Capillary electrophoresis for monitoring of carboxylic, phenolic and amino acids in bioprocesses

Bioprocess technology is a multidisciplinary industry that combines knowledge of biology and chemistry with process engineering. It is a growing industry because its applications have an important role in the food, pharmaceutical, diagnostics and chemical industries. In addition, the current pressure to decrease our dependence on fossil fuels motivates new, innovative research in the replacement of petrochemical products. Bioprocesses are processes that utilize cells and/or their components in the production of desired products. Bioprocesses are already used to produce fuels and chemicals, especially ethanol and building-block chemicals such as carboxylic acids. In order to enable more efficient, sustainable and economically feasible bioprocesses, the raw materials must be cheap and the bioprocesses must be operated at optimal conditions. It is essential to measure different parameters that provide information about the process conditions and the main critical process parameters including cell density, substrate concentrations and products. In addition to offline analysis methods, online monitoring tools are becoming increasingly important in the optimization of bioprocesses. Capillary electrophoresis (CE) is a versatile analysis technique with no limitations concerning polar solvents, analytes or samples. Its resolution and efficiency are high in optimized methods creating a great potential for rapid detection and quantification. This work demonstrates the potential and possibilities of CE as a versatile bioprocess monitoring tool. As a part of this study a commercial CE device was modified for use as an online analysis tool for automated monitoring. The work describes three offline CE analysis methods for the determination of carboxylic, phenolic and amino acids that are present in bioprocesses, and an online CE analysis method for the monitoring of carboxylic acid production during bioprocesses. The detection methods were indirect and direct UV, and laser-induced frescence. The results of this work can be used for the optimization of bioprocess conditions, for the development of more robust and tolerant microorganisms, and to study the dynamics of bioprocesses.

Use of fiber-reinforced composite framework and thermochromic pigment in maxillofacial prostheses

The purpose of this investigation was to evaluate the possibility to enhance certain qualities of facial prostheses. Polymethyl methacrylate is still being used as base mate¬rial or clip carrier material, but it is hard and heavy, and debonding of the silicone from the acrylic base material is a frequent problem. This thesis aims to evaluate the use of fiber-reinforced composite (FRC) as framework material for maxillofacial silicone prostheses. FRC has been used as reinforcement in removable and fixed partial dentures since the 1990s. This material is lightweight and can be fabricated to compress the margins of the prosthesis slightly, to keep it tightly against the skin during jaw movements and facial expressions. Additionally, the use of a thermochromic pigment, colorless in room temperature and red in a cold environment, was studied in order to evaluate the possibility of using this color changing pigment in facial prostheses to mimic the color change of facial skin in cold weather. The tensile bond strength between pre-impregnated, unidirectional FRC and maxillofacial silicone elastomer was studied. Three different bonding agents or primers were compared. Bond strength was improved by one of the primers and by roughening the surface. The effect of a skin compressing glass fiber-reinforced composite framework on facial skin blood flow was studied by using a face mask, constructed with a compression pad corresponding to the outer margin of a glass fiber-reinforced framework beam of a facial prosthesis. The skin blood flow of ten healthy volunteers, aged 23-25 years, was measured during touch, light, and moderate compression of the skin, by using laser Doppler imaging technique. None of the compressions showed any marked effects on local skin blood flow. There were no significant differences between blood flow during compression and at baseline. Maxillofacial silicone elastomer was colored intrinsically with conventional color pigments: a control group containing only conventional pigments was compared to two test groups with 0.2 wt% and 0.6 wt% thermochromic pigment added. The color of the material was measured with a spectrophotometer in room temperature and after storage in a freezer. The color stability of the maxillofacial silicone elastomer colored with thermo¬chromic pigment was evaluated by artificial aging. The color dif¬ference of the L* (lightness) and a* values (redness), comparing color after the samples were stored at room temperature and in a freezer (-19°C), was statistically significant for both 0.2 wt% and 0.6 wt% thermo¬chromic pigment groups. The differences in the b* values (yellowness) were statistically significant for the 0.6 wt% group. Exposure to ultraviolet (UV) radiation led to visually noticeable and statistically signifi¬cant color changes (ΔE) in all color values in both test groups. The specimens containing thermochromic pigment were very sensitive to UV radiation. In conclusion, a framework of fiber-reinforced composite can successfully be bonded to maxillofacial silicone elastomer, and a framework beam, compressing the facial skin, did not remarkably alter the skin blood flow on healthy, young adults. The thermochromic pigment showed color change in maxillofacial silicone elastomer. However, artificial aging showed that it was too sensitive to UV radiation to be used, as such, in maxillofacial prostheses.

Matrix Mind : on managerial reframing in multidimensional problem solving

Det är inte ovanligt att man i organisationer ställs inför problem som inte kan hanteras inom befintlig organisationsstruktur. Skälen kan vara att frågorna har många – ibland konflikterande – dimensioner och perspektiv som samtidigt måste beaktas. I den här avhandlingen studeras vilka tankemönster och föreställningar som fanns när grupper av chefer försökte lösa komplexa problem, som inte hade en naturlig organisatorisk hemvist och hur de omsatte dessa tankemönster i handling. Vad kännetecknade det ledarskap som utövades under problemlösningsprocessen? Avhandlingens empiri hämtas från ledarutvecklingsprogram i två internationellt verksamma företag i Sverige, och omfattar 14 verkliga affärsproblem i dessa företag och den process varigenom de löstes. De 14 seminarierna utgör exempel på hur mångdimensionella frågeställningar framgångsrikt hanteras utanför den befintliga organisationsstrukturen. Studien ger, genom att adressera frågor kring tankesätt och ledningsprocesser, en djupare förståelse för förutsättningarna för detta, och lyfter särskilt fram betydelsen av ett ledarskap som inbegriper begreppen intervention, förmåga och omtolkning. Som ett samlat begrepp introduceras bilden att utöva ledarskapet utifrån ett matrix mind. Att påverka strukturer (i vid mening) och därigenom de förmågor som utvecklas, är del i detta ledarskap. Det sker genom interventioner (ingrepp som påverkar relationer i t ex en grupp) och baserades i den aktuella empirin på uppfattningar om värdet av problematisering, erfarenhetsutbyte och av ett språk, som både beskriver och anger inriktning för aktiviteter. Interventioner i strukturer (och till dem knutna processer) beskriver dock bara delvis detta ledarskap. Att leda med ett matrix mind innefattar också ett nyfiket och kreativt förhållningssätt, och att utifrån detta leda omtolkning av problem. I empirin finns flera exempel på detta. Avhandlingen avser att ge ett bidrag inom såväl organisations- som ledarskapsteori.

Design and validation of stateful composite RESTful web services

A web service is a software system that provides a machine-processable interface to the other machines over the network using different Internet protocols. They are being increasingly used in the industry in order to automate different tasks and offer services to a wider audience. The REST architectural style aims at producing scalable and extensible web services using technologies that play well with the existing tools and infrastructure of the web. It provides a uniform set of operation that can be used to invoke a CRUD interface (create, retrieve, update and delete) of a web service. The stateless behavior of the service interface requires that every request to a resource is independent of the previous ones facilitating scalability. Automated systems, e.g., hotel reservation systems, provide advanced scenarios for stateful services that require a certain sequence of requests that must be followed in order to fulfill the service goals. Designing and developing such services for advanced scenarios with REST constraints require rigorous approaches that are capable of creating web services that can be trusted for their behavior. Systems that can be trusted for their behavior can be termed as dependable systems. This thesis presents an integrated design, analysis and validation approach that facilitates the service developer to create dependable and stateful REST web services. The main contribution of this thesis is that we provide a novel model-driven methodology to design behavioral REST web service interfaces and their compositions. The behavioral interfaces provide information on what methods can be invoked on a service and the pre- and post-conditions of these methods. The methodology uses Unified Modeling Language (UML), as the modeling language, which has a wide user base and has mature tools that are continuously evolving. We have used UML class diagram and UML state machine diagram with additional design constraints to provide resource and behavioral models, respectively, for designing REST web service interfaces. These service design models serve as a specification document and the information presented in them have manifold applications. The service design models also contain information about the time and domain requirements of the service that can help in requirement traceability which is an important part of our approach. Requirement traceability helps in capturing faults in the design models and other elements of software development environment by tracing back and forth the unfulfilled requirements of the service. The information about service actors is also included in the design models which is required for authenticating the service requests by authorized actors since not all types of users have access to all the resources. In addition, following our design approach, the service developer can ensure that the designed web service interfaces will be REST compliant. The second contribution of this thesis is consistency analysis of the behavioral REST interfaces. To overcome the inconsistency problem and design errors in our service models, we have used semantic technologies. The REST interfaces are represented in web ontology language, OWL2, that can be part of the semantic web. These interfaces are used with OWL 2 reasoners to check unsatisfiable concepts which result in implementations that fail. This work is fully automated thanks to the implemented translation tool and the existing OWL 2 reasoners. The third contribution of this thesis is the verification and validation of REST web services. We have used model checking techniques with UPPAAL model checker for this purpose. The timed automata of UML based service design models are generated with our transformation tool that are verified for their basic characteristics like deadlock freedom, liveness, reachability and safety. The implementation of a web service is tested using a black-box testing approach. Test cases are generated from the UPPAAL timed automata and using the online testing tool, UPPAAL TRON, the service implementation is validated at runtime against its specifications. Requirement traceability is also addressed in our validation approach with which we can see what service goals are met and trace back the unfulfilled service goals to detect the faults in the design models. A final contribution of the thesis is an implementation of behavioral REST interfaces and service monitors from the service design models. The partial code generation tool creates code skeletons of REST web services with method pre and post-conditions. The preconditions of methods constrain the user to invoke the stateful REST service under the right conditions and the post condition constraint the service developer to implement the right functionality. The details of the methods can be manually inserted by the developer as required. We do not target complete automation because we focus only on the interface aspects of the web service. The applicability of the approach is demonstrated with a pedagogical example of a hotel room booking service and a relatively complex worked example of holiday booking service taken from the industrial context. The former example presents a simple explanation of the approach and the later worked example shows how stateful and timed web services offering complex scenarios and involving other web services can be constructed using our approach.

Leaf contents of nitrogen and phenolic compounds and their bearing with the herbivore damage to Tibouchina pulchra Cogn. (Melastomataceae), under the influence of air pollutants from industries of Cubatão, São Paulo

The Atlantic Forest on the slopes of Serra do Mar around Cubatão (São Paulo, Brazil) has been affected by massive emissions of pollutants from the local growing industrial complex. The effects of air pollution on the amounts of leaf nitrogen, total soluble phenols and total tannins of Tibouchina pulchra Cogn., a common species in the area of Cubatão, were investigated, as well as the possible influence of the altered parameters on the leaf area damaged by herbivores. Fully expanded leaves were collected at two sites: the valley of Pilões river (VP), characterized by a vegetation virtually not affected by air pollution and taken as a reference; and valley of Mogi river (VM), close to the core region of the industrial complex, and severely affected by air pollution. No differences were observed for any parameters between samples collected in the summer and winter in both sites. On the other hand, compared to VP, individuals growing in VM presented higher amounts of nitrogen and lower amounts of total soluble phenols and total tannins, as well as higher percentages of galls per leaf and higher leaf area lost to herbivores. Regression analysis revealed that the increase in leaf area lost to herbivores can be explained by the increase of the content of nitrogen and decrease in the contents of total soluble phenols and total tannins. Although significant, the coefficients of explanation found were low for all analyses, suggesting that other biotic or abiotic factors are likely to influence leaf attack by herbivores.

Predicting variety composite means without diallel crossing

Prediction of variety composite means was shown to be feasible without diallel crossing the parental varieties. Thus, the predicted mean for a quantitative trait of a composite is given by: Yk = a1 sigmaVj + a2sigmaTj + a3 - a4, with coefficients a1 = (n - 2k)/k²(n - 2); a2 = 2n(k - 1)/k²(n - 2); a3 = n(k - 1)/k(n - 1)(n - 2); and a4 = n²(k - 1)/k(n - 1)(n - 2); summation is for j = 1 to k, where k is the size of the composite (number of parental varieties of a particular composite) and n is the total number of parent varieties. Vj is the mean of varieties and Tj is the mean of topcrosses (pool of varieties as tester), and and are the respective average values in the whole set. Yield data from a 7 x 7 variety diallel cross were used for the variety means and for the "simulated" topcross means to illustrate the proposed procedure. The proposed prediction procedure was as effective as the prediction based on Yk = - ( -)/k, where and refer to the mean of hybrids (F1) and parental varieties, respectively, in a variety diallel cross. It was also shown in the analysis of variance that the total sum of squares due to treatments (varieties and topcrosses) can be orthogonally partitioned following the reduced model Yjj’ = mu + ½(v j + v j’) + + h j+ h j’, thus making possible an F test for varieties, average heterosis and variety heterosis. Least square estimates of these effects are also given

Concanavalin A-reactive nuclear matrix glycoprotein

The binding capacity of concanavalin A (Con A) to condensed euchromatin and heterochromatin was investigated in chicken erythrocyte nuclei (CEN), mouse liver cells, Zea mays mays meristematic cells and Drosophila melanogaster polytene chromosomes after 4 N HCl hydrolysis to determine whether binding was preferentially occurring in bands and heterochromatin. Dry mass (DM) variation was investigated in CEN by interference microscopy. Feulgen and Con A reactions were employed for all materials to correlate the loci of the two reactions. Quantifications and topological verifications were carried out by video image analysis (high performance cytometry). It was observed that 4 N HCl hydrolysis caused an important DM loss in CEN leaving a level corresponding to the average DNA DM content. In this material, Con A binding was restricted to the nuclear envelope, which reinforces the idea of the absence of a nuclear matrix in these cells. The other cell types exhibited a correspondence of Feulgen-positive and Con A-reactive areas. The Con A reaction was highly positive in the condensed chromatin areas and heterochromatin. This fact led us to speculate that Con A-positive proteins may play a role in the chromatin condensation mechanism, endowing this structure with physico-chemical stability towards acid hydrolysis and contributing to its rheological properties.

Flavonoids and other phenolic compounds: characterization and interactions with lepidopteran and sawfly larvae

This thesis focuses on flavonoids, a subgroup of phenolic compounds produced by plants, and how they affect the herbivorous larvae of lepidopterans and sawflies. The first part of the literature review examines different techniques to analyze the chemical structures of flavonoids and their concentrations in biological samples. These techniques include, for example, ultraviolet-visible spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy. The second part of the literature review studies how phenolic compounds function in the metabolism of larvae. The harmful oxidation reactions of phenolic compounds in insect guts are also emphasized. In addition to the negative effects, many insect species have evolved the use of phenolic compounds for their own benefit. In the experimental part of the thesis, high concentrations of complex flavonoid oligoglycosides were found in the hemolymph (the circulatory fluid of insects) of birch and pine sawflies. The larvae produced these compounds from simple flavonoid precursors present in the birch leaves and pine needles. Flavonoid glycosides were also found in the cocoon walls of sawflies, which suggested that flavonoids were used in the construction of cocoons. The second part of the experimental work studied the modifications of phenolic compounds in conditions that mimicked the alkaline guts of lepidopteran larvae. It was found that the 24 plant species studied and their individual phenolic compounds had variable capacities to function as oxidative defenses in alkaline conditions. The excrements of lepidopteran and sawfly species were studied to see how different types of phenolics were processed by the larvae. These results suggested that phenolic compounds were oxidized, hydrolyzed, or modified in other ways during their passage through the digestive tract of the larvae.

Cellular and matrix interactions during the development of T lymphocytes

The thymus contains an extensive extracellular matrix. Although thymocytes express integrins capable of binding to matrix molecules, the functional significance of the matrix for T cell development is uncertain. We have shown that the matrix is associated with thymic fibroblasts which are required for the CD44+ CD25+ stage of double negative (CD4-8-) thymocyte development. The survival of cells at this stage is dependent on IL-7 and we propose that the role of fibroblasts is to present, via the matrix, IL-7 to developing T cells.

Expression of extracellular matrix components and their receptors in the central nervous system during experimental Toxoplasma gondii and Trypanosoma cruzi infection

Alterations in extracellular matrix (ECM) expression in the central nervous system (CNS) usually associated with inflammatory lesions have been described in several pathological situations including neuroblastoma and demyelinating diseases. The participation of fibronectin (FN) and its receptor, the VLA-4 molecule, in the migration of inflammatory cells into the CNS has been proposed. In Trypanosoma cruzi infection encephalitis occurs during the acute phase, whereas in Toxoplasma infection encephalitis is a chronic persisting process. In immunocompromised individuals such as AIDS patients, T. cruzi or T. gondii infection can lead to severe CNS damage. At the moment, there are no data available regarding the molecules involved in the entrance of inflammatory cells into the CNS during parasitic encephalitis. Herein, we characterized the expression of the ECM components FN and laminin (LN) and their receptors in the CNS of T. gondii- and T. cruzi-infected mice. An increased expression of FN and LN was detected in the meninges, leptomeninges, choroid plexus and basal lamina of blood vessels. A fine FN network was observed involving T. gondii-free and T. gondii-containing inflammatory infiltrates. Moreover, perivascular spaces presenting a FN-containing filamentous network filled with a4+ and a5+ cells were observed. Although an increased expression of LN was detected in the basal lamina of blood vessels, the CNS inflammatory cells were a6-negative. Taken together, our results suggest that FN and its receptors VLA-4 and VLA-5 might be involved in the entrance, migration and retention of inflammatory cells into the CNS during parasitic infections.

Regulatory roles of microtubule-associated proteins in neuronal morphogenesis. Involvement of the extracellular matrix

As a result of recent investigations, the cytoskeleton can be viewed as a cytoplasmic system of interconnected filaments with three major integrative levels: self-assembling macromolecules, filamentous polymers, e.g., microtubules, intermediate filaments and actin filaments, and supramolecular structures formed by bundles of these filaments or networks resulting from cross-bridges between these major cytoskeletal polymers. The organization of this biological structure appears to be sensitive to fine spatially and temporally dependent regulatory signals. In differentiating neurons, regulation of cytoskeleton organization is particularly relevant, and the microtubule-associated protein (MAP) tau appears to play roles in the extension of large neuritic processes and axons as well as in the stabilization of microtubular polymers along these processes. Within this context, tau is directly involved in defining neuronal polarity as well as in the generation of neuronal growth cones. There is increasing evidence that elements of the extracellular matrix contribute to the control of cytoskeleton organization in differentiating neurons, and that these regulations could be mediated by changes in MAP activity. In this brief review, we discuss the possible roles of tau in mediating the effects of extracellular matrix components on the internal cytoskeletal arrays and its organization in growing neurons.

Extracellular matrix molecules play diverse roles in the growth and guidance of central nervous system axons

Axon growth and guidance represent complex biological processes in which probably intervene diverse sets of molecular cues that allow for the appropriate wiring of the central nervous system (CNS). The extracellular matrix (ECM) represents a major contributor of molecular signals either diffusible or membrane-bound that may regulate different stages of neural development. Some of the brain ECM molecules form tridimensional structures (tunnels and boundaries) that appear during time- and space-regulated events, possibly playing relevant roles in the control of axon elongation and pathfinding. This short review focuses mainly on the recognized roles played by proteoglycans, laminin, fibronectin and tenascin in axonal development during ontogenesis.

Adhesion of the human pathogen Sporothrix schenckii to several extracellular matrix proteins

The pathogenic fungus Sporothrix schenckii is the causative agent of sporotrichosis. This subcutaneous mycosis may disseminate in immunocompromised individuals and also affect several internal organs and tissues, most commonly the bone, joints and lung. Since adhesion is the first step involved with the dissemination of pathogens in the host, we have studied the interaction between S. schenckii and several extracellular matrix (ECM) proteins. The binding of two morphological phases of S. schenckii, yeast cells and conidia, to immobilized type II collagen, laminin, fibronectin, fibrinogen and thrombospondin was investigated. Poly (2-hydroxyethyl methacrylate) (poly-HEMA) was used as the negative control. Cell adhesion was assessed by ELISA with a rabbit anti-S. schenckii antiserum. The results indicate that both morphological phases of this fungus can bind significantly to type II collagen, fibronectin and laminin in comparison to the binding observed with BSA (used as blocking agent). The adhesion rate observed with the ECM proteins (type II collagen, fibronectin and laminin) was statistically significant (P<0.05) when compared to the adhesion obtained with BSA. No significant binding of conidia was observed to either fibrinogen or thrombospondin, but yeast cells did bind to the fibrinogen. Our results indicate that S. schenckii can bind to fibronectin, laminin and type II collagen and also show differences in binding capacity according to the morphological form of the fungus.

Functions of the extracellular matrix and matrix degrading proteases during tumor progression

Cell interactions with extracellular matrices are important to pathological changes that occur during cell transformation and tumorigenesis. Several extracellular matrix proteins including fibronectin, thrombospondin-1, laminin, SPARC, and osteopontin have been suggested to modulate tumor phenotype by affecting cell migration, survival, or angiogenesis. Likewise, proteases including the matrix metalloproteinases (MMPs) are understood to not only facilitate migration of cells by degradation of matrices, but also to affect tumor formation and growth. We have recently demonstrated an in vivo role for the RGD-containing protein, osteopontin, during tumor progression, and found evidence for distinct functions in the host versus the tumor cells. Because of the compartmentalization and temporal regulation of MMP expression, it is likely that MMPs may also function dually in host stroma and the tumor cell. In addition, an important function of proteases appears to be not only degradation, but also cleavage of matrix proteins to generate functionally distinct fragments based on receptor binding, biological activity, or regulation of growth factors.