Type II aromatic polyketide biosynthetic tailoring enzymes: diversity and adaptation in Streptomyces secondary metabolism.

Members of the bacterial genus Streptomyces are well known for their ability to produce an exceptionally wide selection of diverse secondary metabolites. These include natural bioactive chemical compounds which have potential applications in medicine, agriculture and other fields of commerce. The outstanding biosynthetic capacity derives from the characteristic genetic flexibility of Streptomyces secondary metabolism pathways: i) Clustering of the biosynthetic genes in chromosome regions redundant for vital primary functions, and ii) the presence of numerous genetic elements within these regions which facilitate DNA rearrangement and transfer between non-progeny species. Decades of intensive genetic research on the organization and function of the biosynthetic routes has led to a variety of molecular biology applications, which can be used to expand the diversity of compounds synthesized. These include techniques which, for example, allow modification and artificial construction of novel pathways, and enable gene-level detection of silent secondary metabolite clusters. Over the years the research has expanded to cover molecular-level analysis of the enzymes responsible for the individual catalytic reactions. In vitro studies of the enzymes provide a detailed insight into their catalytic functions, mechanisms, substrate specificities, interactions and stereochemical determinants. These are factors that are essential for the thorough understanding and rational design of novel biosynthetic routes. The current study is a part of a more extensive research project (Antibiotic Biosynthetic Enzymes; www.sci.utu.fi/projects/biokemia/abe), which focuses on the post-PKS tailoring enzymes involved in various type II aromatic polyketide biosynthetic pathways in Streptomyces bacteria. The initiative here was to investigate specific catalytic steps in anthracycline and angucycline biosynthesis through in vitro biochemical enzyme characterization and structural enzymology. The objectives were to elucidate detailed mechanisms and enzyme-level interactions which cannot be resolved by in vivo genetic studies alone. The first part of the experimental work concerns the homologous polyketide cyclases SnoaL and AknH. These catalyze the closure of the last carbon ring of the tetracyclic carbon frame common to all anthracycline-type compounds. The second part of the study primarily deals with tailoring enzymes PgaE (and its homolog CabE) and PgaM, which are responsible for a cascade of sequential modification reactions in angucycline biosynthesis. The results complemented earlier in vivo findings and confirmed the enzyme functions in vitro. Importantly, we were able to identify the amino acid -level determinants that influence AknH and SnoaL stereoselectivity and to determine the complex biosynthetic steps of the angucycline oxygenation cascade of PgaE and PgaM. In addition, the findings revealed interesting cases of enzyme-level adaptation, as some of the catalytic mechanisms did not coincide with those described for characterised homologs or enzymes of known function. Specifically, SnoaL and AknH were shown to employ a novel acid-base mechanism for aldol condenzation, whereas the hydroxylation reaction catalysed by PgaM involved unexpected oxygen chemistry. Owing to a gene-level fusion of two ancestral reading frames, PgaM was also shown to adopt an unusual quaternary sturucture, a non-covalent fusion complex of two alternative forms of the protein. Furthermore, the work highlighted some common themes encountered in polyketide biosynthetic pathways such as enzyme substrate specificity and intermediate reactivity. These are discussed in the final chapters of the work.

Reações de contração de anel promovidas por sais de tálio(III)

Thallium(III) salts promote a number of different reactions useful in organic synthesis. In this paper, the ring contraction of ketones and olefins, mediated by thallium(III) salts, is exhaustively reviewed.

Venäjän sähkömarkkinareformi

Työn tarkoituksessa on luoda kokonaisvaltainen kuva Venäjän sähkösektorista ja siellä suoritettavasta reformista. Työ johdattelee lukijan aiheeseen kuvaamalla Venäjän sähkösektorin- ja markkinoiden tilaa ennen reformia. Tässä on tärkeää ymmärtää, kuinka kulutus ja tuotanto kohtasivat. Tämän jälkeen siirrytään selvittämään reformin syitä, suuntia ja päämääriä. Reformin kolme pääkohtaa ovat lainsäädännöllisen viitekehyksen luominen, sähkösektorin uudelleenjärjestely sekä uusien sähkömarkkinoiden kehittäminen ja täytäntöönpano. Uudelleenjärjestely on valtava prosessi, joka koskettaa koko Venäjän sähkömarkkinoita. Tämän prosessin keskeisenä tekijänä tosin on lähes monopoliasemaa nauttiva Unified Energy System of Russia. Tätä prosessia pyritään kuvaamaan alusta asti tähän päivään saakka. Toinen merkittävä prosessi on ollut uuden sähkömarkkinamallin kehittäminen ja tämän käyttöönotto. Työssä kuvataan tämän uuden mallin toimintaa ja rakennetta, ja työn loppupuolella pyritään selvittämään Venäjän sähkösektorin tulevaisuuden suunnitelmia, haasteita ja ennusteita.

Consumo y convergencia mediática de la prensa local en Cataluña

Los nuevos hábitos de consumo de medios en el marco de la convergencia digital están propiciando un cambio en las estrategias de los medios de comunicación. Mientras unos usuarios siguen prefiriendo la radio, la televisión y la prensa tradicional para informarse otros participan de la experiencia digital en internet (blogs, redes sociales...) y dispositivos móviles, etc. En este panorama es complicado precisar dónde nace y termina el contenido o hasta qué punto es la participación del usuario quien (re) construye la información; así los contenidos mutan y se diluyen en varios soportes. En este contexto, la prensa local también vive las consecuencias del nuevo paradigma y asume los retos del reacomodo del sector ante la nueva realidad. Esta comunicación presenta las conclusiones del estudio cualitativo con entrevistas en profundidad a los responsables de medios locales en Cataluña sobre sus usuarios, contenidos y soportes en el marco de la convergencia digital.

Lipídios modificados obtidos a partir de gordura do leite, óleo de girassol e ésteres de fitosteróis para aplicação em spreads

The objective of this paper was to evaluate the modifications in milkfat properties with the addition of sunflower oil (SO) and phytosterol esters (PE) and chemical interesterification. Fatty acid composition, softening point and consistency were determined. The saturation degree of milkfat decreased with the addition of SO and PE. Consequently, milkfat presented lower softening point and consistency. Chemical interesterification caused an increase in softening point due to the formation of higher amounts of trissaturated triacylglycerols with rearrangement. The incorporation of unsaturated fatty acids from SO and PE by milkfat triacylglycerols after chemical reaction caused linearization of consistency curves.

Energia, meio ambiente e economia: o Brasil no contexto mundial

The cycle of fossil fuels as an energy source for mankind is approaching its end. Finite resources, coupled with greenhouse gas, have led to an increased effort in the search for alternative renewable energy sources. Brazil has a leading position, due to a 46% participation of renewable sources in its primary energy supply, compared to the global average of 12%. The expansion of the renewable sources in Brazil depends on medium and long term planning, and a large volume of investments. The present financial crisis will have major effects in the energy market. Despite a negative initial impact, it is expected that the rearrangement of the financial system will ultimately lead to an expansion in the use of renewable energy sources. Brazil is a tropical country, with the largest biodiversity in our planet and excellent conditions to expand the use of all forms of renewable sources.

Adsorção do corante azul de metileno em partículas de argilominerais: análise dos tamanhos das partículas

Interactions of cationic dye methylene blue (MB) with clay particles in aqueous suspension have been extensively studied. As already known, the number of natural negative charges on the clay modifies significantly the particle sizes dispersed in water and therefore the nature of the interaction with the dye. This work evaluated with UV-Vis spectroscopy method how the clay particle sizes weighted on the adsorption and rearrangement of the dye molecules in aqueous system. The results obtained from light-scattering measurements confirmed that larger particles are found in suspensions containing the high-charged clays as the visible absorption band related to the MB aggregates (570 nm) on these suspensions prevailed.

Argumenttien kirjo

Kirja-arvio

Functional Study of Oncogenic Transcription Factor ERG and its Signaling in Prostate Cancer

TMPRSS2–ERG is the most frequent type of genomic rearrangement present in prostate tumors, in which the 5- prime region of the TMPRSS2 gene is fused to the ERG oncogene. TMPRSS2, containing androgen response elements (AREs), is regulated by androgens in the prostate. The truncated TMPRSS2-ERG fusion transcript is overexpressed in half of the prostate cancer patients. The formation of TMPRSS2-ERG transcript is an early event in prostate carcinogenesis and previous in vivo and in vitro studies have shown ectopic ERG expression to be associated with increased cell invasion. However, the molecular function of ERG and its role in cell signaling is poorly understood. In this study, genomic rearrangement of ERG with TMPRSS2 was studied by using comparative genomic hybridization (CGH) in prostate cancer samples. The biological processes associated with the ERG oncogene expression in prostate epithelial cells were studied, and the results were compared with findings observed in clinical prostate tumor samples. The gene expression data indicated that increased WNT signaling and loss of cell adhesion were a characteristic of TMPRSS2- ERG fusion positive prostate tumor samples. Up- regulation of WNT pathway genes were present in ERG positive prostate tumors, with frizzled receptor 4 (FZD4) presenting with the highest association with ERG overexpression, as verified by quantitative reverse transcription-PCR, immunostaining, and immunoblotting in TMPRSS2-ERG positive VCaP prostate cancer cells. Furthermore, ERG and FZD4 silencing increased cell adhesion by inducing active β1-integrin and E-cadherin expression in VCaP cells. Furthermore, we found a novel inhibitor, 4-(chloromethyl) benzoyl chloride which inhibited the WNT signaling and induced similar phenotypic effects as observed after ERG or FZD4 down regulation in VCaP cells. In conclusion, this work deepens our understanding on the complex oncogenic mechanisms of ERG in prostate cancer that may help in developing drugs against TMPRSS2-ERG positive tumors.

Toimenpidesuosituksia ilmavoimille lentopahoinvoinnin vähentämiseksi LentoRuk:lla

Lentopahoinvointi alentaa tai jopa vie kokonaan sotilaslentäjän toimintakyvyn. Sitä esiintyy lähinnä vain koulutuksen aikana sotilaslento-oppilailla. Tällöin se hidastaa ja vaikeuttaa koulutuksen etenemistä. Lentopahoinvointi on normaalia ja sen mahdollistavat ihmisen terveiden aistien heikkoudet. Heikkouksiensa johdosta aistit eivät aisti lentokoneen liikkeitä oikein. Virheellisistä liikeaistimuksista syntyy ristiriitoja ja asentoharhoja ihmisen keskushermostossa. Kaksi hyväksytyintä teoriaa siitä, miten nämä harhat aiheuttavat liikesairauden (eli lentopahoinvoinnin), ovat subjektiivisen vertikaalin (subjective vertical) ja sensorisen konfliktin (sensory rearrangement / conflict) teoriat. Kun teorian mukaan liikesairautta aiheuttava ärsyke ilmenee, henkilö alkaa tuntea olonsa epämukavaksi. Liikkeen jatkuessa olo pahenee ja johtaa lopulta oksentamiseen. Lentopahoinvointia voi ehkäistä koulutusjärjestelyillä, omilla ja lennonopettajantoimilla lennonaikana sekä lääkkeillä. Nykyisten lääkkeiden käyttö ei tule kyseeseen lentopalveluksessa. Teoriakoulutuksella ja asenteilla on suuri vaikutus lentopahoinvoinnin ennaltaehkäisyyn. Tehokkain käytännön menetelmä on antaa lento-oppilaan ohjata konetta itse, koska hän voi paremmin ennakoida lentokoneen liikkeitä. Lentopahoinvointi poistuu henkilön tottuessa lentokoneen liikkeisiin. Tottuminen poistuu ajan kuluessa, jos henkilö ei lennä. Tauon jälkeen lentopahoinvointia taas ilmenee, mutta uudelleen tottuminen on nopeaa. Huono sää lisää lentopahoinvointia.

A Farewell to Flat Biology. Three-dimensional Cell Culture Models in Cancer Drug Target Identification and Validation

Cells of epithelial origin, e.g. from breast and prostate cancers, effectively differentiate into complex multicellular structures when cultured in three-dimensions (3D) instead of conventional two-dimensional (2D) adherent surfaces. The spectrum of different organotypic morphologies is highly dependent on the culture environment that can be either non-adherent or scaffold-based. When embedded in physiological extracellular matrices (ECMs), such as laminin-rich basement membrane extracts, normal epithelial cells differentiate into acinar spheroids reminiscent of glandular ductal structures. Transformed cancer cells, in contrast, typically fail to undergo acinar morphogenic patterns, forming poorly differentiated or invasive multicellular structures. The 3D cancer spheroids are widely accepted to better recapitulate various tumorigenic processes and drug responses. So far, however, 3D models have been employed predominantly in the Academia, whereas the pharmaceutical industry has yet to adopt a more widely and routine use. This is mainly due to poor characterisation of cell models, lack of standardised workflows and high throughput cell culture platforms, and the availability of proper readout and quantification tools. In this thesis, a complete workflow has been established entailing well-characterised 3D cell culture models for prostate cancer, a standardised 3D cell culture routine based on high-throughput-ready platform, automated image acquisition with concomitant morphometric image analysis, and data visualisation, in order to enable large-scale high-content screens. Our integrated suite of software and statistical analysis tools were optimised and validated using a comprehensive panel of prostate cancer cell lines and 3D models. The tools quantify multiple key cancer-relevant morphological features, ranging from cancer cell invasion through multicellular differentiation to growth, and detect dynamic changes both in morphology and function, such as cell death and apoptosis, in response to experimental perturbations including RNA interference and small molecule inhibitors. Our panel of cell lines included many non-transformed and most currently available classic prostate cancer cell lines, which were characterised for their morphogenetic properties in 3D laminin-rich ECM. The phenotypes and gene expression profiles were evaluated concerning their relevance for pre-clinical drug discovery, disease modelling and basic research. In addition, a spontaneous model for invasive transformation was discovered, displaying a highdegree of epithelial plasticity. This plasticity is mediated by an abundant bioactive serum lipid, lysophosphatidic acid (LPA), and its receptor LPAR1. The invasive transformation was caused by abrupt cytoskeletal rearrangement through impaired G protein alpha 12/13 and RhoA/ROCK, and mediated by upregulated adenylyl cyclase/cyclic AMP (cAMP)/protein kinase A, and Rac/ PAK pathways. The spontaneous invasion model tangibly exemplifies the biological relevance of organotypic cell culture models. Overall, this thesis work underlines the power of novel morphometric screening tools in drug discovery.

Terpenoid transformations over gold catalysts

Nowadays biomass transformation has a great potential for the synthesis of value-added compounds with a wide range of applications. Terpenoids, extracted from biomass, are inexpensive and renewable raw materials which often have a biological activity and are widely used as important organic platform molecules in the development of new medicines as well as in the synthesis of fine chemicals and intermediates. At the same time, special attention is devoted to the application of gold catalysts to fine chemical synthesis due to their outstanding activity and/or selectivity for transformations of complex organic compounds. Conversion of renewable terpenoids in the presence of gold nanoparticles is one of the new and promising directions in the transformation of biomass to valuable chemicals. In the doctoral thesis, different kinds of natural terpenoids, such as α-pinene, myrtenol and carvone were selected as starting materials. Gold catalysts were utilized for the promising routes of these compounds transformation. Investigation of selective α-pinene isomerization to camphene, which is an important step in an industrial process towards the synthesis of camphor as well as other valuable substrates for the pharmaceutical industry, was performed. A high activity of heterogeneous gold catalysts in the Wagner-Meerwein rearrangement was demonstrated for the first time. Gold on alumina carrier was found to reach the α-pinene isomerization conversion up to 99.9% and the selectivity of 60-80%, thus making this catalyst very promising from an industrial viewpoint. A detailed investigation of kinetic regularities including catalyst deactivation during the reaction was performed. The one-pot terpene alcohol amination, which is a promising approach to the synthesis of valuable complex amines having specific physiological properties, was investigated. The general regularities of the one-pot natural myrtenol amination in the presence of gold catalysts as well as a correlation between catalytic activity, catalyst redox treatment and the support nature were obtained. Catalytic activity and product distribution were shown to be strongly dependent on the support properties, namely acidity and basicity. The gold-zirconia (Au/ZrO2) catalyst pretreated under oxidizing atmosphere was observed to be rather active, resulting in the total conversion of myrtenol and the selectivity to the corresponding amine of about 53%. The reaction kinetics was modelled based on the mechanistic considerations with the catalyst deactivation step incorporated in the mechanism. Carvone hydrogenation over a gold catalyst was studied with the general idea of investigating both the activity of gold catalysts in competitive hydrogenation of different functional groups and developing an approach to the synthesis of valuable carvone derivatives. Gold was found to promote stereo- and chemoselective carvone hydrogenation to dihydrocarvone with a predominant formation of the trans-isomer, which generally is a novel synthetic method for an industrially valuable dihydrocarvone. The solvent effect on the catalytic activity as well as on the ratio between trans- and cis-dihydrocarvone was evaluated.

Occurrence of Akodon cursor (Rodentia, Cricetidae) with 14, 15 and 16 chromosome cytotypes in the same geographic area in Southern Brazil

The karyotype of Akodon cursor (initially identified as A. arviculoides) had been reported with chromosomal numbers 14 and 15 in the South and Southeast and 16 in Northeastern Brazil. We found the three cytotypes in a region of Southern Brazil. The G-band patterns of these specimens were the same as those from southeastern and northeastern regions. Seventeen different combinations of chromosomes due to a complex rearrangement in pair 1 and pericentric inversions in pairs 2 and 3 were identified. Seven of these combinations are new to in the literature.

Chromosome studies in some Stevia. Cav. (Compositae) species from Southern Brazil

Karyotypes of six species of the genus Stevia from Southern Brazil were studied, utilizing root tip metaphases. All species were diploid with 2n = 22 chromosomes. It was possible to identify each species by chromosome morphology. The basic chromosome number for Brazilian species of Stevia is X = 11. This number is also found in almost all South American species. We suggest that in Stevia there is an evolutionary trend toward chromosomal rearrangement, caused mainly by pericentric inversions. It was found that, in addition to aneuploidy and polyploidy, chromosomal rearrangements are common in the tribe Eupatorieae.

The Lebanese mutation as an important cause of familial hypercholesterolemia in Brazil

Familial hypercholesterolemia (FH) is a common autosomal disorder that affects about one in 500 individuals in most Western populations and is caused by a defect in the low-density-lipoprotein receptor (LDLr) gene. In this report we determined the molecular basis of FH in 59 patients from 31 unrelated Brazilian families. All patients were screened for the Lebanese mutation, gross abnormalities of the LDLr gene, and the point mutation in the codon 3500 of the apolipoprotein B-100 gene. None of the 59 patients presented the apoB-3500 mutation, suggesting that familial defective ApoB-100 (FDB) is not a major cause of inherited hypercholesterolemia in Brazil. A novel 4-kb deletion in the LDLr gene, spanning from intron 12 to intron 14, was characterized in one family. Both 5' and 3' breakpoint regions were located within Alu repetitive sequences, which are probably involved in the crossing over that generated this rearrangement. The Lebanese mutation was detected in 9 of the 31 families, always associated with Arab ancestry. Two different LDLr gene haplotypes were demonstrated in association with the Lebanese mutation. Our results suggest the importance of the Lebanese mutation as a cause of FH in Brazil and by analogy the same feature may be expected in other countries with a large Arab population, such as North American and Western European countries.