A regulatory network for the efficient control of transgene expression.

BACKGROUND: Expression of heterologous genes in mammalian cells or organisms for therapeutic or experimental purposes often requires tight control of transgene expression. Specifically, the following criteria should be met: no background gene activity in the off-state, high gene expression in the on-state, regulated expression over an extended period, and multiple switching between on- and off-states. METHODS: Here, we describe a genetic switch system for controlled transgene transcription using chimeric repressor and activator proteins functioning in a novel regulatory network. In the off-state, the target transgene is actively silenced by a chimeric protein consisting of multimerized eukaryotic transcriptional repression domains fused to the DNA-binding tetracycline repressor. In the on-state, the inducer drug doxycycline affects both the derepression of the target gene promoter and activation by the GAL4-VP16 transactivator, which in turn is under the control of an autoregulatory feedback loop. RESULTS: The hallmark of this new system is the efficient transgene silencing in the off-state, as demonstrated by the tightly controlled expression of the highly cytotoxic diphtheria toxin A gene. Addition of the inducer drug allows robust activation of transgene expression. In stably transfected cells, this control is still observed after months of repeated cycling between the repressed and activated states of the target genes. CONCLUSIONS: This system permits tight long-term regulation when stably introduced into cell lines. The underlying principles of this network system should have general applications in biotechnology and gene therapy.

Feedback control of DnaA-mediated replication initiation by replisome-associated HdaA protein in Caulobacter.

Chromosome replication in Caulobacter crescentus is tightly regulated to ensure that initiation occurs at the right time and only once during the cell cycle. The timing of replication initiation is controlled by both CtrA and DnaA. CtrA binds to and silences the origin. Upon the clearance of CtrA from the cell, the DnaA protein accumulates and allows loading of the replisome at the origin. Here, we identify an additional layer of replication initiation control that is mediated by the HdaA protein. In Escherichia coli, the Hda protein inactivates DnaA after replication initiation. We show that the Caulobacter HdaA homologue is necessary to restrict the initiation of DNA replication to only once per cell cycle and that it dynamically colocalizes with the replisome throughout the cell cycle. Moreover, the transcription of hdaA is directly activated by DnaA, providing a robust feedback regulatory mechanism that adjusts the levels of HdaA to inactivate DnaA.

On sliding mode control ofhydraulic servo systems and a manipulator

The aim of the study is to developa novel robust controller based on sliding mode control technique for the hydraulic servo system with flexible load and for a flexible manipulator with the lift and jib hydraulic actuators. For the purpose of general control design, a dynamic model is derived describing the principle physical behavior for both the hydraulic servo system and the flexible hydraulic manipulator. The mechanism of hydraulic servo systems is described by basic mathematical equations of fluid powersystems and the dynamics of flexible manipulator is modeled by the assumed modemethod. The controller is constructed so as to track desired trajectories in the presence of model imprecision. Experimental and simulation results demonstratethat sliding mode control has benefits which can be used to guarantee stabilityin uncertain systems and improve the system performance and load tolerance.

Direct to consumer advertising : the case for greater consumer control

A nyone traveling to the United States from countries other than New Zealand will be surprised by the prevalence of health-related advertisements on television, including ads for drugs. Typically, these TV ads follow a pattern: an ad for a burger at only 99 cents, followed by one for a proton-pump inhibitor, then an ad on healthy home-cooked food delivered directly to your home and an ad for a home-based abdominal workout DVD, followed by an ad for a lipid-lowering drug. There are, however, nuances. After 8 pm, the visitor might encounter an ad for the "little blue pill." This sequence sometimes includes an ad featuring antihistamines for allergic rhinitis in spring and one promoting antidepressants in the winter. Such direct-to-consumer advertising (DTCA) of prescription drugs is usual business in the United States and New Zealand but is prohibited in the rest of the world. Why? Because DTCA for prescribing drugs has pros and cons (discussed elsewhere,1-3 including in JGIM4) that are balanced differently in different countries. Constitutional factors-such as the First Amendment protections on speech, including commercial speech, in the United States5 -as well as patient and population safety considerations, which all differ across countries, modulate reactions to DTCA. Additionally, lack of robust data on the impact of DTCA on prescription drug use adds to the confusion. Evidence, though limited, suggests that DTCA increases drug sales. However, whether the increase in sales corrects existing underuse or encourages over/misuse is not clear.

Variaciones dimensionales en piezas plásticas de inyección y análisis de los medios de control

En la industria de la automoción, así como en todas las que se dedican a fabricar piezas, ya sean plásticas o de otros materiales, es tan importante la Producción como la Calidad. No sirve de nada fabricar mucho si no son piezas de una calidad adecuada, y del mismo modo no es rentable fabricar muy poco volumen por mucho que tenga una calidad excelente. Por ello hay que buscar siempre el equilibrio entre ambos conceptos. La finalidad es tener procesos lo más robustos posibles que nos permitan fabricar cantidad con una buena calidad. La finalidad de este trabajo es buscar los parámetros que más afectan en un proceso de inyección, es decir, saber cuáles son los que debemos tener bajo control para lograr una calidad de piezas buena y un proceso estable y controlado. Para ver si el proceso es capaz de lograr ese objetivo utilizamos gráficos basados en la teoría de 6- sigma que nos calculan el coeficiente de capacidad del proceso (Cpk). A su vez analizaremos los sistemas de medida que utilizamos en cada una de las piezas analizadas para evaluar si son los correctos y nos permiten discriminar piezas buenas de malas en el proceso productivo. La conclusión del trabajo es que hay que prestar especial atención en controlar aquellos aspectos de un proceso que nos aportan variación, y no invertir tiempo ni dinero en aquellos otros que no nos aportan valor añadido y que no afectan sustancialmente al proceso. Este análisis por supuesto lleva su coste, que he analizado y plasmado en las conclusiones para saber lo que le cuesta aproximadamente a una empresa realizarlo.

Spontaneous cell polarization: Feedback control of Cdc42 GTPase breaks cellular symmetry.

Spontaneous polarization without spatial cues, or symmetry breaking, is a fundamental problem of spatial organization in biological systems. This question has been extensively studied using yeast models, which revealed the central role of the small GTPase switch Cdc42. Active Cdc42-GTP forms a coherent patch at the cell cortex, thought to result from amplification of a small initial stochastic inhomogeneity through positive feedback mechanisms, which induces cell polarization. Here, I review and discuss the mechanisms of Cdc42 activity self-amplification and dynamic turnover. A robust Cdc42 patch is formed through the combined effects of Cdc42 activity promoting its own activation and active Cdc42-GTP displaying reduced membrane detachment and lateral diffusion compared to inactive Cdc42-GDP. I argue the role of the actin cytoskeleton in symmetry breaking is not primarily to transport Cdc42 to the active site. Finally, negative feedback and competition mechanisms serve to control the number of polarization sites.

SQualTrack: A Tool for Robust Fault Detection

One of the techniques used to detect faults in dynamic systems is analytical redundancy. An important difficulty in applying this technique to real systems is dealing with the uncertainties associated with the system itself and with the measurements. In this paper, this uncertainty is taken into account by the use of intervals for the parameters of the model and for the measurements. The method that is proposed in this paper checks the consistency between the system's behavior, obtained from the measurements, and the model's behavior; if they are inconsistent, then there is a fault. The problem of detecting faults is stated as a quantified real constraint satisfaction problem, which can be solved using the modal interval analysis (MIA). MIA is used because it provides powerful tools to extend the calculations over real functions to intervals. To improve the results of the detection of the faults, the simultaneous use of several sliding time windows is proposed. The result of implementing this method is semiqualitative tracking (SQualTrack), a fault-detection tool that is robust in the sense that it does not generate false alarms, i.e., if there are false alarms, they indicate either that the interval model does not represent the system adequately or that the interval measurements do not represent the true values of the variables adequately. SQualTrack is currently being used to detect faults in real processes. Some of these applications using real data have been developed within the European project advanced decision support system for chemical/petrochemical manufacturing processes and are also described in this paper

A human immune data-informed vaccine concept elicits strong and broad T-cell specificities associated with HIV-1 control in mice and macaques

Background: None of the HIV T-cell vaccine candidates that have reached advanced clinical testing have been able to induce protective T cell immunity. A major reason for these failures may have been suboptimal T cell immunogen designs. Methods: To overcome this problem, we used a novel immunogen design approach that is based on functional T cell response data from more than 1,000 HIV-1 clade B and C infected individuals and which aims to direct the T cell response to the most vulnerable sites of HIV-1. Results: Our approach identified 16 regions in Gag, Pol, Vif and Nef that were relatively conserved and predominantly targeted by individuals with reduced viral loads. These regions formed the basis of the HIVACAT T-cell Immunogen (HTI) sequence which is 529 amino acids in length, includes more than 50 optimally defined CD4+ and CD8+ T-cell epitopes restricted by a wide range of HLA class I and II molecules and covers viral sites where mutations led to a dramatic reduction in viral replicative fitness. In both, C57BL/6 mice and Indian rhesus macaques immunized with an HTI-expressing DNA plasmid (DNA.HTI) induced broad and balanced T-cell responses to several segments within Gag, Pol, and Vif. DNA.HTI induced robust CD4+ and CD8+ T cell responses that were increased by a booster vaccination using modified virus Ankara (MVA.HTI), expanding the DNA.HTI induced response to up to 3.2% IFN-γ T-cells in macaques. HTI-specific T cells showed a central and effector memory phenotype with a significant fraction of the IFN-γ+ CD8+ T cells being Granzyme B+ and able to degranulate (CD107a+). Conclusions: These data demonstrate the immunogenicity of a novel HIV-1 T cell vaccine concept that induced broadly balanced responses to vulnerable sites of HIV-1 while avoiding the induction of responses to potential decoy targets that may divert effective T-cell responses towards variable and less protective viral determinants.

Cyclin-dependent kinases 4 and 6 control tumor progression and direct glucose oxidation in the pentose cycle

Cyclin-dependent kinases CDK4 and CDK6 are essential for the control of the cell cycle through the G1 phase. Aberrant expression of CDK4 and CDK6 is a hall- mark of cancer, which would suggest that CDK4 and CDK6 are attractive targets for cancer therapy. Herein, we report that calcein AM is a potent specific inhibitor of CDK4 and CDK6 in HCT116 human colon adenocarcinoma cells, inhibiting retinoblastoma protein (pRb) phosphorylation and inducing cell cycle arrest in the G1 phase. The metabolic effects of calcein AM (the calcein acetoxymethyl-ester) on HCT116 cells were also evaluated and the flux between the oxidative and non-oxidative branches of the pentose phos-phate pathway was significantly altered. To elucidate whe-ther these metabolic changes were due to the inhibition of CDK4 and CDK6, we also characterized the metabolic profile of a CDK4, CDK6 and CDK2 triple knockout of mouse embryonic fibroblasts. The results show that the metabolic profile associated with the depletion of CDK4, CDK6 and CDK2 coincides with the metabolic changes induced by calcein AM on HCT116 cells, thus confirming that the inhibition of CDK4 and CDK6 disrupts the balance between the oxidative and non-oxidative branches of the pentose phosphate pathway. Taken together, these results indicate that low doses of calcein can halt cell division and kill tumor cells. Thus, selective inhibition of CDK4 and CDK6 may be of greater pharmacological interest, since inhibitors of these kinases affect both cell cycle progression and the robust metabolic profile of tumors.

Control d'un sistema de marcatge làser de caixes

Avui en dia, l’alta competitivitat que existeix al mercat, fa que les empreses hagin d’esprémer al màxim les seves possibilitats per no quedar-se enrere. Un dels processos en que aquest fet hi és més present és el productiu. L’empresa JCM Technologies també engloba aquest camp i és en un dels seus processos productius on aquest projecte pren part. L’objectiu d’aquest projecte final de carrera ha estat desenvolupar un sistema per poder marcar caixes mitjançant un làser de CO2 i un automatisme manipulador de caixes. D’aquesta manera aquest procés productiu té una durada molt inferior a l’antic procés, que consistia en enganxar una etiqueta al lloc on ara és marcat pel làser. Per satisfer els objectius, s’ha creat una aplicació de Windows que per mitjà d’una interfície gràfica, permet a l’usuari realitzar els passos necessaris per fer el marcatge. Primerament es recullen les dades procedents de la comanda; seguidament es seleccionen les que s’han de marcar a les caixes i s’envien al làser mitjançant comunicació sèrie; una vegada aquesta inicialització ha finalitzat correctament, s’engega la seqüència de marcatge de les caixes, que en marcarà la quantitat indicada. Aquest procés de marcatge consisteix en supervisar l’estat en que es troben certes senyals, procedents de l’automatisme i del làser, i depenent d’aquestes generar-ne unes altres. Aconseguint així realitzar el procés de marcatge de cada caixa. Com a conclusions cal dir, que els objectius s’han complert, ja que s’ha aconseguit un procés de marcatge ràpid i robust. També s’ha aconseguit que les parts de configuració de l’aplicació, i de les caixes siguin de fàcil manipulació. Per tant, amb l’acompliment dels objectius d’aquest projecte, aconseguim completar el sistema de marcatge, i permetre que les caixes siguin marcades de forma correcta, ràpida i eficient.

Green tea in transdermal formulation: HPLC method for quality control and in vitro drug release assays

RP-HPLC based analytical method for use in both quality control of green tea in a semisolid formulation and for in vitro drug release assays was developed and validated. The method was precise (CV < 5%), accurate (recovery between 98% and 102%), linear (R² > 0.99), robust, and specific for the determination of epigallocatechin 3-gallate (EGCG), caffeine (CAF), and gallic acid (GA). In a diffusion cell chamber, the release rate of EGCG was 8896.01 µg cm-2. This data showed that EGCG will be able to exert its systemic activity when delivered though the transdermal formulation, due to its good flux rates with the synthetic membrane.

Control of transients in water distribution networks by H¥ Control

In this work, a controller for regulating the transients in water distribution networks is established. The control technique is the H¥ Control. The developed controller is applied to a water distribution network and the results of this application demonstrate that the technique allowed the establishment of a robust controller, capable of attenuating the disturbances in a suitable way, being effective in controlling the oscillations of the state variables in question.

Cortical and autonomic modulation of attentional control /

Whereas the role of the anterior cingulate cortex (ACC) in cognitive control has received considerable attention, much less work has been done on the role of the ACC in autonomic regulation. Its connections through the vagus nerve to the sinoatrial node of the heart are thought to exert modulatory control over cardiovascular arousal. Therefore, ACC is not only responsible for the implementation of cognitive control, but also for the dynamic regulation of cardiovascular activity that characterizes healthy heart rate and adaptive behaviour. However, cognitive control and autonomic regulation are rarely examined together. Moreover, those studies that have examined the role of phasic vagal cardiac control in conjunction with cognitive performance have produced mixed results, finding relations for specific age groups and types of tasks but not consistently. So, while autonomic regulatory control appears to support effective cognitive performance under some conditions, it is not presently clear just what factors contribute to these relations. The goal of the present study was, therefore, to examine the relations between autonomic arousal, neural responsivity, and cognitive performance in the context of a task that required ACC support. Participants completed a primary inhibitory control task with a working memory load embedded. Pre-test cardiovascular measures were obtained, and ontask ERPs associated with response control (N2/P3) and error-related processes (ERN/Pe) were analyzed. Results indicated that response inhibition was unrelated to phasic vagal cardiac control, as indexed by respiratory sinus arrhythmia (RSA). However, higher resting RSA was associated with larger ERN ampUtude for the highest working memory load condition. This finding suggests that those individuals with greater autonomic regulatory control exhibited more robust ACC error-related responses on the most challenging task condition. On the other hand, exploratory analyses with rate pressure product (RPP), a measure of sympathetic arousal, indicated that higher pre-test RPP (i.e., more sympathetic influence) was associated with more errors on "catch" NoGo trials, i.e., NoGo trials that simultaneously followed other NoGo trials, and consequently, reqviired enhanced response control. Higher pre-test RPP was also associated with smaller amplitude ERNs for all three working memory loads and smaller ampUtude P3s for the low and medium working memory load conditions. Thus, higher pretest sympathetic arousal was associated with poorer performance on more demanding "catch" NoGo trials and less robust ACC-related electrocortical responses. The findings firom the present study highlight tiie interdependence of electrocortical and cardiovascular processes. While higher pre-test parasympathetic control seemed to relate to more robust ACC error-related responses, higher pre-test sympathetic arousal resulted in poorer inhibitory control performance and smaller ACC-generated electrocortical responses. Furthermore, these results provide a base from which to explore the relation between ACC and neuro/cardiac responses in older adults who may display greater variance due to the vulnerabihty of these systems to the normal aging process.

A Beacon based Localization System for Autonomous Mobile Robots and its Applications in Traffic and Transport Control

ACCURATE sensing of vehicle position and attitude is still a very challenging problem in many mobile robot applications. The mobile robot vehicle applications must have some means of estimating where they are and in which direction they are heading. Many existing indoor positioning systems are limited in workspace and robustness because they require clear lines-of-sight or do not provide absolute, driftfree measurements.The research work presented in this dissertation provides a new approach to position and attitude sensing system designed specifically to meet the challenges of operation in a realistic, cluttered indoor environment, such as that of an office building, hospital, industrial or warehouse. This is accomplished by an innovative assembly of infrared LED source that restricts the spreading of the light intensity distribution confined to a sheet of light and is encoded with localization and traffic information. This Digital Infrared Sheet of Light Beacon (DISLiB) developed for mobile robot is a high resolution absolute localization system which is simple, fast, accurate and robust, without much of computational burden or significant processing. Most of the available beacon's performance in corridors and narrow passages are not satisfactory, whereas the performance of DISLiB is very encouraging in such situations. This research overcomes most of the inherent limitations of existing systems.The work further examines the odometric localization errors caused by over count readings of an optical encoder based odometric system in a mobile robot due to wheel-slippage and terrain irregularities. A simple and efficient method is investigated and realized using an FPGA for reducing the errors. The detection and correction is based on redundant encoder measurements. The method suggested relies on the fact that the wheel slippage or terrain irregularities cause more count readings from the encoder than what corresponds to the actual distance travelled by the vehicle.The application of encoded Digital Infrared Sheet of Light Beacon (DISLiB) system can be extended to intelligent control of the public transportation system. The system is capable of receiving traffic status input through a GSM (Global System Mobile) modem. The vehicles have infrared receivers and processors capable of decoding the information, and generating the audio and video messages to assist the driver. The thesis further examines the usefulness of the technique to assist the movement of differently-able (blind) persons in indoor or outdoor premises of his residence.The work addressed in this thesis suggests a new way forward in the development of autonomous robotics and guidance systems. However, this work can be easily extended to many other challenging domains, as well.

Optimizing Robust Quantum Gates in Open Quantum Systems

We are currently at the cusp of a revolution in quantum technology that relies not just on the passive use of quantum effects, but on their active control. At the forefront of this revolution is the implementation of a quantum computer. Encoding information in quantum states as “qubits” allows to use entanglement and quantum superposition to perform calculations that are infeasible on classical computers. The fundamental challenge in the realization of quantum computers is to avoid decoherence – the loss of quantum properties – due to unwanted interaction with the environment. This thesis addresses the problem of implementing entangling two-qubit quantum gates that are robust with respect to both decoherence and classical noise. It covers three aspects: the use of efficient numerical tools for the simulation and optimal control of open and closed quantum systems, the role of advanced optimization functionals in facilitating robustness, and the application of these techniques to two of the leading implementations of quantum computation, trapped atoms and superconducting circuits. After a review of the theoretical and numerical foundations, the central part of the thesis starts with the idea of using ensemble optimization to achieve robustness with respect to both classical fluctuations in the system parameters, and decoherence. For the example of a controlled phasegate implemented with trapped Rydberg atoms, this approach is demonstrated to yield a gate that is at least one order of magnitude more robust than the best known analytic scheme. Moreover this robustness is maintained even for gate durations significantly shorter than those obtained in the analytic scheme. Superconducting circuits are a particularly promising architecture for the implementation of a quantum computer. Their flexibility is demonstrated by performing optimizations for both diagonal and non-diagonal quantum gates. In order to achieve robustness with respect to decoherence, it is essential to implement quantum gates in the shortest possible amount of time. This may be facilitated by using an optimization functional that targets an arbitrary perfect entangler, based on a geometric theory of two-qubit gates. For the example of superconducting qubits, it is shown that this approach leads to significantly shorter gate durations, higher fidelities, and faster convergence than the optimization towards specific two-qubit gates. Performing optimization in Liouville space in order to properly take into account decoherence poses significant numerical challenges, as the dimension scales quadratically compared to Hilbert space. However, it can be shown that for a unitary target, the optimization only requires propagation of at most three states, instead of a full basis of Liouville space. Both for the example of trapped Rydberg atoms, and for superconducting qubits, the successful optimization of quantum gates is demonstrated, at a significantly reduced numerical cost than was previously thought possible. Together, the results of this thesis point towards a comprehensive framework for the optimization of robust quantum gates, paving the way for the future realization of quantum computers.