In this study, potential of high hydrostatic pressure (HHP) and essential oils (EOs) as natural antimicrobials was evaluated to produce E. coli safe and quality beef product. First, the individual and combined effects of antimicrobial activity (minimum inhibitory concentration) of basil, black cumin, cilantro, cumin, fenugreek, ginger, oregano, black pepper, rosemary, thyme, turmeric oil emulsions on E. coli ATCC 25922 with and without HHP treatment were evaluated. Cumin, oregano and thyme EOs showed highest antimicrobial activity against E. coli ATCC 25922. The synergy of selected EOs against E. coli ATCC 25922 was determined using the checkerboard method to obtain fractional inhibitory concentration index. Although their combinations did not show synergy, they expressed synergy when combined with HHP (400 MPa, 10 min, 20 °C) and the best combination was cumin and oregano EOs with HHP. Effects of HHP and EO combinations on inactivation of E. coli ATCC 25922 in beef were investigated using response surface methodology (RSM). Statistical analysis showed the model was significant for predicting log reduction with high accuracy. The significant model terms were pressure and time. Compared to control, HHP/EO treated samples showed no-post growth when stored up to 120 days at 4°C. Presented results suggests that the combination of HHP and antimicrobials has not only improved the process parameters (lowered pressure, time, and EO concentration) but also prevented recovery of E. coli ATCC 25922 during storage. RSM was employed to analyze the synergistic effects of HHP and EOs on beef quality (color, texture and lipid oxidation). Color indices were significantly affected by pressure, time and their interactions. Above 400 MPa the discoloration was similar to cooked beef and EO addition did not help color improvement. However, EOs showed significant antioxidant activity on both treated and untreated samples during storage. In conclusion, there is a great potential of HHP and EO combinations to enhance pathogen inactivation while keeping the quality of beef. Moreover, presence of EOs can prolong the shelf life of pressure treated beef. Therefore, the combination of HHP and EO is very promising for meat industry. / Ph. D. / Meat is a natural source of protein, essential vitamins, which makes it a nutrient-rich source of a healthy diet as well as an ideal environment for food-borne pathogens and spoilage bacteria. It is therefore essential to preserve very perishable meat products in terms of microbial contamination. As an alternative to many preservation methods such as chilling, canning, curing, smoking, dehydrating and heat treatment, a non-thermal mild food preservation technology of high hydrostatic pressure processing (HHP) is proposed for inactivating the most common meat contaminant bacteria of <i>E. coli</i>. Essential oils (EOs) can provide a solution for pasteurization requirements and reducing quality losses associated with HHP treatment. In this study the synergistic effect of selected EOs (basil, black cumin, cilantro, cumin, fenugreek, ginger, oregano, black pepper, rosemary, thyme, turmeric oil emulsions) and HHP technology on inactivation of <i>E. coli</i> ATCC 25922 on contaminated meat cuts were investigated. Experimental design and statistical analysis were conducted using response surface methodology (RSM). Combination of HHP/EO treated samples showed no-post growth of <i>E. coli</i> ATCC 25922 when meat samples were stored up to 120 days at 4°C. Presented results are suggesting that HHP in combination with EOs has increased the log reduction of <i>E.coli</i> and as well, decreased the quality losses (color, lipid oxidation textural analysis) compared to control samples where HHP is applied alone. As a conclusion, this study shows that there is a great potential of HHP and EO combinations to enhance pathogen inactivation while keeping the quality of beef.

This diploma deals is occupied with design of the heavy rotary table with hydrostatic bearing of table. In introductory part of deals the design part are described, for exampel. tipes of bearings, drive and etc. In other parts the design of rotary table with hydrostatic bearings is described.

This diploma thesis deals with an optimization of hydrostatic slide of the large-portal gantry machine FRU (TOS Kuřim). First part of the thesis concerns with an analysis of the current practice in hydrostatic guides in the field of heavy gantry machines, an usage of alternative materials for a construction of carrying parts of machines, and possibilities of current simulation methods. Aim of the second part of this thesis is a brief clarification of the principle of mathematical optimization, and creation of general methodology of construction optimization by means of system analysis. Optimization calculations were processed by ANSYS Workbench 11.0. There is achieved adjusted and optimized design of the hydrostatic slide as the result of the thesis. Moreover, the comparison of key deformations was added. Created methodology is applicable in both an area of construction of gantry machines, and a wide spectrum of construction fields.

The diploma thesis deals with design of an experimental test bench for recovery energy simulation at machines with hydrostatic energy transmission. This work describes choice of connection and selection of hydrostatic and other components. A first part of the thesis deals with calculation and design of the flywheel whose is its rotational kinetic energy corresponds in a certain scale to translational kinetic energy of the experimental vehicle. The thesis outlines the efficiency of transformation of rotational kinetic energy to pressure energy its accumulation and subsequent conversion back to rotational kinetic energy. Precise determination of the effectiveness of hydrostatic recovery is one of the objectives of experiments that will be implemented at the test bench. The task of the measurement and physical-technical analysis to be conducted at the manufactured test bench is primarily to obtain data at for the precise control of the hydrostatic energy recovery system to maximize its efficiency.

In the current study a variable speed pump controlled hydrostatic circuit where an underlapped shuttle valve is utilized to compensate the unequal flow rate of a single rod actuator is analyzed. Parameters of the shuttle valve are included in the system analysis, rather than treating it as an ideal switching element as handled in literature. A linearized model of the system is obtained. An inverse kinematic model, which calculates the required pump drive speed for a desired actuator speed and given pilot pressure input, is formed. A numerical stability analysis program is developed, and the stability of all possible shuttle valve spool positons is determined. The theoretical findings are validated by non-linear simulation model responses.

Wave Energy Converters (WEC) are used to transform energy stored in ocean waves into electrical energy. One type of WECs consists of buoyant bodies. To extract energy from their motion, hydraulic cylinders can be used to generate hydraulic power. For conversion into electric power various systems have been analysed in literature. However, the focus was put on efficiency and rigorous analyses of the system behaviour are still missing. In this paper an exemplary system consisting of two hydraulic cylinders, switchable check valves, accumulators and three motor-generator sets is analysed with help of simulation and measurement. This exemplary system is called WavePOD and was installed at the Institute for Fluid Power Drives and Controls (IFAS) of RWTH Aachen University together with Aquamarine Power and Bosch Rexroth for testing. In this paper the data collected during various test phases is used for system analysis and for validating the simulation. The simulation model is presented. The system’s response to various switching operations is investigated. Comparing the simulation with measurements validates the system`s dynamic model.

Presented in this study is a wide-ranging investigation into the use of properties of balance in an operational numerical weather prediction context. Initially, a joint numerical and observational study is undertaken. We used the Unified Model (UM), the suite of atmospheric and oceanic prediction software used at the UK Met Office (UKMO), to locate symmetric instabilities (SIs), an indicator of imbalanced motion. These are areas of negative Ertel potential vorticity (in the Northern hemisphere) calculated on surfaces of constant potential temperature. Once located, the SIs were compared with satellite and aircraft observational data. As a full three-dimensional calculation of Ertel PV proved outwith the scope of this study we calculated the two-dimensional, vertical component of the absolute vorticity, to assess the inertial stability criterion. We found that at the synoptic scale in the atmosphere, if there existed a symmetric instability, it was dominated by an inertial instability. With the appropriate observational data, evidence of inertial instability from the vertical component of the absolute vorticity, predicted by the UM was found at 12km horizontal grid resolution. Varying the horizontal grid resolution allowed the estimation of a grid length scale, above which, the inertial instability was not captured by the observational data, of approximately 20km. Independently, aircraft data was used to estimate that horizontal grid resolutions above 20-25km should not model any features of imbalance providing a real world estimate of the lower bound of the grid resolution that should be employed by a balanced atmospheric prediction model. A further investigation of the UM concluded that the data assimilation scheme and time of initialisation had no effect on the generation of SIs. An investigation was then made into the robustness of balanced models in the shallow water context, employing the contour-advective semi-Lagrangian (CASL) algorithm, Dritschel & Ambaum (1997), a novel numerical algorithm that exploits the underlying balance observed within a geophysical flow at leading order. Initially two algorithms were considered, which differed by the prognostic variables employed. Each algorithm had their three-time-level semi-implicit time integration scheme de-centred to mirror the time integration scheme of the UM. We found that the version with potential vorticity (PV), divergence and acceleration divergence, CA[subscript(δ,γ)], as prognostic variables preserved the Bolin-Charney balance to a much greater degree than the model with PV, divergence and depth anomaly CA[subscript(tilde{h},δ)], as prognostic variables. This demonstrated that CA[subscript(δ,γ)] was better equipped to benefit from de-centring, an essential property of any operational numerical weather prediction (NWP) model. We then investigate the robustness of CA[subscript(δ,γ)] by simulating flows with Rossby and Froude number O(1), to find the operational limits of the algorithm. We also investigated increasing the efficiency of CA[subscript(δ,γ)] by increasing the time-step Δt employed while decreasing specific convergence criteria of the algorithm while preserving accuracy. We find that significant efficiency gains are possible for predominantly mid-latitude flows, a necessary step for the use of CA[subscript(δ,γ)] in an operational NWP context. The study is concluded by employing CASL in the non-hydrostatic context under the Boussinesq approximation, which allows weak stratification to be considered, a step closer to physical reality than the shallow water case. CASL is compared to the primitive equation pseudospectral (PEPS) and vorticity-based pseudospectral (VPS) algorithms, both as the names suggest, spectral-based algorithms, which again differ by the prognostic variables employed. This comparison is drawn to highlight the computational advantages that CASL has over common numerical methods used in many operational forecast centres. We find that CASL requires significantly less artificial numerical diffusion than its pseudospectral counterparts in simulations of Rossby number ~O(1). Consequently, CASL obtains a much less diffuse, more accurate solution, at a lower resolution and therefore lower computational cost. At low Rossby number, where the flow is strongly influence by the Earth's rotation, it is found that CASL is the most cost-effective method. In addition, CASL also preserves a much greater proportion of balance, diagnosed with nonlinear quasigeostrophic balance (NQG), another significant advantage over its pseudospectral counterparts.

A partir de medidas isotérmicas e isobáricas de susceptibilidade magnética diferencial, determinamos o diagrama de fases magnéticas do metamagneto Ni(NO3)2.2H2O, para pressões hidrostáticas de até cerca de 11 kbar. Determinamos o campo de transição à temperatura nula Hc(0,P) e à temperatura de Néel, ambos apresentando um crescimento aproximadamente linear com P. A partir de efeitos de desmagnetização, pudemos determinar o ponto de união das linhas de primeira e segunda ordem de cada diagrama, e para P=0 pudemos caracterizá-lo com um ponto tricrítico, com temperatura tricrítica Tt=3.70K. Observamos ainda a existência de uma transição metamagnética induzida por pressão para valores do intervalo de 0 a 8 kbar. Usando a Aproximação de Campo Médio num modelo elástico aproximado para metamagnetos com S=1, pudemos obter expressões para TN(P) e Hc(0,P), ambas apresentando uma dependência linear em P, em bom acordo com os dados experimentais. Usando este modelo obtivemos também uma expressão analítica para Tt(P). Uma vez que TN(P), Hc(0,P) e Tt(P) dependem dos parâmetros de \"exchange\", pudemos determinar a dependência de tais parâmetros com a pressão. Também através deste modelo simples, pudemos compreender que a transição metamagnética induzida por pressão está provavelmente associada a uma forte dependência do campo cristalino com a pressão / From isothermical and isobaric differential magnetic susceptibility measurements we have determined the magnetic phase diagram of the metamagnetic system Ni(NO3)2.2H2O, for several hydrostatic pressures up to 11 kbar. The zero temperature transition field Hc(0,P) and the Néel temperature TN(P) were determined, both increasing almost linearly with P. From demagnetizing effects we were able to determine the joint point of the first and second order lines of each diagram, which for P=0 is a tricritical point, with tricritical temperature Tt=3.70K. We also observed a pressure induced metamagnetic transition in the range from 0 to 8 kbar. Using Mean Field Theory in an approximated elastic S=1 metamagnetic model, we could obtain expressions for TN(P) and Hc(0,P), both showing linear dependence on P, in agreement with experimental data. We could also obtain, using this model, an analytical expression for Tt(P). Once TN(P), Hc(0,P) and Tt(P) are dependent on the exchange parameters, we cou1d obtain the pressure dependence of these parameters. From this simple model we could understand that the pressure induced metamagnetic transition is probably associated with a strong variation of the crystalline field with pressure

No presente trabalho estudamos a renaturação sob alta pressão hidrostática de uma forma mutante da proteína verde fluorescente (enhanced GFP, eGFP), a qual somente emite fluorescência característica quando enovelada na sua forma nativa. A abordagem do presente estudo foi focada no controle da bioatividade da proteína recombinante, a fluorescência, como alternativa à determinação de solubilidade da proteína, fator que não é um indicador ideal de enovelamento proteico adequado. A ação da alta pressão na solubilização dos corpos de inclusão (CI) de eGFP produzidos em bactérias E. coli recombinantes e no enovelamento da proteína foi estudada. A compressão dos CI de eGFP em 2,4 kbar durante 30 minutos promoveu a dissociação dos agregados. No entanto, a incubação nesta condição não favoreceu o enovelamento da eGFP. O processo de renaturação foi avaliado em diversas condições de descompressão após a dissociação em 2,4 kbar. Durante a descompressão gradual, o aumento da fluorescência foi obtido em pressões que variaram entre a pressão atmosférica e 1,38kbar. Os níveis mais elevados de fluorescência de eGFP foram obtidos por incubação durante várias horas a níveis de pressão entre 0,35 e 0,69 kbar. Esta condição de pressão se mostrou favorável à renaturação de eGFP e é possível que também possa ser utilizada para favorecer o enovelamento de outras proteínas monoméricas. Ainda utilizando a eGFP como modelo, verificamos que os CI desta proteína produzidos por bactérias cultivadas em menor temperatura (37ºC) possuem maior quantidade de proteína recombinante apresentando a fluorescência característica em 509 nm, ou seja, na sua forma nativa, do que os CI expressos em temperaturas mais elevadas (42ºC e 47ºC). A análise realizada por espectroscopia de infravermelho (FT-IR) também demonstrou que os CI produzidos em temperaturas mais brandas possuem maior grau de estruturas secundárias semelhantes às da proteína na sua forma nativa. Além disso, os CI produzidos a 37ºC também são mais facilmente solubilizados pela ação da alta pressão do que aqueles produzidos em maior temperatura. Conforme esperado, a renaturação da eGFP a partir de CI produzidos a 37ºC foi 25 vezes mais eficiente do que a obtida utilizando CI produzidos a 47ºC. No presente estudo demonstramos também que a dissociação dos agregados exercida pela ação da alta pressão (2,4 kbar) pode ser amplificada quando em associação com a incubação em baixa temperatura (-9ºC) e que a combinação destas duas propriedades físicas eleva a solubilização dos agregados em CI, com a consequente elevação dos rendimentos de renaturação de eGFP. Mostramos ainda no presente estudo que a cinética de renaturação de eGFP em 0,69 kbar é proporcional à temperatura de incubação (entre 10ºC e 50ºC). O nível mais elevado de fluorescência foi obtido quando a renaturação de eEGP foi realizada a 20ºC. A taxa de maturação do cromóforo da eGFP é mais fortemente afetada pela temperatura do que a taxa de enovelamento da proteína. Em conclusão, a temperatura de produção dos CI, a temperatura de dissociação dos agregados e a temperatura de enovelamento podem afetar muito o rendimento e a cinética da renaturação de eGFP em alta pressão. Os resultados do presente estudo podem abrir novas perspectivas para melhorias no processo de enovelamento de proteínas a partir de CI utilizando alta pressão. Também neste trabalho descrevemos a renaturação das proteínas de Xac, PilB e os produtos dos genes XAC2810 e XAC3272 nunca antes obtidas na forma solúvel. Os rendimentos de solubilização destas três proteínas foram muito altos, entre 75% e 89%. A proteína PilB renaturada em alta pressão apresentou atividade ATPasica elevada, o que nunca antes foi demonstrado para a PilB de Xac. / In the present work we studied the refolding under high hydrostatic pressure of a mutant form of the green fluorescent protein (eGFP), which only emits the green characteristic fluorescence when in the native folded state. The approach of the present study was focused on controlling the bioactivity of the recombinant protein, the fluorescence, as an alternative for the determination of protein solubility, which is not an ideal indicator of proper protein folding. We studied the action of high pressure in the solubilization of the inclusion bodies (IB) of eGFP produced in bacteria E. coli and in the folding of this protein. The compression of a suspension of eGFP IB at 2.4 kbar for 30 minutes promoted dissociation of aggregates. However, the eGFP folding, monitored by the fluorescence at 509 nm, does not occur in this pressure level. The process of eGFP refolding was evaluated under various decompression conditions after dissociation of the IB at 2.4 kbar. During the gradual decompression, the increase in fluorescence was achieved at pressures ranging between atmospheric pressure and 1.38 kbar. The higher levels of eGFP fluorescence were obtained by incubation for several hours at pressure levels between 0.35 and 0.69 kbar. It is possible that the pressure condition that proved favorable for refolding of eGFP can also be used to favor the folding of other monomeric proteins. Using eGFP as a model, we also found that the IB produced by bacteria grown in a relatively low temperature (37ºC) is more fluorescent, presenting a higher amount of recombinant protein with the characteristic fluorescence at 509 nm, i.e., in its native form, than the IB expressed at higher temperatures (42ºC and 47ºC). The analysis by infrared spectroscopy (FT-IR) also demonstrated that the IB produced at milder temperatures have a higher degree of secondary structure similar to the protein in its native form. Furthermore, the IB produced at 37ºC are also more readily solubilized by the action of high pressure than those produced at the higher temperatures. As expected, the folding of eGFP from IB produced at 37ºC was 25 times more efficient than that obtained using IB produced at 47ºC. In this study we demonstrated that the dissociation of aggregates exerted by the action of high pressure (2.4 kbar) can be amplified by combination with incubation at low temperature (-9ºC) and the association of these two physical properties can be used to increase the solubilization of the aggregates in IB, with a consequent increase in the yield of eGFP refolding. In the present study we also showed that the kinetics of refolding of eGFP is proportional to temperature (10ºC 50ºC). The higher level of fluorescence was obtained when the refolding of eGFP was performed at 20°C. The rate of maturation of the eGFP chromophore is more strongly affected by temperature than the rate of folding of the protein. In conclusion, the temperature of production of IB, the temperature of dissociation of aggregates and the folding temperature can greatly affect the yield and kinetics of refolding of eGFP at high pressure. The results of this study may open new perspectives for improvements in the process of protein folding from IB using high pressure. In this paper we also describe the refolding of the proteins of Xac, PilB and the gene products XAC2810 and XAC3272, which have never before been achieved in soluble form. The yields of solubilization/refolding of these three proteins were very high, between 75% and 89%. The protein PilB refolded at high pressure presented high ATPase activity, which has never been shown for the PilB of Xac.

A produção de proteínas recombinantes é uma ferramenta essencial para a indústria biotecnológica e suporta a expansão da pesquisa biológica moderna. Uma variedade de hospedeiros pode ser utilizada para produzir estas proteínas e dentre eles, as bactérias E. coli são as hospedeiras mais utilizadas. No entanto, a expressão heteróloga de genes em E. coli frequentemente resulta em um processo de enovelamento incompleto que leva ao acúmulo de agregados insolúveis, conhecidos como corpos de inclusão (CI). Altas pressões hidrostáticas são capazes de desfavorecer interações intermoleculares hidrofóbicas e eletrostáticas, levando à dissociação dos agregados e por isso são úteis para solubilizar e renaturar proteínas agregadas em CI. O presente trabalho teve como objetivo o estudo do processo de desagregação dos CI e de renaturação das proteínas oligoméricas subunidade B da toxina colérica (CTB) e região globular da fibra adenoviral (RGFA) utilizando altas pressões hidrostáticas. A toxina colérica (CT) é composta por uma subunidade A e cinco subunidades B combinadas em uma holotoxina AB5. A CTB é a porção pentamérica não tóxica da CT, responsável pela ligação da holotoxina ao receptor gangliosídeo GM1. A fibra do adenovírus é uma proteína homotrimérica que forma parte do capsídeo viral, organizada em três regiões: a cauda N-terminal, a haste central e a região C-terminal (região globular). A RGFA se liga à proteína de membrana CAR nas células hospedeiras e promove a internalização do vírus. Os estudos apresentados neste trabalho demonstraram que a alta pressão hidrostática foi eficaz na desagregação dos CI da CTB e da RGFA. As condições de renaturação foram otimizadas utilizando-se diferentes proporções do par redox glutationa oxidada e reduzida, concentrações de agentes caotrópicos, presença de aditivos e esquemas diferenciados de compressão/descompressão daqueles previamente descritos na literatura. CTB solúvel e pentamérica foi obtida pela compressão da suspensão de CI a 2,4 kbar por 16 horas em tampão TrisHCl 50 mM pH 8,5, 1 mM de tween 20 e descompressão direta seguida de incubação em pressão atmosférica. O rendimento de renaturação da CTB solúvel e pentamérica foi de até 45 % e 288 mg de CTB/litro de cultura bacteriana. Esta proteína apresentou estrutura regular e atividade biológica. RGFA trimérica foi obtida pela compressão da suspensão de CI em tampão TrisHCl 50 mM pH 8,0 e 0,5 M de L-arginina a 2,4 kbar por 1,5 horas e 0,4 kbar por 16 horas antes da completa descompressão. O rendimento de proteína solúvel trimérica da RGFA foi de 4 %, porém não foi possível obter a atividade biológica desta proteína. / The production of recombinant proteins is an essential tool for the biotechnology industry and supports the expansion of modern biological research. Recombinant proteins can be produced by a variety of hosts and among them the bacteria E. coli is the most commonly used. However, the expression of heterologous genes in E. coli often results in an incomplete folding process that leads to the accumulation of insoluble aggregates known as inclusion bodies (IB). The application of high hydrostatic pressure impairs intermolecular hydrophobic and electrostatic interactions of proteins in solution, leading to dissociation of aggregates and is therefore useful tool to solubilize and refold aggregated proteins in IB. This work aimed to study the process of disaggregation of IB and refolding of oligomeric proteins the B subunit of cholera toxin (CTB) and the globular region of the adenoviral fiber (RGFA) using high hydrostatic pressure. The cholera toxin (CT) comprises one A subunit and five B subunits, combined in the AB5 holotoxin. The pentameric CTB is non-toxic moiety of CT which is responsible for binding to the receptor ganglioside GM1 holotoxin. The adenovirus fiber is a homotrimeric protein wich forms part of the viral capsid and it is organized into three regions: the N-terminal tail, the central rod and the C-terminal region (globular region). The RGFA binds to membrane protein CAR in host cells and promotes the internalization of virus. The studies presented here demonstrate that high hydrostatic pressure was effective in the disaggregation of the CTB and RGFA IB. The refolding conditions were optimized using different proportions of the redox couple oxidated and reduced glutathione, concentrations of chaotropic agents, presence of additives and pressure/decompression schemes distinguished from the previously described in the literature. Soluble pentameric CTB was obtained when the suspension of IB were compressed at 2.4 kbar for 16 hours in 50 mM of Tris-HCl buffer pH 8.5, 1 mM of tween 20, followed by direct decompression and incubation at atmospheric pressure. The yield of refolded soluble pentameric CTB was up to 45 % and 288 mg of CTB/ liter of bacterial culture. This protein was shown to presented regular structure and biological activity. Trimeric RGFA was obtained by compression of the suspension of IB in 50 mM of Tris-HCl buffer pH 8.0, 0.5M L-arginine at 2.4 kbar for 1.5 hours and at 0.4 kbar for 16 hours prior to the complete decompression. The yield of soluble trimeric RGFA was 4 %, however this protein did not present biological activity.