Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system

Lactic acid bacteria (LAB) are Gram-positive bacteria and are generally regarded as safe (GRAS) organisms. Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles. To develop such live vaccines, a better control of protein secretion is required. We developed an efficient secretion system in the model LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc) was used as the reporter protein. We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Secretion efficiency (SE) of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coronavirus (BCV) epitope-protein fusion (BCV-Nuc). BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

Simulation of wind powered hydraulic heating system

The objective of this master’s thesis was to design and simulate a wind powered hydraulic heating system that can operate independently in remote places where the use of electricity is not possible. Components for the system were to be selected in such a way that the conditions for manufacture, use and economic viability are the as good as possible. Savonius rotor was chosen for wind turbine, due to its low cut in speed and robust design. Savonius rotor produces kinetic energy in wide wind speed range and it can withstand high wind gusts. Radial piston pump was chosen for the flow source of the hydraulic heater. Pump type was selected due to its characteristics in low rotation speeds and high efficiency. Volume flow from the pump is passed through the throttle orifice. Pressure drop over the orifice causes the hydraulic oil to heat up and, thus, creating thermal energy. Thermal energy in the oil is led to radiator where it conducts heat to the environment. The hydraulic heating system was simulated. For this purpose a mathematical models of chosen components were created. In simulation wind data gathered by Finnish meteorological institute for 167 hours was used as input. The highest produced power was achieved by changing the orifice diameter so that the rotor tip speed ratio follows the power curve. This is not possible to achieve without using electricity. Thus, for the orifice diameter only one, the optimal value was defined. Results from the simulation were compared with investment calculations. Different parameters effecting the investment profitability were altered in sensitivity analyses in order to define the points of investment profitability. Investment was found to be profitable only with high average wind speeds.

Designing of Balanced Performance Measurement System to Purchasing Process

The performance measurement produces information about the operation of the business process. On the basis of this information performance of the company can be followed and improved. Balanced performance measurement system can monitor performance of several perspectives and business processes can be led according to company strategy. Major part of the costs of a company is originated from purchased goods or services are an output of the buying process emphasising the importance of a reliable performance measurement of purchasing process. In the study, theory of balanced performance measurement is orientated and framework of purchasing process performance measurement system is designed. The designed balanced performance measurement system of purchasing process is tested in case company paying attention to the available data and to other environmental enablers. The balanced purchasing performance measurement system is tested and improved during the test period and attention is paid to the definition and scaling of objectives. Found development initiatives are carried out especially in the scaling of indicators. Finally results of the study are evaluated, conclusions and additional research areas proposed.

JNK regulates dendrite and spine architecture in the central nervous system influencing spatial learning and motor tasks

JNK1 is a MAP-kinase that has proven a significant player in the central nervous system. It regulates brain development and the maintenance of dendrites and axons. Several novel phosphorylation targets of JNK1 were identified in a screen performed in the Coffey lab. These proteins were mainly involved in the regulation of neuronal cytoskeleton, influencing the dynamics and stability of microtubules and actin. These structural proteins form the dynamic backbone for the elaborate architecture of the dendritic tree of a neuron. The initiation and branching of the dendrites requires a dynamic interplay between the cytoskeletal building blocks. Both microtubules and actin are decorated by associated proteins which regulate their dynamics. The dendrite-specific, high molecular weight microtubule associated protein 2 (MAP2) is an abundant protein in the brain, the binding of which stabilizes microtubules and influences their bundling. Its expression in non-neuronal cells induces the formation of neurite-like processes from the cell body, and its function is highly regulated by phosphorylation. JNK1 was shown to phosphorylate the proline-rich domain of MAP2 in vivo in a previous study performed in the group. Here we verify three threonine residues (T1619, T1622 and T1625) as JNK1 targets, the phosphorylation of which increases the binding of MAP2 to microtubules. This binding stabilizes the microtubules and increases process formation in non-neuronal cells. Phosphorylation-site mutants were engineered in the lab. The non-phosphorylatable mutant of MAP2 (MAP2- T1619A, T1622A, T1625A) in these residues fails to bind microtubules, while the pseudo-phosphorylated form, MAP2- T1619D, T1622D, Thr1625D, efficiently binds and induces process formation even without the presence of active JNK1. Ectopic expression of the MAP2- T1619D, T1622D, Thr1625D in vivo in mouse brain led to a striking increase in the branching of cortical layer 2/3 (L2/3) pyramidal neurons, compared to MAP2-WT. The dendritic complexity defines the receptive field of a neuron and dictates the output to the postsynaptic cells. Previous studies in the group indicated altered dendrite architecture of the pyramidal neurons in the Jnk1-/- mouse motor cortex. Here, we used Lucifer Yellow loading and Sholl analysis of neurons in order to study the dendritic branching in more detail. We report a striking, opposing effect in the absence of Jnk1 in the cortical layers 2/3 and 5 of the primary motor cortex. The basal dendrites of pyramidal neurons close to the pial surface at L2/3 show a reduced complexity. In contrast, the L5 neurons, which receive massive input from the L2/3 neurons, show greatly increased branching. Another novel substrate identified for JNK1 was MARCKSL1, a protein that regulates actin dynamics. It is highly expressed in neurons, but also in various cancer tissues. Three phosphorylation target residues for JNK1 were identified, and it was demonstrated that their phosphorylation reduces actin turnover and retards migration of these cells. Actin is the main cytoskeletal component in dendritic spines, the site of most excitatory synapses in pyramidal neurons. The density and gross morphology of the Lucifer Yellow filled dendrites were characterized and we show reduced density and altered morphology of spines in the motor cortex and in the hippocampal area CA3. The dynamic dendritic spines are widely considered to function as the cellular correlate during learning. We used a Morris water maze to test spatial memory. Here, the wild-type mice outperformed the knock-out mice during the acquisition phase of the experiment indicating impaired special memory. The L5 pyramidal neurons of the motor cortex project to the spinal cord and regulate the movement of distinct muscle groups. Thus the altered dendrite morphology in the motor cortex was expected to have an effect on the input-output balance in the signaling from the cortex to the lower motor circuits. A battery of behavioral tests were conducted for the wild-type and Jnk1-/- mice, and the knock-outs performed poorly compared to wild-type mice in tests assessing balance and fine motor movements. This study expands our knowledge of JNK1 as an important regulator of the dendritic fields of neurons and their manifestations in behavior.

Finance-Led Growth Regime no Brasil: estatuto teórico, evidências empíricas e consequências macroeconômicas

This paper analyzes the Brazilian growth pattern during the post-liberalization period, emphasizing the structural links between finance and productive capital accumulation. The results indicate a finance-led growth regime in the period 2004-2008, under a very specific financialization process. The first part is a survey of the international literature, which defines the financialization concept and its relevance for understanding Brazilian economic problems. The next part provides a historical overview on the structural changes that made possible the development of financial-led regimes. The paper also applies an empirical analysis of some selected Brazilian macroeconomic indicators.

New insights into small molecules inhibitors and protein-protein interactions of VirB8 : a critical conserved component of the type IV secretion system.

Les systèmes bactériens de sécrétion de type IV (T4SS) sont constitués d’un ensemble de 8 à 12 protéines conservées. Ces dernières sont utilisées lors de la translocation de protéines, la translocation de complexes ADN-protéines mais aussi pour le transport de ces derniers au travers de la membrane cellulaire. Les T4SS, en tant que facteurs de virulence pour beaucoup de pathogènes comme Brucella suis, sont donc d’excellents modèles cibles pour le développement de médicaments d’antivirulence. Ces médicaments, en privant le pathogène de son facteur essentiel de virulence : le T4SS, constituent une alternative ou encore une amélioration des traitements antibiotiques utilisés actuellement. VirB8, un facteur d’assemblage conservé dans le T4SS, forme des dimères qui sont importants pour la fonction des T4SS dans ces pathogènes. De par ses interactions multiples, VirB8 est un excellent modèle pour l’analyse des facteurs d’assemblage mais aussi en tant que cible de médicaments qui empêcheraient son interaction avec d’autres protéines et qui, in fine, désarmeraient les bactéries en les privant de leur fonctions essentielles de virulence. À ce jour, nous savons qu’il existe un équilibre monomère-dimère et un processus d’homodimerization de VirB8 dont l’importance est vitale pour la fonctionnement biologique des T4SSs. En se basant sur des essais quantitatifs d’interaction, nous avons identifié (i) des sites potentiels d’interaction avec d’autres protéines VirB du T4SS mais aussi (ii) isolé des petites molécules inhibitrices afin de tester la fonction protéique de VirB8. Afin de déterminer les acides aminés importants pour l’hétérodimérization de VirB8 avec VirB10, nous avons effectué des expériences de mutagenèse aléatoire, de phage display et d’arrimage moléculaire in silico. Ces expériences ont démontré l’importance de trois acides aminés localisés sur le feuillet β : R160, S162, T164 et I165. Ces derniers seraient importants pour l’association de VirB8 avec VirB10 étant donné que leur mutagenèse entraine une diminution de la formation du complexe VirB8-VirB10. L’objectif actuel de notre projet de recherche est de pouvoir mieux comprendre mais aussi d’évaluer le rôle de VirB8 dans l’assemblage du T4SS. Grace à un méthode de criblage adaptée à partir de la structure de VirB8, nous avons pu identifié une petite molécule inhibitrice BAR-068, qui aurait un rôle prometteur dans l’inhibition du T4SS. Nous avons utilisé la spectroscopie par fluorescence, l’essai à deux hybrides, le cross-linking et la cristallographie afin de déterminer le mécanisme d'interaction existant entre VirB8 et BAR-068. Ces travaux pourraient permettre de nombreuses avancées, notamment en termes de compréhension des mécanismes d’inhibition du T4SS. Notre objectif ultime est de pouvoir caractériser la séquence d’évènements essentiels à l’assemblage et au fonctionnement du T4SS. De manière globale, notre projet de recherche permettrait de révéler les grands principes d’assemblage des protéines membranaires, les processus de sécrétion de protéines chez les bactéries mais aussi de proposer une nouvelle stratégie lors du développement de drogues antimicrobiennes.

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.

Design and development of an LED based fiber optic evanescent wave sensor for simultaneous detection of chromium and nitrite traces in water

The design and development of a cost-effective, simple, sensitive and portable LED based fiber optic evanescent wave sensor for simultaneously detecting trace amounts of chromium and nitrite in water are presented. In order to obtain the desired performance, the middle portions of two multimode plastic clad silica fibers are unclad and are used as the sensing elements in the two arms of the sensor. Each of the sensor arms is sourced by separate super bright green LEDs, which are modulated in a time-sharing manner and a single photo detector is employed for detecting these light signals. The performance and characteristics of this system clearly establish the usefulness of the technique for detecting very low concentrations of the dissolved contaminants.

Jmodels: Java models of earth system regulation

The JModel suite consists of a number of models of aspects of the Earth System. They can all be run from the JModels website. They are written in the Java language for maximum portability, and are capable of running on most computing platforms including Windows, MacOS and Unix/Linux. The models are controlled via graphical user interfaces (GUI), so no knowledge of computer programming is required to run them. The models currently available from the JModels website are: Ocean phosphorus cycle Ocean nitrogen and phosphorus cycles Ocean silicon and phosphorus cycles Ocean and atmosphere carbon cycle Energy radiation balance model (under development) The main purpose of the models is to investigate how material and energy cycles of the Earth system are regulated and controlled by different feedbacks. While the central focus is on these feedbacks and Earth System stabilisation, the models can also be used in other ways. These resources have been developed by: National Oceanography Centre, Southampton project led by Toby Tyrrell and Andrew Yool, focus on how the Earth system works.

Colombian Medical Education System: A Hundred Years Behind

Almost one hundred years ago the Carnegie Foundation for the Advancement of Teaching authorized a study and report about the medical education of the United States and Canada directed by Mr. Abraham Flexner an education expert of the time. This report turned out to be one of the most important documents of the medical education revolution that took place by that time in North America and that led it to become what it is today. Almost a century after that, Colombian medical education has reached an outstanding similarity to the system described in the Flexner report. The present article highlights the parallel between North America’s medical education situation a hundred years ago and Colombia’s actual medical education situation. We present here some notions about the actual education system based on what was described on 1910 and which we consider, constitutes the current medical education situation on our country and possibly on many Latin American countries.

Phosphorus dynamics in the ditch system of a restored peat wetland

The restoration of wetlands as bird habitats often involves the maintenance of a fluctuating water regime by careful, localised ditch water management using pumps and sluices. However, there is evidence in the literature to Suggest that alternate flood/drainage cycles can accelerate nutrient cycling and transport within the soil and, therefore, pose a threat to water quality through the process of eutrophication. This study focused on the dynamics and losses of soil P in a recently re-wetted, eutrophic fen peat developed on alluvium ill South west England. During the 2-year Study (2001 and 2002), soil water tensiometry revealed that the field water table (fluctuating annually between +20 and 60 cm relative to ground level) was extensively influenced across most of the 8.4 ha field site by the management of the adjacent ditch water levels. This conservation-led, prescribed water balance was facilitated by the high hydraulic conductivity (1.1 x 10(-s) ms(-1)) of the lower (70-140 cm), degraded layer of peat. However, only during a 7-day period of water table drawdown by intermittent pump drainage, approximately 45 g ha(-1) of dissolved reactive P (DRP) entered the pumped ditch from the field via this degraded layer. Summer rainfall events >35 mm d(-1) also coincided with significant peaks ill ditch water P concentration (up to 200 mu g L-1 DRP). Even larger peaks (Up to 700 mu g L-1 DRP) Occurred With the annual onset of autumn reflooding. These episodic P loss events pose a serious potential threat to biological water quality. (C) 2009 Elsevier B.V. All rights reserved.

Hygiene assessment system (HAS) scores: an analysis of the available data from English slaughterhouses

As control systems have developed and the implications of poor hygienic practices have become better known, the evaluation of the hygienic status of premises has become more critical. The assessment of the overall status of premises hygiene call provide useful management data indicating whether the premises are improving or whether, whilst still meeting legal requirements, they might be failing to maintain previously high standards. Since the creation, for the United Kingdom, of the meat hygiene service (MHS), one of the aims of the service was to monitor hygiene on different premises to provide a means of comparing standards and to identify and encourage improvements. This desire led to the implementation of a scoring system known as the hygiene assessment system (HAS). This paper analyses English slaughterhouses HAS scores between 1998 and 2005 outlining the main incidents throughout this period, Although rising initially, the later results displayed a clear decrease in the general hygiene scores. These revealing results coincide with the start of a new meat inspection system where, after several years of discussion, risk based inspection is finally coming to a reality within Europe. The paper considers the implications of these changes in the way hygiene standards will be monitored in the future.

The Spanish system of food controls - its administration and enforcement

Following a number of major food safety problems in Europe, including in particular the issues of BSE and dioxin, consumers have become increasingly concerned about food safety. This has led authorities in Europe to revise their systems of food control. The establishment of the European Food Safety Authority (EFSA) is one of the main structural changes made at the moment within the European Union, and similar action at national level has been or is being taken by many EU member states. In Spain a law creating the Spanish Agency of Food Safety has been approved. This has general objectives that include the promotion of food security and offering guarantees and the provision of objective information to consumers and food businesses in the Spanish agrifood sector. This paper reviews the general structure of the current food control system in Spain. At a national level this involves three different Ministries. Spain however also has a devolved system involving Autonomous Communities the paper considers Castilla y Leon as an example. In conclusion the paper recognises that Spain has a complex system for food control. and considers that it will take time before a full evaluation of the new system is possible. (C) 2003 Elsevier Ltd. All rights reserved.

Requirements analysis for an e-government system to support multi-organisational collaborative groups

We present a conceptual architecture for a Group Support System (GSS) to facilitate Multi-Organisational Collaborative Groups (MOCGs) initiated by local government and including external organisations of various types. Multi-Organisational Collaborative Groups (MOCGs) consist of individuals from several organisations which have agreed to work together to solve a problem. The expectation is that more can be achieved working in harmony than separately. Work is done interdependently, rather than independently in diverse directions. Local government, faced with solving complex social problems, deploy MOCGs to enable solutions across organisational, functional, professional and juridical boundaries, by involving statutory, voluntary, community, not-for-profit and private organisations. This is not a silver bullet as it introduces new pressures. Each member organisation has its own goals, operating context and particular approaches, which can be expressed as their norms and business processes. Organisations working together must find ways of eliminating differences or mitigating their impact in order to reduce the risks of collaborative inertia and conflict. A GSS is an electronic collaboration system that facilitates group working and can offer assistance to MOCGs. Since many existing GSSs have been primarily developed for single organisation collaborative groups, even though there are some common issues, there are some difficulties peculiar to MOCGs, and others that they experience to a greater extent: a diversity of primary organisational goals among members; different funding models and other pressures; more significant differences in other information systems both technologically and in their use than single organisations; greater variation in acceptable approaches to solve problems. In this paper, we analyse the requirements of MOCGs led by local government agencies, leading to a conceptual architecture for an e-government GSS that captures the relationships between 'goal', 'context', 'norm', and 'business process'. Our models capture the dynamics of the circumstances surrounding each individual representing an organisation in a MOCG along with the dynamics of the MOCG itself as a separate community.

Rifaximin modulates the colonic microbiota of patients with Crohn's disease: an in vitro approach using a continuous culture colonic model system.

Rifaximin, a rifamycin derivative, has been reported to induce clinical remission of active Crohn's disease (CD), a chronic inflammatory bowel disorder. In order to understand how rifaximin affects the colonic microbiota and its metabolism, an in vitro human colonic model system was used in this study. We investigated the impact of the administration of 1800 mg/day of rifaximin on the faecal microbiota of four patients affected by colonic active CD [Crohn's disease activity index (CDAI > 200)] using a continuous culture colonic model system. We studied the effect of rifaximin on the human gut microbiota using fluorescence in situ hybridization, quantitative PCR and PCR–denaturing gradient gel electrophoresis. Furthermore, we investigated the effect of the antibiotic on microbial metabolic profiles, using 1H-NMR and solid phase microextraction coupled with gas chromatography/mass spectrometry, and its potential genotoxicity and cytotoxicity, using Comet and growth curve assays. Rifaximin did not affect the overall composition of the gut microbiota, whereas it caused an increase in concentration of Bifidobacterium, Atopobium and Faecalibacterium prausnitzii. A shift in microbial metabolism was observed, as shown by increases in short-chain fatty acids, propanol, decanol, nonanone and aromatic organic compounds, and decreases in ethanol, methanol and glutamate. No genotoxicity or cytotoxicity was attributed to rifaximin, and conversely rifaximin was shown to have a chemopreventive role by protecting against hydrogen peroxide-induced DNA damage. We demonstrated that rifaximin, while not altering the overall structure of the human colonic microbiota, increased bifidobacteria and led to variation of metabolic profiles associated with potential beneficial effects on the host.