Helix-coil-transition in solid polypeptides

Abstract Poly(L-glutamic acid) (PLGA) was synthesized by living anionic ring-opening polymerization of the NCA monomer, which was obtained by reacting diphosgene with an amino acid derivative. The chemical structures and thermal properties were characterized by 1H-NMR, 13C-NMR, TGA and DSC. XRD powder patterns found to be amorphous for all polymers obtained. The molecular weights could be determined under severe limitations due to low solubility and high aggregation tendency. The secondary structure of the PLGA films was analyzed in the solid state by IR spectroscopy; the order was determined mainly by XRD. Uniform bulk films (1-5 µm) were produced by drop-casting of PLGA solutions in TFA on silica. The XRD film analysis indicated the absence of a long range order or an orientation even if a helical microstructure was confirmed by IR spectroscopy. The coil solvent TFA delivered constantly a helical or a β-sheet structure in the solid state depending on the water content of the solvent which was observed for the first time to exhibit a high influence on the crystallization process for PLGA. Temperature dependent in-situ IR measurements were examined to analyze if a helix-coil transition occurs, but there could be no solvent system determined, which resulted in a disordered coil structure in the solid state. General parameters like solvent systems, evaporation conditions, concentration, substrates etc. were analyzed. New crystallizations were obtained on silica prepared by drop-casting of solutions of PLGA in DMF, DMA, TMU, NMP, and pyridine/water mixtures, respectively. PSCBC in DMF, CDCl3/TFA-d, and PSBC in CDCl3/TFA-d exhibited the same crystalline diffraction patterns like PLGA. The long range order in the X-ray diffraction pattern is proven by extremely sharp crystalline signals, which are not changing the shape or the position of the peak by increasing the temperature up to 160°C. The substrate seems to play a decisive role because the crystalline structures were not obtainable on glass. The crystal structure consists probably of two different layered structures based on the intensity ratios of the two series of crystalline signals in the X-ray diffraction patterns. The source of the layered structure remains unclear and needs further studies to investigate the spatial arrangement of the chains in more detail. The secondary structure was still not changing upon heating even if a highly crystalline diffraction pattern occurs. Concluding that even the newly investigated crystallization did not show a helix-coil transition in the solid state by annealing, the phenomenon known in solution has to be claimed as unachievable in the solid state based on the results of this work. A remaining open question represents the observation that the same crystalline pattern can be reproducibly prepared with exhibiting two different ordered secondary structures (helix and β-sheet). After the investigation that the evaporation time cannot be decisive for the crystal growth, the choice of a strong hydrogen bonding interrupting solvent is most probably the key to support and induce the crystallization process.

Zur Struktur und Funktion der Typ-3-Kupferproteine sowie der Lichtsammlerkomplexe LHCI und LHCII

In der vorliegenden Arbeit wurden die bioinformatischen Methoden der Homologie-Modellierung und Molekularen Modellierung dazu benutzt, die dreidimensionalen Strukturen verschiedenster Proteine vorherzusagen und zu analysieren. Experimentelle Befunde aus Laborversuchen wurden dazu benutzt, die Genauigkeit der Homologie-Modelle zu erhöhen. Die Ergebnisse aus den Modellierungen wurden wiederum dazu benutzt, um neue experimentelle Versuche vorzuschlagen. Anhand der erstellten Modelle und bekannten Kristallstrukturen aus der Protein-Datenbank PDB wurde die Struktur-Funktionsbeziehung verschiedener Tyrosinasen untersucht. Dazu gehörten sowohl die Tyrosinase des Bakteriums Streptomyces als auch die Tyrosinase der Hausmaus. Aus den vergleichenden Strukturanalysen der Tyrosinasen resultierten Mechanismen für die Monophenolhydroxylase-Aktivität der Tyrosinasen sowie für den Import der Kupferionen ins aktive Zentrum. Es konnte der Beweis geführt werden, daß die Blockade des CuA-Zentrums tatsächlich der Grund für die unterschiedliche Aktivität von Tyrosinasen und Catecholoxidasen ist. Zum ersten Mal konnte mit der Maus-Tyrosinase ein vollständiges Strukturmodell einer Säugetier-Tyrosinase erstellt werden, das dazu in der Lage ist, die Mechanismen bekannter Albino-Mutationen auf molekularer Ebene zu erklären. Die auf der Basis des ermittelten 3D-Modells gewonnenen Erkenntnisse über die Wichtigkeit bestimmter Aminosäuren für die Funktion wurde durch gerichtete Mutagenese an der rekombinant hergestellten Maus-Tyrosinase getestet und bestätigt. Weiterhin wurde die Struktur der Tyrosinase des Krebses Palinurus elephas durch eine niedrigaufgelöste 3D-Rekonstruktion aus elektronenmikroskopischen Bildern aufgeklärt. Der zweite große Themenkomplex umfasst die Strukturanalyse der Lichtsammlerkomplexe LHCI-730 und LHCII. Im Falle des LHCII konnte der Oligomerisierungszustand der LHCMoleküle mit diskreten Konformationen des N-Terminus korreliert werden. Auch hier kam eine Kombination von Homologie-Modellierung und einer experimentellen Methode, der Elektronen-Spin-Resonanz-Messung, zum Einsatz. Die Änderung des Oligomerisierungszustands des LHCII kontrolliert den Energiezufluß zu den Photosystemen PS I und PS II. Des Weiteren wurde ein vollständiges Modell des LHCI-730 erstellt, um die Auswirkungen gerichteter Mutagenese auf das Dimerisierungsverhalten zu untersuchen. Auf Basis dieses Modells wurden die Wechselwirkungen zwischen den Monomeren Lhca1 und Lhca4 evaluiert und potentielle Bindungspartner identifiziert.

Pubblici poteri e signorie di castello nella romagna nord-occidentale (secoli XI-XIII)

La presente ricerca di dottorato consiste in un’analisi di carattere politico ed istituzionale dei poteri signorili e territoriali, collegati a distretti castrensi, documentati nella Romagna nord-occidentale durante il pieno medioevo. L’indagine mira a ricostruire, principalmente attraverso fonti documentarie, alcune delle quali inedite, la geografia dei poteri in un’area sub-regionale, con particolare attenzione al fenomeno della signoria rurale, dei poteri comitali e dell’incastellamento. Partendo dallo studio di una realtà locale, la ricerca arriva a sviluppare argomentazioni di carattere generale, ricollegandosi al dibattito storiografico sui poteri signorili e l’incastellamento. La ricerca risulta incentrata sui soggetti politici, laici ed ecclesiastici, detentori dei castelli e dei poteri pubblici nella Bassa Romagna, in primo luogo gli arcivescovi di Ravenna, i vescovi e i conti di Imola, le famiglie comitali di Cunio, Bagnacavallo e Donigallia nei secoli XI-XIII. L'attenzione si concentra, in particolare, sulla fase del cosiddetto “secondo incastellamento” e sui decenni a cavaliere tra XII e XIII secolo, con il tentativo di espansione dei comuni nel contado e la formalizzazione dei poteri dei signori rurali da parte dei sovrani svevi. Proprio alla complessa interazione con il mondo cittadino e allo stretto rapporto dei Cunio e dei Malvicini con la corte di Federico II viene dato ampio spazio nei capitoli conclusivi del presente lavoro.

Stabilization of Juxta-Physeal Distal Tibial and Fibular Fractures in a Juvenile Tiger Using a Hybrid Circular-Linear External Fixator

OBJECTIVE: To report stabilization of closed, comminuted distal metaphyseal transverse fractures of the left tibia and fibula in a tiger using a hybrid circular-linear external skeletal fixator. STUDY DESIGN: Clinical report. ANIMAL: Juvenile tiger (15 months, 90 kg). METHODS: From imaging studies, the tiger had comminuted distal metaphyseal transverse fractures of the left tibia and fibula, with mild caudolateral displacement and moderate compression. Multiple fissures extended from the fractures through the distal metaphyses, extending toward, but not involving the distal tibial and fibular physes. A hybrid circular-linear external skeletal fixator was applied by closed reduction, to stabilize the fractures. RESULTS: The fractures healed and the fixator was removed 5 weeks after stabilization. Limb length and alignment were similar to the normal contralateral limb at hospital discharge, 8 weeks after surgery. Two weeks later, the tiger had fractures of the right tibia and fibula and was euthanatized. Necropsy confirmed pathologic fractures ascribed to copper deficiency. CONCLUSION: Closed application of the hybrid construct provided sufficient stability to allow this 90 kg tiger's juxta-articular fractures to heal with minimal complications and without disrupting growth from the adjacent physes.

Cryoultramicrotomy

Cryoultramicrotomy allows the sectioning of vitrified biological samples. These biological samples are preserved at the atomic level and represent the real structure at the moment of freezing. Cryoultramicrotomy produces ultra-thin cryosections that are investigated in a cryoelectron microscope. The necessity of working during the whole preparation at temperatures less than −140°C results in some difficulties, including the cryosection transfer from the knife-edge to the electron microscropy grid; the grid handling in the cryochamber and the grid transfer into the cryoholder of the electron microscope. Furthermore, ice crystal contamination (from air humidity) can obscure the structures of interest in the sections. It is mainly know-how and experience that will prevent the contamination of ice crystals and the recrystallization of the sections during the manipulations. Here, we describe the tips, tricks, the tools, and methods that help to overcome these burdens and pave the path for successful cryoultramicrotomy.

Effects of elevated atmospheric CO2 and O3 on wood density, antomical proerties and decomposition of Northern Hardwoods

Anthropogenic activities continue to drive atmospheric CO2 and O3 concentrations to levels higher than during the pre-industrial era. Accumulating evidence indicates that both elevated CO2 and elevated O3 could modify the quantity and biochemistry of woody plant biomass. Anatomical properties of woody plants are largely influenced by the activity of the cambium and the growth characteristics of wood cells, which are in turn influenced by a range of environmental factors. Hence, alterations in the concentrations of atmospheric CO2 and / or O3 could also impact wood anatomical properties. Many fungi derive their metabolic resources for growth from plant litter, including woody tissue, and therefore modifications in the quantity, biochemistry and anatomical properties of woody plants in response to elevated CO2 and / or O3 could impact the community of wood-decaying fungi and rates of wood decomposition. Consequently carbon and nutrient cycling and productivity of terrestrial ecosystem could also be impacted. Alterations in wood structure and biochemistry of woody plants could also impact wood density and subsequently impact wood quality. This dissertation examined the long term effects of elevated CO2 and / or O3 on wood anatomical properties, wood density, wood-decaying fungi and wood decomposition of northern hardwood tree species at the Aspen Free-Air CO2 and O3 Enrichment (Aspen FACE) project, near Rhinelander, WI, USA. Anatomical properties of wood varied significantly with species and aspen genotypes and radial position within the stem. Elevated CO2 did not have significant effects on wood anatomical properties in trembling aspen, paper birch or sugar maple, except for marginally increasing (P < 0.1) the number of vessels per square millimeter. Elevated O3 marginally or significantly altered vessel lumen diameter, cell wall area and vessel lumen area proportions depending on species and radial position. In line with the modifications in the anatomical properties, elevated CO2 and O3, alone, significantly modified wood density but effects were species and / or genotype specific. However, the effects of elevated CO2 and O3, alone, on wood anatomical properties and density were ameliorated when in combination. Wood species had a much greater impact on the wood-decaying fungal community and initial wood decomposition rate than did growth or decomposition of wood in elevated CO2 and / or O3. Polyporales, Agaricales, and Russulales were the dominant orders of fungi isolated. Based on the current results, future higher levels of CO2 and O3 may have moderate effects on wood quality of northern hardwoods, but for utilization purposes these may not be considered significant. However, wood-decaying fungal community composition and decomposition of northern hardwoods may be altered via shifts in species and / or genotype composition under future higher levels of CO2 and O3.

MITEP: REFLECTIVE JOURNEY

This research looks at the use of the Interactive Student Notebook (ISN) in the math classroom and the impact on student achievement as part of the MiTEP program. A reflective critical analysis of the MiTEP program discusses impact on teacher pedagogy, leadership, and connections to people and resources. The purpose of the study stemmed from the lack of student retention, poor organizational skills, and the students’ inability to demonstrate college readiness skills such as how to study, completing homework, and thinking independently. Motivation also stemmed from teacher frustration. The research was conducted at Linden Grove Middle School in Kalamazoo Michigan in a strategic math class. Twenty-two sixth graders, thirty-two seventh graders, and forty eighth graders were part of the study.Students were given the Strategic Math Inventory (SMI) test in week 1 of the class and again at the end of a 12 week marking period. Students participated in an attitude survey to record their feelings about the use of the ISN in the strategic math classroom. The data compared the control group (the previous year’s [2012-2013] growth data) to the experimental group, the current year’s (2013-2014) growth data. Both groups were statistically similar in that the mean average was about a 4th grade level equivalency and the groups had similar numbers of grade level students. The significant findings were in the amount of growth made using the ISN. The control group started with a mean average of 586.6 and ended with a mean average of 697.1, making about one year’s growth from a 4th to a 5th grade level equivalency. The experimental group started with a mean average of 585.2 and ended with a mean average of 744.2, making about two years growth from a 4th to a 6th grade level equivalency. This is double the growth of the control group. The Cohen’s test resulted in a score of 0.311 which describes that the teaching method, the use of the ISN in the math classroom had a medium impact on student growth.

Mineralization of calcium phosphate crystals in starch template inducing a brushite kidney stone biomimetic composite

Dicalcium phosphate dihydrate (brushite) and octacalcium phosphate (OCP) crystals are precursors of hydroxyapatite (HAp) for tooth enamel, dentine, and bones formation in living organisms. Here, we introduce a new method for biomimicking brushite and OCP in starch using single and double diffusion techniques. Brushite and OCP crystals were grown by precipitation in starch after gelation. The obtained materials were analyzed by infrared spectroscopy (IR), scanning electron microscopy (SEM), X-ray diffraction (XRD), and confocal laser scanning microscopy (CLSM). IR spectra demonstrate starch inclusion by peak shifts in the 2900–3500 cm–1 region. SEM showed two different morphologies: plate-shaped and needle-like crystals. Calcium phosphate/starch aggregates bear strong resemblance to prismatic brushite kidney stones. This may open up a clue to understand the mechanism of kidney stone formation.

Mutations in the cofilin partner Aip1/Wdr1 cause autoinflammatory disease and macrothrombocytopenia.

A pivotal mediator of actin dynamics is the protein cofilin, which promotes filament severing and depolymerization, facilitating the breakdown of existing filaments, and the enhancement of filament growth from newly created barbed ends. It does so in concert with actin interacting protein 1 (Aip1), which serves to accelerate cofilin's activity. While progress has been made in understanding its biochemical functions, the physiologic processes the cofilin/Aip1 complex regulates, particularly in higher organisms, are yet to be determined. We have generated an allelic series for WD40 repeat protein 1 (Wdr1), the mammalian homolog of Aip1, and report that reductions in Wdr1 function produce a dramatic phenotype gradient. While severe loss of function at the Wdr1 locus causes embryonic lethality, macrothrombocytopenia and autoinflammatory disease develop in mice carrying hypomorphic alleles. Macrothrombocytopenia is the result of megakaryocyte maturation defects, which lead to a failure of normal platelet shedding. Autoinflammatory disease, which is bone marrow-derived yet nonlymphoid in origin, is characterized by a massive infiltration of neutrophils into inflammatory lesions. Cytoskeletal responses are impaired in Wdr1 mutant neutrophils. These studies establish an essential requirement for Wdr1 in megakaryocytes and neutrophils, indicating that cofilin-mediated actin dynamics are critically important to the development and function of both cell types.

Virus-like particle-mediated intracellular delivery of mRNA cap analog with in vivo activity against hepatocellular carcinoma.

UNLABELLED Adenovirus dodecahedron (Dd), a nanoparticulate proteinaceous biodegradable virus-like particle (VLP), was used as a vector for delivery of an oncogene inhibitor to hepatocellular carcinoma (HCC) rat orthotopic model. Initiation factor eIF4E is an oncogene with elevated expression in human cancers. Cell-impermeant eIF4E inhibitor, cap structure analog (cap) and anti-cancer antibiotic doxorubicin (Dox) were delivered as Dd conjugates. Dd-cap and Dd-dox inhibited cancer cell culture proliferation up to 50 and 84%, respectively, while with free Dox similar results could be obtained only at a 5 times higher concentration. In animal HCC model the combination treatment of Dd-cap/Dd-dox caused 40% inhibition of tumor growth. Importantly, the level of two pro-oncogenes, eIF4E and c-myc, was significantly diminished in tumor sections of treated rats. Attachment to Dd, a virus-like particle, permitted the first demonstration of cap analog intracellular delivery and resulted in improved doxorubicin delivery leading to statistically significant inhibition of HCC tumor growth. FROM THE CLINICAL EDITOR Adenovirus dodecahedron, a nanoparticulate proteinaceous biodegradable virus-like particle was used in this study as a vector for the concomitant delivery of cap structure analog and doxorubicine to hepatocellular carcinoma in a rat model, resulting in significant inhibition of tumor growth.