Uma das principais áreas de pesquisa em Biologia de Sistemas refere-se à descoberta de redes biológicas a partir de conjuntos de dados de microarrays. Estas redes consistem de um grande número de genes cujos níveis de expressão afetam os outros genes de vários modos. Nesta tese, apresenta-se uma nova maneira de analisar os conjuntos de dados de microarrays, com base nos diferentes tipos de ciclos encontrados entre os genes das redes de co-expressão construídas com dados quantificados obtidos a partir dos microarrays. A entrada do método de análise é formada pelos dados brutos, um conjunto de genes de interesse (por exemplo, genes de uma via conhecida) e uma função (ativador ou inibidor) destes genes. A saída do método é um conjunto de ciclos. Um ciclo é um caminho fechado com todos os vértices (exceto o primeiro e o último) distintos. Graças à nova forma de encontrar relações entre os genes, é possível uma interpretação mais robusta das correlações dos genes, porque os ciclos estão associados a mecanismos de feedback, que são muito comuns em redes biológicas. A hipótese é que feedbacks negativos permitem encontrar relações entre os genes que podem ajudar a explicar a estabilidade do processo regulatório dentro da célula. Ciclos de feedback positivo, por outro lado, podem mostrar a quantidade de desequilíbrio de uma determinada célula em um determinado momento. A análise baseada em ciclos permite identificar a relação estequiométrica entre os genes da rede. Esta metodologia proporciona uma melhor compreensão da biologia do tumor. Portanto, as principais contribuições desta tese são: (i) um novo método de análise baseada em ciclos; (ii) um novo método de classificação; (iii) e, finalmente, aplicação dos métodos e a obtenção de resultados práticos. A metodologia proposta foi utilizada para analisar os genes de quatro redes fortemente relacionadas com o câncer - apoptose, glicólise, ciclo celular e NF B - em tecidos do tipo mais agressivo de tumor cerebral (Gliobastoma multiforme - GBM) e em tecidos cerebrais saudáveis. A maioria dos pacientes com GBM morrem em menos de um ano, essencialmente nenhum paciente tem sobrevivência a longo prazo, por isso estes tumores têm atraído atenção significativa. Os principais resultados nesta tese mostram que a relação estequiométrica entre genes envolvidos na apoptose, glicólise, ciclo celular e NF B está desequilibrada em amostras de GBM em comparação as amostras de controle. Este desequilíbrio pode ser medido e explicado pela identificação de um percentual maior de ciclos positivos nas redes das primeiras amostras. Esta conclusão ajuda a entender mais sobre a biologia deste tipo de tumor. O método de classificação baseado no ciclo proposto obteve as mesmas métricas de desempenho como uma rede neural, um método clássico de classificação. No entanto, o método proposto tem uma vantagem significativa em relação às redes neurais. O método de classificação proposto não só classifica as amostras, fornecendo diagnóstico, mas também explica porque as amostras foram classificadas de uma certa maneira em termos dos mecanismos de feedback que estão presentes/ausentes. Desta forma, o método fornece dicas para bioquímicos sobre possíveis experiências laboratoriais, bem como sobre potenciais genes alvo de terapias. / One of the main research areas in Systems Biology concerns the discovery of biological networks from microarray datasets. These networks consist of a great number of genes whose expression levels affect each other in various ways. We present a new way of analyzing microarray datasets, based on the different kind of cycles found among genes of the co-expression networks constructed using quantized data obtained from the microarrays. The input of the analysis method is formed by raw data, a set of interest genes (for example, genes from a known pathway) and a function (activator or inhibitor) of these genes. The output of the method is a set of cycles. A cycle is a closed walk, in which all vertices (except the first and last) are distinct. Thanks to the new way of finding relations among genes, a more robust interpretation of gene correlations is possible, because cycles are associated with feedback mechanisms that are very common in biological networks. Our hypothesis is that negative feedbacks allow finding relations among genes that may help explaining the stability of the regulatory process within the cell. Positive feedback cycles, on the other hand, may show the amount of imbalance of a certain cell in a given time. The cycle-based analysis allows identifying the stoichiometric relationship between the genes of the network. This methodology provides a better understanding of the biology of tumors. As a consequence, it may enable the development of more effective treatment therapies. Furthermore, cycles help differentiate, measure and explain the phenomena identified in healthy and diseased tissues. Cycles may also be used as a new method for classification of samples of a microarray (cancer diagnosis). Compared to other classification methods, cycle-based classification provides a richer explanation of the proposed classification, that can give hints on the possible therapies. Therefore, the main contributions of this thesis are: (i) a new cycle-based analysis method; (ii) a new microarray samples classification method; (iii) and, finally, application and achievement of practical results. We use the proposed methodology to analyze the genes of four networks closely related with cancer - apoptosis, glucolysis, cell cycle and NF B - in tissues of the most aggressive type of brain tumor (Gliobastoma multiforme – GBM) and in healthy tissues. Because most patients with GBMs die in less than a year, and essentially no patient has long-term survival, these tumors have drawn significant attention. Our main results show that the stoichiometric relationship between genes involved in apoptosis, glucolysis, cell cycle and NF B pathways is unbalanced in GBM samples versus control samples. This dysregulation can be measured and explained by the identification of a higher percentage of positive cycles in these networks. This conclusion helps to understand more about the biology of this tumor type. The proposed cycle-based classification method achieved the same performance metrics as a neural network, a classical classification method. However, our method has a significant advantage with respect to neural networks. The proposed classification method not only classifies samples, providing diagnosis, but also explains why samples were classified in a certain way in terms of the feedback mechanisms that are present/absent. This way, the method provides hints to biochemists about possible laboratory experiments, as well as on potential drug target genes.

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Glutamat ist der wichtigste exzitatorische Neurotransmitter im Zentralnervensystem der Säugetiere. Die präzise Kontrolle des extrazellulären Glutamatspiegels ist für eine normale synaptische Transmission wichtig und erforderlich, um die Neurone vor Exzitotoxizität zu schützen. Im Gehirn sorgen vor allem verschiedene hochaffine Na+-abhängige Glutamattransporter für diese Kontrolle. In der vorliegenden Arbeit wurde deshalb untersucht, welchen Einfluß die Inhibition der Glutamattransporter auf die exzitatorische synaptische Transmission in Schicht III, einer Region in der bei Alzheimer-Demenz, Schizophrenie und Epilepsie häufig Zellschädigungen und Zellverluste beobachtet werden, und Schicht V des medialen entorhinalen Kortex (mEC) hat. Extrazelluläre Messungen in den Schichten III und V der Ratte zeigten, daß die verwendeten Transport-Inhibitoren signifikant die negativen Feldpotentialkomponenten beider Schichten reduzierten. Schichtspezifische Unterschiede konnten dabei nicht festgestellt werden, was auf eine ähnliche Glutamatregulation in beiden Schichten schließen läßt. Für die anschließenden intrazellulären und patch-clamp Messungen wurden aus diesem Grund nur noch Neurone der Schicht III untersucht. Beide Transport-Inhibitoren (L-trans-2,4-PDC und DL-TBOA) reduzierten die Amplituden der pharmakologisch isolierbaren EPSPs/EPSCs ohne die Kinetik zu beeinflussen. Diese reduzierende Wirkung konnte durch trans-(±)-ACPD, einen Agonisten der Gruppe I und II metabotropen Glutamatrezeptoren (mGluRs), nachgeahmt werden. Die Vorinkubation der Hirnschnitte mit dem unspezifischen Gruppe I und II mGluR-Antagonisten MCPG verhinderte die durch trans-(±)-ACPD hervorgerufene Amplitudenreduktion und auch den reduzierenden Effekt der beiden Transport-Inhibitoren. In nachfolgenden Experimenten mit dem spezifischen Gruppe II mGluR-Antagonisten EGLU konnte dieser zwar die durch L-trans-2,4-PDC hervorgerufene Wirkung verhindern, nicht aber den durch DL-TBOA vermittelten Effekt, was auf eine Aktivierung von Gruppe I mGluRs hinweist. Zusätzlich führte die Applikation von DL-TBOA zu einer signifikanten Veränderung des Doppelpuls-Index, was auf einen präsynaptischen Wirkmechanismus hinweist. Die Applikation von L-trans-2,4-PDC hingegen hatte keinen Effekt auf den Doppelpuls-Index. Die Ergebnisse der vorliegenden Arbeit sprechen dafür, daß beide Transport-Inhibitoren die erregende synaptische Transmission über eine Aktivierung präsynaptischer metabotroper Glutamatrezeptoren der Gruppen I und II hemmen. Dabei konnte festgestellt werden, daß diese Hemmung unter Applikation von DL-TBOA die präsynaptische Transmitterausschüttung über einen negativen Rückkopplungsmechanismus durch Aktivierung von Gruppe I mGluRs vermindert, während L-trans-2,4-PDC seine Wirkung vor allem über eine Aktivierung der Gruppe II vermittelt. Dabei kann davon ausgegangen werden, daß L-trans-2,4-PDC in der benutzten Konzentration die mGluRs der Gruppe II direkt aktivieren kann und der Effekt nicht nur präsynaptisch vermittelt wird. / Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system. The precise control of extracellular glutamate is crucial for the maintenance of normal synaptic transmission and the prevention of excitotoxicity. High-affinity glutamate transporters ensure termination of glutamatergic neurotransmission and keep the synaptic glutamate concentration below excitotoxic levels. In layer III, a region that is especially prone to cell damage in Alzheimer's disease, schizophrenia and epilepsy, and layer V of the medial entorhinal cortex (mEC) effects of blocking glutamate uptake on excitatory synaptic transmission were studied. Extracellular recordings in rat brain slices revealed that application of glutamate uptake inhibitors significantly reduced stimulus-induced negative field potentials in both, layer III and V of the mEC. This effect showed no significant differences in both layers suggesting a similar glutamate regulation in layer III and V. Therefore, only layer III neurons of the mEC were used for the subsequent intracellular and patch-clamp recordings. Two competitive glutamate transporter antagonists, DL-TBOA and L-trans-2,4-PDC, reduced the amplitude of pharmacologically isolated EPSPs/EPSCs without changing the time course of the events. This effect was mimicked by trans-(±)-ACPD, an agonist of group I and II metabotropic glutamate receptors (mGluRs). The competitive group I and II mGluR antagonist MCPG blocked the depression of the EPSC amplitude induced by trans-(±)-ACPD and also masked the effect of either DL-TBOA or L-trans-2,4-PDC. Furthermore, EGLU, which selectively antagonizes group II mGluRs, masked the effect of L-trans-2,4-PDC but not that of DL-TBOA, indicating an involvement of group I mGluRs in the latter case. Finally, DL-TBOA significantly enhanced the paired-pulse index, suggesting a presynaptic mechanism for the depression of EPSP/EPSC amplitude, whereas application of L-trans-2,4-PDC had no significant effect on the paired-pulse behaviour. The present study shows that both transport inhibitors depress pharmacologically isolated EPSPs/EPSCs in layer III neurons of the mEC in combined entorhinal-hippocampal slices. This effect seems to be mediated via activation of different groups of mGluRs. The results suggest that DL-TBOA causes a negative feedback on glutamate release via indirect activation of presynaptic group I mGluRs, possibly due to an accumulation of glutamate, whereas application of L-trans-2,4-PDC most likely leads to an activation of presynaptic group II mGluRs reducing Ca2+-independent release. The latter might be due to a direct action of L-trans-2,4-PDC at these receptors. The present data suggest that blockade of glutamate transport in the mEC does not lead to an excessive accumulation of glutamate because of a counteractive autoinhibiting mechanism.

Zentralnervöse Lernvorgänge hängen wesentlich von der Plastizität der synaptischen Übertragung ab. Synapsen verändern ihre Effizienz sowohl durch Änderungen in der Freisetzungswahrscheinlichkeit von Neurotransmittern als auch durch Variabilität der postsynaptischen Rezeptorausstattung. Darüberhinaus gibt es seit einiger Zeit Hinweise, dass auch die Konzentration des Neurotransmitters in synaptischen Terminalen variieren kann und dadurch die Freisetzungswahrscheinlichkeit von Vesikeln verändert wird. Um den Zusammenhang zwischen der Füllung synaptischer Vesikel und ihrer Dynamik im Vesikelzyklus besser zu verstehen, habe ich zunächst ein Computermodell entwickelt. Ich habe gefunden, dass nur eine Modifikation des Nachschubs von Vesikeln aus der Reservepopulation in die Population unmittelbar freisetzbarer Vesikel die experimentell gemessene Abhängigkeit der Freisetzungswahrscheinlichkeit von der präsynaptischen Transmitterkonzentration reproduzieren kann, nicht jedoch ein direkter Effekt auf die Freisetzungsrate. Einer der im Modell simulierten Mechanismen für den beobachteten Effekt des vesikulären Füllungszustandes auf die Vesikelfreisetzung besteht in einer Rückwirkung des freigesetzten Transmitters auf ionotrope Autorezeptoren. Diesen Mechanismus habe ich anschliessend an GABAergen Synapsen in der CA3-Region des Hippocampus untersucht. Tatsächlich habe ich durch patch-clamp Messungen herausgefunden, dass sowohl die Antwortwahrscheinlichkeit auf extrazelluläre Stimulation als auch die Frequenz von spontanen Vesikelfreisetzungen nach Applikation des GABAA Rezeptor-Agonisten Muscimol signifikant verringert ist. Diese Befunde weisen darauf hin, dass GABA seine eigene Freisetzung über ionotrope Autorezeptoren hemmt. Direkte Messungen der Vesikelfreisetzungen sind an GABAergen Synapsen nur durch bildgebende Verfahren möglich. Mit Hilfe von Zwei-Photonen Mikroskopie ist es mir erstmalig gelungen, im Hirnschnitt die spontane Fusion von Vesikeln zu untersuchen. Diese Methode könnte für eine Reihe von Fragestellungen relevant sein. Dabei hat sich gezeigt, dass nach Applikation von Muscimol die spontane Abnahme der Fluoreszenz vorher angefärbter synaptischer Vesikel ausschliesslich in perisomatischen (überwiegend GABAergen) synaptischen Boutons signifikant verringert ist. Zusammenfassend habe ich bei der experimentellen Prüfung von Vorhersagen eines Computermodells durch patch-clamp Untersuchungen und durch eine neue Methode mit funktioneller Bildgebung Hinweise auf funktionelle präsynaptische GABAA Rezeptoren an GABAergen Terminalen der CA3-Region des Hippocampus gefunden, die eine negative Rückwirkung auf die Freisetzung von Vesikeln ausüben. / Learning processes depend on synaptic plasticity. Synaptic efficacy depends both on the probability of transmitter release and on the variability of postsynaptic receptors. Furthermore, recent data suggest that the transmitter concentration in presynaptic terminals may be an additional variable influencing the release probability of synaptic vesicles. To better understand the relationship between vesicular filling and vesicular dynamics, I first developed a computer model. I found that only a modification of the replenishment of the readily releasable pool from a reserve pool reproduces the experimentally observed dependence of vesicular release on the presynaptic transmitter concentration, but not a direct effect on the release rate. One of the simulated mechanisms consists in a feedback of released transmitter on ionotropic autoreceptors. I investigated this mechanism in the CA3 region of the hippocampus. Indeed, using patch-clamp recordings I observed that application of the GABAA receptor agonist muscimol decreases significantly the response probability to extracellular stimulation as well as the frequency of spontaneous transmitter release. These results suggest that GABA inhibits its own release via ionotropic autoreceptors. Direct measurements of GABAergic vesicular release are only possible by imaging techniques. Using two-photon microscopy, I was (to my knowledge) the first one to investigate spontaneous vesicle fusion in brain slices. This method could be relevant to a variety of topics. I found out that application of muscimol leads to a significant decrease of fusion-associated fluorescence decay in perisomatic (mostly GABAergic) synaptic boutons. Taken together, I used patch-clamp recordings and a new application of two-photon microscopy to verify predictions of a computer model. The results suggest a negative feedback of GABA release via presynaptic GABAA receptors in the CA3 region of the hippocampus.

Cette thèse a pour objectif d’étudier certains aspects du traitement et de l’acquisition de la morphologie verbale du russe. Le but de ce travail est double. Premièrement, nous avons étudié le traitement d’une alternance consonantique, la palatalisation, par des adultes russophones. Ce processus morphonologique mène à l’allomorphie des radicaux dans plusieurs classes verbales dont les verbes subissent la surgénéralisation dans le langage des enfants. Deuxièmement, nous avons testé l’effet de la rétroaction négative présentée dans l’input, surtout l’effet durable, sur l’élimination des erreurs de surgénéralisation fréquentes chez les enfants en russe. Dans la première étude, nous présentons des données expérimentales sur le traitement des emprunts et des non-mots. Plus particulièrement, cette étude vise à répondre à la question de savoir comment la palatalisation de consonnes dentales et vélaires est traitée par des locuteurs adultes du russe. Les résultats montrent que la palatalisation est semi-productive en fonction des facteurs suivants: a) la distribution des allomorphes à l’intérieur du paradigme, et b) la productivité des classes verbales. Nous supposons que la différence dans le traitement de la palatalisation chez les adultes devrait être reflétée dans le langage des enfants. Notre deuxième article présente les résultats de l’étude sur les effets de la rétroaction négative dans l’acquisition de la morphologie flexionnelle en russe. Pour ce faire nous avons mené une série de tâches induites auprès d’enfants russophones âgés de 3 à 4 ans. Des verbes sensibles à la surgénéralisation en yod /j/, une erreur typique des enfants de cet âge, ont été utilisés comme stimuli. Les participants ont été divisés en quatre groupes selon le type de rétroaction (correction, question de clarification et répétition) auquel ils étaient exposés. Dans chaque groupe de participants, nous avons observé une amélioration significative en production cible de formes verbales avec le temps. Cependant aucune différence significative n’a été trouvée concernant le type de rétroaction. Ces résultats suggèrent que la rétroaction négative ne joue pas un rôle important dans le processus d’acquisition. Ensemble, les deux études représentent une nouvelle contribution à la discussion sur les processus irréguliers en morphologie et le phénomène de surgénéralisation, ainsi que sur le (non) rôle de la rétroaction dans l’élimination des formes surgénéralisées dans le langage des enfants. / This thesis aims at studying certain aspects of Russian verb morphology processing and acquisition. The goal was two-fold: first, we investigated the productivity of morphonological alternations that lead to irregular verb stem allomorphy among adult speakers of Russian. The verbs in the study are known to undergo overregularization in Russian child speech. Second, we tested the (potentially) lasting effect of negative feedback on the retreat from overregularization errors in children. In the first paper, we present experimental data on the processing of loanwords and nonce words that focus on a morphonological alternation (palatalization) in Russian. This study addresses the issue of how stem allomorphy involving palatalization of the velar/palatal and dental/palatal types in the Russian verb system is processed by adults. Processing of palatalization is shown to be quite variable and to depend on: (i) different distribution of allomorphs (past/non-past or 1Sg./other forms) within the verb paradigm, and (ii) overall productivity of verbal classes. We also hypothesized that these differences should be reflected in child language verb morphology acquisition. The study presented in the second article investigates negative feedback effects on inflectional morphology acquisition in Russian. With that goal in mind, we conducted a series of elicited tasks with Russian speaking children aged from 3 to 4 years. Verbs which undergo overregularization in the non-past tense resulting from applying the yod /j/-pattern (typical errors for children of this age) were used as stimuli. Four groups of participants were formed accordingly to three types of feedback (Correction, Clarification question and Repetition), and a control group without feedback. Our results revealed a significant effect of time on target verb form production. However, no significant difference was observed as a function of feedback type, or even where there was no feedback. This finding supports the general hypothesis that negative feedback is not an important factor of language acquisition. Altogether, the results presented in this thesis provide new insights on irregular processes in Russian verb morphology, as well as on the inefficiency of negative feedback in the acquisition of L1 morphology.

Homeostasis is an indispensable phenomenon in the maintenance of living organisms. Genetic defects which disrupt negative feedback processes can impact homeostatic regulation, potentially resulting in disease. To uncover the molecular mechanisms governing these and other diseases potentially related to defective homeostasis, I used the Drosophila neuromuscular junction as a model system. I characterized two potential mechanisms that regulate homeostasis within the nervous system. First, in Drosophila larval motor neurons, ligand activation of Drosophila metabotropic glutamate receptor A (DmGluRA) mediates a Phosphoinositide 3-kinase (PI3K)-dependent downregulation of neuronal activity, but the mechanism by which mGluR activates PI3K remains incompletely understood. Here, I identified Ca 2+ /Calmodulin-dependant protein kinase II (CaMKII) and the Focal adhesion kinase (DFak) as critical intermediates in the DmGluRA-dependent activation of PI3K at Drosophila motor nerve terminals. I found that transgene-induced CaMKII inhibition or the DFak CG1 null mutation each block the ability of glutamate application to activate PI3K in larval motor nerve terminals, whereas transgene-induced CaMKII activation increases PI3K activity in motor nerve terminals in a DFak-dependent manner, even in the absence of glutamate application. I conclude that the activation of PI3K by DmGluRA is mediated by CaMKII and DFak. Second, I observed that Push, a putative E3-ubiquitin ligase and Ca 2+ /Calmodulin binding protein, regulates both neurotransmitter release and retrograde signaling in the Drosophila neuromuscular junction. I found that RNAi-mediated Push inhibition in the neuron increases but, in the muscle decreases, neurotransmitter release. Similar results were obtained from RNAi knock down of PLCβ and IP3R, which mediates Ca 2+ release from the endoplasmic reticulum. I conclude that Push mediation of the ubiquitin proteasome system may be important in the regulation of PLCβ/IP3R-mediated intracellular Ca 2+ release, and that this Ca 2+ release in the neuron inhibits neurotransmitter release, but in the muscle activates neurotransmitter release via a retrograde signal.

Microwave limiting amplifiers are the key components of Instantaneous Frequency Measurement (IFM) systems. Limiting amplifiers provide constant output power level in a wide input dynamic range and over a broad frequency band. Moreover, limiting amplifiers are high gain devices that are used to bring very low input power levels to a constant output power level. Besides, limiting amplifiers are required to provide minimum small signal gain ripple in order not to reduce the sensitivity of the IFM system over the operating frequency band. In this thesis work, a high gain, medium power, 2-18 GHz limiting amplifier module is designed, simulated, fabricated and measured. First, a 3-stage cascaded amplifier with 27 dB small signal gain is designed and fabricated. The 3-stage amplifier is composed of a novel cascaded combination of negative feedback and distributed amplifiers that provides the minimum small signal gain ripple and satisfactory input and output return losses inside 2-18 GHz frequency band. Then, the designed two 3-stage amplifiers and one 4-stage amplifier are cascaded to constitute a limiting amplifier module with minimum 80 dB small signal gain. The designed 10-stage limiting amplifier module also includes an analog voltage controllable attenuator to be used for compensating the gain variations resulting from temperature changes. The fabricated 10-stage limiting amplifier module provides 20 +/- 1.2 dBm output power level and excellent small signal gain flatness, +/- 2.2 dB, over 2-18 GHz frequency range.

The developmental processes of lymphopoiesis generate mature B lymphocytes from hematopoietic stem cells through increasingly restricted intermediates. Networks of transcription factors regulate these cell fate choices and are composed of both ubiquitously expressed and B lineage-specific factors. E-protein transcription factors are encoded by the three genes E2A, E2-2 (SEF2-1), and HEB. The E2A gene is required for B cell development and encodes the alternatively spliced proteins E12 and E47. During B lymphocyte development, the cells have to pass several checkpoints verifying the functionality of their antigen receptors. Early in the development, the expression of a pre-B cell receptor (pre-BCR) with membrane-bound immunoglobulin (Ig) heavy chain protein associated with surrogate light chain (SLC) proteins is a critical checkpoint that monitors for functional Ig heavy chain rearrangement. Signaling from the pre-BCR induces survival and a limited clonal expansion. Here it is shown that pre-BCR signaling rapidly down-regulates the SLCs l5 and VpreB and also the co-receptor CD19. Ca2+ signaling and E2A were shown to be essential for this regulation. E2A mutated in its binding site for the Ca2+ sensor protein calmodulin (CaM), and thus with CaM-resistant DNA binding, makes l5, VpreB and CD19 expression resistant to the inhibition following pre-BCR stimulation. Thus, Ca2+ down-regulates SLC and CD19 gene expression upon pre-BCR stimulation through inhibition of E2A by Ca2+/CaM. A general negative feedback regulation of the pre-BCR proteins as well as many co-receptors and proteins in signal pathways from the receptor was also shown. After the ordered recombination of Ig heavy chain gene segments, also Ig light chain gene segments are recombined together to create antibody diversity. The recombinations are orchestrated by the recombination activating gene (RAG) enzymes, other enzymes that cleave/mutate/assemble DNA of the Ig loci, and the transcription factor Pax5. A key feature of the immune system is the concept that one lymphocyte has only one antigen specificity that can be selected for or against. This requires that only one of the alleles of genes for Ig chains is made functional. The mechanism of this allelic exclusion has however been an enigma. Here pre-BCR signaling was shown to down-regulate several components of the recombination machinery including RAG1 and RAG2 through CaM inhibition of E2A. Furthermore, E2A, Pax5 and the RAGs were shown to be in a complex bound to key sequences on the IgH gene before pre-BCR stimulation and instead bound to CaM after this stimulation. Thus, the recombination complex is directly released through CaM inhibition of E2A. Upon encountering antigens, B cells must adapt to produce a highly specific and potent antibody response. Somatic hypermutation (SH), which introduces point mutations in the variable regions of Ig genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both SH and CSR. The AID enzyme has to be tightly controlled as it is a powerful mutagen. BCR signaling, which signals that good antibody affinity has been reached, was shown to inhibit AID gene expression through CaM inhibition of E2A.  SH increases the antigen binding strength by many orders of magnitude. Each round of SH leads to one or a few mutations, followed by selection for increased affinity. Thus, BCR signaling has to enable selection for successive improvements in antibodies (Ab) over an extremely broad range of affinities. Here the BCR is shown to be subject to general negative feedback regulation of the receptor proteins as well as many co-receptors and proteins in signal pathways from the receptor. Thus, the BCR can down-regulate itself to enable sensitive detection of successive improvements in antigen affinity. Furthermore, the feedback inhibition of the BCR signalosome and most of its protein, and most other gene regulations by BCR stimulation, is through inhibition of E2A by Ca2+/CaM. Differentiation to Ab-secreting plasmablasts and plasma cells is antigen-driven. The interaction of antigen with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. Genome-wide analysis showed that differentiation of B cells to Ab-secreting cell is induced by BCR stimulation through very fast regulatory events, and induction of IRF-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1 and Spi-B gene expressions were identified as immediate early events. Ca2+ signaling through CaM inhibition of E2A was essential for these rapid down-regulations of immediate early genes after BCR stimulation in initiation of plasma cell differentiation.

La biologie moléculaire et, plus spécifiquement, la régulation de l’expression génique ont été révolutionnées par la découverte des microARN (miARN). Ces petits ARN d’une vingtaine de nucléotides sont impliqués dans la majorité des processus cellulaires et leur expression est dérégulée dans plusieurs maladies, comme le cancer. Un miARN reconnaît ses cibles principalement par son noyau, ce qui lui permet de réguler simultanément la traduction de centaines d’ARN messagers. Nos travaux ont montré l’existence d’une boucle de rétro-activation négative, entre deux miARN du polycistron miR-17-92 et trois facteurs de transcription de la famille E2F. E2F1, 2 et 3 induisent la transcription de miR-20 et miR-17 qui par la suite inhibent leur traduction. Nos résultats suggèrent l’implication de cette boucle dans la résistance à l’apoptose induite par E2F1 dans les cellules du cancer de la prostate, ce qui expliquerait en partie le potentiel oncogénique du polycistron miR-17-92. L’étude de ce motif de régulation nous a donc permis de réaliser le potentiel incroyable qu’ont les miARN à inhiber la traduction de plusieurs gènes. Basé sur les règles de reconnaissance des miARN, nous avons développé et validé MultiTar. Cet outil bioinformatique permet de trouver la séquence d’un miARN artificiel ayant le potentiel d’inhiber la traduction de gènes d’intérêts choisis par l’utilisateur. Afin de valider MultiTar, nous avons généré des multitargets pouvant inhiber l’expression des trois E2F, ce qui nous a permis de comparer leur efficacité à celle de miR-20. Nos miARN artificiels ont la capacité d’inhiber la traduction des E2F et de neutraliser leur fonction redondante de la progression du cycle cellulaire de façon similaire ou supérieur à miR-20. La fonctionnalité de notre programme, ouvre la voie à une stratégie flexible pouvant cibler le caractère multigénique de différents processus cellulaires ou maladies complexes, tel que le cancer. L’utilisation de miARN artificiels pourrait donc représenter une alternative intéressante aux stratégies déjà existantes, qui sont limitées à inhiber des cibles uniques. En plus d’élucider un réseau de régulation complexe impliquant les miARN, nous avons pu tirer profit de leur potentiel d’inhibition par la conception de miARN artificiels. / miRNAs are powerful regulators of gene expression in mammals. These small RNAs of around 20 nucleotides are involved in several cellular processes and diseases. MiRNAs recognize their targets mainly by a region comprising nucleotides 2-8, known as the seed. This characteristic gives them the potential to inhibit hundreds of messenger RNAs. Our first goal was to better characterize the complex network involving miRNAs in the regulation of gene expression. To achieve this, we studied the relation between a family of transcription factors, the E2Fs, and a family of miRNAs, the miR-17-92 cluster. Our results suggest a negative feedback loop involving miR-17, miR-20a, E2F1, E2F2 and E2F3. In this loop E2F1, 2 and 3 activate the transcription of the two miRNAs that inhibit their translation in return. The inhibition of the antiapoptotic function of E2F1 by miR-17 and miR-20 in a prostate cancer context, could explain the oncogenic potential of the miR-17-92 cluster that was previously reported. Studying the miR-20/E2F feedback loop made us realize how powerful was the ability of miRNAs to inhibit several targets. To overcome the lack of efficient tools able to inhibit simultaneously the expression of multiple genes, our second goal was to develop MultiTar, an algorithm able to design artificial miRNAs that target a set of predetermined genes. MultiTar was validated in silico, using known targets of endogenous miRNAs and in vivo, taking advantage of our experience with the E2F context. We designed artificial miRNAs against E2F1-3 and expressed them both in normal human fibroblasts and prostate cancer cells where they inhibited cell proliferation and induced cellular senescence. The observed phenotypes were precisely those known for inhibiting E2F activities. Hence, MultiTar can efficiently design artificial micro RNAs able to target multiple genes and is thus a flexible tool that can address the issue of multigenic diseases and complex cellular processes. The use of multitargets could be an alternative to overcome the limits of drugs or siRNAs that are designed generally to regulate only one target.

<p>\abstract</p><p>Transcription and translation describe the flow of genetic information from DNA to mRNA to protein. Recent studies show that at a single cell level, these processes are stochastic, which results in the variation of the number of mRNA and proteins even under identical environmental conditions. Because the number of mRNA and protein in each single cell are actually very small, these variations can be crucial for cellular function in diverse contexts, such as development, stress response, immunological and nervous system function. Most studies examine the origin and effects of stochastic gene expression using computer simulations. My goal is to develop a theoretical framework to study activity-dependent gene expression using simplified models that capture essential features. </p><p>I have examined the dynamics of stochastic gene regulation in three contexts. First, I examine how fluctuations in promoter accessibility lead to "bursty" transcription, during which genes are turned "on" or "off" stochastically. I describe a mathematical formalism to represent bursty gene expression in a coarse-grained manner as a Markov process and derive a master equation for the time evolution of the probability distribution of the number of mRNA molecules. This allows us to examine how transcript number responds to time varying stimuli. This model forms a basic building block for understanding the signal transmission and noise of the transcription process to time varying inputs as would be sensed by cells in dynamic environments. In addition to synthesis, gene expression is subject to additional modes of regulation. One such mechanism that controls transcript numbers is by microRNAs (miRNAs), which pair with target mRNAs to repress protein production following transcription. Although hundreds of miRNAs have been identified in mammalian genomes, the function of miRNA-based repression in the context of gene regulation networks still remains unclear. I explore the functional roles of feedback regulation by miRNAs and show that protein fluctuations strongly depend on the mode of miRNA-mediated repression. I discuss the functional implications of protein fluctuations arising from miRNA-mediated repression on gene regulatory networks. Finally, I examine the impact of fluctuations on alternative splicing, which is a major source for proteomic complexity in higher eukaryotes. Although the proteins regulating alternative splicing have been extensively studied, little is known about how noise arising from the stochastic nature of alternative splicing contributes to the entire gene expression process. I explore the functional roles and noise properties of alternative splicing, focusing on the case of exon skipping and intron retention. I show that while the overall counts of the mRNAs of the two isoforms are independent and Poisson distributed, diffusion and binding of the splicing factors contributes to the variance in the abundance of the isoforms. </p><p>Noise in gene expression may be of particular relevance in the nervous system. Environmental stimuli drive the rapid remodeling of neural circuitry in part by inducing the activation of genes to make proteins that modify neuronal excitability and connectivity, ultimately influencing higher order brain function. Finally, I examine the implications of our studies for activity dependent gene expression in the nervous system.</p> / Dissertation