This study investigates the occurrence of di-butyl phthalate (DBP) and di-ethylhexyl phthalate (DEHP) in raw cow's milk. The idea was to compare level of contamination in manually and mechanically obtained milk. Samples were collected at a farm in the Vysočina region in January and April 2013. The concentrations of DBP and DEHP were determined by high performance liquid chromatography. Throughout the experiment a statistically significantly higher content of DBP than DEHP was observed. The concentration of DBP ranged from 0,57 to 16,79 mg.kg-1 of milk, DEHP ranged from undetectable values to 0,39 mg.kg-1 of milk. The amount of phthalates in the mechanically obtained milk was approximately two times higher than in the milk milked by hand, probably due to migration from the contact materials of the milking equipment. In April a slight decrease in the concentration values was observed, but the values did not differ statistically significantly from the values measured in January. The presence of phthalates in milk on the primary production level is a shining example of their wide application, which burdens the environment and the food chain.

Os microrganismos podem apresentar-se tanto em forma de vida livre como aderidos a uma superfície ou interface ar-líquido, formando comunidades complexas e dinâmicas conhecidas como biofilmes. Nos últimos anos, com o avanço das pesquisas em nível molecular, foi identificado que a maioria das bactérias utilizam guanosina monofosfato (3´-5´)-cíclica dimérica (c-di-GMP) como um segundo mensageiro. De forma geral, essa molécula controla a sinalização celular, virulência, comunicação entre células e a expressão de proteínas relacionadas com o fenótipo de biofilmes, em resposta à sua concentração intracelular. Sua síntese e degradação são controladas respectivamente por diguanilto ciclases (DGCs) contendo domínio GGDEF e fosfodiesterases (PDEs) que possuem os domínios EAL ou HD-GYP. Em Pseudomonas aeruginosa (PA14) foi identificada uma nova classe de receptor específico para c-di-GMP, a proteína transmembranar PelD, cuja porção citoplasmática contém os domínios GAF e GGDEF degenerado. Sua modulação através desse dinucleotídeo controla a produção de exopolissacarídeos pelos componentes do conservado operon pel e influencia diretamente na capacidade de formação de biofilmes. Devido à escassez de dados a respeito dos eventos moleculares do mecanismo de sinalização mediado por c-di-GMP, este trabalho teve como objetivo principal a caracterização biofísica/estrutural da proteína PelD, bem como o reconhecimento de interação entre este ligante e a porção citoplasmática da proteína. Diversas construções solúveis de PelD foram clonadas e expressas, sendo que a construção compreendendo os resíduos 176-455 (PelD176-455) foi cristalizada com sucesso e teve sua estrutura determinada por iodo-SAD. O modelo final apresentou os dois domínios com enovelamentos característicos das famílias GAF e GGDEF, sendo a interface inter-domínios composta majoritariamente por resíduos hidrofóbicos. Visando uma compreensão das bases moleculares de reconhecimento e ativação de PelD por c-di-GMP, uma estrutura em complexo com o ligante foi resolvida. Como esperado, o dinucleotídeo foi encontrado no sítio inibitório do domínio GGDEF, onde o motivo R367xxD370 e o resíduo R402 são responsáveis pela maior parte das interações com c-di-GMP. No entanto, nenhuma grande mudança estrutural foi observada entre as formas apo e holo de PelD, ao contrário de outros sistemas efetores tal como LapD e domínios PilZ. Curiosamente, apenas uma molécula de c-di-GMP foi encontrada no sítio, contrastando com a forma dimérica intercalada normalmente ligada aos sítios inibitórios de domínios GGDEF, tais como em PleD e WspR. Estudos de ITC confirmaram a estequiometria 1:1 em solução. Isso mostra a versatilidade dos diversos receptores já identificados até o momento, frente à ligação desse dinucleotídeo. Estudos de bioinformática identificaram uma potencial região de coiled-coil na hélice juxtamembrana de PelD, resíduos 115-160, provavelmente responsável pela homodimerização. Visando uma comprovação experimental dessa hipótese, uma construção contendo toda a porção citoplasmática, PelD111-455, foi expressa e purificada. Estudos comparativos de dicroísmo circular e ultracentrifugação analítica entre as construções PelD176-455 e PelD111-455 realmente demonstraram que os resíduos extras presentes em PelD111-455 formam uma hélice-α e são responsáveis pela dimerização da porção citoplasmática da proteína. De modo geral, os resultados aqui apresentados não só contribuirão para o entendimento dos mecanismos de regulação das vias de sinalização mediadas por c-di-GMP como, em longo prazo, poderão levar ao desenvolvimento de agentes contra infecções bacterianas. / Microorganisms may be presented either in planktonic free-swimming life-style or adhered to surfaces, forming a complex and dynamic community known as biofilm. In recent years, with the progress of research at the molecular level, it was identified that the majority of bacteria use guanosine monophosphate (3\'-5 \')-cyclic dimeric (c-di-GMP) as a second messenger. Generally, this molecule controls the cell signaling, virulence, communication between cells and expression of proteins related to the phenotype of biofilms in response to its intracellular concentration. Its synthesis and degradation are controlled respectively by diguanylate cyclases (DGC) containing the GGDEF domain and phosphodiesterases (PDE) with the domains EAL or HD-GYP. In Pseudomonas aeruginosa (PA14) a novel class of receptor specific for c-di-GMP has been identified, the transmembrane protein PelD, which contains a GAF and degenerate GGDEF domains in the cytoplasmic portion. Its modulation through this dinucleotide controls the production of exopolysaccharides by the components of the conserved operon pel and directly influences the ability of biofilm formation. Due to the paucity of data about the molecular events of the signaling mechanism mediated by c-di-GMP, this study aimed to characterize biophysically and structurally the protein PelD. Various soluble constructions of PelD were cloned and expressed, and the construction comprising the residues 176-455 (PelD176-455) was successfully crystallized and its structure was determined by iodine-SAD. The final model showed the two characteristic domains of families GAF and GGDEF, and the inter-domain interface composed primarily of hydrophobic residues. Seeking an understanding of the molecular basis of recognition and activation of PelD by c-di-GMP, a structure in complex with the ligand was solved. As expected, the dinucleotide was found at the inhibitory site of the GGDEF domain, where the motif R367xxD370 and the residue R402 are responsible for most of the interactions with c-di-GMP. However, no major structural change was observed between the apo and holo forms of PelD, unlike other effector systems such as LapD and domains PilZ. Interestingly, only one molecule of c-di-GMP was present on the site, in contrast to the dimeric intercalated form normally found at I-sites GGDEF domains, such as PleD and in WspR. ITC studies confirmed the 1:1 stoichiometry in solution. This shows the versatility of the various receptors identified so far, compared to the binding of dinucleotide. Bioinformatics studies have identified a potential coiled-coil region in the juxtamembrane helix of PelD, residues 115-160, probably responsible for homodimerization. Aiming at an experimental confirmation of this hypothesis, a construct containing the full cytoplasmic portion, PelD111-455 was expressed and purified. Comparative studies of circular dichroism and analytical ultracentrifugation between constructs PelD176-455 and PelD111-455 indeed demonstrated that the extra residues present in PelD111-455 form an α-helix and are responsible for dimerization of the cytoplasmic portion of the protein. Overall, the results presented here not only contribute to the understanding of the mechanisms of regulation of signaling pathways mediated by c-di-GMP as in the long run, may lead to the development of agents against bacterial infections.

Recentemente, uma nova molécula de sinalização bacteriana, o AMP dimérico cíclico (c-di-AMP) emergiu como um regulador central dos processos fisiológicos essenciais, tais como a homeostase celular, verificação da integridade do DNA e virulência bacteriana, entre outros. O c-di-AMP é produzido a partir da condensação de duas moléculas de adenosina trifosfato (ATP) por proteínas denominadas diadenilato ciclases, que contém o domínio DisA_N, também denominado DAC. Existem 2842 sequências de proteínas que contém o domínio DAC, provenientes de 2386 organismos encontradas no banco de dados Protein Families Database (Pfam). Essas proteínas são divididas em subfamílias sendo as três subfamílias mais abundantes: DacA (69,1%), proteínas de membrana associadas a sinalização intracelular de alterações decorrentes do meio externo; DisA (24,1%), primeira diadenilato ciclase a ser amplamente estudada, é uma proteína intracelular encontrada na forma de octâmeros ativos em solução, a qual, indiretamente, controla a divisão celular através da verificação da integridade do DNA e DacB (5,5%), proteínas citoplasmáticas expressa, particularmente, durante a formação de esporos bacterianos. Uma característica interessante é que a maioria dos organismos contém uma única e essencial proteína com domínio DAC. Os organismos que contém duas ou mais proteínas-DAC, tais como Clostridium e Bacillus spp., são uma exceção. Em Staphylococcus aureus (S. aureus), um patógeno humano oportunista e responsável por inúmeras doenças infecciosas, uma única diadenilato ciclase é encontrada pendurada na porção interna da membrana celular (Sau_DacA). A atividade desta proteína é potencialmente regulada através da interação direta com uma proteína YbbR-like, que contém um domínio sensor extracelular. Sau_DacA conserva todos os elementos-chave de uma diadenilato ciclase bacteriana, e por ser a única presente em S. aureus, revela-se um excelente alvo de estudo para o desenvolvimento de novos fins terapêuticos. No entanto, até o presente momento, existem poucas informações em relação a estrutura proteica, ao mecanismo de síntese de c-di-AMP e regulação do mecanismo de síntese de nucleotídeo destas proteínas, sendo, portanto, neste aspectos que o presente trabalho pretendeu contribuir. Através de uma série de ensaios, estruturais, calorimétricos, espectroscópicos e bioquímicos, aliados a mutações sítio-dirigidas, identificou-se a relevância de uma conformação dimérica para a estabilidade conformacional e térmica para a proteína ser funcionalmente ativa, assim como a importância dos motivos conservados DGA (Aspartato-Glicina-Alanina) e RHR (Arginina-Histidina-Arginina) para a atividade da Sau_DacA. O loop L5 localizado entre o sítio ativo e a interface dimérica mostrou-se relevante, uma vez que nele é encontrado o motivo DGA - de ligação ao ATP - e o mesmo encontra-se estabilizado em uma posição favorável para ligação do ATP, apenas na conformação dimérica da proteína. Nossos resultados aliados a dados da literatura possibilitaram a proposição de um mecanismo de síntese de c-di-AMP que deve ocorrer via encontro face-a-face de dois sítios de ligação de ATP presentes em dímeros proteicos distintos, podendo a taxa de síntese de o nucleotídeo sofrer interferência via interação proteína-proteína com a proteína receptora de sinal Sau_YbbR. Desta forma, contribuímos para uma melhor compreensão da estrutura e função da Sau_DacA, possibilitando o uso desta como alvo para o desenvolvimento de novos fármacos, uma vez que é sabido que a biossíntese de c-di-AMP é essencial para a maioria dos patógenos que o sintetizam. / Recently, a new bacterial signaling molecule, the dimeric cyclic AMP (c-di-AMP) has emerged as a central regulator of essential physiological processes, such as cell wall homeostasis, DNA integrity and bacterial virulence, among others. C-di-AMP is synthesized from two molecules of adenosine triphosphate (ATP) by proteins containing DisA_N domain, also called diadenilato cyclases (DACs). A survey in the Protein Families Database database (Pfam) found 2842 protein sequences containing the DAC domain, from 2386 different organisms. These proteins are divided into subfamilies and the three most abundant are: DacA (69,1%), a membrane protein associated with intracellular signaling resulting from an external environment change; DisA (24,1%), the first and most widely studied diadenilate cyclase, an intracellular protein found as active octamers in solution which indirectly controls cell division by DNA integrity verification; and DacB (5,5%), a cytoplasmic proteins, particularly expressed during bacterial spores formation. An interesting feature is that most organisms contain just a single and essential DAC-protein. Organisms containing two or more DAC-containing proteins, such as Clostridium and Bacillus spp., are exceptions. In Staphylococcus aureus (S. aureus), an opportunistic human pathogen responsible for some life-threating diseases, there is a single membrane attached diadenilate cyclase, hanging in the inner portion of the cell membrane (Sau_DacA). The activity of this protein is potentially regulated through direct interaction with YbbR, which contains an extracellular sensor domain. Sau_DacA conserves all key elements of bacterial di-adenylate cyclase, and for being the only di-adenylate cyclase from S. aureus, proves to be an excellent study target for new therapeutic purposes. However, to date, there is a lack of information about structure, c-di-AMP synthesis mechanism and regulation of nucleotide synthesis by Sau_DacA. Therefore, in this context the present work aims to contribute. Through a series of structural, calorimetric, spectroscopic and biochemical assays combined with site-directed mutations, we solved the structure of a soluble construct of Sau_DacA and identified a dimeric interface relevance for the conformational and thermal stability to the protein. This dimer is functionally active and highlights the importance of conserved motifs DGA (Aspartate-Glycine-Alanine) and RHR (Arginine-Histidine-Arginine) for the activity of Sau_DacA. The L5 loop, located between the active site and the dimer interface where is allocated the ATP binding motif (DGA), is stabilized in a favorable position for ATP binding, just in protein dimeric conformation. Our results combined with literary allowed us infer the synthesis of c-di-AMP occurs by face-to-face encounter of two distinct ATP binding site and its rate of synthesis could be regulated through direct protein interaction with. In this way, we contribute to a better understanding of Sau_DacA structure and function, assisting in its use as a target for new drugs development since it is known the biosynthesis of c-di-AMP is essential for most pathogens that synthesize.

A formação de biofilmes bacterianos é um fenômeno bem conhecido, caracterizado pela formação de uma comunidade bacteriana estática, embebida em uma matriz exopolimérica, regulada pela molécula sinalizadora c-di-GMP. Os domínios proteicos que catalisam a síntese (GGDEF) e degradação (EAL e HD-GYP) de c-di-GMP estão presentes em grande quantidade em quase todos os genomas bacterianos sequenciados até hoje. Dentre as diversas proteínas envolvidas nas vias de sinalização mediadas por esse nucleotídeo, uma grande parcela são proteínas transmembranares que possuem ambos domínios GGDEF e EAL. Funcionalmente, esses domínios se apresentam em todas combinações: ambos degenerados ou conservados e combinações GGDEF-degenerado/EAL-conservado ou vice-versa. Enquanto que domínios conservados potencialmente apresentam atividade catalítica, os degenerados geralmente convertem-se em domínios estruturais ou receptores de c-di-GMP. Embora recentes estudos estruturais revelaram detalhes de proteínas com ambos domínios degenerados (LapD) ou ativos (MorA), pouco se sabe sobre uma das combinações mais representativas: GGDEF-degenerado/EAL-conservado. Nesse trabalho, realizamos um estudo estrutural e funcional da proteína STM3615 de Salmonella enterica, que apresenta um domínio periplasmático de função desconhecida, seguido pelos domínios citoplasmáticos HAMP, GGDEF-degenerado e EAL-conservado. Através de diferentes construções citoplasmáticas solúveis de STM3615, confirmamos que essa proteína apresenta atividade fosfodiesterase, mesmo quando o domínio EAL encontra-se isolado. Corroborando com sua atividade catalítica, estudos em solução, tais como SAXS e cromatografia de exclusão molecular, mostraram que o EAL isolado de STM3615 é dimérico, um pré-requisito para ser ativo. Utilizando uma construção com os domínios GGDEF-EAL determinamos sua estrutura cristalográfica a uma resolução de 2,5 Å. Comparada com proteínas de arquitetura próxima, como o receptor de c-di-GMP LapD de Pseudomonas fluorescens, ou a enzima bifuncional MorA de Pseudomonas aeruginosa, sua estrutura se assemelha muito mais a essa última. Em particular, a hélice que conecta os domínios GGDEF e EAL possui a mesma extensão que a de MorA, maiores que a encontrada em LapD. Como a hélice pequena de LapD está relacionada com sua plasticidade conformacional interdomínios, a estrutura apresentada nesse trabalho sugere as proteínas dual domain cataliticamente ativas (EAL-mono ou bifuncionais) sejam estruturalmente rígidas. Combinando esses resultados com uma análise computacional feita em outras 150 sequências representativas de proteínas dual domain, propomos mecanismos catalíticos distintos para as enzimas bifuncionais e as EAL-monofuncionais. Enquanto que essas últimas formam dímeros estáveis através do domínio EAL, numa conformação apta para interagir e degradar c-di-GMP, as enzimas bifuncionais apresentam transições oligoméricas mediadas por interação de c-di-GMP com EAL, impondo atividades ciclase (GGDEF) e fosfodiesterase (EAL) excludentes. Por fim, baseados nesses mecanismos e na arquitetura de STM3615, ainda especulamos mecanismos funcionais in vivo compatíveis com o tema emergente de interações proteicas e localização do sinal nas vias de sinalização mediadas por c-di-GMP. / The formation of bacterial biofilms is a well-established phenomenon regulated by the signaling molecule c-di-GMP, characterized by the establishment of a static bacterial community embedded in a exopolymeric matrix. The domains responsible for the synthesis (GGDEF) or degradation (EAL and HD-GYP) of c-di-GMP are present in multiple proteins in nearly all bacterial genomes sequenced to date. Among the multiple and structurally diverse proteins involved in c-di-GMP signaling and biosynthesis, a large class are transmembrane proteins bearing both EAL and GGDEF domains. Functionally, these domains are presented in all combinations: both degenerate or conserved and combinations GGDEF-degenerated/EAL-conserved or vice versa. While the predicted conserved domains exhibit catalytic activity, the degenerate usually converted into structural domains or c-di-GMP receptors. While structural studies have revealed details of proteins with both domains degenerated (LapD) or conserved (MorA), little is known about one of the most representative combinations: GGDEF-degenerated/EAL-conserved. In this work, we conducted a structural and functional study of Salmonella enterica STM3615 protein, which has a periplasmic domain of unknown function, followed by cytoplasmic domains HAMP, GGDEF-degenerated and EAL-conserved. Through different soluble cytoplasmic constructs of STM3615, we confirmed that this protein has phosphodiesterase activity, even with the isolated EAL domain. In agreement with its catalytic activity, solution studies, such as SAXS and size exclusion chromatography, showed that STM3615 isolated EAL is dimeric, a prerequisite for phosphodiesterase activity. Using a construct with the isolated EAL-GGDEF domains, we determine its crystal structure to a resolution of 2.5 Å. Compared to the architectural closed c-di-GMP receptor LapD from Pseudomonas fluorescens and the bifunctional enzyme MorA from Pseudomonas aeruginosa, STM3615 structure is more similar to the latter. In particular, the α-helix connecting the domains GGDEF and EAL has similar extension, longer than the helix found in LapD. Given that this helix in LapD is essential for its inter-domain conformational plasticity, the structure presented in this study suggests the dual domain catalytically active proteins are structurally rigid. Combining these results with a computational analysis with 150 representative sequences containing the tandem GGDEF-EAL domains, we propose distinct catalytic mechanisms for bifunctional and monofunctional EAL enzymes. While the latter form stable dimers through the EAL domain, a conformation prompted to interact and degrade c-di-GMP, the bifunctional enzymes present oligomeric transitions mediated by interaction of c-di-GMP with EAL domain, imposing excluding cyclase (GGDEF) or phosphodiesterase (EAL) activities. Finally, based on these mechanisms and STM3615 architecture, we also speculated about functional mechanisms in vivo consistent with the emerging theme of protein interactions and localized signal involved in signaling pathways mediated by c-di-GMP.

A formação de biofilmes bacterianos é um fenômeno bem conhecido, caracterizado pela formação de uma comunidade bacteriana estática, embebida em uma matriz exopolimérica, regulada pela molécula sinalizadora c-di-GMP. Os domínios proteicos que catalisam a síntese (GGDEF) e degradação (EAL e HD-GYP) de c-di-GMP estão presentes em grande quantidade em quase todos os genomas bacterianos sequenciados até hoje. Dentre as diversas proteínas envolvidas nas vias de sinalização mediadas por esse nucleotídeo, uma grande parcela são proteínas transmembranares que possuem ambos domínios GGDEF e EAL. Funcionalmente, esses domínios se apresentam em todas combinações: ambos degenerados ou conservados e combinações GGDEF-degenerado/EAL-conservado ou vice-versa. Enquanto que domínios conservados potencialmente apresentam atividade catalítica, os degenerados geralmente convertem-se em domínios estruturais ou receptores de c-di-GMP. Embora recentes estudos estruturais revelaram detalhes de proteínas com ambos domínios degenerados (LapD) ou ativos (MorA), pouco se sabe sobre uma das combinações mais representativas: GGDEF-degenerado/EAL-conservado. Nesse trabalho, realizamos um estudo estrutural e funcional da proteína STM3615 de Salmonella enterica, que apresenta um domínio periplasmático de função desconhecida, seguido pelos domínios citoplasmáticos HAMP, GGDEF-degenerado e EAL-conservado. Através de diferentes construções citoplasmáticas solúveis de STM3615, confirmamos que essa proteína apresenta atividade fosfodiesterase, mesmo quando o domínio EAL encontra-se isolado. Corroborando com sua atividade catalítica, estudos em solução, tais como SAXS e cromatografia de exclusão molecular, mostraram que o EAL isolado de STM3615 é dimérico, um pré-requisito para ser ativo. Utilizando uma construção com os domínios GGDEF-EAL determinamos sua estrutura cristalográfica a uma resolução de 2,5 Å. Comparada com proteínas de arquitetura próxima, como o receptor de c-di-GMP LapD de Pseudomonas fluorescens, ou a enzima bifuncional MorA de Pseudomonas aeruginosa, sua estrutura se assemelha muito mais a essa última. Em particular, a hélice que conecta os domínios GGDEF e EAL possui a mesma extensão que a de MorA, maiores que a encontrada em LapD. Como a hélice pequena de LapD está relacionada com sua plasticidade conformacional interdomínios, a estrutura apresentada nesse trabalho sugere as proteínas dual domain cataliticamente ativas (EAL-mono ou bifuncionais) sejam estruturalmente rígidas. Combinando esses resultados com uma análise computacional feita em outras 150 sequências representativas de proteínas dual domain, propomos mecanismos catalíticos distintos para as enzimas bifuncionais e as EAL-monofuncionais. Enquanto que essas últimas formam dímeros estáveis através do domínio EAL, numa conformação apta para interagir e degradar c-di-GMP, as enzimas bifuncionais apresentam transições oligoméricas mediadas por interação de c-di-GMP com EAL, impondo atividades ciclase (GGDEF) e fosfodiesterase (EAL) excludentes. Por fim, baseados nesses mecanismos e na arquitetura de STM3615, ainda especulamos mecanismos funcionais in vivo compatíveis com o tema emergente de interações proteicas e localização do sinal nas vias de sinalização mediadas por c-di-GMP. / The formation of bacterial biofilms is a well-established phenomenon regulated by the signaling molecule c-di-GMP, characterized by the establishment of a static bacterial community embedded in a exopolymeric matrix. The domains responsible for the synthesis (GGDEF) or degradation (EAL and HD-GYP) of c-di-GMP are present in multiple proteins in nearly all bacterial genomes sequenced to date. Among the multiple and structurally diverse proteins involved in c-di-GMP signaling and biosynthesis, a large class are transmembrane proteins bearing both EAL and GGDEF domains. Functionally, these domains are presented in all combinations: both degenerate or conserved and combinations GGDEF-degenerated/EAL-conserved or vice versa. While the predicted conserved domains exhibit catalytic activity, the degenerate usually converted into structural domains or c-di-GMP receptors. While structural studies have revealed details of proteins with both domains degenerated (LapD) or conserved (MorA), little is known about one of the most representative combinations: GGDEF-degenerated/EAL-conserved. In this work, we conducted a structural and functional study of Salmonella enterica STM3615 protein, which has a periplasmic domain of unknown function, followed by cytoplasmic domains HAMP, GGDEF-degenerated and EAL-conserved. Through different soluble cytoplasmic constructs of STM3615, we confirmed that this protein has phosphodiesterase activity, even with the isolated EAL domain. In agreement with its catalytic activity, solution studies, such as SAXS and size exclusion chromatography, showed that STM3615 isolated EAL is dimeric, a prerequisite for phosphodiesterase activity. Using a construct with the isolated EAL-GGDEF domains, we determine its crystal structure to a resolution of 2.5 Å. Compared to the architectural closed c-di-GMP receptor LapD from Pseudomonas fluorescens and the bifunctional enzyme MorA from Pseudomonas aeruginosa, STM3615 structure is more similar to the latter. In particular, the α-helix connecting the domains GGDEF and EAL has similar extension, longer than the helix found in LapD. Given that this helix in LapD is essential for its inter-domain conformational plasticity, the structure presented in this study suggests the dual domain catalytically active proteins are structurally rigid. Combining these results with a computational analysis with 150 representative sequences containing the tandem GGDEF-EAL domains, we propose distinct catalytic mechanisms for bifunctional and monofunctional EAL enzymes. While the latter form stable dimers through the EAL domain, a conformation prompted to interact and degrade c-di-GMP, the bifunctional enzymes present oligomeric transitions mediated by interaction of c-di-GMP with EAL domain, imposing excluding cyclase (GGDEF) or phosphodiesterase (EAL) activities. Finally, based on these mechanisms and STM3615 architecture, we also speculated about functional mechanisms in vivo consistent with the emerging theme of protein interactions and localized signal involved in signaling pathways mediated by c-di-GMP.

The Beat rejection of conventional values meant a rejection of marriage, family, and a nine-to-five job, and few women were prepared to make that kind of radical shift in a society that condemned women for behaving the way the Beats behaved. Though she has faced difficulty in getting published, Beat writer Diane Di Prima has been publishing steadily for the past forty years. Di Prima has also lived the life of a Beat, wandering the country, avoiding nine-to-five work and supporting herself with grants, teaching and poetry readings. In spite of her success and adherence to the Beat lifestyle, Di Prima has given birth to five children, all of whom she took with her in her travels. Diane Di Prima has always faced the particular challenge of gaining the acceptance of her male peers amid indifference and hatred toward her sex while not allowing these men to go unanswered.

The thesis Phthalates in yoghurts during storage focuses on monitoring of phthalic acid esters in yogurt samples in relation to their storage period. The analyzed phthalates are di-2-(ethylhexyl)-phthalate (DEHP) and di-n-butyl-phthalate (DBP), which are among the most commonly used polymeric plasticizers, especially polyvinyl chloride. I analyzed samples of yoghurt with 1 % and 5 % chia and bamboo fiber and then subsequently compared them with natural yogurts. In total, I analyzed 120 samples of yoghurt, for which I measured DBP concentrations ranging from 0.2 mg.kg-1 to 7.7 mg.kg-1 of the original raw material and concentration of undetected values of DEHP in range up to 2.6 mg.kg-1 of the original raw material. While analyzing the yoghurt packaging, I measured average DBP values of 174.1 μg.dm-2 (68.0 μg.g-1 plastic) and DEHP values of 26.2 μg.dm-2 (10.2 μg.g-1 plastic). After prior preparation of samples, I used high performance liquid chromatography to do the phthalates analysis.

Illaria Pescini's book provides a detailed inventory of the communal archive of San Casciano Val di Pesa, which contains documents relevant to a territory traditionally under the control of Florence and located southwest of the city. These documents contain information on both local rule and the relationship of the area with the central administrative authority in Florence. Pescini provides a historical summary of the archive and the territory related to it with particular emphasis on its institutional history.

Since its origins, company law has placed itself in the middle of the traditional pair of ‘enterprise’ and ‘liability’, making it possible to exercise business activity while enjoying the benefit of limited liability. At the same time, this opportunity has always fostered the search for stratagems to fully pass on to creditors the risks associated with the entrepreneurial activity. This is a well-known and multiform phenomenon that we shall call ‘eterodirezione abusiva’, an expression which could be approximately rendered as the ‘abuse of the legal personality’ by the ‘dominus’ of the company. Although the vexata quaestio of the abuse of legal personality has been debated among interpreters for over a century, it does not seem to have yet found a definitive answer in the legal system, and indeed continues to encounter partial and largely unsatisfactory solutions in legal terms. In this regard, the need to investigate the phenomenon originates from the empirical observation of recent case law, where (i) the ‘abusive’ connotation of some ‘risk-spreading’ conducts perpetrated through companies is taken for granted and (ii) contradictory views for its repression coexist. More specifically, the hermeneutic approaches and drifts reached up to this point have given rise to widespread uncertainty around the predictability of judicial decisions in this controversial field. While pursuing the goal of finding one unambiguous solution in this multifarious scenario, the work has been structured in four sections. Section I (Overview of the problem) frames the phenomenon of abusive conducts exercised by the dominus over the corporation. In particular, the investigation starts with the analysis of the traditional theories developed around the repression of the abuse of the legal personality, and the focus is placed on the two hermeneutic paths on which case law is currently polarised: the first qualifies the dominus as a holding of a group of companies, and predicates his tort liability; the second depicts the dominus as a shareholder with unlimited liability of a concealed ‘super-company’, and subjects him to personal liability. Section II (The dominus as a holding) subjects the former interpretative trend to criticism. The scrutiny concentrates on the distinction between the ‘group of companies’ as a legal institution and the different phenomenon of abuse of legal personality, a demarcation which leads one to reject judicial attempts to sanction the dominus with tort liability under Article 2497 of the Civil Code. Section III (The dominus as a shareholder in a de facto corporation) investigates the normative bases of -and the many critical issues arising from- the use of the judicial technique called ‘concealed de facto super-company’ as a means of extending the area of personal liability to the dominus of the insolvent corporation. Finally, Section IV (Conclusions) examines the results of the analysis of both hermeneutic paths in a twofold direction. Firstly, in the current and prospective regulatory framework, the all-pervading dialectic between the ‘effectiveness’ of entrepreneurial activity and the ‘formality’ of company law seems to be solved in favour of the latter. Secondly, a bird’s-eye view of the legal system allows one to ascertain the existence of a progressive and ongoing erosion of the role traditionally attributed to both the dogma of inviolability of the legal personality and that of formal imputation of business activity and liability (i.e. spendita del nome). This observation makes it possible to forecast a potential further evolution of the legal system towards a reopening of the debate on the qualification of the dominus as an entrepreneur who can be directly imputed with liability for obligations assumed in the name of the dominated company.

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