Products for the treatment of inflammatory bowel disease: a patent review (2013-2014)

Introduction: Inflammatory bowel disease (IBD) consists of Crohn's disease, ulcerative colitis and an unspecific IBD. The unclear etiology of IBD is a limiting factor that complicates the development of new pharmacological treatments and explains the high frequency of refractory patients to current drugs, including both conventional and biological therapies. In view of this, recent progress on the development of novel patented products to treat IBD was reviewed.Areas covered: Evaluation of the patent literature during the period 2013 - 2014 focused on chemical compounds, functional foods and biological therapy useful for the treatment of IBD.Expert opinion: Majority of the patents are not conclusive because they were based on data from unspecific methods not related to intestinal inflammation and, when related to IBD models, few biochemical and molecular evaluations that could be corroborating their use in human IBD were presented. On the other hand, methods and strategies using new formulations of conventional drugs, guanylyl cyclase C peptide agonists, compounds that influence anti-adhesion molecules, mAbs anti-type I interferons and anti-integrin, oligonucleotide antisense Smad7, growth factor neuregulin 4 and functional foods, particularly fermented wheat germ with Saccharomyces cerevisiae, are promising products for use in the very near future.

Computation of (3)J(HH) coupling constants with a combination of density functional theory and semiempirical calculations. Application to complex molecules

This work evaluates the efficiency of economic levels of theory for the prediction of (3)J(HH) spin-spin coupling constants, to be used when robust electronic structure methods are prohibitive. To that purpose, DFT methods like mPW1PW91. B3LYP and PBEPBE were used to obtain coupling constants for a test set whose coupling constants are well known. Satisfactory results were obtained in most of cases, with the mPW1PW91/6-31G(d,p)//B3LYP/6-31G(d,p) leading the set. In a second step. B3LYP was replaced by the semiempirical methods PM6 and RM1 in the geometry optimizations. Coupling constants calculated with these latter structures were at least as good as the ones obtained by pure DFT methods. This is a promising result, because some of the main objectives of computational chemistry - low computational cost and time, allied to high performance and precision - were attained together. (C) 2012 Elsevier B.V. All rights reserved.

Within-plant isoprene oxidation confirmed by direct emissions of oxidation products methyl vinyl ketone and methacrolein

Isoprene is emitted from many terrestrial plants at high rates, accounting for an estimated 1/3 of annual global volatile organic compound emissions from all anthropogenic and biogenic sources combined. Through rapid photooxidation reactions in the atmosphere, isoprene is converted to a variety of oxidized hydrocarbons, providing higher order reactants for the production of organic nitrates and tropospheric ozone, reducing the availability of oxidants for the breakdown of radiatively active trace gases such as methane, and potentially producing hygroscopic particles that act as effective cloud condensation nuclei. However, the functional basis for plant production of isoprene remains elusive. It has been hypothesized that in the cell isoprene mitigates oxidative damage during the stress-induced accumulation of reactive oxygen species (ROS), but the products of isoprene-ROS reactions in plants have not been detected. Using pyruvate-2-13C leaf and branch feeding and individual branch and whole mesocosm flux studies, we present evidence that isoprene (i) is oxidized to methyl vinyl ketone and methacrolein (iox) in leaves and that iox/i emission ratios increase with temperature, possibly due to an increase in ROS production under high temperature and light stress. In a primary rainforest in Amazonia, we inferred significant in plant isoprene oxidation (despite the strong masking effect of simultaneous atmospheric oxidation), from its influence on the vertical distribution of iox uptake fluxes, which were shifted to low isoprene emitting regions of the canopy. These observations suggest that carbon investment in isoprene production is larger than that inferred from emissions alone and that models of tropospheric chemistry and biotachemistryclimate interactions should incorporate isoprene oxidation within both the biosphere and the atmosphere with potential implications for better understanding both the oxidizing power of the troposphere and forest response to climate change.

Application of chromatographic and spectroscopic techniques in the evaluation of the lipid fraction of animal products

Lipolysis and oxidation of lipids in foods are the major biochemical and chemical processes that cause food quality deterioration, leading to the characteristic, unpalatable odour and flavour called rancidity. In addition to unpalatability, rancidity may give rise to toxic levels of certain compounds like aldehydes, hydroperoxides, epoxides and cholesterol oxidation products. In this PhD study chromatographic and spectroscopic techniques were employed to determine the degree of rancidity in different animal products and its relationship with technological parameters like feeding fat sources, packaging, processing and storage conditions. To achieve this goal capillary gas chromatography (CGC) was employed not only to determine the fatty acids profile but also, after solid phase extraction, the amount of free fatty acids (FFA), diglycerides (DG), sterols (cholesterol and phytosterols) and cholesterol oxidation products (COPs). To determine hydroperoxides, primary products of oxidation and quantify secondary products UV/VIS absorbance spectroscopy was applied. Most of the foods analysed in this study were meat products. In actual fact, lipid oxidation is a major deterioration reaction in meat and meat products and results in adverse changes in the colour, flavour and texture of meat. The development of rancidity has long recognized as a serious problem during meat handling, storage and processing. On a dairy product, a vegetal cream, a study of lipid fraction and development of rancidity during storage was carried out to evaluate its shelf-life and some nutritional features life saturated/unsaturated fatty acids ratio and phytosterols content. Then, according to the interest that has been growing around functional food in the last years, a new electrophoretic method was optimized and compared with HPLC to check the quality of a beehive product like royal jelly. This manuscript reports the main results obtained in the five activities briefly summarized as follows: 1) comparison between HPLC and a new electrophoretic method in the evaluation of authenticity of royal jelly; 2) study of the lipid fraction of a vegetal cream under different storage conditions; 3) study of lipid oxidation in minced beef during storage under a modified atmosphere packaging, before and after cooking; 4) evaluation of the influence of dietary fat and processing on the lipid fraction of chicken patties; 5) study of the lipid fraction of typical Italian and Spanish pork dry sausages and cured hams.

Architectures and design patterns for functional design of logic control and diagnostics in industrial automation

Recently in most of the industrial automation process an ever increasing degree of automation has been observed. This increasing is motivated by the higher requirement of systems with great performance in terms of quality of products/services generated, productivity, efficiency and low costs in the design, realization and maintenance. This trend in the growth of complex automation systems is rapidly spreading over automated manufacturing systems (AMS), where the integration of the mechanical and electronic technology, typical of the Mechatronics, is merging with other technologies such as Informatics and the communication networks. An AMS is a very complex system that can be thought constituted by a set of flexible working stations, one or more transportation systems. To understand how this machine are important in our society let considerate that every day most of us use bottles of water or soda, buy product in box like food or cigarets and so on. Another important consideration from its complexity derive from the fact that the the consortium of machine producers has estimated around 350 types of manufacturing machine. A large number of manufacturing machine industry are presented in Italy and notably packaging machine industry,in particular a great concentration of this kind of industry is located in Bologna area; for this reason the Bologna area is called “packaging valley”. Usually, the various parts of the AMS interact among them in a concurrent and asynchronous way, and coordinate the parts of the machine to obtain a desiderated overall behaviour is an hard task. Often, this is the case in large scale systems, organized in a modular and distributed manner. Even if the success of a modern AMS from a functional and behavioural point of view is still to attribute to the design choices operated in the definition of the mechanical structure and electrical electronic architecture, the system that governs the control of the plant is becoming crucial, because of the large number of duties associated to it. Apart from the activity inherent to the automation of themachine cycles, the supervisory system is called to perform other main functions such as: emulating the behaviour of traditional mechanical members thus allowing a drastic constructive simplification of the machine and a crucial functional flexibility; dynamically adapting the control strategies according to the different productive needs and to the different operational scenarios; obtaining a high quality of the final product through the verification of the correctness of the processing; addressing the operator devoted to themachine to promptly and carefully take the actions devoted to establish or restore the optimal operating conditions; managing in real time information on diagnostics, as a support of the maintenance operations of the machine. The kind of facilities that designers can directly find on themarket, in terms of software component libraries provides in fact an adequate support as regard the implementation of either top-level or bottom-level functionalities, typically pertaining to the domains of user-friendly HMIs, closed-loop regulation and motion control, fieldbus-based interconnection of remote smart devices. What is still lacking is a reference framework comprising a comprehensive set of highly reusable logic control components that, focussing on the cross-cutting functionalities characterizing the automation domain, may help the designers in the process of modelling and structuring their applications according to the specific needs. Historically, the design and verification process for complex automated industrial systems is performed in empirical way, without a clear distinction between functional and technological-implementation concepts and without a systematic method to organically deal with the complete system. Traditionally, in the field of analog and digital control design and verification through formal and simulation tools have been adopted since a long time ago, at least for multivariable and/or nonlinear controllers for complex time-driven dynamics as in the fields of vehicles, aircrafts, robots, electric drives and complex power electronics equipments. Moving to the field of logic control, typical for industrial manufacturing automation, the design and verification process is approached in a completely different way, usually very “unstructured”. No clear distinction between functions and implementations, between functional architectures and technological architectures and platforms is considered. Probably this difference is due to the different “dynamical framework”of logic control with respect to analog/digital control. As a matter of facts, in logic control discrete-events dynamics replace time-driven dynamics; hence most of the formal and mathematical tools of analog/digital control cannot be directly migrated to logic control to enlighten the distinction between functions and implementations. In addition, in the common view of application technicians, logic control design is strictly connected to the adopted implementation technology (relays in the past, software nowadays), leading again to a deep confusion among functional view and technological view. In Industrial automation software engineering, concepts as modularity, encapsulation, composability and reusability are strongly emphasized and profitably realized in the so-calledobject-oriented methodologies. Industrial automation is receiving lately this approach, as testified by some IEC standards IEC 611313, IEC 61499 which have been considered in commercial products only recently. On the other hand, in the scientific and technical literature many contributions have been already proposed to establish a suitable modelling framework for industrial automation. During last years it was possible to note a considerable growth in the exploitation of innovative concepts and technologies from ICT world in industrial automation systems. For what concerns the logic control design, Model Based Design (MBD) is being imported in industrial automation from software engineering field. Another key-point in industrial automated systems is the growth of requirements in terms of availability, reliability and safety for technological systems. In other words, the control system should not only deal with the nominal behaviour, but should also deal with other important duties, such as diagnosis and faults isolations, recovery and safety management. Indeed, together with high performance, in complex systems fault occurrences increase. This is a consequence of the fact that, as it typically occurs in reliable mechatronic systems, in complex systems such as AMS, together with reliable mechanical elements, an increasing number of electronic devices are also present, that are more vulnerable by their own nature. The diagnosis problem and the faults isolation in a generic dynamical system consists in the design of an elaboration unit that, appropriately processing the inputs and outputs of the dynamical system, is also capable of detecting incipient faults on the plant devices, reconfiguring the control system so as to guarantee satisfactory performance. The designer should be able to formally verify the product, certifying that, in its final implementation, it will perform itsrequired function guarantying the desired level of reliability and safety; the next step is that of preventing faults and eventually reconfiguring the control system so that faults are tolerated. On this topic an important improvement to formal verification of logic control, fault diagnosis and fault tolerant control results derive from Discrete Event Systems theory. The aimof this work is to define a design pattern and a control architecture to help the designer of control logic in industrial automated systems. The work starts with a brief discussion on main characteristics and description of industrial automated systems on Chapter 1. In Chapter 2 a survey on the state of the software engineering paradigm applied to industrial automation is discussed. Chapter 3 presentes a architecture for industrial automated systems based on the new concept of Generalized Actuator showing its benefits, while in Chapter 4 this architecture is refined using a novel entity, the Generalized Device in order to have a better reusability and modularity of the control logic. In Chapter 5 a new approach will be present based on Discrete Event Systems for the problemof software formal verification and an active fault tolerant control architecture using online diagnostic. Finally conclusive remarks and some ideas on new directions to explore are given. In Appendix A are briefly reported some concepts and results about Discrete Event Systems which should help the reader in understanding some crucial points in chapter 5; while in Appendix B an overview on the experimental testbed of the Laboratory of Automation of University of Bologna, is reported to validated the approach presented in chapter 3, chapter 4 and chapter 5. In Appendix C some components model used in chapter 5 for formal verification are reported.

Structural and functional analysis of the hepatitis C virus non-structural protein 5A

Das Hepatitis C Virus (HCV) ist ein umhülltes RNA Virus aus der Familie der Flaviviridae. Sein Genom kodiert für ein ca. 3000 Aminosäuren langes Polyprotein, welches co- und posttranslational in seine funktionellen Einheiten gespalten wird. Eines dieser viralen Proteine ist NS5A. Es handelt sich hierbei um ein stark phosphoryliertes Protein, das eine amphipatische α-Helix im Amino-Terminus trägt, welche für die Membran-Assoziation von NS5A verantwortlich ist. Welche Rolle die Phosphorylierung für die Funktion des Proteins spielt, bzw. welche Funktion NS5A überhaupt ausübt, ist zur Zeit noch unklar. Beobachtungen lassen Vermutungen über eine Funktion von NS5A bei der Resistenz infizierter Zellen gegenüber Interferon-alpha zu. Weiterhin wird vermutet, das NS5A als Komponente des membranständigen HCV Replikasekomplexes an der RNA Replikation beteiligt ist. Das Ziel dieser Doktorarbeit war es, die Funktion von NS5A für die RNA Replikation zu untersuchen. Zu diesem Zweck wurde eine Serie von Phosphorylierungsstellen-Mutanten generiert, die auf Ihre Replikationsfähigkeit und den Phosphorylierungsstatus hin untersucht wurden. Wir fanden, dass bestimmte Serin-Substitutionen im Zentrum von NS5A zu einer gesteigerten RNA Replikation führten, bei gleichzeitig reduzierter NS5A Hyperphosphorylierung. Weiterhin studierten wir den Einfluß von Mutationen in der Amino-terminalen amphipatischen α-Helix von NS5A auf die RNA-Replikation, sowie Phosphorylierung und subzelluläre Lokalisation des Proteins. Wir fanden, dass geringfügige strukturelle Veränderungen der amphipatischen Helix zu einer veränderten subzellulären Lokalisation von NS5A führten, was mit einer reduzierten oder komplett inhibierten RNA Replikation einherging. Zudem interferierten die strukturellen Veränderungen mit der Hyperphosphorylierung des Proteins, was den Schluß nahe legt, dass die amphipatische Helix eine wichtige strukturelle Komponente des Proteins darstellt, die für die korrekte Faltung und Phosphorylierung des Proteins essentiell ist. Als weitere Aspekte wurden die Trans-Komplementationsfähigkeit der verschiedenen viralen Komponenten des HCV Replikasekomplexes untersucht, sowie zelluläre Interaktionspartner von NS5A identifiziert. Zusammenfassend zeigen die Ergebnisse dieser Doktorarbeit, dass NS5A eine wichtige Rolle bei der RNA-Replikation spielt. Diese Funktion wird wahrscheinlich über den Phosphorylierungszustand des Proteins reguliert.

Functional components and lipid quality assessment in vegetable foods: analytical and technological approaches

Lipids are important components that contribute very significantly to nutritional and technological quality of foods because they are the least stable macro-components in foods, due to high susceptibility to oxidation. When rancidity take place, it makes food unhealthy and unacceptable for consumers. Thus, the presence of antioxidants, naturally present of added to foods, is required to enhance shelf life of foods. Moreover, antioxidant like phenolic compounds play an important role in human health enhancing the functionality of foods. The aim of this PhD project was the study of lipid quality and lipid oxidation in different vegetable foods focusing on analytical and technological aspects in order to figure out the effects of lipid composition and bioactive compounds (phenolic compounds, omega-3 fatty acids and dietary fiber) addition on their shelf life. In addition, bioavailability and antioxidant effects of phenolic compounds in human and animals, respectively, were evaluated after consumption of vegetable foods. The first section of the work was focused on the evaluation of lipid quality impact on technological behaviour of vegetable foods. Because of that, cocoa butter with different melting point were evaluated by chromatographic techniques (GC, TLC) and the sample with the higher melting point showed the presence of fatty acids, triglycerides, 2-monoglycerides and FT-IR profile different from genuine cocoa butter, meaning an adding of foreign fat (lauric-fat) not allowed by the law. Looking at lipid quality of other vegetable foods, an accelerated shelf life test (OXITEST®), was used to evaluate of lipid stability to oxidation in tarallini snacks made up using different lipid matrices (sunflower oil, extravirgin olive oil and a blend of extravirgin olive oil and lard). The results showed a good ability of OXITEST® to discriminate between lipid unsaturation and different cooking times, without any samples fat extraction. In the second section, the role of bioactive compounds on cereal based food shelf life was studied in different bakeries by GC, spectrophotometric methods and capillary electrophoresis. It was examined the relationships between phenolic compounds, added with flour, and lipid oxidation of tarallini and frollini. Both products showed an increase in lipid oxidation during storage and antioxidant effects on lipid oxidation were not as expected. Furthermore, the influence of enrichment in polyunsaturated fatty acids on lipid oxidation of pasta was evaluated. The results proved that LC n-3 PUFA were not significantly implicated in the onset of oxidation in spaghetti stored under daylight and accelerated oxidation in a laboratory heater. The importance of phenolic compounds as antioxidant in humans and rats was also studied, by HPLC/MS in the latter section. For this purpose, apigenin and apigenin glycosides excretion was investigated in six women’s urine in a 24 hours study. After a single dose of steamed artichokes, both aglicone and glucuronide metabolites were recovered in 24 h urine. Moreover, the effect of whole grain durum wheat bread and whole grain Kamut® khorasan bread in rats were evaluated. Both cereals were good sources of antioxidants but Kamut® bread fed animals had a better response to stress than wheat durum fed, especially when a sourdough bread was supplied.

Milk and dairy products: evaluation of bioactive components by analytical techniques

Milk and dairy products are important source of bioactive compounds useful to satisfy the nutritional and physiological needs of any newborns of mammalian species and useful to guarantee adequate growth and development of infants as well as provide a complete nourishment of adults. Physico-chemical, nutritional and organoleptic properties of the main constituents and the “minor” components have a crucial role in the quality of milk and milk products. Although in the past decades dietary milk fat was often regarded as harmful for the human health, recent researches suggest that milk contains specific fatty acids with nutritional and physiological health benefits. For these reasons, a major attention is given to the quantity and quality of total fat intake. In the recent years, as a result of the new concept of multifunctional agriculture and the changing behaviours about diet, consumer demands in favor of high-quality, security and safety dairy products are increased. Moreover, milk proteins and milk-derived bioactive peptides are recognized to have a high nutritive value, several health-promoting functional activities and excellent technological properties. Accordingly, growing interest in the development of functional dairy products and preparation of infant formulae for babies who cannot be breast-fed, has been give in order to meet the specific consumer’s requests. This manuscript presents the main results obtained during my PhD research aimed to evaluate the main bioactive lipids and proteins in milk and dairy products using innovative analytical techniques. The experimental section of this manuscript is divided in two sections where are reported the main results obtained during my research activities on dairy products and human milks in order to characterize their bioactive compounds for functional food applications.

Possibilities for the healthy and nutritional improvement of confectionery and sweet products.

The overall objective of this PhD was to investigate the possibility to increase the nutritional value of confectionary products by the use of natural ingredients with healthy functions. The first part of the thesis focused on the possible substitution of the most characteristic component of confectionary products, i.e. refined sugar. Many natural whole sweetening alternatives are available, though not widely used; the use of molasses, the byproduct of sugar beet and cane production, still rich in healthy components as minerals and phytochemicals is hereby discussed; after having verified molasses effectiveness in oxidative stress counteraction on liver cultured cells, the higher antioxidant capacity of a sweet food prepared with molasses instead of refined sugar was confirmed. A second step of the project dealt with another main ingredient of various sweet products, namely wheat. Particularly, the exploitation of soft and durum wheat byproducts could be another sustainable strategy to improve the healthy value of confectionery. The isolation of oligosaccharides with bioactive functions form different fractions of the wheat milling stream was studied and the new ingredients were shown to have a high dietary fiber and antioxidants content. As valid alternative, product developers should consider the appealing and healthy addition of ancient grains flour to sweet baked goods. The possibility of substituting the modern whole durum wheat with the ancient Kamut® khorasan was considered, and the antioxidant and anti-inflammatory effects of these grains were evaluated and compared both in vitro and in vivo on rats. Finally, since high consumption of confectionery is a risk factor for obesity, a possible strategy for the counteraction of this disease was investigated. The ability of three bioactives in inhibiting adipocytes differentiation was investigated. In fact, theoretically, compounds able to influence adipogenesis could be used in the formulation of functional sweet products and contribute to prevent obesity.

Functional silicones and silicone-containing block copolymers

Polysiloxanes can be synthesized and subsequently modified (i) by the attachment of small molecules that change the properties of the silicone in such a way that it becomes more hydrophilic, but under the premise that this does not go together with a loss of the silicone-specific features. This can be done by adding hydrophilic sidechains to a polysiloxane. Polyethers like poly(ethylene glycol) or hyperbranched polyether-polyols are suitable in this regard. In order to assure that the silicone properties retain, these side groups can be attached to only one part of the polysiloxane backbone, which results in a block copolymer that consists of a common polysiloxane and a second block of the modified structure. (ii) Polysiloxanes can be equipped with functional groups that are capable of initializing polymerization of a different monomer (macroinitiator approach). For example, hydroxyl groups are used to initiate the ring opening polymerization of cyclic esters, or ATRP macroinitiators can be synthesized to add a second block via controlled radical polymerization. Stimuli responsive polymers like poly(oligoethylene glycol methacrylate) (POEGMA) can be added via this route to create “smart” siloxane-containing block copolymers that respond to certain stimuli. rnAn important premise for all synthetic routes is to achieve the targeted structure in a process as simple as possible, because facile availability of the material is crucial with regard to industrial applicability of the invented products. rnConcerning characterization of the synthesized macromolecules, emphasize is put on their (temperature dependent) aggregation behavior, which can be investigated by several microscopic and scattering methods, their behavior at the interface between silicone oils and water and their thermal properties.rnrn

Synthetic routes toward functional block copolymers and bioconjugates via RAFT polymerization

Synthetic Routes toward Functional Block Copolymers and Bioconjugates via RAFT PolymerizationrnSynthesewege für funktionelle Blockcopolymere und Biohybride über RAFT PolymerisationrnDissertation von Dipl.-Chem. Kerstin T. WissrnIm Rahmen dieser Arbeit wurden effiziente Methoden für die Funktionalisierung beider Polymerkettenenden für Polymer- und Bioanbindung von Polymeren entwickelt, die mittels „Reversible Addition-Fragmentation Chain Transfer“ (RAFT) Polymerisation hergestellt wurden. Zu diesem Zweck wurde ein Dithioester-basiertes Kettentransferagens (CTA) mit einer Aktivestereinheit in der R-Gruppe (Pentafluorphenyl-4-phenylthiocarbonylthio-4-cyanovaleriansäureester, kurz PFP-CTA) synthetisiert und seine Anwendung als universelles Werkzeug für die Funktionalisierung der -Endgruppe demonstriert. Zum Einen wurde gezeigt, wie dieser PFP-CTA als Vorläufer für die Synthese anderer funktioneller CTAs durch einfache Aminolyse des Aktivesters genutzt werden kann und somit den synthetischen Aufwand, der üblicherweise mit der Entwicklung neuer CTAs verbunden ist, reduzieren kann. Zum Anderen konnte der PFP-CTA für die Synthese verschiedener Poly(methacrylate) mit enger Molekulargewichtsverteilung und wohl definierter reaktiver -Endgruppe verwendet werden. Dieses Kettenende konnte dann erfolgreich mit verschiedenen primären Aminen wie Propargylamin, 1-Azido-3-aminopropan und Ethylendiamin oder direkt mit den Amin-Endgruppen verschiedener Peptide umgesetzt werden.rnAus der Reaktion des PFP-CTAs mit Propargylamin wurde ein Alkin-CTA erhalten, der sich als effizientes Werkzeug für die RAFT Polymerisation verschiedener Methacrylate erwiesen hat. Der Einbau der Alkin-Funktion am -Kettenende wurde mittels 1H und 13C NMR Spektroskopie sowie MALDI TOF Massenspektroskopie bestätigt. Als Modelreaktion wurde die Kopplung eines solchen alkin-terminierten Poly(di(ethylenglykol)methylethermethacrylates) (PDEGMEMA) mit azid-terminiertem Poly(tert-butylmethacrylat), das mittels Umsetzung einer Aktivester-Endgruppe erhalten wurde, als kupferkatalysierte Azid-Alkin-Cycloaddition (CuAAC) durchgeführt. Die Aufarbeitung des resultierenden Diblockcopolymers durch Fällen ermöglichte die vollständige Abtrennung des Polymerblocks 1, der im Überschuss eingesetzt wurde. Darüber hinaus blieb nur ein sehr kleiner Anteil (< 2 Gew.-%) nicht umgesetzten Polymerblocks 2, was eine erfolgreiche Polymeranbindung und die Effizienz der Endgruppen-Funktionalisierung ausgehend von der Aktivester--Endgruppe belegt.rnDie direkte Reaktion von stimuli-responsiven Polymeren mit Pentafluorphenyl(PFP)ester-Endgruppen, namentlich PDEGMEMA und Poly(oligo(ethylenglykol)methylethermethacrylat), mit kollagen-ähnlichen Peptiden ergab wohl definierte Polymer-Peptid-Diblockcopolymere und Polymer-Peptid-Polymer-Triblockcopolymer unter nahezu quantitativer Umsetzung der Endgruppen. Alle Produkte konnten vollständig von nicht umgesetztem Überschuss des Homopolymers befreit werden. In Analogie zu natürlichem Kollagen und dem nicht funktionalisierten kollagen-ähnlichen Peptid bilden die PDEGMEMA-basierten, entschützten Hybridcopolymere Trimere mit kollagen-ähnlichen Triple-Helices in kalter wässriger Lösung, was mittels Zirkular-Dichroismus-Spektroskopie (CD) nachgewiesen werden konnte. Temperaturabhängige CD-Spektroskopie, Trübungsmessungen und dynamische Lichtstreuung deuteten darauf hin, dass sie bei höheren Temperaturen doppelt stimuli-responsive Überstrukturen bilden, die mindestens zwei konformative Übergänge beim Aufheizen durchlaufen. Einer dieser Übergänge wird durch den hydrophoben Kollaps des Polymerblocks induziert, der andere durch Entfalten der kollagen-ähnlichen Triple-Helices.rnAls Ausweitung dieser synthetischen Strategie wurde homotelecheles PDEGMEMA mit zwei PFP-Esterendgruppen dargestellt, wozu der PFP-CTA für die Funktionalisierung der -Endgruppe und die radikalische Substitution des Dithioesters durch Behandlung mit einem Überschuss eines funktionellen AIBN-Derivates für die Funktionalisierung der -Endgruppe ausgenutzt wurde. Die Umsetzung der beiden reaktiven Kettenenden mit dem N-Terminus eines Peptidblocks ergab ein Peptid-Polymer-Peptid Triblockcopolymer.rnSchließlich konnten die anorganisch-organischen Hybridmaterialien PMSSQ-Poly(2,2-diethoxyethylacrylat) (PMSSQ-PDEEA) und PMSSQ-Poly(1,3-dioxolan-2-ylmethylacrylat) (PMSSQ-PDMA) für die Herstellung robuster, peptid-reaktiver Oberflächen durch Spin Coaten und thermisch induziertes Vernetzen angewendet werden. Nach saurem Entschützen der Acetalgruppen in diesen Filmen konnten die resultierenden Aldehydgruppen durch einfaches Eintauchen in eine Lösung mit einer Auswahl von Aminen und Hydroxylaminen umgesetzt werden, wodurch die Oberflächenhydrophilie modifiziert werden konnte. Darüber hinaus konnten auf Basis der unterschiedlichen Stabilität der zwei hier verglichenen Acetalgruppen Entschützungsprotokolle für die exklusive Entschützung der Diethylacetale in PMSSQ-PDEEA und deren Umsetzung ohne Entschützung der zyklischen Ethylenacetale in PMSSQ-PDMA entwickelt werden, die die Herstellung multifunktioneller Oberflächenbeschichtungen z.B. für die Proteinimmobilisierung ermöglichen.

Transcriptional regulation and functional analysis of the Wilms' tumor gene WT1

The Wilms' tumor gene, WT1, encodes a zinc finger transcription factor which functions as a tumor suppressor. Defects in the WT1 gene can result in the development of nephroblastoma. WT1 is expressed during development, primarily in the metanephric kidney, the mesothelial lining of the abdomen and thorax, and the developing gonads. WT1 expression is tightly regulated and is essential for renal development. The WT1 gene encodes a protein with a proline-rich N-terminus which functions as a transcriptional repressor and C-terminus contains 4 zinc fingers that mediate DNA binding. WT1 represses transcription from a number of growth factors and growth factor receptors. WT1 mRNA undergoes alternative splicing at two sites, resulting in 4 mRNA species and polypeptide products. Exon 5, encoding 17 amino acids is alternatively spliced, and is located between the transcriptional repression domain and the DNA binding domain. The second alternative splice is the terminal 9 nucleotides of zinc finger 3, encoding the tripeptide Lys-Thr-Ser (KTS). The presence or absence of KTS within the zinc fingers of WT1 alters DNA binding.^ I have investigated transcriptional regulation of WT1, characterizing two means of repressing WT1 transcription. I have cloned a transcriptional silencer of the WT1 promoter which is located in the third intron of the WT1 gene. The silencer is 460 bp in length and contains an Alu repeat. The silencer functions in cells of non-renal origin.^ I have found that WT1 protein can autoregulate the WT1 promoter. Using the autoregulation of the WT1 promoter as a functional assay, I have defined differential consensus DNA binding motifs of WT1 isoforms lacking and containing the KTS tripeptide insertion. With these refined consensus DNA binding motifs, I have identified two additional targets of WT1 transcriptional repression, the proto-oncogenes bcl-2 and c-myc.^ I have investigated the ability of the alternatively spliced exon 5 to influence cell growth. In cell proliferation assays, isoforms of WT1 lacking exon 5 repress cell growth. WT1 isoforms containing exon 5 fail to repress cell growth to the same extent, but alter the morphology of the cells. These experiments demonstrate that the alternative splice isoforms of WT1 have differential effects on the function of WT1. These findings suggest a role for the alternative splicing of WT1 in metanephric development. ^

A FUNCTIONAL VARIANT OF HLA-DR1 DELINEATED BY T-LYMPHOCYTE CLONE REACTIVITY, PROTEIN CONFORMATION, AND GENOMIC RESTRICTION SITES

This research characterized a serologically indistinguishable form of HLA-DR1 that: (1) cannot stimulate some DR1-restricted or specific T-lymphocyte clones; (2) displays an unusual electrophoretic pattern on two dimensional gels; and (3) is marked by a polymorphic restriction site of the alpha gene. Inefficient stimulation of some DR1-restricted clones was a property of DR1$\sp{+}$ cells that shared HLA-B14 on the same haplotype and/or were carriers of 21-hydroxylase (21-OH) deficiency. Nonclassical 21-OH deficiency frequently demonstrates genetic linkage with HLA-B14;DR1 haplotypes and associates with duplications of C4B and one 21-OH gene. Cells having both stimulatory (DR1$\sb{\rm n}$) and nonstimulatory (DR1$\sb{\rm x}$) parental haplotypes did not mediate proliferation of these clones. However, heterozygous DR1$\sb{\rm x}$, 2 and DR1$\sb{\rm x}$, 7 cells were efficient stimulators of DR2 and DR7 specific clones, respectively, suggesting that a trans acting factor may modify DR1 alleles or products to yield a dominant DR1$\sb{\rm x}$ phenotype. Incompetent stimulator populations did not secrete an intercellular soluble or contact dependent suppressor factor nor did they express interleukin-2 receptors competing for T-cell growth factors. Two dimensional gel analysis of anti-DR immunoprecipitates revealed, in addition to normal DR$\alpha$ and DR$\beta$ chains, a 50kD species from DR1$\sb{\rm x}$ but not from the majority of DR1$\sb{\rm n}$ or non-DR1 cells. The 50kD structure was stable under reducing conditions in SDS and urea, had antigenic homology with DR, and dissociated after boiling into 34kD and 28kD peptide chains apparently identical with DR$\alpha$ and DR$\beta$ as shown by limited digest peptide maps. N-linked glycosylation and sialation of DRgp50 appeared to be unchanged from normal DR$\alpha$ and DR$\beta$. Bg1II digestion and $DR\alpha$ probing of DR1$\sb{\rm x}$ genomic DNA revealed a 4.5kb fragment while DR1$\sb{\rm n}$ DNA yielded 3.8 and 0.76kb fragments; all restriction sites mapped to the 3$\sp\prime$ untranslated region of $DR\alpha$. Collectively, these data suggest that DRgp50 represents a novel combinatorial association between constitutive chains of DR that may interfere with or compete for normal T cell receptor recognition of DR1 as both an alloantigen and restricting element. Furthermore, extensive chromosomal abnormalities previously mapped to the class III region of B14;DR1 haplotypes may extend into the adjacent class II region with consequent intrusion on immune function. ^

Characterization of cytochrome P450 4F subfamily: Functional role and response in inflammation

CYP4F subfamily comprises a group of enzymes that metabolize LTB4 to biologically less active metabolites. These inactive hydroxy products are incapable of chemotaxis and recruitment of inflammatory cells. This has led to a hypothesis that CYP4Fs may modulate inflammatory conditions serving as a signal of resolution. ^ We investigated the regulation of rat CYP4F gene expression under various inflammatory prompts including a bacterial lipopolysaccharide (LPS) treated model system, controlled traumatic brain injury (TBI) model as well as using direct cytokine challenges. CYP4Fs showed an isoform specific response to LPS. The pro-inflammatory cytokines IL-1β, IL-6 and TNF-α produced an overall inductive CYP4F response whereas IL-10, an anti-inflammatory cytokine, suppressed CYP4F gene expression in primary hepatocytes. The molecular mechanism behind IL-6 mediated CYP4F induction was partially STAT3 dependent. ^ An alternate avenue of triggering the inflammatory cascade is TBI, which is known to cause several secondary effects leading to multiorgan dysfunction syndrome. The results from this study elicited that trauma to the brain can produce acute inflammatory changes in organs distant from the injury site. Local production of LTB4 after CNS injury caused mobilization of inflammatory cells such as neutrophils to the lung. In the resolution phase, CYP4F expression increased with time along with the associated activity causing a decline in LTB4 concentration. This marked a significant reduction in neutrophil recruitment to the lung which led to subsequent recovery and repair. In addition, we showed that CYP4Fs are localized primarily in pulmonary endothelium. We speculate that the temporally regulated LTB4 clearance in the endothelium may be a novel target for treatment of pulmonary inflammation following injury. ^ In humans, several CYP4F isoforms have been identified and shown to metabolize LTB4 and other endogenous eicosanoids. However, the specific activity of the recently cloned human CYP4F11 is unknown. In the final part of this thesis, CYP4F11 protein was expressed in yeast in parallel to CYP4F3A. To our surprise, CYP4F11 displayed a different substrate profile than CYP4F3A. CYP4F3A metabolized eicosanoids while CYP4F11 was a better catalyst for therapeutic drugs. Thus, besides their endogenous function in clearing inflammation, CYP4Fs also may play a part in drug metabolism. ^

Plant Functional Type (PFT) map over Siberia derived from GlobCover 2005 dataset, link to dataset in NetCDF format

High-latitude ecosystems play an important role in the global carbon cycle and in regulating the climate system and are presently undergoing rapid environmental change. Accurate land cover data sets are required to both document these changes as well as to provide land-surface information for benchmarking and initializing Earth system models. Earth system models also require specific land cover classification systems based on plant functional types (PFTs), rather than species or ecosystems, and so post-processing of existing land cover data is often required. This study compares over Siberia, multiple land cover data sets against one another and with auxiliary data to identify key uncertainties that contribute to variability in PFT classifications that would introduce errors in Earth system modeling. Land cover classification systems from GLC 2000, GlobCover 2005 and 2009, and MODIS collections 5 and 5.1 are first aggregated to a common legend, and then compared to high-resolution land cover classification systems, vegetation continuous fields (MODIS VCFs) and satellite-derived tree heights (to discriminate against sparse, shrub, and forest vegetation). The GlobCover data set, with a lower threshold for tree cover and taller tree heights and a better spatial resolution, tends to have better distributions of tree cover compared to high-resolution data. It has therefore been chosen to build new PFT maps for the ORCHIDEE land surface model at 1 km scale. Compared to the original PFT data set, the new PFT maps based on GlobCover 2005 and an updated cross-walking approach mainly differ in the characterization of forests and degree of tree cover. The partition of grasslands and bare soils now appears more realistic compared with ground truth data. This new vegetation map provides a framework for further development of new PFTs in the ORCHIDEE model like shrubs, lichens and mosses, to represent the water and carbon cycles in northern latitudes better. Updated land cover data sets are critical for improving and maintaining the relevance of Earth system models for assessing climate and human impacts on biogeochemistry and biophysics.