Metabolic studies on the nitrophile yeast Rhodotorula glutinis DSBCA06

Nitrogen is the most abundant element in atmosphere and fundamental component of proteins, nucleic acids and other essential molecules. In the past century the industrial use of nitrogen compounds has grown exponentially causing widespread pollution. Nitrogen pollution has wide-ranging impacts including contributions to global warming, acid rains and eutrophication. Reduction of nitrogen use in industry and agriculture coupled whit remediation treatments could represent a solution. To this purpose we isolated from environmental samples a nitrophile strain capable of removing nitrogen compounds efficiently from the medium. Through the molecular characterization, we identified the strain as a Rhodotorula glutinis that we called DSBCA06. We examined the main metabolic features of the strain, also to determine the best growing conditions. At the same time, the ability of the strain to grow in presence of high nitrite concentrations was assayed, being a relevant feature poorly studied earlierfor other environmental yeasts. The ability of the strain to grow in presence of heavy metal cations was also tested, showing a noticeable tolerance. The cost of bioremediation treatments is often a problem. One of the way to obviate this is to produce valuable secondary metabolites, capable of positively impact the cost of the processes. In this context the ability of the strain to produce carotenoids, natural molecules with antioxidant properties used for food production, cosmetic and pharmaceutical industry, has been evaluated. The strain Rhodotorula glutinis DSBCA06 showed interesting features suggesting its possible use in bioremediation or industrials process for production of secondary metabolites such as lipids and carotenoids

European Union funding Research Development and Innovation projects on Smart Cities.: The state of the art in 2019

European cities currently host 72% of the European population, which probably will rise to 80% by 2050. European Union, Member States, National and Regions Authorities and different type of stakeholders have worked - and keep on doing - together to promote a sustainable urban development and to adapt policies to the needs of cities, thus make visible improvements to the daily lives of people. According to this approach, many Member States decided to pool resources at european level, achieving more than by acting alone. It is thanks to the coordinated approach of European Union and Member State that Research Development & Innovation boost smart cities and smart specialization strategies as two novelties that have been adopted by policymakers

Building envelope design as a contribution for improvement of urban spaces and social housing environmental quality

The design of building envelope and the definition of its elements, can influence both the quality of the external spaces perception and the living standard referred to internal building spaces. This improvement depends by the planning of some component design. Particularly, solar shadings and integrated plant solutions, also thanks to an increasing consequential interest about the issue and the legislative and normative evolution, represent factors able to be involved both in the performance and morphological quality of building envelope (improvement of energy efficiency and living quality of internal spaces), which can influence the perception of environment. A study about this questions has been conducted through the elaboration of a system of Best Pratices, a Code of Practice, for the new Plans of Zone of Rome Municipality. The indications contained in the Code takes in examination the integration-mitigation and facilities connection of solar collectors in the building design, and the possibility of integration between solar shading and collector elements, customized like a support tool for the sustainable design of building envelope. The design of building envelope, reported to morphological and technological issues, can assume particular importance in the definition of living quality. Design of closures, developed through some indications referred to its technological components, can influence both the quality perception of external living spaces through the morphological definition of building, and the life quality of internal spaces by the implementation of energy efficiency of building system. Solar shading in particular, also thanks to the increasing consequential interest in the evolution of legislation about the argument, more in the future will represent a fundamental element for design and the increment of performance and morphological quality of building enclosure

A implementação da audiência de conciliação ou mediação prevista no Código de Processo Civil e seus aspectos nas Varas Cíveis de Criciúma/SC

Trabalho de Conclusão de Curso, apresentado para obtenção do grau de Bacharel no curso de Direito da Universidade do Extremo Sul Catarinense, UNESC.No ano de 2015, houve uma mudança completa aos aplicadores do direito com o advento do Novo Código de Processo Civil, que entrou em vigência no ano de 2016. Este trouxe várias alterações, inclusive a modificação no que tange ao início do processo cível, com a inclusão de uma audiência inicial de conciliação ou mediação, na qual as partes tem a possibilidade de solucionar o conflito sem que seja necessário prosseguir com a lide no Poder Judiciário. Contudo, há doutrinadores que apresentam entendimentos de que esta audiência contém pontos negativos para a prática judiciária, uma vez que ocasiona uma superlotação nas pautas de cada Vara Cível. Porém, por outro lado, há de se reconhecer que, se houver o acordo na referida audiência, não haverá a necessidade de dar andamento ao feito, de modo que o Fórum, a Vara Cível, bem como o Poder Judiciário como um todo, não ficarão sobrecarregados com os processos pendentes para julgamento. Assim, no presente trabalho realizou-se um estudo de caso na Comarca de Criciúma/SC, em suas Varas Cíveis, bem como na Unidade Judiciaria de Cooperação (UJC), efetuando uma comparação entre as audiências realizadas, analisando-se o êxito, ou não, dos referidos acordos realizados na sessão, com o intuito de analisar a efetividade da audiência de conciliação ou mediação inserida pelo Novo Código de Processo Civil. O método de pesquisa utilizado foi o dedutivo, em pesquisa teórica e qualitativa com emprego de material bibliográfico e documental legal

Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation

Leucine-rich repeat kinase 2 (LRRK2) kinase activity is increased in several pathogenic mutations, including the most common mutation, G2019S, and is known to play a role in Parkinson’s disease (PD) pathobiology. This has stimulated the development of potent, selective LRRK2 kinase inhibitors as one of the most prevailing disease-modifying therapeutic PD strategies. Although several lines of evidence support beneficial effects of LRRK2 kinase inhibitors, many questions need to be answered before clinical applications can be envisaged. Using six different LRRK2 kinase inhibitors, we show that LRRK2 kinase inhibition induces LRRK2 dephosphorylation and can reduce LRRK2 protein levels of overexpressed wild type and G2019S, but not A2016T or K1906M, LRRK2 as well as endogenous LRRK2 in mouse brain, lung and kidney. The inhibitor-induced reduction in LRRK2 levels could be reversed by proteasomal inhibition, but not by lysosomal inhibition, while mRNA levels remained unaffected. In addition, using LRRK2 S910A and S935A phosphorylation mutants, we show that dephosphorylation of these sites is not required for LRRK2 degradation. Increasing our insight in the molecular and cellular consequences of LRRK2 kinase inhibition will be crucial in the further development of LRRK2-based PD therapies

Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field

Heterologous expression of three enzymes forstructural studies

In questo lavoro di tesi ci si \ue8 occupati dell\u2019espressione, della purificazione e della cristallizzazione di tre enzimi (bile acid CoA:amino acid N-acyltransferase, BAAT; farnesyl cysteine-carboxyl methyltransferase, Ste14; e stearoyl-CoA desaturase, SCD) con lo scopo finale di determinarne la struttura tridimensionale mediante analisi di diffrazione di raggi X. I cDNA delle proteine in questione sono stati clonati in vettori per l\u2019espressione in sistemi eterologhi. Le scelta del sistema di espressione opportuno per la produzione su larga scala \ue8 stata condotta dopo una valutazione della resa, della stabilit\ue0 ed integrit\ue0 del prodotto proteico. La proteina BAAT umana \ue8 stata espressa utilizzando il sistema procariotico E.coli. La proteina ricombinante \ue8 stata purificata tramite due cromatografie di affinit\ue0 al nichel e gel filtrazione. L\u2019integrit\ue0 del campione ottenuto \ue8 stata controllata attraverso spettrometria di massa e l\u2019omogeneit\ue0 mediante DLS. E\u2019 stata condotta anche una valutazione dell\u2019attivit\ue0 enzimatica verificando la presenza dei prodotti mediante spettrometria di massa. Oltre alla proteina wild type \ue8 stato prodotto il mutante privo di attivit\ue0 enzimatica (C235A) per procedere con le prove di cristallizzazione in presenza del substrato. I campioni ottenuti si presentano sufficientemente puri, stabili ed omogenei per allestire le prove di cristallizzazione. Le prove di cristallizzazione hanno dato esito positivo con la proteina mutata, anche se i cristalli fino ad ora ottenuti non sono idonei per gli esperimenti di diffrazione di raggi X. La proteina di membrana Ste14 di C.glabrata \ue8 stata individuata come buon candidato per la produzione su larga scala al fine di condurre studi strutturali attraverso l\u2019utilizzo di un sistema high-throughput pubblicato da Drew et al., 2008. Il sistema prevede l\u2019espressione eterologa in S. cerevisiae; la proteina di interesse viene prodotta in fusione alla GFP permettendo la valutazione del livello di espressione e la stabilit\ue0 nei diversi detergenti attraverso misure di fluorescenza sull\u2019estratto cellulare. L\u2019enzima \ue8 stato purificato attraverso due cromatografie di affinit\ue0 al nichel e gel filtrazione in presenza sia del detergente DDM sia del detergente LDAO. Il profilo della gel filtrazione suggerisce che il campione \ue8 \u2018monodisperso\u2019 e quindi adeguato per l\u2019allestimento di prove di cristallizzazione. Ad oggi non sono per\uf2 stati ottenuti cristalli adatti ad esperimenti di diffrazione. E\u2019 stato infine messo appunto un protocollo di espressione e purificazione per l\u2019enzima transmembrana umano SCD. La proteina ricombinante \ue8 stata espressa in cellule di insetto e purificata in presenza del detergente DDM attraverso IMAC. La resa e la purezza del prodotto proteico ad oggi non \ue8 ancora ottimale ma la quantit\ue0 di enzima \ue8 sufficiente per allestire prove di cristallizzazione e per condurre studi biochimici e strutturali.This thesis work was aimed at the expression, purification and crystallization of three human proteins (bile acid CoA:amino acid N-acyltransferase, BAAT; farnesyl cysteine-carboxyl methyltransferase, Ste14; and stearoyl-CoA desaturase, SCD) in order to determine their three-dimensional structure using X-ray crystallography. The cDNA sequences were cloned into specific vectors for overexpression in heterologous systems. The choice of the best expression system was made considering the protein yield, stability and integrity before scaling up. Human BAAT was expressed in E.coli. The recombinant protein was purified by IMAC, reverse IMAC and gel filtration. The integrity of the sample was assessed by mass spectrometry and its homogeneity using DLS. The enzymatic activity was controlled verifying the presence of the products by mass spectrometry. In addition to the wild type protein, the catalytic mutant (C235A) was cloned and expressed to carry out crystallization trials in the presence of the substrate. The samples obtained are sufficiently pure, stable and homogeneous to set up crystallization trials. Crystals were obtained only with the mutant but they are not sufficiently ordered for X-ray diffraction experiments. Using the high-throughput system published by Drew et al., 2008, the membrane protein Ste14 from C.glabrata was selected as a good candidate for a large scale production in order to conduct structural studies. The system uses S.cerevisiae to overexpress membrane proteins; the protein of interest is cloned into a GFPfusion vector allowing to estimate the expression level and the stability in several detergents by measuring fluorescence directly in cell extracts. The enzyme was purified by IMAC, reverse IMAC and gel filtration in the presence of the detergents DDM and LDAO. The gel filtration profile indicates that the sample is \u2018monodisperse\u2019 and hence adequate to set crystallization trials. Up to now no suitable crystals were obtained. Finally, a protocol for the expression and purification of the human membrane enzyme SCD was developed. The recombinant protein was expressed in insect cells and purified in the presence of the detergent DDM by IMAC. Until now the yield and the purity obtained are not optimal but a sufficient quantity of the enzyme can be extracted to carry out crystallization trials aimed at structural and biochemical studies

Obtenção e avaliação das propriedades elétricas de misturas físicas de poliuretano termoplástico com aditivos semicondutores

Dissertação (mestrado) - Universidade Fderal de Santa Catarina, Centro Tecnologico, Programa de Pós-Graduação em Ciência e Engenharia de Materiais, Florianópolis, 2010A obtenção de materiais poliméricos com a combinação das propriedades de polímeros de engenharia e propriedades elétricas de materiais condutores ou semicondutores tem atraído a atenção de vários grupos de pesquisa devido ao grande potencial em aplicações tecnológicas. Várias estratégias têm sido adotadas para obter materiais com tais propriedades, uma técnica muito utilizada é a preparação de misturas físicas de polímeros isolantes e materiais condutores ou semicondutores. Neste trabalho, foram preparadas misturas físicas de poliuretano termoplástico (TPU) com polianilina dopada com ácido dodecilbenzeno sulfônico (PAni.DBSA), negro de fumo condutor (NFC) e NFC aditivado com PAni.DBSA (NFC-PAni.DBSA), respectivamente, em um misturador de dois cilindros. A PAni e NFCPAni foram sintetizadas a partir da polimerização em emulsão da anilina na presença do ácido dodecilbenzeno sulfônico (DBSA) e negro de fumo condutor, respectivamente. A PAni.DBSA, NFC e NFCPAni. DBSA foram caracterizados por análise termogravimétrica (TGA), espectroscopia de absorção no infravermelho com transformada de Fourier (FTIR) e no ultravioleta-visível (UV-vis), microscopia eletrônica de varredura (MEV) e a condutividade elétrica avaliada pelo método padrão quatro pontas. As blendas de TPU/PAni.DBSA apresentaram condutividade elétrica da ordem de 10-6 S.cm-1 para concentrações de 20% em massa de PAni.DBSA e limiar de percolação inferior aos compósitos de TPU/NFC e TPU/NFC-PAni.DBSA. Análises de FTIR mostraram que existe interação entre os grupos (NH) da polianilina com os grupos (C=O) presentes no poliuretano para as misturas de TPU/PAni.DBSA e TPU/NFC-PAni.DBSA. Entretanto, por esta técnica não foi possível verificar interações entre os componentes presentes na mistura de TPU/NFC. As micrografias das amostras de TPU/PAni.DBSA e TPU/NFC-PAni.DBSA obtidas por MEV evidenciaram que as partículas de PAni.DBSA e NFC-PAni.DBSA estão bem dispersas e distribuídas na matriz de TPU, porém não foi possível evidenciar os caminhos condutores os quais são responsáveis pelo aumento da condutividade elétrica da mistura. Ensaios de absorção de radiação eletromagnética mostraram que as misturas obtidas neste trabalho são capazes de absorver ondas eletromagnéticas.To obtain polymeric materials by combining the properties of engineering polymers and electrical properties of conductor or semiconductor materials has attracted the attention of several groups of researchers due to the great potential for technological applications. Several strategies have been adopted to obtain materials with such properties. A technique often used is the preparation of physical mixtures of insulating polymers and conductors or semiconductors materials. In this work were prepared physical mixtures of thermoplastic polyurethane (TPU) with polyaniline doped with acid dodecylbenzene sulfonic (PAni.DBSA), carbon black conductor (NFC) and NFC modified with PAni.DBSA (NFC-PAni.DBSA), respectively in a two cylinders mixer. PAni and NFC-PAni.DBSA were synthesized through emulsion polymerization of aniline in the presence of dodecylbenzene sulfonic acid (DBSA) and carbon black conductor, respectively. The PAni.DBSA, NFC and NFC-PAni.DBSA were characterized by thermo gravimetric analysis (TGA), absorption spectroscopy by Fourier transform infrared (FTIR) and in the ultraviolet-visible spectroscopy (UV-vis), scanning electron microscopy (MEV) and electrical conductivity evaluated by the standard method four corners. The Blends of TPU/PAni.DBSA showed electrical conductivity of 10-6 S.cm-1 at 20 wt.% of PAni.DBSA content, and threshold of percolation less than TPU/NFC and TPU/NFC-PAni.DBSA composites. FTIR analyzes showed that there is an interaction between groups (NH) of polyaniline with groups (C=O) present in polyurethane in the TPU/PAni.DBSA and TPU/NFC-PAni.DBSA mixtures. However, by using this technique it was not possible to verify interactions between the components present in the mixture of TPU/NFC. The micrographs of samples of TPU/PAni.DBSA and TPU/NFC-PAni.DBSA obtained by MEV evidenced that Pani.DBSA and NFC-PAni.DBSA particles are well dispersed and distributed in the TPU matrix. However it was not possible to evidence the conductor paths which are responsible for increasing the electric conductivity of the mixture. Experiments using absorption of electromagnetic radiation revealed that mixtures obtained in this work are able to absorb electromagnetic waves

Industrialised constructive systems for residential buildings: the CCCabita system

The integration between the working reality of the building site, scientific research and industrial production encourages the creation of technological systems capable of providing new forms of housing that meet planning requirements on issues related to sustainability, speed of construction and containment of costs arising from the various demands of the market. Open industrialised systems represent the transposition into the constructive field of technological research in the productive chain and they restore the synthesis on the front of the possibilities of development of industrial systems in construction, ensuring the adoption of the most innovative technical solutions and favouring the wider variability of the different housing models

Genetic, structural, and molecular insights into the function of ras of complex proteins domains

Ras of complex proteins (ROC) domains were identified in 2003 as GTP binding modules in large multidomain proteins from Dictyostelium discoideum. Research into the function of these domains exploded with their identification in a number of proteins linked to human disease, including leucine-rich repeat kinase 2 (LRRK2) and death-associated protein kinase 1 (DAPK1) in Parkinson’s disease and cancer, respectively. This surge in research has resulted in a growing body of data revealing the role that ROC domains play in regulating protein function and signaling pathways. In this review, recent advances in the structural informa- tion available for proteins containing ROC domains, along with insights into enzymatic function and the integration of ROC domains as molecular switches in a cellular and organismal context, are explored