Individual cells in a genetically identical population, exposed to the same environment, can show great variation in their protein expression levels. This is due to noise, which is inherent in many biological processes, due in part to the low molecule numbers and probabilistic interactions which lead to stochasticity. Much of the work in the field of noise and its propagation in gene expression networks, whether it is experimental, modelling or theoretical, has been conducted on networks/systems that occur within a single cell. However, cells do not exist solely in isolation and understanding how cells are able to coordinate their behaviour despite this noise is an interesting area of expansion for the field. In this study, a synthetic intercellular communication system was designed that allows the investigation of how noise is propagated in intercellular communication. The communication system consists of separate sender and receiver cells incorporating components of the Lux quorum sensing system of Vibrio fischeri. The sender cell was designed so that the production of the signalling molecule, 3-oxohexanoyl homoserine lactone, is able to be controlled by addition of isopropyl-β-D-thio-galactoside (IPTG) and monitored via a reporter gene. The receiver cell was designed with a dual reporter system to enable the response of the cell to the signalling molecule to be monitored and the intrinsic and extrinsic noise contributions to the total noise to be calculated. Sender and the receiver cells were engineered in Escherichia coli. The functionality of the receiver cells was tested in the presence of known concentrations of the signalling molecule. The population response and the noise characteristics of the receiver cells in the homogeneous environment were determined from single cell measurements. The functionality of the sender cells was tested in the presence of a range of IPTG concentrations and the induction of expression from the LacI-repressible promoter was monitored. Mathematical models of the system were developed. Stochastic simulations of the models were used to investigate any unexplained behaviour seen in the characterisation of the cells. The full functionality of the intercellular communication system was then tested by growing the receiver in the collected media of the induced sender cells. The response of the receiver cells to the signalling molecule in the media was again characterised using single cell measurements of the reporter expression levels. The analysis of mixed populations of the sender and receiver cells was hampered by the technical limitations of the instruments used for the single cell measurements. Difficulties were encountered in simultaneous and specific measurement of the three reporter genes. Two methods for overcoming this issue were proposed using microscopy, and one of these methods was shown to have potential in overcoming the issue.

A proteína colibistina (CB), uma Rho GEF neuro-especifica, apresenta papel importante na formação e funcionamento das sinapses inibitórias do sistema nervoso central por interagir com a proteína scaffold gefirina e com receptores GABAA e promover o agrupamento e transporte dessas proteínas para a membrana pós-sináptica. Recentemente, nosso grupo de pesquisa identificou interação de CB com um complexo proteico que controla o início da tradução em eucariotos (complexo eIF3), o que sugeriu pela primeira vez que essa proteína pode estar envolvida também na regulação da tradução em células neurais. Ainda, já havia sido descrito que gefirina, parceira funcional de CB, interage com mTOR, uma quinase que desempenha papel fundamental no controle do início da tradução. Contudo, até o momento não havia estudos adicionais investigando o papel de CB neste cenário. Assim sendo, o presente trabalho teve como objetivo investigar o envolvimento da proteína CB no controle do início da síntese proteica mediada pela via de sinalização mTORC1. Foram utilizados dois modelos experimentais: i) um sistema de expressão heteróloga - superexpressão de CB em células HEK293T, e ii) um modelo endógeno de expressão - células neuroprogenitoras derivadas de células-tronco pluripotentes induzidas (iNPCs) provenientes de indivíduos controles e de um paciente com deleção no gene da CB. Por meio de experimentos de coimunoprecipação nós verificamos que CB interage fisicamente com mTOR nos dois modelos experimentais utilizados. Ainda, nossos resultados mostraram que CB parece modular a atividade da via mTORC1, e nas iNPCs derivadas do paciente a ausência de CB leva a um aumento na ativação desta via de sinalização. Em concordância com esses resultados, nós observamos aumento em neo-síntese proteica nas iNPCs provenientes do paciente, o que pode ser um mecanismo patofisiológico contribuindo para as alterações cognitivas e comportamentais observadas no paciente. Embora estudos adicionais sejam necessários para melhor entender os mecanismos moleculares deste controle de início de tradução mediado por CB, nós sugerimos um modelo no qual CB, por interagir fisicamente com mTOR e eIF3, sequestra estas proteínas e impede que mTOR ative seus alvos e desencadeie a formação do complexo de inicio de tradução. Em conclusão, nossos resultados oferecem novas evidências do envolvimento de CB no controle da síntese proteica / Collybistin (CB), a neural specific RhoGEF, plays key roles in inhibitory synapse formation and function that cluster and localize the scaffold protein gephyrin and GABAA receptors to the neural postsynaptic membrane. We have recently reported that CB interacts with a protein complex that controls translation initiation in eukaryotic cells (eIF3 complex), which suggested for the first time that this protein may also act as regulator of protein synthesis in neural cells. Moreover, it has been previously described that gephyrin, the CB functional partner, interacts with mTOR, a kinase that plays a pivotal role in the control of translation initiation. However, until now there were no further studies investigating the role of CB in this scenario. The purpose of this study was to investigate if CB is involved in the control of translational initiation mediated by the mTORC1 signaling pathway. Two experimental models were used: i) a heterologous expression system - overexpression of CB in HEK293T cells, and ii) an endogenous expression model - neural progenitor cells derived from induced pluripotent stem cells (iNPCs) from control individuals and a patient with a deletion of the entire CB gene. We performed coimmunoprecipitation experiments and verified that CB physically interacts with mTOR both in 293T cells and in control iNPCs. In addition, our results show that CB appears to modulate the activity of mTORC1 pathway, and the absence of CB leads to increased mTORC1 signaling activation in patient\' iNPCs. In agreement with these results, we observed increased de novo cap-dependent translation in patient cells, which could be a pathophysiological mechanism contributing to cognitive and behavioral abnormalities observed in the patient. Although further studies are needed to better understand the molecular details of CB-mediated translational control, we suggest a model whereby CB, by physically interacting with mTOR and eIF3, sequesters these proteins, thereby preventing both the ability of mTOR to activate its targets and the formation of the translational initiation complex. In conclusion, our results offer new insights into the role of CB in protein synthesis control

Wnt/β-catenin signaling is activated in the heart after myocardial infarction (MI). This study aims to investigate if β-catenin deletion affects post-MI ion channel gene alterations and ventricular tachycardias (VT). MI was induced by permanent ligation of left anterior descending artery in wild-type (WT) and cardiomyocyte-specific β-catenin knockout (KO) mice. KO mice showed reduced susceptibility to VT (18% vs. 77% in WT) at 8 weeks after MI, associated with reduced scar size and attenuated chamber dilation. qPCR analyses of both myocardial tissues and purified cardiomyocytes demonstrated upregulation of Wnt pathway genes in border and infarct regions after MI, including Wnt ligands (such as Wnt4) and receptors (such as Fzd1 and Fzd2). At 1 week after MI, cardiac sodium channel gene (Scn5a) transcript was reduced in WT but not in KO hearts, consistent with previous studies showing Scn5a inhibition by Wnt/β-catenin signaling. At 8 weeks after MI when Wnt genes have declined, Scn5a returned to near sham levels and K⁺ channel gene downregulations were not different between WT and KO mice. This study demonstrated that VT susceptibility in the chronic phase after MI is reduced in mice with cardiomyocyte-specific β-catenin deletion primarily through attenuated structural remodeling, but not ion channel gene alterations.

Development in VoIP technology is connected with the rapid growth of quality and data rate of the Internet connection. The application of the VoIP technology has become one of the key areas in telecommunication and networking. The main task of this thesis was to explore the questions of IP telephony and voice transmission over data networks according to H.323, SIP and Cisco architectures. Next focus was on designing and implementing experimental workplaces of mentioned architectures and their converged solutions. The purpose of the first chapter is to introduce the theory background of architectures required for implementation of real-time services in data networks and their comparison. The key objectives of the second and third part are to design and implement experimental workplaces containing signaling and proxy servers and hardware or software IP phones of several architectures. Illustration examples of different network topologies are included to demonstrate designed and realized VoIP solutions. Several tests of established communication are done using a packet analyzer. These tests examine duplex data transmission over IP. This article also contains two laboratory exercises designed according to gained practical experiences. The laboratory exercises provide an overview of theory and clearly indicate the possibilities of IP telephony.

This study re-examines a theoretical scenario introduced by Kelly Bedard in which increased university access leads to an increase in high school dropouts due to the decreased wage premium of a high school diploma caused by talent departing to higher education. The goal for this empirical study is to expand upon the theoretical framework introduced by Bedard in order to determine whether job market signalling is present in the European labour market. In line with Bedard, we theorise that if signalling holds true, secondary education graduates will decrease when access to tertiary education increases. To test this we construct 3 linear regression models to analyse a panel data set constructed of data gathered by Eurostat. Our research question is: Does increased enrolment in tertiary education have a negative effect on participation in secondary education? Our results differ from earlier studies as they indicate that increased enrolment in European tertiary education correlates with increased participation in secondary education. Ergo, we do not prove the presence of signalling in the European labour market. Our results support continued policy efforts with the aim of increasing participation at all levels of education as we find no evidence of a trade-off between university access and secondary schooling graduate rates.

Notch est le récepteur d’une voie de communication intercellulaire, conservée au cours de l’évolution et impliquée dans de nombreux processus développementaux. Chez Drosophila melanogaster, la spécification et la division des précurseurs des organes sensoriels (SOPs) sont gouvernées par l’activation différentielle de la voie de signalisation Notch. Cette activation est dépendante de l’interaction entre le récepteur Notch et les ligands Delta/Serrate et LAG-2. Cette interaction favorise le clivage protéolytique du récepteur Notch puis la libération et la translocation du domaine intracellulaire dans le noyau de la cellule receveuse du signal. L’activation de Notch est étroitement régulée dans le temps et dans l’espace et est sous le contrôle du trafic intracellulaire. Toutefois, la localisation exacte de l’interaction entre le ligand et le récepteur demeure encore débattue.Précédemment, la protéine Stratum, prédite pour avoir un rôle de facteur d’échange nucléotidique (GEF), fut identifiée comme régulateur de la voie de signalisation Notch. Ici, nous montrons que la perte de Stratum induit des phénotypes Notch associés à une délocalisation du co-facteur de Notch, Sanpodo, au pôle apical des cellules et dans le réseau trans- golgien, avec Notch et Delta. De plus, nous montrons que Rab8 est délocalisée en absence de Stratum et la perte de Rab8 récapitule les phénotypes Notch observés dans le mutant strat. Ensemble, nous résultats indiquent que Stratum et Rab8 régulent la voie de signalisation Notch en contrôlant à la fois le tri et le transport polarisé de Notch, Sanpodo et Delta à la sortie de l’appareil de Golgi. / Notch is the receptor of an evolutionarily conserved intercellular communication pathway, involved in numerous developmental processes. In Drosophila melanogaster, the specification and the division of sensory organ precursors (SOPs) are governed by the differential activation of the Notch signalling pathway. This activation depends on the interaction between the Notch receptor and its ligands Delta/Serrate and LAG-2. This interaction induces the proteolytic cleavage of the Notch receptor, the release and the translocation of the intracellular domain in the nucleus of the signal-receiving cell. The Notch activation is tightly regulated in time and in space and is controlled by intracellular trafficking. However, the exact localisation of the interaction remains debated.Previously, Stratum, predicted to be a guanine exchange factor (GEF), was identified as a regulator of the Notch signalling pathway. Here we show that the loss of Stratum induces Notch phenotypes associated with a mislocalization of the Notch co- factor, Sanpodo, at the apical pole of cells and in the trans-golgi network, with Notch and Delta. Moreover we show that Rab8 is mislocalized in the absence of Stratum and the loss of Rab8 recapitules Notch phenotypes observed in the strat mutant. Together our results indicate that Stratum and Rab8 regulate the Notch signalling pathway by controlling both the sorting and the transport of Notch, Sanpodo and Delta at the exit of the Golgi apparatus.

Bacterial bodies (colonies) can develop complex patterns of color and structure. These patterns may arise as a result of both colony-autonomous processes (self-patterning) and environmental influences, including those generated by neighbor bodies. We have studied the interplay of intra-colony signaling (self-patterning) and inter-colony influences in related clones of Serratia rubidaea on rich media. We show that the mutual influencing of colonies, present in a common morphospace, is communicated by at least two putative signals. A model accounting for some aspects of colony morphogenesis and inter-colony interactions is proposed. Key words bacteria; Serratia sp.; airborne signals; colony morphogenesis

Extracellular adenosin-5'-triphosphate (ATP) is an important signalling molecule. Cells of eukaryotic tissues release ATP and express responding purinergic receptors. Ionotropic P2X receptors are trimeric ion channels permeable for K+, Na+ and Ca2+ ions. Each subunit consists of two transmembrane domains (TM1 and TM2), an extracellular loop and intracellular N- and C- termini. The transmembrane region is formed by six helical domains. According to the known crystal structure of zfP2X4 receptor, TM1 helixes are oriented peripherally and stabilize TM2 helixes which form the ion gate. However, eletrophysiological studies revealed that TM1 might also participate in channel gating and forming of the ion pore in the open state. The aim of this work was to investigate the role of TM1 in the process of desensitization of rat P2X4 receptor using cystein-scanning mutagenesis. Mutation of two residues (in Asn32 and Tyr42) prolonged desensitization of P2X4 receptor. Moreover, experiments with a partial agonist α,β-methylenadenosin-5'-triphosphate (αβ-meATP) proved that conformation change of TM domains in the process of desensitization is independent on conformation change caused by an agonist binding. Conserved residue Tyr42 is located in the proximity of TM2 of neighbouring subunit. It probably interacts with Met336...

Please read the abstract on page 3. Copyright 1975, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Underwood, R 1975, Social behaviour of the Eland (Tayrotragus Oryx) on Loskop Dam Nature Reserve, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-02212012-134828 / > E12/4/130/gm / Dissertation (MSc)--University of Pretoria, 1975. / Zoology and Entomology / unrestricted

Background: The canonical Wnt/β-catenin pathway is activated in a variety of heart diseases, such as myocardial infarction and cardiac hypertrophy, that are associated with altered ion channel expressions and increased risk of cardiac arrhythmias. Previous work from our lab has demonstrated that the Wnt/β-catenin signalling (Wnt signalling) inhibits sodium (Na+) current in rat cardiomyocytes. In this project, we aim to investigate the mechanisms that underlie the inhibition of Na+ current by Wnt signalling in both rat and human cardiomyocytes. Results: In both neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), activation of the Wnt/β-catenin signalling led to reduced level of Na+ channel gene transcript (Scn5a), channel protein (Nav1.5) and channel current density. This suggests that reduced Scn5a expression is likely the primary mechanism for reduced Na+ current. In addition, we found that activation of the Wnt/β-catenin signalling in both NRVMs and iPSC-CMs upregulated Tbx3 transcript and protein levels, which is a transcription factor that is known to suppress Scn5a transcription. In NRVMs, siRNA-mediated Tbx3 knockdown attenuated (by ~30%) Wnt-induced reductions in Scn5a and Nav1.5 levels. Conclusions: Our findings are consistent with the conclusion that Wnt/β-catenin signalling inhibits Na+ current in both rat and human cardiomyocytes by reducing Scn5a levels, with Tbx3 as one of the mediators.