Study of Basic Wood Decay Mechanisms and Their Biotechnological Applications

The overall objective of this thesis was to gain further understanding of the non-enzymatic mechanisms involved in brown-rot wood decay, especially the role of pH, oxalic acid, and low molecular catecholate compounds on the dissolution and reduction of iron, and the formation of reactive oxygen species. Another focus of this study will be the potential application of a biomimetic free radical generating system inspired from fungi wood decay process, especially the non-enzymatic mechanism. The possible pathways of iron uptake and iron redox cycling in non-enzymatic brown-rot decay were investigated in this study. UV-Vis spectroscopy and HPLC were employed to study the kinetics and pathways of the interaction between iron and model catecholate compounds under different pH and chelator/iron molar ratio conditions. Iron chelation and reduction during early non-enzymatic wood decay processes have been studied in this thesis. The results indicate that the effects of the chelator/iron ratio, the pH, and other reaction parameters on the hydroxyl radical generation in a Fenton type system can be determined using ESR spin-trapping techniques. Data also support the hypothesis that superoxide radicals are involved in chelator-mediated Fenton processes. The mechanisms involved in free radical activation of Thermal Mechanical Pulp fibers were investigated. The activation of TMP fibers was evaluated by ESR measurement of free phenoxy radical generation on solid fibers. The results indicate that low molecular weight chelators can improve Fenton reactions, thus in turn stimulating the free radical activation of TMP fibers. A mediated Fenton system was evaluated for decolorization of several types of dyes. The result shows that the Fenton system mediated by a catecholate-type chelator effectively reduced the color of a diluted solution of synthetic dyes after 90 minutes of treatment at room temperature. The results show that compared to a neat Fenton process, the mediated Fenton decolorization process increased the production, and therefore the effective longevity, of hydroxyl radical species to increase the decolorization efficiency.

The little-studied cluster Berkeley 90 I. LS III +46 11: a very massive O3.5 If* + O3.5 If* binary

Context. It appears that most (if not all) massive stars are born in multiple systems. At the same time, the most massive binaries are hard to find owing to their low numbers throughout the Galaxy and the implied large distances and extinctions. Aims. We want to study LS III +46 11, identified in this paper as a very massive binary; another nearby massive system, LS III +46 12; and the surrounding stellar cluster, Berkeley 90. Methods. Most of the data used in this paper are multi-epoch high S/N optical spectra, although we also use Lucky Imaging and archival photometry. The spectra are reduced with dedicated pipelines and processed with our own software, such as a spectroscopic-orbit code, CHORIZOS, and MGB. Results. LS III +46 11 is identified as a new very early O-type spectroscopic binary [O3.5 If* + O3.5 If*] and LS III +46 12 as another early O-type system [O4.5 V((f))]. We measure a 97.2-day period for LS III +46 11 and derive minimum masses of 38.80 ± 0.83 M⊙ and 35.60 ± 0.77 M⊙ for its two stars. We measure the extinction to both stars, estimate the distance, search for optical companions, and study the surrounding cluster. In doing so, a variable extinction is found as well as discrepant results for the distance. We discuss possible explanations and suggest that LS III +46 12 may be a hidden binary system where the companion is currently undetected.

Defining the Fluid framework

In this position paper we present the developing Fluid framework, which we believe offers considerable advantages in maintaining software stability in dynamic or evolving application settings. The Fluid framework facilitates the development of component software via the selection, composition and configuration of components. Fluid's composition language incorporates a high-level type system supporting object-oriented principles such as type description, type inheritance, and type instantiation. Object-oriented relationships are represented via the dynamic composition of component instances. This representation allows the software structure, as specified by type and instance descriptions, to change dynamically at runtime as existing types are modified and new types and instances are introduced. We therefore move from static software structure descriptions to more dynamic representations, while maintaining the expressiveness of object-oriented semantics. We show how the Fluid framework relates to existing, largely component based, software frameworks and conclude with suggestions for future enhancements. © 2007 IEEE.

Using a simulation model for capacity management

A simulation model has been constructed of a valve manufacturing plant with the aim of assessing capacity requirements in response to a forecast increase in demand. The plant provides a weekly cycle of valves of varying types, based on a yearly production plan. Production control is provided by a just-in-time type system to minimise inventory. The simulation model investigates the effect on production lead time of a range of valve sequences into the plant. The study required the collection of information from a variety of sources, and a model that reflected the true capabilities of the production system. The simulation results convinced management that substantial changes were needed in order to meet demand. The case highlights the use of simulation in enabling a manager to quantify operational scenarios and thus provide a rational basis on which to take decisions on meeting performance criteria.

Practical secure information flow in programming languages

If we classify variables in a program into various security levels, then a secure information flow analysis aims to verify statically that information in a program can flow only in ways consistent with the specified security levels. One well-studied approach is to formulate the rules of the secure information flow analysis as a type system. A major trend of recent research focuses on how to accommodate various sophisticated modern language features. However, this approach often leads to overly complicated and restrictive type systems, making them unfit for practical use. Also, problems essential to practical use, such as type inference and error reporting, have received little attention. This dissertation identified and solved major theoretical and practical hurdles to the application of secure information flow. ^ We adopted a minimalist approach to designing our language to ensure a simple lenient type system. We started out with a small simple imperative language and only added features that we deemed most important for practical use. One language feature we addressed is arrays. Due to the various leaking channels associated with array operations, arrays have received complicated and restrictive typing rules in other secure languages. We presented a novel approach for lenient array operations, which lead to simple and lenient typing of arrays. ^ Type inference is necessary because usually a user is only concerned with the security types for input/output variables of a program and would like to have all types for auxiliary variables inferred automatically. We presented a type inference algorithm B and proved its soundness and completeness. Moreover, algorithm B stays close to the program and the type system and therefore facilitates informative error reporting that is generated in a cascading fashion. Algorithm B and error reporting have been implemented and tested. ^ Lastly, we presented a novel framework for developing applications that ensure user information privacy. In this framework, core computations are defined as code modules that involve input/output data from multiple parties. Incrementally, secure flow policies are refined based on feedback from the type checking/inference. Core computations only interact with code modules from involved parties through well-defined interfaces. All code modules are digitally signed to ensure their authenticity and integrity. ^

Language-based Techniques for Practical and Trustworthy Secure Multi-party Computations

Secure Multi-party Computation (MPC) enables a set of parties to collaboratively compute, using cryptographic protocols, a function over their private data in a way that the participants do not see each other's data, they only see the final output. Typical MPC examples include statistical computations over joint private data, private set intersection, and auctions. While these applications are examples of monolithic MPC, richer MPC applications move between "normal" (i.e., per-party local) and "secure" (i.e., joint, multi-party secure) modes repeatedly, resulting overall in mixed-mode computations. For example, we might use MPC to implement the role of the dealer in a game of mental poker -- the game will be divided into rounds of local decision-making (e.g. bidding) and joint interaction (e.g. dealing). Mixed-mode computations are also used to improve performance over monolithic secure computations. Starting with the Fairplay project, several MPC frameworks have been proposed in the last decade to help programmers write MPC applications in a high-level language, while the toolchain manages the low-level details. However, these frameworks are either not expressive enough to allow writing mixed-mode applications or lack formal specification, and reasoning capabilities, thereby diminishing the parties' trust in such tools, and the programs written using them. Furthermore, none of the frameworks provides a verified toolchain to run the MPC programs, leaving the potential of security holes that can compromise the privacy of parties' data. This dissertation presents language-based techniques to make MPC more practical and trustworthy. First, it presents the design and implementation of a new MPC Domain Specific Language, called Wysteria, for writing rich mixed-mode MPC applications. Wysteria provides several benefits over previous languages, including a conceptual single thread of control, generic support for more than two parties, high-level abstractions for secret shares, and a fully formalized type system and operational semantics. Using Wysteria, we have implemented several MPC applications, including, for the first time, a card dealing application. The dissertation next presents Wys*, an embedding of Wysteria in F*, a full-featured verification oriented programming language. Wys* improves on Wysteria along three lines: (a) It enables programmers to formally verify the correctness and security properties of their programs. As far as we know, Wys* is the first language to provide verification capabilities for MPC programs. (b) It provides a partially verified toolchain to run MPC programs, and finally (c) It enables the MPC programs to use, with no extra effort, standard language constructs from the host language F*, thereby making it more usable and scalable. Finally, the dissertation develops static analyses that help optimize monolithic MPC programs into mixed-mode MPC programs, while providing similar privacy guarantees as the monolithic versions.

Trace Oblivious Program Execution

The big data era has dramatically transformed our lives; however, security incidents such as data breaches can put sensitive data (e.g. photos, identities, genomes) at risk. To protect users' data privacy, there is a growing interest in building secure cloud computing systems, which keep sensitive data inputs hidden, even from computation providers. Conceptually, secure cloud computing systems leverage cryptographic techniques (e.g., secure multiparty computation) and trusted hardware (e.g. secure processors) to instantiate a “secure” abstract machine consisting of a CPU and encrypted memory, so that an adversary cannot learn information through either the computation within the CPU or the data in the memory. Unfortunately, evidence has shown that side channels (e.g. memory accesses, timing, and termination) in such a “secure” abstract machine may potentially leak highly sensitive information, including cryptographic keys that form the root of trust for the secure systems. This thesis broadly expands the investigation of a research direction called trace oblivious computation, where programming language techniques are employed to prevent side channel information leakage. We demonstrate the feasibility of trace oblivious computation, by formalizing and building several systems, including GhostRider, which is a hardware-software co-design to provide a hardware-based trace oblivious computing solution, SCVM, which is an automatic RAM-model secure computation system, and ObliVM, which is a programming framework to facilitate programmers to develop applications. All of these systems enjoy formal security guarantees while demonstrating a better performance than prior systems, by one to several orders of magnitude.

Multivariate orthogonal polynomials and integrable systems

Multivariate orthogonal polynomials in D real dimensions are considered from the perspective of the Cholesky factorization of a moment matrix. The approach allows for the construction of corresponding multivariate orthogonal polynomials, associated second kind functions, Jacobi type matrices and associated three term relations and also Christoffel-Darboux formulae. The multivariate orthogonal polynomials, their second kind functions and the corresponding Christoffel-Darboux kernels are shown to be quasi-determinants as well as Schur complements of bordered truncations of the moment matrix; quasi-tau functions are introduced. It is proven that the second kind functions are multivariate Cauchy transforms of the multivariate orthogonal polynomials. Discrete and continuous deformations of the measure lead to Toda type integrable hierarchy, being the corresponding flows described through Lax and Zakharov-Shabat equations; bilinear equations are found. Varying size matrix nonlinear partial difference and differential equations of the 2D Toda lattice type are shown to be solved by matrix coefficients of the multivariate orthogonal polynomials. The discrete flows, which are shown to be connected with a Gauss-Borel factorization of the Jacobi type matrices and its quasi-determinants, lead to expressions for the multivariate orthogonal polynomials and their second kind functions in terms of shifted quasi-tau matrices, which generalize to the multidimensional realm, those that relate the Baker and adjoint Baker functions to ratios of Miwa shifted tau-functions in the 1D scenario. In this context, the multivariate extension of the elementary Darboux transformation is given in terms of quasi-determinants of matrices built up by the evaluation, at a poised set of nodes lying in an appropriate hyperplane in R^D, of the multivariate orthogonal polynomials. The multivariate Christoffel formula for the iteration of m elementary Darboux transformations is given as a quasi-determinant. It is shown, using congruences in the space of semi-infinite matrices, that the discrete and continuous flows are intimately connected and determine nonlinear partial difference-differential equations that involve only one site in the integrable lattice behaving as a Kadomstev-Petviashvili type system. Finally, a brief discussion of measures with a particular linear isometry invariance and some of its consequences for the corresponding multivariate polynomials is given. In particular, it is shown that the Toda times that preserve the invariance condition lay in a secant variety of the Veronese variety of the fixed point set of the linear isometry.

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb. © 2007 Springer-Verlag.

Immune response to insulin and changes in the gut immune system in children with or at risk for type 1 diabetes

Type 1 diabetes (T1D) is considered to be an autoimmune disease. The cause of T1D is the destruction of insulin-producing β-cells in the pancreatic islets. The autoimmune nature of T1D is characterized by the presence of autoreactive T-cells and autoantibodies against β-cell molecules. Insulin is the only β-cell-specific autoantigen associated with T1D but the insulin autoantibodies (IAAs) are difficult to measure with proper sensitivity. T-cell assays for detection of autoreactive T-cells, such as insulin-specific T-cells, have also proven to be difficult to perform. The genetic risk of T1D is associated with the HLA gene region but the environmental factors also play an important role. The most studied environmental risk factors of T1D are enteroviruses and cow's milk which both affect the immune system through the gut. One hypothesis is that the insulin-specific immune response develops against bovine insulin in cow's milk during early infancy and later spreads to include human insulin. The aims of this study were to determine whether the separation of immunoglobulin (Ig)G from plasma would improve the sensitivity of the IAA assay and how insulin treatment affects the cellular immune response to insulin in newly diagnosed patients. Furthermore, the effect of insulin concentration in mother's breast milk on the development of antibodies to dietary insulin in the child was examined. Small intestinal biopsies were also obtained from children with T1D to characterize any immunological changes associated with T1D in the gut. The isolation of the IgG fraction from the plasma of T1D patients negative for plasma IAA led to detectable IAA levels that exceeded those in the control children. Thus the isolation of IgG may improve the sensitivity of the IAA assay. The effect of insulin treatment on insulin-specific T-cells was studied by culturing peripheral blood mononuclear cells with insulin. The insulin stimulation induced increased expression of regulatory T-cell markers, such as Foxp3, in those patients treated with insulin than in patients examined before initiating insulin treatment. This finding suggests that insulin treatment in patients with T1D stimulates regulatory T-cells in vivo and this may partly explain the difficulties in measuring autoantigen-specific T-cell responses in recently diagnosed patients. The stimulation of regulatory T-cells by insulin treatment may also explain the remission period often seen after initiating insulin treatment. In the third study we showed that insulin concentration in mother's breast milk correlates inversely with the levels of bovine insulin-specific antibodies in those infants who were exposed to cow's milk proteins in their diet, suggesting that human insulin in breast milk induces tolerance to dietary bovine insulin. However, in infants who later developed T1D-associated autoantibodies, the insulin concentration in their mother's breast milk was increased. This finding may indicate that in those children prone to β-cell autoimmunity, breast milk insulin does not promote tolerance to insulin. In the small intestinal biopsies the presence of several immunological markers were quantified with the RT-PCR. From these markers the expression of the interleukin (IL)-18 cytokine was significantly increased in the gut in patients with T1D compared with children with celiac disease or control children. The increased IL-18 expression lends further support for the hypothesis that the gut immune system is involved in the pathogenesis of T1D.

Type III Secretion System of Phytopathogenic Bacterium Pseudomonas syringae:From Gene to Function

The type III secretion system (T3SS) is an essential requirement for the virulence of many Gram-negative bacteria which infect plants, animals and men. Pathogens use the T3SS to deliver effector proteins from the bacterial cytoplasm to the eukaryotic host cells, where the effectors subvert host defenses. The best candidates for directing effector protein traffic are the bacterial type III-associated appendages, called needles or pili. In plant pathogenic bacteria, the best characterized example of a T3SS-associated appendage is the HrpA pilus of the plant pathogen Pseudomonas syringae pv. tomato DC3000. The components of the T3SS in plant pathogens are encoded by a cluster of hrp (hypersensitive reaction and pathogenicity) genes. Two major classes of T3SS-secreted proteins are: harpin proteins such as HrpZ which are exported into extracellular space, and avirulence (Avr) proteins such as AvrPto which are translocated directly to the plant cytoplasm. This study deals with the structural and functional characterization of the T3SS-associated HrpA pilus and the T3SS-secreted harpins. By insertional mutagenesis analysis of HrpA, we located the optimal epitope insertion site in the amino-terminus of HrpA, and revealed the potential application of the HrpA pilus as a carrier of antigenic determinants for vaccination. By pulse-expression of proteins combined with immuno-electron microscopy, we discovered the Hrp pilus assembly strategy as addition of HrpA subunits to the distal end of the growing pilus, and we showed for the first time that secretion of HrpZ occurs at the tip of the pilus. The pilus thus functions as a conduit delivering proteins to the extracellular milieu. By using phage-display and scanning-insertion mutagenesis methods we identified a conserved HrpZ-binding peptide and localized the peptide-binding site to the central domain of HrpZ. We also found that the HrpZ specifically interacts with a host bean protein. Taken together, the current results provide deeper insight into the molecular mechanism of T3SS-associated pilus assembly and effector protein translocation, which will be helpful for further studies on the pathogenic mechanisms of Gram-negative bacteria and for developing new strategies to prevent bacterial infection.

Innate immune system and adiponectin in diabetic nephropathy in type 1 diabetes

Introduction: The pathogenesis of diabetic nephropathy remains a matter of debate, although strong evidence suggests that it results from the interaction between susceptibility genes and the diabetic milieu. The true pathogenetic mechanism remains unknown, but a common denominator of micro- and macrovascular complications may exist. Some have suggested that low-grade inflammation and activation of the innate immune system might play a synergistic role in the pathogenesis of diabetic nephropathy. Aims of the study: The present studies were undertaken to investigate whether low-grade inflammation, mannan-binding lectin (MBL) and α-defensin play a role, together with adiponectin, in patients with type 1 diabetes and diabetic nephropathy. Subjects and methods: This study is part of the ongoing Finnish Diabetic Nephropathy Study (FinnDiane). The first four cross-sectional substudies of this thesis comprised 194 patients with type 1 diabetes divided into three groups (normo-, micro-, and macroalbuminuria) according to their albumin excretion rate (AER). The fifth substudy aimed to determine whether baseline serum adiponectin plays a role in the development and progression of diabetic nephropathy. This follow-up study included 1330 patients with type 1 diabetes and a mean follow-up period of five years. The patients were divided into three groups depending on their AER at baseline. As a measure of low-grade inflammation, highly sensitive CRP (hsCRP) and α-defensin were measured with radio-immunoassay, and interleukin-6 (IL-6) with high- sensitivity enzyme immuno-assay. Mannan-binding lectin and adiponectin were determined with time-resolved immunofluorometric assays. The progression of albuminuria from one stage to the other served as a measure of the progression of diabetic nephropathy. Results: Low-grade inflammatory markers, MBL, adiponectin, and α-defensin were all associated with diabetic nephropathy, whereas MBL, adiponectin, and α-defensin per se were unassociated with low-grade inflammatory markers. AER was the only clinical variable independently associated with hsCRP. AER, HDL-cholesterol and the duration of diabetes were independently associated with IL-6. HbA1c was the only variable independently associated with MBL. The estimated glomerular filtration rate (eGFR), AER, and waist-to-hip ratio were independently associated with adiponectin. Systolic blood pressure, HDL-cholesterol, total cholesterol, age, and eGFR were all independently associated with α-defensin. In patients with macroalbuminuria, progression to end-stage renal disease (ESRD) was associated with higher baseline adiponectin concentrations. Discussion and conclusions: Low-grade inflammation, MBL, adiponectin, and defensin were all associated with diabetic nephropathy in these cross-sectional studies. In contrast however, MBL, adiponectin, and defensin were not associated with low-grade inflammatory markers per se. Nor was defensin associated with MBL, which may suggest that these different players function in a coordinated fashion during the deleterious process of diabetic nephropathy. The question of what causes low-grade inflammation in patients with type 1 diabetes and diabetic nephropathy, however, remains unanswered. We could observe in our study that glycemic control, an atherosclerotic lipid profile, and waist-to-hip ratio (WHR) were associated with low-grade inflammation in the univariate analysis, although in the multivariate analysis, only AER, HDL-cholesterol, and the duration of diabetes, as a measure of glycemic load, proved to be independently associated with inflammation. Notably, all these factors are modifiable with changes in lifestyle and/or with a targeted medication. In the follow-up study, elevated serum adiponectin levels at baseline predicted the progression from macroalbuminuria to ESRD independently of renal function at baseline. This observation does not preclude adiponectin as a favorable factor during the process of diabetic nephropathy, since the rise in serum adiponectin concentrations may remain a mechanism by which the body compensates for the demands created by the diabetic milieu.