The Use Of Amido- And Imido-Functionalized Alpha Hydroxy Acids In The Creation Of Biodegradable Polyesters: An Exploration Of Va

The purpose of this thesis was to synthesize biodegradable polyesters from a wide array of functionalized ¿-hydroxy acids. The initial strategy was to use amido-functionalized ¿-hydroxy acids and 2-bromopropanoyl bromide to form amido-functionalized cyclic diesters. Then, the resulting cyclic diesters would be used in ring opening polymerization to create biodegradable polyesters. However, the spontaneous rapid degradation of the secondary amido-functionalized cyclic diester structure, as seen with 2-benzamido-hydroxyacetic acid, limited ring formation to tertiary amido-functionalized ¿-hydroxy acids. Also, the hydrophilic nature of most ¿-hydroxy acids allowed water into the crystal structure of the ¿-hydroxy acid. Then, when the ¿-hydroxy acid was used in ring forming reactions, the associated water deactivated reactive reagents and limited cyclic diester synthesis. These issues led to the synthesis of hydrophobic and tertiary amido- and imido-functionalized ¿-hydroxy acids, 2-phthalimido-2-hydroxyacetic acid and 2-(1-oxoisoindolin-2-yl) hydroxyacetic acid. The new ¿-hydroxy acids were used in two new polymerization techniques, melt polycondensation and solution polymerization, instead of ring open polymerization. Melt polycondensation and solution polymerization had shown previous success in forming oligomers of amido-functionalized ¿-hydroxy acids. Melt polycondensation was conducted by heating the monomer past its melting temperature under reduced pressure. The uncatalyzed melt polycondensation of 2-(1-oxoisoindolin-2-yl) hydroxyacetic acid created polyesters (¿ 960 g/mol). The scandium(III) trifluoromethanesulfonate enhanced melt polycondensation polymerization created slightly larger oligomers (¿ 1340 g/mol). However, 2-phthalimido-2-hydroxyacetic acid was not compatible with melt polycondensation because thermal degradation occurred. Thus, solution polymerization was conducted via Steglich esterification. Only oligomeric functionalized polyesters were formed (¿ 1060 g/mol). Future work should focus on optimization of the catalyst and the reaction conditions to obtain higher molecular weight polyesters. Also, 2-(1-oxoisoindolin-2-yl) hydroxyacetic acid should be utilized in the cyclic diester synthesis technique.

Growth response to climate and drought change along an aridity gradient in the southernmost Pinus nigra relict forests

Tree populations at the rear edge of species distribution are sensitive to climate stress and drought. However, growth responses of these tree populations to those stressors may vary along climatic gradients. To analyze growth responses to climate and drought using dendrochronology in rear-edge Pinus nigra populations located along an aridity gradient. Tree-ring width chronologies were built for the twentieth century and related to monthly climatic variables, a drought index (Standardized Precipitation-Evapotranspiration Index), and two atmospheric circulation patterns (North Atlantic and Western Mediterranean Oscillations). Growth was enhanced by wet and cold previous autumns and warm late winters before tree-ring formation. The influence of the previous year conditions on growth increased during the past century. Growth was significantly related to North Atlantic and Western Mediterranean Oscillations in two out of five sites. The strongest responses of growth to the drought index were observed in the most xeric sites. Dry conditions before tree-ring formation constrain growth in rear-edge P. nigra populations. The comparisons of climate-growth responses along aridity gradients allow characterizing the sensitivity of relict stands to climate warming.

Molecular mechanism by which the Escherichia coli nucleoid occlusion factor, SlmA, keeps cytokinesis in check

Cell division or cytokinesis is one of the most fundamental processes in biology and is essential for the propagation of all living species. In Escherichia coli, cell division occurs by ingrowth of the membrane envelope at the cell center and is orchestrated by the FtsZ protein. FtsZ self-assembles into linear protofilaments in a GTP dependent manner to form a cytoskeletal scaffold called the Z-ring. The Z-ring provides the framework for the assembly of the division apparatus and determines the site of cytokinesis. The total amount of FtsZ molecules in a cell significantly exceeds the concentration required for Z-ring formation. Hence, Z-ring formation must be highly regulated, both temporally and spatially. In particular, the assembly of Z-rings at the cell poles and over chromosomal DNA must be prevented. These inhibitory roles are played by two key regulatory systems called the Min and nucleoid occlusion (NO) systems. In E. coli, Min proteins oscillate from pole to pole; the net result of this oscillatory process is the formation of a zone of FtsZ inhibition at the cell poles. However, the replicated nucleoid DNA near the midcell must also be protected from bisection by the Z-ring which is ensured by NO. A protein called SlmA was shown to be the effector of NO in E. coli. SlmA was identified in a screen designed to isolate mutations that were lethal in the absence of Min, hence the name SlmA (synthetic lethal with a defective Min system). Furthers SlmA was shown to bind DNA and localize to the nucleoid fraction of the cell. Additionally, light scattering experiments suggested that SlmA interacts with FtsZ-GTP and alters its polymerization properties. Here we describe studies that reveal the molecular mechanism by which SlmA mediates NO in E. coli. Specifically, we determined the crystal structure of SlmA, identified its DNA binding site specificity, and mapped its binding sites on the E. coli chromosome by chromatin immuno-precipitation experiments. We went on to determine the SlmA-FtsZ structure by small angle X-ray scattering and examined the effect of SlmA-DNA on FtsZ polymerization by electron microscopy. Our combined data show how SlmA is able to disrupt Z-ring formation through its interaction with FtsZ in a specific temporal and spatial manner and hence prevent nucleoid guillotining during cell division.

Molecular mechanism by which the nucleoid occlusion factor, SlmA, keeps cytokinesis in check.

In Escherichia coli, cytokinesis is orchestrated by FtsZ, which forms a Z-ring to drive septation. Spatial and temporal control of Z-ring formation is achieved by the Min and nucleoid occlusion (NO) systems. Unlike the well-studied Min system, less is known about the anti-DNA guillotining NO process. Here, we describe studies addressing the molecular mechanism of SlmA (synthetic lethal with a defective Min system)-mediated NO. SlmA contains a TetR-like DNA-binding fold, and chromatin immunoprecipitation analyses show that SlmA-binding sites are dispersed on the chromosome except the Ter region, which segregates immediately before septation. SlmA binds DNA and FtsZ simultaneously, and the SlmA-FtsZ structure reveals that two FtsZ molecules sandwich a SlmA dimer. In this complex, FtsZ can still bind GTP and form protofilaments, but the separated protofilaments are forced into an anti-parallel arrangement. This suggests that SlmA may alter FtsZ polymer assembly. Indeed, electron microscopy data, showing that SlmA-DNA disrupts the formation of normal FtsZ polymers and induces distinct spiral structures, supports this. Thus, the combined data reveal how SlmA derails Z-ring formation at the correct place and time to effect NO.

Study of the cellular mechanism of Sunitinib mediated inactivation of activated hepatic stellate cells and its implications in angiogenesis

The development of hepatocellular carcinomas from malignant hepatocytes is frequently associated with intra- and peritumoral accumulation of connective tissue arising from activated hepatic stellate cells (HSC). Inhibition of receptor tyrosine kinase (RTK) signaling showed promise in the treatment of hepatocellular carcinoma. However, there is a lack of knowledge about the effects of RTK inhibitors on the tumor supportive cells. We performed in vitro experiments to study whether Sunitinib, a platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) RTKs' inhibitor, could block both activated HSC functions and angiogenesis and thus prevent the progression of cirrhotic liver to hepatocellular carcinoma. In immortalized human activated HSC LX-2, treatment with Sunitinib 100 nM blocked collagen synthesis by 47%, as assessed by Sirius Red staining, attenuated HSC contraction by 65%, and reduced cell migration by 28% as evaluated using a Boyden's chamber, without affecting cell viability, measured by Trypan blue staining, and apoptosis, measured by propidium iodide (PI) incorporation assay. Our data revealed that Sunitinib treatment blocked the transdifferentiation of primary human HSC (hHSC) to activated myofibroblast-like cells by 65% without affecting hHSC apoptosis and migration. In in vitro angiogenic assays, Sunitinib 100 nM reduced endothelial cells (EC) ring formation by 46% and tube formation by 68%, and decreased vascular sprouting in aorta ring assay and angiogenesis in vascular bed of chick embryo. In conclusion, the present study demonstrates that the RTK inhibitor Sunitinib blocks the activation of HSC and angiogenesis suggesting its potential as a drug candidate in pathological conditions like liver fibrosis and hepatocellular carcinoma.

Site and Age Condition the Growth Responses to Climate and Drought of Relict Pinus nigra subsp. salzmannii Populations in Southern Spain

To assess if tree age may modulate the main climatic drivers of radial growth, two relict Pinus nigra subsp. salzmannii populations (Maria, most xeric site; Magina, least xeric site) were sampled in southern Spain near the limits of the species range. Tree-ring width residual chronologies for two age groups (mature trees, age <= 100 years (minimum 40 years); old trees, age > 100 years) were built to evaluate their responses to climate by relating them to monthly precipitation and temperature and a drought index (DRI) using correlation and response functions. We found that drought is the main driver of growth of relict P. nigra populations, but differences between sites and age classes were also observed. First, growth in the most xeric site depends on the drought severity during the previous autumn and the spring of the year of tree-ring formation, whereas in the relatively more mesic site growth is mainly enhanced by warm and wet conditions in spring. Second, growth of mature trees responded more to drought severity than that of old trees. Our findings indicate that drought severity will mainly affect growth of relict P. nigra populations dominated by mature trees in xeric sites. This conclusion may also apply to similar mountain Mediterranean conifer relicts.

Exploring Growth Variability and Crown Vitality of Sessile Oak (Quercus Petraea) in the Czech Republic

Unraveling climatic effects on growth of oak - Europe’s most ecologically and economically important forest species - has been the subject of many recent studies; however, more insight based on field data is necessary to better understand the relationship between climate and tree growth and to adapt forest management strategies to future climate change. In this report, we explore the influence of temperature, precipitation and drought variability on the productivity and vitality of oak stands in the Czech Highlands. We collected 180 cores from mature oaks (Quercus petraea) at four forest stands in the Czech Drahany Highlands. Standard dendromethods were used for sample preparation, ring width measurements, cross-dating, chronology development, and the assessment of growth-climate response patterns. Crown vitality was also evaluated, using the modified ICP Forests methodology. Late spring precipitation totals between May and June as well as the mean July temperature for the year of ring formation were found to be the most important factors for oak growth, whereas crown condition was significantly affected by spring and summer drought. This study is rep-resentative for similar bio-ecological habitats across Central Europe and can serve as a dendroclima-tological blueprint for earlier periods for which detailed meteorological information is missing .

Heritabilities of health traits in Swiss Warmblood horses

REASONS FOR PERFORMING THE STUDY: There is a lack of evidence regarding genetic parameters of health traits in Swiss Warmblood horses. OBJECTIVES: To estimate heritabilities of equine sarcoid disease, horn quality of the hooves, prognathism and increased filling of talocrural joints as a possible indicator for osteochondrosis in Swiss Warmblood horses examined at the field tests for 3-year-olds between 2005 and 2013. STUDY DESIGN: Retrospective analysis of breed society database. METHODS: Swiss Warmblood horses were examined clinically by 13 veterinarians at field tests in Switzerland between 2005 and 2013. The presence of sarcoids, horn quality of the hooves, incisor occlusion and increased joint filling were assessed and recorded. Records of 3715 horses were integrated in a pedigree comprising 217,282 horses. Variance components and heritabilities were estimated on the liability scale using MTGSAM. RESULTS: The prevalences of the examined traits were rather low, ranging from 2.4 to 13.0%. Heritabilities estimated were 0.21 ± 0.07 for the occurrence of sarcoids, 0.04 ± 0.02 for hooves with markedly brittle and friable horn quality, 0.03 ± 0.01 for hooves with marked growth ring formation, 0.06 ± 0.03 for prognathism and 0.08 ± 0.04 for increased filling of the talocrural joint (an indicator of possible osteochondrosis). The influence of the examiner on the variance of these observations was considerable. CONCLUSIONS: With the exception of equine sarcoid disease, estimates for the heritabilities for the traits examined here were low. A standardised examination protocol may reduce the variance due to the examiner. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Genetic and functional analyses of cell division proteins FtsZ and FtsA in Escherichia coli

In the current model for bacterial cell division, the FtsZ protein forms a ring that marks the division plane, creating a cytoskeletal framework for the subsequent action of other essential division proteins such as FtsA and ZipA. The putative protein complex ultimately generates the division septum. The essential cell division protein FtsZ is a functional and structural homolog of eukaryotic tubulin, and like tubulin, FtsZ hydrolyzes GTP and self-assembles into protein filaments in a strictly GTP-dependent manner. FtsA shares sequence similarity with members of the ATPase superfamily that include actin, but its actual function remains unknown. To test the division model and elucidate functions of the division proteins, this dissertation primarily focuses on the analysis of FtsZ and FtsA in Escherichia coli. ^ By tagging with green fluorescent protein, we first demonstrated that FtsA also exhibits a ring-like structure at the potential division site. The localization of FtsA was dependent on functional FtsZ, suggesting that FtsA is recruited to the septum by the FtsZ ring. In support of this idea, we showed that FtsA and FtsZ directly interact. Using a novel E. coli in situ assay, we found that the FtsA-FtsZ interaction appears to be species-specific, although an interspecies interaction could occur between FtsA and FtsZ proteins from two closely related organisms. In addition, mutagenesis of FtsA revealed that no single domain is solely responsible for its septal localization or interaction with FtsZ. To explore the function of FtsA, we purified FtsA protein and demonstrated that it has ATPase activity. Furthermore, purified FtsA stimulates disassembly of FtsZ polymers in a sedimentation assay but does not affect GTP hydrolysis of FtsZ. This result suggests that in the cell, FtsA may function similarly in regulating dynamic instability of the FtsZ ring during the cell division process. ^ To elucidate the structure-function relationship of FtsZ, we carried out thorough genetic and functional analyses of the mutagenized FtsZ derivatives. Our results indicate that the conserved N-terminal domain of FtsZ is necessary and sufficient for FtsZ self-assembly and localization. Moreover, we discovered a critical role for an extreme C-terminal domain of FtsZ that consists of only 12 residues. Truncated FtsZ derivatives lacking this domain, though able to polymerize and localize, are defective in ring formation in vivo as well as interaction with FtsA and ZipA. Alanine scanning mutagenesis of this region pinpointed at least five residues necessary for the function of FtsZ. Studies of protein levels and protein-protein interactions suggested that these residues may be involved in regulating protein stability and/or FtsZ-FtsA interactions. Interestingly, two of the point mutants exhibited dominant-negative phenotypes. ^ In summary, results from this thesis work have provided additional support for the division machinery model and will contribute to a better understanding of the coordinate functions of FtsA and FtsZ in the cell division process. ^

Mid-Pleistocene Mg/Ca, temperature and stable isotopes from Globigerinoides ruber white and Globorotalia inflata of sediment core GeoB3801-6 from the subtropical South Atlantic

The glacial marine isotope stage 14 (MIS 14) appears in many climate records as an unusually warm glacial. During this period an almost monospecific, up to 1.5 m thick, laminated layer of the giant diatom Ethmodiscus rex has been deposited below the South Atlantic Subtropical Gyre. This oligotrophic region is today less favorable for diatom growth with sediments typically consisting of calcareous nannofossil oozes. We have reconstructed temperatures and the stable oxygen isotopic compositions of sea surface and thermocline water (d18Ow) from planktonic foraminiferal (Globigerinoides ruber and Globorotalia inflata) Mg/Ca and stable oxygen isotopes to test whether perturbations in surface ocean conditions contributed to the deposition of the diatom layer at ~530 kyr B.P. Temperatures and d18Ow values reconstructed from this diatom ooze interval are highly variable, with maxima similar to interglacial values. Since the area of the Ethmodiscus oozes resembles the region where Agulhas rings are present, we interpret these hydrographic changes to reflect the varying influence of warm and saline water of Indian Ocean origin that entered the Subtropical Gyre trapped in Agulhas rings. The formation of the Ethmodiscus oozes is associated with a period of maximum Agulhas leakage and a maximum frequency of Agulhas ring formation caused by a termination-type position of the Subtropical Front during the unusual warm MIS 14. The input of silica through the Agulhas rings enabled the shift in primary production from calcareous nannoplankton to diatoms, leading to the deposition of the massive diatom oozes.

Morphogenetic Effects of Neuregulin (Neu Differentiation Factor) in Cultured Epithelial Cells

Neuregulin, or neu differentiation factor, induces cell proliferation or differentiation through interaction with members of the ErbB family of receptor tyrosine kinases. We report that neuregulin can also induce profound morphogenic responses in cultured epithelial cells of different origins. These effects include scattering of small epithelial islands and rearrangement of larger cell islands into ordered ring-shaped arrays with internal lumens. The ring-forming cells are interconnected by cadherin- and β-catenin-containing adherens junctions. In confluent cultures, neuregulin treatment induces formation of circular lumenlike gaps in the monolayer. Both cell scattering and ring formation are accompanied by a marked increase in cell motility that is independent of hepatocyte growth factor/scatter factor and its receptor (c-Met). Affinity-labeling experiments implied that a combination of ErbB-2 with ErbB-3 mediates the morphogenic signal of neuregulin in gastric cells. Indeed, a similar morphogenic effect could be reconstituted in nonresponsive cells by coexpression of ErbB-2 and -3. We conclude that a heterodimer between the kinase-defective neuregulin receptor, ErbB-3, and the coreceptor, ErbB-2, mediates the morphogenetic action of neuregulin.

Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods

A novel protein superfamily with over 600 members was discovered by iterative profile searches and analyzed with powerful bioinformatics and information visualization methods. Evidence exists that these proteins generate a radical species by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. The superfamily (named here Radical SAM) provides evidence that radical-based catalysis is important in a number of previously well- studied but unresolved biochemical pathways and reflects an ancient conserved mechanistic approach to difficult chemistries. Radical SAM proteins catalyze diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation and protein radical formation. They function in DNA precursor, vitamin, cofactor, antibiotic and herbicide biosynthesis and in biodegradation pathways. One eukaryotic member is interferon-inducible and is considered a candidate drug target for osteoporosis; another is observed to bind the neuronal Cdk5 activator protein. Five defining members not previously recognized as homologs are lysine 2,3-aminomutase, biotin synthase, lipoic acid synthase and the activating enzymes for pyruvate formate-lyase and anaerobic ribonucleotide reductase. Two functional predictions for unknown proteins are made based on integrating other data types such as motif, domain, operon and biochemical pathway into an organized view of similarity relationships.

Poly(3-hexylthiophene) based block copolymers prepared by "click" chemistry

p-Conjugated block copolymers have been prepared from terminal azide functionalized polystyrenes (PS) and alkyne functionalized poly(3- hexylthiophene)s (P3HT) via a copper(I) catalyzed Huisgen [3 + 2] dipolar cycloaddition reaction. The functionalized a-azido-PS homopolymer was prepared by atom transfer radical polymerization from a specifically designed initiator bearing the azide function, whereas ?-ethynyl-P3HT and a,?-pentynyl-P3HT were synthesized by a modified Grignard metathesis polymerization using alkynyl Grignard derivatives. The electronic environment of the alkynyl end groups was shown to be decisive in determining triazole ring formation.

Substrate and catalyst effects in C-H insertion reactions of α-diazoacetamides

Intramolecular C–H insertion reactions of α-diazocarbonyl compounds typically proceed with preferential five-membered ring formation. However, the presence of a heteroatom such as nitrogen can activate an adjacent C–H site toward insertion resulting in regiocontrol issues. In the case of α-diazoacetamide derivatives, both β- and γ-lactam products are possible owing to this activating effect. Both β- and γ-lactam products are powerful synthetic building blocks in the area of organic synthesis, as well as a common scaffold in a range of natural and pharmaceutical products and therefore C–H insertion reactions to form such compounds are attractive processes.

The unusual formation of methyl alpha-(5,6-dimethoxycarbonyl-2,3-dimethoxyazepin-7-ylidene)-alpha-[5-methoxycarbonyl-2,3-dimethoxypyrid-6-yl)acetate during the pyrolysis of ''azido-meta-hemipinate'' - First example of a reaction involving a concomitant ring expansion and ring extrusion

he ortho methoxycarbonyl substituent constitutes a sole exception in the ring closure reactions of ortho substituted aryl azides, as it provides no rate acceleration to this reaction. Pyrolysis of ''azido-meta-hemipinate'', an aryl azide containing such a substituent, led us to the title compound, a new azepinylidenepyridylacetic ester, whose structure has been established unambiguously by a single crystal X-ray diffraction study. This is the first report of a reaction involving both a ring expansion to an azaheptafulvalene and a ring extrusion to a pyridyl ring residue.