Title from first page of PDF document. Includes bibliographical references (p. 26-29).

Four type II toxin-antitoxin (TA) systems were previously identified in Burkholderia pseudomallei K96243. Type II TA toxins are able to induce cell growth arrest or death by interfering with key processes within the organism. BPSS0390-0391 is one of the TA systems previously identified and has homology to hicBA system in Acinetobacter baumannii. B. pseudomallei HicA is able to cause a reduction in the number of culturable cells after expression in E. coli. This study aimed to characterise B. pseudomallei HicA in three ways: by inducing expression of HicA in bacterial species other than E. coli, by identifying amino acids in HicA involved in toxicity and neutralisation by the antitoxin HicB and by examining the interaction of HicA with other TA antitoxins identified within B. pseudomallei genome. A broad host range plasmid encoding BPSS0390 was transformed into a range of Gram negative bacteria including Yersinia pseudotuberculosis IP32953, Vibrio vulnificus E64MW, Salmonella enterica serovar Typhimurium SL1344 and Burkholderia thailandensis E264. Expression of BPSS0390 was toxic in all bacterial species tested, despite the presence of antitoxin BPSS0391 homologues in some species. Unregulated expression in E. coli resulted in the appearance of escape mutants encoding non-toxic variants of HicA. An alanine scanning mutagenesis study of HicA identified 20 mutants where toxicity was abolished despite high levels of expression, but identified no mutants that affected TA complex formation. Finally an existing co-expression assay was modified to examine interactions between HicA and other type II TA antitoxins in B. pseudomallei. The assay revealed no interaction between HicA and non-cognate antitoxins and clarified the role of IPTG as an inhibitor of PBAD promoter on the arabinose operon.

abstract: Synthetic gene networks have evolved from simple proof-of-concept circuits to complex therapy-oriented networks over the past fifteen years. This advancement has greatly facilitated expansion of the emerging field of synthetic biology. Multistability is a mechanism that cells use to achieve a discrete number of mutually exclusive states in response to environmental inputs. However, complex contextual connections of gene regulatory networks in natural settings often impede the experimental establishment of the function and dynamics of each specific gene network. In this work, diverse synthetic gene networks are rationally designed and constructed using well-characterized biological components to approach the cell fate determination and state transition dynamics in multistable systems. Results show that unimodality and bimodality and trimodality can be achieved through manipulation of the signal and promoter crosstalk in quorum-sensing systems, which enables bacterial cells to communicate with each other. Moreover, a synthetic quadrastable circuit is also built and experimentally demonstrated to have four stable steady states. Experiments, guided by mathematical modeling predictions, reveal that sequential inductions generate distinct cell fates by changing the landscape in sequence and hence navigating cells to different final states. Circuit function depends on the specific protein expression levels in the circuit. We then establish a protein expression predictor taking into account adjacent transcriptional regions’ features through construction of ~120 synthetic gene circuits (operons) in Escherichia coli. The predictor’s utility is further demonstrated in evaluating genes’ relative expression levels in construction of logic gates and tuning gene expressions and nonlinear dynamics of bistable gene networks. These combined results illustrate applications of synthetic gene networks to understand the cell fate determination and state transition dynamics in multistable systems. A protein-expression predictor is also developed to evaluate and tune circuit dynamics. / Dissertation/Thesis / Doctoral Dissertation Biomedical Engineering 2017

L’adénocarcinome pancréatique (PDA) présente une résistance accrue aux chimiothérapies. Un concept propose que sa composition cellulaire participe à ce processus en limitant l’accès aux drogues tout en modulant les capacités des cellules tumorales. En effet, les cellules non tumorales, principalement mésenchymateuses (CAFs) et immunitaires, représentent 70% de la masse tumorale et forment le microenvironnement intra-tumoral ou stroma. L’impact du stroma dans le développement et la progression des PDA se trouve être au centre d’un large champ d’investigations cliniques. Notre première étude a porté sur un facteur neurotrophique, Slit2, impliqué dans la guidance axonale est sécrété par les CAFs. Ce dernier induit une augmentation de la migration des cellules de Schwann et des changements morphologiques et quantitatifs des cellules neuronales. Ainsi, les nerfs se retrouvent plus nombreux et de taille plus importante dans la tumeur comparée à un pancréas sain, c’est ce qu’on appelle le remodelage neural. Notre second travail a permis d’identifier un complexe multi-protéique (ANXA6/LRP1/TSP1), associé au trafic vésiculaire, présent uniquement dans le compartiment stromal et plus particulièrement dans les CAFs. Ce complexe est porté par des vésicules extracellulaires et procure un avantage prolifératif et pro-migratoire aux cellules tumorales. Les données obtenues au cours de mon travail de thèse constituent un rationnel fort pour étudier le potentiel thérapeutique des éléments permettant le dialogue entre les différents compartiments de la tumeur dans le but de sensibiliser les cellules tumorales aux chimiothérapies et ainsi d’améliorer la survie des patients. / Pancreatic adenocarcinoma (PDA) is particularly resistant to current therapies. A concept suggests that its cellular composition participates in this process, limiting drugs access and affecting tumor cells behavior. Indeed, non-tumor cells, mainly mesenchymal (including Cancer Associated Fibroblasts, CAFs) and immune cells display over 70% of the tumor mass and form the intra-tumoral microenvironment or stroma. The impact of stroma in PDA development and progression is at the center of many clinical investigations. Firstly, we studied a neurogenic factor, Slit2, implicated in axon guidance pathway and secreted by CAFs. Slit2 increases Schawnn cells migration and morphologic changes of neural cells. Indeed, nerve size and density are increased in a tumor compared to a healthy pancreas, that is called, neural remodeling. Secondly, we worked on a multi-proteic complex (ANXA6/LRP1/TSP1), associated to vesicular trafficking, only expressed in stromal compartment, and mainly in CAFs. This complex is present in extracellular vesicles and confers proliferative and pro-migratory capacities to tumor cells. Data obtained during my thesis constitute an important rationale to target the crosstalk between tumor and stromal compartment, in order to sensitize tumor cells to chemotherapy and improve patient survival.

Les jasmonates et l'éthylène sont d'importants signaux de régulation du développement des plantes et de réponse aux stress biotiques et abiotiques. La production de jasmonates est induite à la suite d'une blessure mécanique ou des agents pathogènes. L'acide jasmonique et l'éthylène agissent en synergie sur l'activation de l'expression des gènes de défense tels que PDF1.2. Le Facteur de Réponse à l'Ethylène 1 (ERF1) est un intégrateur clé de ces signaux hormonaux chez Arabidopsis. ERF1 appartient à la superfamille des facteurs de transcription AP2/ERF, lesquels jouent un rôle crucial dans le développement et la réponse aux stress. Hevea brasiliensis est la seule source commerciale de caoutchouc naturel, lequel est synthétisé dans les cellules laticifères. Le latex s'écoule du tronc des hévéas après la saignée. L'éthéphon, un générateur d'éthylène, est un stimulant exogène adopté largement dans les plantations d'hévéa pour améliorer la production de latex en prolongeant l'écoulement de latex et en stimulant le métabolisme des cellules requis pour la régénération du latex. Les jasmonates sont aussi impliqués dans la formation des laticifères. Etant donné l'implication de l'éthylène et de l'acide jasmonique dans la réponse coordonnée à la saignée et à la stimulation par l'éthéphon chez Hevea brasiliensis, leur interaction est supposée jouer un rôle important dans la production de latex.L'objectif de cette thèse est de découvrir les régulateurs clés de l'interaction entre la blessure, le jasmonate et l'éthylène chez Hevea brasiliensis. A travers l'analyse de l'expression de 25 gènes impliqués dans les voies de transduction du jasmonate, de l'éthylène et dans le métabolisme cellulaire, nous avons montré que des voies de réponse dépendantes et indépendantes à l'éthylène et au jasmonate coexistent chez Hevea brasiliensis. La régulation temporelle influence aussi l'expression des gènes. L'étude s'est alors focalisée sur les facteurs de transcription de la superfamille des AP2/ERF. A partir de bases de données de séquences transcriptomiques de différents tissus obtenu par pyroséquençage, 173 membres AP2/ERF ont été identifiés chez Hevea brasiliensis. Cette superfamille est divisée en 3 familles majeures : AP2, ERF et RAV. Soixante six membres sont exprimés dans le latex ce qui suggère qu'ils ont une fonction importante dans le métabolisme des laticifères. En plus du microARN 172 connu pour cibler les transcrits AP2/ERF, six autres microARNs ont été prédits pour inhiber les transcrits de cette superfamille. L'identification de l'orthologue à AtERF1 a été aussi menée chez Hevea brasiliensis. L'expression de 14 gènes HbERF du groupe IX a été étudiée en réponse à la blessure, au méthyl jasmonate et à l'éthylène. L'accumulation relative des transcrits est remarquable pour trois gènes : HbERF-IXc4, HbERF-IXc5 et HbERF-IXc6. Ces gènes candidats ont été caractérisés pour la localisation subcellulaire et la trans-activation du promoteur du gène PDF1.2. La fusion traductionnelle HbERF-IXc4::GFP a révélé que HbERF-IXc4 code pour une protéine nucléaire comme les facteurs de transcription. Le HbERF-IXc5 induit la plus forte activation du promoteur du gene PDF1.2 qui est un gène de défense induit fortement par AtERF1 et ORA59. Ces résultats suggèrent que HbERF-IXc5 est l'orthologue à AtERF1 chez Hevea brasiliensis, lequel est impliqué dans la communication des voies de signalisation de l'éthylène et du jasmonate. L'identification des transcrits AP2/ERF chez Hevea brasiliensis, et la caractérisation des ERFs du groupe IX apportent les bases générales pour étudier la régulation moléculaire de la production de latex en réponse aux stress et de la différentiation des cellules laticifères. Nos résultats suggèrent que HbERF-IXc5 est un intégrateur essentiel des voies de signalisation éthylène et jasmonate chez Hevea brasiliensis. / Jasmonates and ethylene are important signals in regulating the plant development and metabolism, and in response to biotic and abiotic stresses. Production of jasmonates is induced by mechanical wounding and pathogens. Jasmonic acid and ethylene are synergistically required to activate the expression of some defence related genes such as PDF1.2. Ethylene Response Factor 1 (ERF1) was demonstrated as a key integrator in the signal interaction in Arabidopsis. ERF1 belongs to AP2/ERF transcription factors superfamily, which plays a crucial role in plant development and response to biotic and abiotic stresses. Hevea brasiliensis is the sole source of natural rubber, which is synthesized in latex cells. Latex is expelled out after tapping the soft bark. Ethephon, an ethylene releaser, is an exogenous stimulant adopted widely in the rubber plantation for improving latex yield by prolonging latex flow and by stimulating the metabolism required for the latex regeneration. Jasmonates are also involved in the laticifer formation. Given the involvement of ethylene and jasmonic acid in the coordinated response to tapping and ethephon stimulation in Hevea brasiliensis, their interaction is considered to play an important role in latex production. The objective of this thesis is aiming to discover the key regulators in the interaction of wounding, jasmonate and ethylene in Hevea brasiliensis. Through the expression analysis on one group of 25 genes involved in the jasmonate and ethylene and cellular metabolism, we discovered that jasmonate and ethylene dependent and independent response coexist in Hevea brasiliensis. Temporal regulation can also have an influence on the gene expression. We then focused the study on the AP2/ERF transcription factor superfamily. Based on new generation of sequencing data, we identified 173 AP2/ERF members from several Hevea brasiliensis transcripts libraries. This superfamily is divided into 3 major families: AP2, ERF and RAV. Sixty six members are expressed in latex which may indicate that they have an important function in the latex metabolism. In addition to the microRNA 172, which is known to target AP2/ERF transcripts, six other microRNAs were predicted to inhibit transcripts of this superfamily. The identification of the AtERF1 orthologous gene was further conducted in Hevea brasiliensis. Expression analysis of 14 HbERF genes from the group IX was studied in response to wounding, methyl jasmonate and ethylene. A remarkable relative transcript accumulation was observed for genes HbERF-IXc4, HbERF-IXc5 and HbERF-IXc6. These candidate genes were further analysed for subcellular localization and trans-activation of the promoter of the PDF1.2 gene. The translational fusion HbERF-IXc4::GFP revealed that HbERF-IXc4 encoded a nuclear targeted protein like transcription factor. The HbERF-IXc5 was shown to mediate the activation of the PDF1.2 promoter, which is a defence gene dramatically induced by AtERF1 and ORA59. For that reason, HbERF-IXc5 is suggested to be AtERF1 ortholog gene in Hevea brasiliensis, which is at the crosstalk of jasmonic acid and ethylene signalling pathways. This identification of the Hevea brasiliensis AP2/ERF transcripts and the characterization of the ERF group IX provide general basis for studying the molecular regulation of both latex production in response to abiotic stresses and differentiation of latex cells. Our results suggested that the HbERF-IXc5 is an essential integrator of the jasmonic acid and ethylene signalling pathways in Hevea.

Hippo signaling is a tumor suppressive signaling pathway that controls organ size by regulating cellular proliferation, apoptosis, and differentiation during development, regeneration, and homeostasis. The Hippo pathway inhibits transcriptional co-activators and Hippo pathway effectors YAP/TAZ, activation of which is often seen in cancer. Within the adult mammalian intestine, homeostasis of which requires intricate reciprocal interaction between the gut epithelium and adjacent mesenchyme, the Hippo-YAP pathway is crucial for intestinal epithelial homeostasis and regeneration. However, its role in adult mesenchymal homeostasis remains poorly understood. Here, I genetically dissect the role of mesenchymal Hippo-YAP signaling in adult intestinal homeostasis. I find that deletion of core kinases LATS1/2 or YAP activation in mesenchymal progenitor cells, but not terminally differentiated cells, disrupts signaling in the stem cell niche and mesenchymal homeostasis by inducing mesenchymal overgrowth and suppressing smooth muscle actin expression. Furthermore, inhibition of Hippo signaling in Gli1+ mesenchymal progenitors, the main source of Wnt ligands within the stem cell niche, stimulates Wnt ligand production and subsequent epithelial Wnt pathway activation, thereby driving epithelial regeneration following DSS-mediated injury as well as exacerbating APC-mediated tumorigenesis. Altogether, our data reveal a previously underappreciated requirement and the underlying mechanism for stromal Hippo-YAP signaling in adult intestinal homeostasis.

As we move deep into nanometer regime of CMOS VLSI (45nm node and below), the device noise margin gets sharply eroded because of continuous lowering of device threshold voltage together with ever increasing rate of signal transitions driven by the consistent demand for higher performance. Sharp erosion of device noise margin vastly increases the likelihood of intermittent failures (also known as parametric failures) during device operation as opposed to permanent failures caused by physical defects introduced during manufacturing process. The major sources of intermittent failures are capacitive crosstalk between neighbor interconnects, abnormal drop in power supply voltage (also known as droop), localized thermal gradient, and soft errors caused by impact of high energy particles on semiconductor surface. In nanometer technology, these intermittent failures largely outnumber the permanent failures caused by physical defects. Therefore, it is of paramount importance to come up with efficient test generation and test application methods to accurately detect and characterize these classes of failures. Soft error rate (SER) is an important design metric used in semiconductor industry and represented by number of such errors encountered per Billion hours of device operation, known as Failure-In-Time (FIT) rate. Soft errors are rare events. Traditional techniques for SER characterization involve testing multiple devices in parallel, or testing the device while keeping it in a high energy neutron bombardment chamber to artificially accelerate the occurrence of single events. Motivated by the fact that measurement of SER incurs high time and cost overhead, in this thesis, we propose a two step approach: hii a new filtering technique based on amplitude of the noise pulse, which significantly reduces the set of soft error susceptible nodes to be considered for a given design; followed by hiii an Integer Linear Program (ILP)-based pattern generation technique that accelerates the SER characterization process by 1-2 orders of magnitude compared to the current state-of-the-art. During test application, it is important to distinguish between an intermittent failure and a permanent failure. Motivated by the fact that most of the intermittent failures are temporally sparse in nature, we present a novel design-for-testability (DFT) architecture which facilitates application of the same test vector twice in a row. The underlying assumption here is that a soft fail will not manifest its effect in two consecutive test cycles whereas the error caused by a physical defect will produce an identically corrupt output signature in both test cycles. Therefore, comparing the output signature for two consecutive applications of the same test vector will accurately distinguish between a soft fail and a hard fail. We show application of this DFT technique in measuring soft error rate as well as other circuit marginality related parametric failures, such as thermal hot-spot induced delay failures. A major contribution of this thesis lies on investigating the effect of multiple sources of noise acting together in exacerbating the noise effect even further. The existing literature on signal integrity verification and test falls short of taking the combined noise effects into account. We particularly focus on capacitive crosstalk on long signal nets. A typical long net is capacitively coupled with multiple aggressors and also tend to have multiple fanout gates. Gate leakage current that originates in fanout receivers, flows backward and terminates in the driver causing a shift in driver output voltage. This effect becomes more prominent as gate oxide is scaled more aggressively. In this thesis, we first present a dynamic simulation-based study to establish the significance of the problem, followed by proposing an automatic test pattern generation (ATPG) solution which uses 0-1 Integer Linear Program (ILP) to maximize the cumulative voltage noise at a given victim net due to crosstalk and gate leakage loading in conjunction with propagating the fault effect to an observation point. Pattern pairs generated by this technique are useful for both manufacturing test application as well as signal integrity verification for nanometer designs. This research opens up a new direction for studying nanometer noise effects and motivates us to extend the study to other noise sources in tandem including voltage drop and temperature effects.

No description available.

The master thesis deals with G.fast and G.mgfast transmission technologies, including their parameters. The work deals with the principle of vector DMT modulation and the possibility of time duplex TDD and full-duplex FDX in two-way communication used in these technologies. The following is a description of the line using the KHM model, which is suitable for simulations in the transmission band using G.fast and G.mgfast technologies. Subsequently, the disturbing effects of crosstalk at the near end of NEXT and the far end of FEXT and their elimination with these technologies are discussing. Part of the work explains supporting calculations to determine the SNR and bit allocation to calculate the baud rate. The work describes the methods of compensation of crosstalk FEXT and NEXT, which affect the resulting baud rate. The work also includes an application for simulation of transmission speed as a function of distance for G.fast and G.mgfast technologies, allowing changing input parameters and adjusting the transmission bandwidth based on G.9700 and G.9701 standards. Also, in work, an application is created to display the compensation of the influence of the transmitted signal crosstalk FEXT and NEXT, which allow the import of measured crosstalk between individual participants. The issue of influencing crosstalk for accurate measurements in the laboratory is also discussing. An application in the MATLAB environment is creating to display the measured characteristics.

In multitasking, the execution of a prioritized task is in danger of crosstalk by the secondary task. Task shielding allows minimizing this crosstalk. However, the locus and temporal dynamics of crosstalk effects and further sources of influence on the execution of the prioritized task are to-date only vaguely understood. Here we combined a dual-task paradigm with an action dynamics approach and studied how and according to which temporal characteristics crosstalk, previously experienced interference and previously executed responses influenced participants' mouse movements in the prioritized task's execution. Investigating continuous mouse movements of the prioritized task, our results indicate a continuous crosstalk from secondary task processing until the endpoint of the movement was reached, although the secondary task could only be executed after finishing execution of the prioritized task. The motor movement in the prioritized task was further modulated by previously experienced interference between the prioritized and the secondary task. Furthermore, response biases from previous responses of the prioritized and the secondary task in movements indicate different sources of such biases. The bias by previous responses to the prioritized task follows a sustained temporal pattern typical for a contextual reactivation, while the bias by previous responses to the secondary task follows a decaying temporal pattern indicating residual activation of previously activated spatial codes.