturbation de ce processus se traduit par un spectre de défauts cardiaques congénitaux. Les cellules situées dans les domaines antérieur et postérieur du SHF forment deux sous-populations de progéniteurs qui contribuent respectivement au myocarde du ventricule droit et de la voie efférente au pôle artériel et au myocarde auriculaire au pôle veineux. Des expériences d’analyses clonales, de lignages génétiques et de marquage au DiI ont montré que ces populations dérivent de progéniteurs communs situés dans le CPM postérieur. Mon projet a consisté à étudier les mécanismes régulant la contribution des cellules du SHF aux deux pôles du cœur. J’ai montré que Tbx1 est requis pour l’addition des cellules du SHF postérieur au pôle veineux, nécessaire à la septation atrioventriculaire. J’ai également démontré que l’activation de Tbx5 dans les cellules du SHF postérieur entraine la répression du programme antérieur Tbx1-dépendant et conduit à la formation d’une frontière nette séparant les populations de progéniteurs contribuant aux pôles artériel et veineux. L’expression de Tbx5 est dépendante de la voie de signalisation de l’acide rétinoïque (AR) et indispensable à la septation atrioventriculaire. L’AR contribue aussi au développement du muscle trapèze, dérivé du CPM postérieur et lié de façon clonale au pôle veineux du cœur. Ainsi l’AR est requis pour le développement de cellules aux destins myogéniques divergents dans le CPM postérieur. / In vertebrates, the embryonic heart tube grows by addition of Second Heart Field (SHF) progenitor cells from cardiopharyngeal mesoderm (CPM) to both the arterial and venous poles. Perturbation of this process results in a failure of maximal heart tube elongation and a spectrum of congenital heart defects. Distinct anterior and posterior SHF subpopulations contribute to right ventricular and outflow tract myocardium at the arterial pole and atrial myocardium at the venous pole, respectively. However, clonal retrospective analysis, genetic lineage and DiI labelling experiments have shown that a common progenitor population in the posterior CPM contributes to both subpulmonary arterial and venous pole myocardium. My project investigated the mechanisms regulating the contribution of SHF cells to alternate cardiac poles. I showed that the murine homologue of the 22q11.2 deletion syndrome gene Tbx1 is required for posterior SHF cells addition to the venous pole and subsequent atrioventricular septation, in addition to outflow tract development. I also demonstrated a retinoic acid (RA) signaling dependent activation of Tbx5 in Tbx1 expressing posterior SHF cells necessary for atrioventricular septation. This is followed by a Tbx1 and Tbx5 dependent downregulation of the anterior SHF program resulting in the formation of a sharp boundary between arterial and venous pole progenitor populations. RA is also required for branchiomeric neck skeletal muscle development, including the trapezius that is clonally related to the venous pole of the heart and originates in the posterior CPM. RA is thus required for development of divergent myogenic cell fates within the posterior CPM.

Chez les vertébrés et les invertébrés les neurones sont souvent produits par division cellulaire asymétrique. Ce processus est notamment régulé par certains composants de la voie de signalisation Wnt. Cependant, comment les ligands Wnt, des molécules sécrétées activatrices de la voie Wnt, régulent ces divisions n’est pas compris. Le but de ma thèse est d’analyser le rôle des ligands Wnt et de leurs récepteurs dans les divisions asymétriques des précurseurs neuronaux en utilisant C. elegans comme organisme modèle. Chez C. elegans, les précurseurs neuronaux se divisent asymétriquement le long de l’axe antéro-posterieur. Le laboratoire d’accueil a montré que ces divisions sont régulées par un composant nucléaire de la voie Wnt, la beta-caténine, qui s’accumule spécifiquement dans le noyau de la cellule fille postérieure suite à la division asymétrique. Durant ma thèse, j’ai analysé le rôle de composants extracellulaires et corticaux dans ces divisions en utilisant le lignage cholinergique AIY comme lignage test. J’ai tout d’abord observé que les précurseurs neuronaux sont allongés le long de l’axe antéro-postérieur avant leur division. Trois ligands Wnt (CWN-1, CWN-2 et MOM-2) sont transcrits de façon plus importante dans la région postérieure de l’embryon. Via des expériences de perte et gain de fonction j’ai montré que les ligands Wnt régulent l’orientation des divisions ainsi que l’asymétrie d’identité des cellules filles. J’ai également identifié un rôle pour le récepteur de Wnt MOM-5 et la protéine corticale APC au cours des divisions asymétriques. MOM-5 est enrichi au pôle postérieur et APC au pôle antérieur des précurseurs neuronaux avant leur division. / In both vertebrates and invertebrates neurons are often produced by asymmetric cell divisions. Some components of the Wnt pathway have been implicated in this process. However, how Wnt ligands, secreted activators of the Wnt pathway, regulate these divisions is not understood. The aim of my PhD is to analyze the role of Wnt ligands and of their receptors in neuronal precursor asymmetric divisions using C. elegans as a model organism. In the C. elegans embryo, neuronal precursors divide asymmetrically along the antero-posterior axis. The host laboratory has shown that these asymmetric divisions are regulated by a nuclear component of the Wnt pathway, beta-catenin, which accumulates specifically in the nucleus of the posterior daughter cell following asymmetric cell division. During my PhD, I analyzed the role of extracellular and cortical components in the asymmetric divisions of neuronal precursors using the AIY cholinergic lineage as a test lineage. I first observed that neuronal precursors are elongated along the antero-posterior axis before their division. Three Wnt ligands (CWN-1, CWN-2 and MOM-2) are transcribed at a higher level in the posterior region of the embryo. Using loss and gain of function experiments, I have observed that the Wnt ligands regulate the orientation of the divisions as well as the asymmetry in the identity of the daughter cells. I also identified a role for the Wnt receptor MOM-5 and the cortical protein APC during these asymmetric divisions. MOM-5 is enriched at the posterior pole and APC at the anterior pole of the neuronal precursors before their divisions.

No description available.