Epstein-Barr Virus Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

ELM-induced transient tungsten melting in the JET divertor

The original goals of the JET ITER-like wall included the study of the impact of an all W divertor on plasma operation (Coenen et al 2013 Nucl. Fusion 53 073043) and fuel retention (Brezinsek et al 2013 Nucl. Fusion 53 083023). ITER has recently decided to install a full-tungsten (W) divertor from the start of operations. One of the key inputs required in support of this decision was the study of the possibility of W melting and melt splashing during transients. Damage of this type can lead to modifications of surface topology which could lead to higher disruption frequency or compromise subsequent plasma operation. Although every effort will be made to avoid leading edges, ITER plasma stored energies are sufficient that transients can drive shallow melting on the top surfaces of components. JET is able to produce ELMs large enough to allow access to transient melting in a regime of relevance to ITER.

Transient W melt experiments were performed in JET using a dedicated divertor module and a sequence of I-P = 3.0 MA/B-T = 2.9 T H-mode pulses with an input power of P-IN = 23 MW, a stored energy of similar to 6 MJ and regular type I ELMs at Delta W-ELM = 0.3 MJ and f(ELM) similar to 30 Hz. By moving the outer strike point onto a dedicated leading edge in the W divertor the base temperature was raised within similar to 1 s to a level allowing transient, ELM-driven melting during the subsequent 0.5 s. Such ELMs (delta W similar to 300 kJ per ELM) are comparable to mitigated ELMs expected in ITER (Pitts et al 2011 J. Nucl. Mater. 415 (Suppl.) S957-64).

Although significant material losses in terms of ejections into the plasma were not observed, there is indirect evidence that some small droplets (similar to 80 mu m) were released. Almost 1 mm (similar to 6 mm(3)) of W was moved by similar to 150 ELMs within 7 subsequent discharges. The impact on the main plasma parameters was minor and no disruptions occurred. The W-melt gradually moved along the leading edge towards the high-field side, driven by j x B forces. The evaporation rate determined from spectroscopy is 100 times less than expected from steady state melting and is thus consistent only with transient melting during the individual ELMs. Analysis of IR data and spectroscopy together with modelling using the MEMOS code Bazylev et al 2009 J. Nucl. Mater. 390-391 810-13 point to transient melting as the main process. 3D MEMOS simulations on the consequences of multiple ELMs on damage of tungsten castellated armour have been performed.

These experiments provide the first experimental evidence for the absence of significant melt splashing at transient events resembling mitigated ELMs on ITER and establish a key experimental benchmark for the MEMOS code.

Effect of polyethylenimine, a cell permeabilizer, on the photosensitized destruction of algae by methylene blue and nuclear fast red

The present study reports the effect a cell permeabilizer, polyethylenimine (PEI) has on the photodynamic effect of methylene blue (MB) and nuclear fast red (NFR) in the presence of hydrogen peroxide (H₂O₂). The photosensitized destruction of the algae Chlorella vulgaris under irradiation with visible light is examined. The photodynamic effect was investigated under aerobic and anaerobic conditions. The presence of a permeabilizer during the photosensitized destruction of C. vulgaris does not enhance the activity of the MB, MB/H₂O₂ system or the NFR, NFR/H₂O ₂ system under aerobic conditions. However under anaerobic conditions we have determined that when a cell permeabilizer was added to the MB/H ₂O₂ system, the photosensitized destruction of C. vulgaris proceeded via a combination of Type I and Type II mechanisms. The presence of PEI enforces MB/H₂O₂ to be active toward the destruction of C. vulgaris whether oxygen is present or absent. Under aerobic and anaerobic conditions the activity of NFR was suppressed in the presence of PEI as a result of electrostatic interactions between the photosensitizer and the cell permeabilizer. The decrease in fluorescence recorded is indicative of destruction of the chlorophyll a pigment. 

Three motAB stator gene products in Bdellovibrio bacteriovorus contribute to motility of a single flagellum during predatory and prey-independent growth

The predatory bacterium Bdellovibrio bacteriovorus uses flagellar motility to locate regions rich in Gram-negative prey bacteria, colliding and attaching to prey and then ceasing flagellar motility. Prey are then invaded to form a "bdelloplast" in a type IV pilus-dependent process, and prey contents are digested, allowing Bdellovibrio growth and septation. After septation, Bdellovibrio flagellar motility resumes inside the prey bdelloplast prior to its lysis and escape of Bdellovibrio progeny. Bdellovibrio can also grow slowly outside prey as long flagellate host-independent (HI) cells, cultured on peptone-rich media. The B. bacteriovorus HD100 genome encodes three pairs of MotAB flagellar motor proteins, each of which could potentially form an inner membrane ion channel, interact with the FliG flagellar rotor ring, and produce flagellar rotation. In 2004, Flannagan and coworkers (R. S. Flannagan, M. A. Valvano, and S. F. Koval, Microbiology 150:649-656, 2004) used antisense RNA and green fluorescent protein (GFP) expression to downregulate a single Bdellovibrio motA gene and reported slowed release from the bdelloplast and altered motility of the progeny. Here we inactivated each pair of motAB genes and found that each pair contributes to motility, both predatorily, inside the bdelloplast and during HI growth; however, each pair was dispensable, and deletion of no pair abolished motility totally. Driving-ion studies with phenamil, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and different pH and sodium conditions indicated that all Mot pairs are proton driven, although the sequence similarities of each Mot pair suggests that some may originate from halophilic species. Thus, Bdellovibrio is a "dedicated motorist," retaining and expressing three pairs of mot genes.

New insights into the causes of human illness due to consumption of azaspiracid contaminated shellfish

Azaspiracid (AZA) poisoning was unknown until 1995 when shellfish harvested in Ireland caused illness manifesting by vomiting and diarrhoea. Further in vivo/vitro studies showed neurotoxicity linked with AZA exposure. However, the biological target of the toxin which will help explain such potent neurological activity is still unknown. A region of Irish coastline was selected and shellfish were sampled and tested for AZA using mass spectrometry. An outbreak was identified in 2010 and samples collected before and after the contamination episode were compared for their metabolite profile using high resolution mass spectrometry. Twenty eight ions were identified at higher concentration in the contaminated samples. Stringent bioinformatic analysis revealed putative identifications for seven compounds including, glutarylcarnitine, a glutaric acid metabolite. Glutaric acid, the parent compound linked with human neurological manifestations was subjected to toxicological investigations but was found to have no specific effect on the sodium channel (as was the case with AZA). However in combination, glutaric acid (1mM) and azaspiracid (50nM) inhibited the activity of the sodium channel by over 50%. Glutaric acid was subsequently detected in all shellfish employed in the study. For the first time a viable mechanism for how AZA manifests itself as a toxin is presented.

Transforming Growth Factor-beta superfamily:evaluation as breast cancer biomarkers and preventive agents

The Transforming Growth Factor-beta (TGFbeta) superfamily of cytokines is comprised of a number of structurally-related, secreted polypeptides that regulate a multitude of cellular processes including proliferation, differentiation and neoplastic transformation. These growth regulatory molecules induce ligand-mediated hetero-oligomerization of distinct type II and type I serine/threonine kinase receptors that transmit signals predominantly through receptor-activated Smad proteins but also induce Smad-independent pathways. Ligands, receptors and intracellular mediators of signaling initiated by members of the TGFbeta family are expressed in the mammary gland and disruption of these pathways may contribute to the development and progression of human breast cancer. Since many facets of TGFbeta and breast cancer have been recently reviewed in several articles, except for discussion of recent developments on some aspects of TGFbeta, the major focus of this review will be on the role of activins, inhibins, BMPs, nodal and MIS-signaling in breast cancer with emphasis on their utility as potential diagnostic, prognostic and therapeutic targets.

Increased expression of the RIα subunit of the cAMP-dependent protein kinase A is associated with advanced stage ovarian cancer

The primary element in the cAMP signal transduction pathway is the cAMP-dependent protein kinase (PKA). Expression of the RIα subunit of type I PKA is elevated in a variety of human tumours and cancer cell lines. The purpose of this study was to assess the prognostic importance of RIα expression in patients with ovarian cancer. We have evaluated the expression of RIα in a panel of human ovarian tumours (n = 40) and five human ovarian cancer cell lines using quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The human ovarian cell lines OAW42 and OTN14 express high endogenous levels of RIα mRNA and protein (at significantly higher mRNA levels than high tissue expressors, P < 0.05). The ovarian cell line A2780 expresses low endogenous levels of RIα mRNA and protein (also at higher mRNA levels than low tissue expressors, P < 0.05). Quantitative RT-PCR revealed no significant difference in RIα mRNA expression between different ovarian histological subtypes in this study. No associations were found between RIα mRNA expression and differentiation state. RIα mRNA expression was significantly associated with tumour stage (P = 0.0036), and this remained significant in univariate analysis (P = 0.0002). A trend emerged between RIα mRNA expression levels and overall survival in univariate analysis (P = 0.051), however, by multivariate analysis, stage remained the major determinant of overall survival (P = 0.0001). This study indicates that in ovarian epithelial tumours high RIα mRNA expression is associated with advanced stage disease. RIα expression may be of predictive value in ovarian cancer and may be associated with dysfunctional signalling pathways in this cancer type.

SN2012ca: a stripped envelope core-collapse SN interacting with dense circumstellar medium

We report optical and near-infrared observations of SN2012ca with the Public ESO Spectroscopy Survey of Transient Objects (PESSTO), spread over one year since discovery. The supernova (SN) bears many similarities to SN1997cy and to other events classified as Type IIn but which have been suggested to have a thermonuclear origin with narrow hydrogen lines produced when the ejecta impact a hydrogen-rich circumstellar medium (CSM). Our analysis, especially in the nebular phase, reveals the presence of oxygen, magnesium and carbon features. This suggests a core-collapse explanation for SN2012ca, in contrast to the thermonuclear interpretation proposed for some members of this group. We suggest that the data can be explained with a hydrogen- and helium-deficient SN ejecta (Type I) interacting with a hydrogen-rich CSM, but that the explosion was more likely a Type Ic core-collapse explosion than a Type Ia thermonuclear one. This suggests that two channels (both thermonuclear and stripped envelope core-collapse) may be responsible for these SN 1997cy-like events.

Autosomal dominant retinitis pigmentosa (ADRP):localization of an ADRP gene to the long arm of chromosome 3

Members of a large pedigree of Irish origin presenting with early onset Type I autosomal dominant retinitis pigmentosa (ADRP) have been typed for D3S47 (C17), a polymorphic marker from the long arm of chromosome 3. Significant, tight linkage of ADRP to D3S47, with a lod score of 14.7 maximizing at 0.00 recombination, has been obtained, hence localizing the ADRP gene (RP1) segregating in this pedigree to 3q.

Induction and antagonism of antiviral responses in respiratory syncytial virus-infected pediatric airway epithelium.

Airway epithelium is the primary target of many respiratory viruses. However, virus induction and antagonism of host responses by human airway epithelium remains poorly understood. To address this, we developed a model of respiratory syncytial virus (RSV) infection based on well- differentiated pediatric primary bronchial epithelial cell cultures (WD-PBECs) that mimics hallmarks of RSV disease in infants. RSV is the most important respiratory viral pathogen in young infants worldwide. We found that RSV induces a potent antiviral state in WD-PBECs that was mediated in part by secreted factors, including interferon lambda-1 (IFNλ1)/IL-29. In contrast, type I interferons were not detected following RSV infection of WD-PBECs., Interferon (IFN) responses in RSV-infected WD-PBECs reflected those in lower airway samples from RSV-hospitalized infants. In view of the prominence of IL-29, we determined whether recombinant IL-29 treatment of WD-PBECs before or after infection abrogated RSV replication. Interestingly, IL-29 demonstrated prophylactic, but not therapeutic, potential against RSV. The absence of therapeutic potential reflected effective RSV antagonism of IFN-mediated antiviral responses in infected cells. Our data are consistent with RSV non-structural proteins 1 and/or 2 perturbing the Jak-STAT signaling pathway, with concomitant reduced expression of antiviral effector molecules, such as MxA/B. Antagonism of Jak-STAT signaling was restricted to RSV-infected cells in WD-PBEC cultures. Importantly, our study provides the rationale to further explore IL-29 as a novel RSV prophylactic.

Tumor necrosis factor receptor expression and signaling in renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC), a tubular epithelial cell (TEC) malignancy, frequently secretes tumor necrosis factor (TNF). TNF signals via two distinct receptors (TNFRs). TNFR1, expressed in normal kidney primarily on endothelial cells, activates apoptotic signaling kinase 1 and nuclear factor-kappaB (NF-kappaB) and induces cell death, whereas TNFR2, inducibly expressed on endothelial cells and on TECs by injury, activates endothelial/epithelial tyrosine kinase (Etk), which trans-activates vascular endothelial growth factor receptor 2 (VEGFR2) to promote cell proliferation. We investigated TNFR expression in clinical samples and function in short-term organ cultures of ccRCC tissue treated with wild-type TNF or specific muteins selective for TNFR1 (R1-TNF) or TNFR2 (R2-TNF). There is a significant increase in TNFR2 but not TNFR1 expression on malignant TECs that correlates with increasing malignant grade. In ccRCC organ cultures, R1-TNF increases TNFR1, activates apoptotic signaling kinase and NF-kappaB, and promotes apoptosis in malignant TECs. R2-TNF increases TNFR2, activates NF-kappaB, Etk, and VEGFR2 and increases entry into the cell cycle. Wild-type TNF induces both sets of responses. R2-TNF actions are blocked by pretreatment with a VEGFR2 kinase inhibitor. We conclude that TNF, acting through TNFR2, is an autocrine growth factor for ccRCC acting via Etk-VEGFR2 cross-talk, insights that may provide a more effective therapeutic approach to this disease.

Specific role of neuronal nitric-oxide synthase when tethered to the plasma membrane calcium pump in regulating the beta-adrenergic signal in the myocardium

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates beta-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser(16) phospholamban (PLB) phosphorylation as well as Ser(22) and Ser(23) cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the beta-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.

Hydraulics of stepped chutes: experimental-numerical-theoretical study

O estudo de escoamentos turbulentos em descarregadores em degraus tem sido um desafio para os investigadores. A macro-rugosidade do leito, a ondulação da superfície livre, a intermitência da localização da secção inicial de entrada de ar e o escoamento bi-fásico a jusante da secção inicial de entrada de ar fazem com que a caracterização do escoamento deslizante sobre turbilhões em descarregadores em degraus não seja simples. Actualmente, é possível combinar técnicas de medição fiáveis com simulações numéricas e análise teórica. Nesta dissertação, o estudo experimental baseia-se em resultados experimentais obtidos em duas instalações experimentais: a instalação A, do Laboratório Nacional de Engenharia Civil (LNEC), dotada de um descarregador em degraus com declive de 1V:0.75H e a instalação B, do Instituto Superior Técnico (IST), dotada de um descarregador em degraus com declive de 1V:2H. Uma sonda de ar, um tubo de Pitot modificado e vários hidrómetros permitiram o estudo do escoamento deslizante sobre turbilhões mono- e bifásico ao longo do descarregador em degraus da instalação A. Na bacia de dissipação de energia, a observação visual do escoamento foi auxiliada pelas leituras efectuadas em tomadas de pressão localizadas na soleira da bacia e pelas leituras da altura do escoamento efectuadas em réguas graduadas localizadas nas paredes da bacia. Na instalação B foram utilizados um tubo de Pitot e vários hidrómetros para estudar a região não arejada do escoamento deslizante sobre turbilhões no descarregador em degraus. O estudo numérico é baseado em simulações numéricas da região não arejada do escoamento deslizante sobre turbilhões sobre descarregadores em degraus com o código comercial de CFD FLOW-3D® de modo a reproduzir as condições ensaiadas experimentalmente. As simulações beneficiaram da técnica de blocos múltiplos (multi-block) num sistema de coordenadas cartesianas, da determinação da superfície livre pelo método TruVOF e da utilização de dois modelos de turbulência: os modelos k- e RNG k-. Por último, o estudo teórico consistiu em desenvolver um modelo simplificado 1D para determinar as características hidráulicas principais do trecho não arejado do escoamento deslizante sobre turbilhões em descarregadores em degraus. O modelo foi desenvolvido a partir das equações de Navier-Stokes, conjuntamente com resultados experimentais e numéricos. Os resultados apresentados nesta dissertação contribuem para o conhecimento do escoamento deslizante sobre turbilhões em descarregadores em degraus, nomeadamente na região não arejada, na secção inicial de entrada de ar e na região arejada. A hidráulica dos dissipadores de energia a jusante de descarregadores em degraus, em particular das bacias tipo III do USBR, é também objecto de estudo. Em relação à região não arejada do escoamento em descarregadores com declive acentuado, e com base em resultados experimentais e numéricos, são propostas expressões para estimar o desenvolvimento da altura equivalente de água, da espessura da camada limite, da concentração média de ar, do coeficiente de energia cinética, da dissipação de energia, do factor de resistência e do coeficiente n da fórmula de Manning. São ainda propostas expressões adimensionais para a energia cinética turbulenta e sua dissipação. Para declives moderados, são propostas expressões para estimar o desenvolvimento da altura equivalente de água, do coeficiente de energia cinética e da energia específica residual. São ainda apresentados valores do expoente 1/N da expressão adimensional da distribuição de velocidades, quer para descarregadores com declive acentuado quer com declive moderado. Em conformidade com outros estudos centrados em escoamentos de parede e com derivações teóricas, para a região não arejada do escoamento em descarregadores em degraus de acentuado declive, observa-se que o factor de resistência depende da macro-rugosidade criada pelos degraus e da geometria da secção transversal e que o coeficiente n da fórmula de Manning aumenta com a rugosidade. A descrição estatística da turbulência do escoamento é igualmente explorada, contribuindo para o conhecimento da estrutura do escoamento. Observou-se que para números de Reynolds rugoso não superiores a 6.8x104 a energia cinética turbulência e a sua dissipação cumprem leis de semelhança. Estas expressões adimensionais estão de acordo com os resultados obtidos por outros autores para escoamentos completamente desenvolvidos em canais abertos e no escoamento em rios com leito de gravilha. Em acréscimo, a taxa de dissipação de energia, quer para descarregadores de declive acentuado quer de moderado declive, é baixa. Por último, observa-se que os valores da média temporal da concentração de ar entre 0 e 1 medidos na região não arejada do escoamento dizem respeito não só ao ar capturado entre ondas de água, na zona de ondulação da superfície livre, mas também ao ar emulsionado no escoamento, i.e., sob a forma de bolhas de ar, quando perto da secção média inicial de entrada de ar, devido à diferença entre localizações instantânea e média temporal. Foram revistas metodologias e fórmulas para estimar a localização da secção inicial de entrada de ar e apresentadas expressões para estimar a concentração média de ar e a altura equivalente de água nessa secção. Relativamente à região de escoamento arejado em descarregadores em degraus com declive acentuado, os resultados experimentais apresentados nesta dissertação permitiram estimar a influência da definição da superfície livre nos parâmetros hidráulicos da região do escoamento arejado e estimar a máxima elevação do escoamento nesta região do escoamento. Com base nos resultados experimentais obtidos na bacia de dissipação de energia do tipo III do USBR localizada a jusante do descarregador em degraus da instalação A, observou-se que os perfis da altura piezométrica e da altura do escoamento tendem a seguir o perfil recomendado pelo USBR para bacias tipo III. A excepção ocorre à entrada da bacia, onde as alturas piezométricas apresentadas nesta dissertação excedem largamente as apresentadas pelo USBR. É ainda observado que, tal como entre as bacias tipo I e tipo III do USBR, o ressalto hidráulico estabiliza muito mais rapidamente numa bacia tipo III a jusante de um descarregador em degraus do que uma bacia tipo I a jusante do mesmo descarregador em degraus. Finalmente, observa-se que os blocos de amortecimento a colocar no descarregador não têm influência visível nos resultados da altura piezométrica nem da altura do escoamento ao longo da bacia. Relativamente às simulações numéricas do escoamento não arejado, a proximidade entre resultados experimentais e numéricos permite validar o modelo teórico e a integração numérica usados no FLOW-3D®. As simulações desenvolvidas também mostraram que o modelo de turbulência k- permite representar as características do escoamento não arejado em descarregadores em degraus, uma vez que não foram observadas diferenças significativas entre as simulações com este modelo e com o modelo RNG k-. Finalmente, observou-se que o modelo de entrada de ar usado no FLOW-3D® é válido para estimar a localização da secção inicial de entrada de ar. Por último, a proximidade entre os resultados obtidos da aplicação do modelo teórico desenvolvido no âmbito desta dissertação e os resultados experimentais indica que as hipóteses e simplificações consideradas no desenvolvimento do modelo são adequadas.

Matéria húmica de sapal: variações estruturais e complexação com o cobre

No presente trabalho foram estudadas as variações na composição, estrutura e comportamento das substâncias húmicas sedimentares (SHS), promovidas pela presença de duas espécies de plantas da família Chenopodiacea, presentes nos sapais da Ria de Aveiro: a Halimione portulacoides e a Arthrocnemum fruticosum. Para além das variações espaciais horizontais, foram investigadas as variações ao longo de um perfil de profundidades. Desta forma, para além da camada superficial (0-10 cm) de sedimentos com diferentes espécies de plantas e sem plantas, também foram investigadas camadas de sedimentos intermédia (20-25 cm) e profunda (45-50 cm). Para a extracção das SHS recorreu-se a uma pequena modificação do método proposto pela International Humic Substances Society (IHSS) para extracção e purificação de substâncias húmicas de solos. As SH extraídas foram caracterizadas através dos métodos espectroscópicos de absorção no ultravioleta visível, de fluorescência molecular síncrona, de absorção no infravermelho com transformadas de Fourier (FTIR) e de RMN 13C em estado sólido. Estas técnicas de caracterização foram complementadas pela análise elementar e termogravimetria. Em algumas amostras, foram ainda determinados o conteúdo em alguns metais por ICP-AES e o conteúdo funcional ácido por titulações potenciométricas. Após exaustiva caracterização das amostras de AH, procedeu-se ao estudo da sua capacidade de complexação com o cobre, recorrendo a titulações monitorizadas por fluorescência molecular síncrona. Os resultados revelaram que a colonização de sedimentos por plantas vasculares superiores conduz a uma alteração profunda no ambiente sedimentar e que esta é reflectida na diagénese das substâncias húmicas sedimentares, tendo-se observado variações a uma pequena escala espacial. O estudo deste tipo de variações nunca tinha sido efectuado em trabalhos anteriores. Nos ácidos fúlvicos detectaram-se diferenças na composição e estrutura das amostras correspondentes às camadas superiores de sedimentos, reflectindo a presença de diferentes colonizações vegetais. Relativamente aos ácidos húmicos, foi possível classificá-los em dois tipos distintos. Os AH do tipo I correspondem aos sedimentos superficiais e intermédios dos locais com plantas. Neste tipo de AH verificou-se uma incorporação de polissacarídeos e estruturas peptídicas, em estados de humificação pouco desenvolvidos. Os AH do tipo II correspondem aos sedimentos mais profundos de todos os locais e aos intermédios do local sem plantas e caracterizam-se por um maior grau de humificação e polimerização, um maior conteúdo aromático e de estruturas conjugadas e um maior conteúdo de grupos carboxílicos. Essas diferenças estruturais conduzem a diferentes comportamentos ácido/base e de complexação com o cobre. Assim, os AH do tipo I, que apresentam sinais espectroscópicos mais baixos associados a grupos carboxílicos e fenólicos, têm menor conteúdo de grupos funcionais ácidos e menor capacidade de complexação com o cobre quando se consideram ligandos característicos de unidades aromáticas conjugadas. Relativamente aos valores de log K dos complexos de AHS com o cobre, as amostras representativas da camada superficial dos sedimentos com plantas apresentaram valores superiores aos das restantes amostras de AH estudadas, cujos valores de log K são similares entre si. Atendendo aos resultados de análise elementar e espectroscópica, este facto pode estar associado à presença de compostos de azoto não peptídicos. Quanto a outros ligandos para o cobre, nomeadamente os representativos de estruturas peptídicas contendo o aminoácido triptofano e estruturas aromáticas simples, não foi possível obter resultados conclusivos. Dado que os ácidos húmicos do tipo I demonstraram um maior conteúdo peptídico e menor conteúdo aromático, o estudo destes ligandos torna-se importante para compreender melhor o efeito da presença de plantas nos sedimentos de sapal sobre o comportamento dos ácidos húmicos sedimentares como ligandos para o cobre. Refere-se ainda que foram detectadas certas especificidades na variação das características do sedimento e das substâncias húmicas com a profundidade nos diferentes locais estudados que não podem ser explicadas meramente com a presença de material vegetal oriundo de diferentes espécies de plantas, requerendo um conhecimento mais aprofundado das comunidades bentónicas e do ambiente físico-químico do sedimento.

Structural characterization and biological activity of polysaccharides from infusions used in the popular tradition

The use of plants with medicinal purposes is an ancient practice still very common in developing regions, and is rapidly spreading in industrialized countries. This fact is evidenced by the large number of ethnobotanical studies found in the literature referring that these plants are often used as decoctions and infusions. In most studies the reported biological activities are attributed to the presence of phenolic compounds, due to their antioxidant properties, and to polysaccharides, with its anti-tumoral properties. In “Trás-os-Montes” region, some of the most popular infusions used by the popular medicine are prepared with the dried leaves of Fraxinus angustifolia, the dried shoots of Mentha suaveolens, and the dried inflorescences of Pterospartum tridentatum. However, there are no studies about the polysaccharides present in these infusions. Thus, through the structural characterization of the polysaccharides present in the infusions of F. angustifolia, M. suaveolens, and P. tridentatum, the present PhD thesis intends to evaluate the possible relation between polysaccharides and the immunostimulatory activity that these infusions might present. In a preliminary phase, infusions of F. angustifolia were prepared according to the popular tradition, and it was observed that the obtained water soluble material contained approximately 85% of material non-retained in C18 cartridges, with hydrophilic characteristics, with the remaining 15% comprising retained-material with hydrophobic characteristics. It was also shown that the infusions only contained between 2 and 4% of high molecular weight material (HMWM), which comprised approximately 30% of carbohydrate material. Sugar and methylation analysis of the HMWM suggested the presence of pectic polysaccharides, together with type II arabinogalactans, mannans, and xyloglucans. However, the amount of material obtained is to low for the fractionation, and structural analysis of the polysaccharides present. The 4 h decoction, divided in two periods of 2 h, with water renewal, allowed to increase the HMWM yield, relatively to the infusions traditional infusions. It was also observed that the decoction also allowed to increase the HMWM proportion of carbohydrate material, due to an increase in the proportion of uronic acid present, although the neutral sugar residues seemed to be detected in similar proportions. Therefore, in all the experiments subsequently performed, the HMWM used was obtained through the decoction of F. angustifolia dried leaves, M. suaveolens dried shoots, and P. tridentatum dried inflorescences. x After the fractionation, through ethanol precipitation, and anion exchange chromatography, of the polysaccharides from the HMWM obtained by the decoction of the vegetable material of the distinct studied plants, it was observed the presence of high proportions of pectic polysaccharides, containing type I arabinogalactans, together with minor proportions of type II arabinogalactans, mannans, and xyloglucans. The presence of pectic polysaccharides in the extracts from F. angustifolia was also evidenced through endo-polygalacturonase treatment, and ESI-MS and ESI-MS/MS experiments. The detection of linked pentose and uronic acid residues, also seemed to suggest the presence of xylogalacturonan domains in the pectic polysaccharides from F. angustifolia. The extracts from F. angustifolia dried leaves also contained type II arabinogalactans that exhibited a higher structural diversity than those detected in the M. suaveolens, and P. tridentatum extracts, particularly in the substitution degree of the galactan backbone, and in the extension of the (1→5)-Araf side chains. Moreover, for all the plants studied, it was also observed that the type II arabinogalactans, extracted during the 2nd 2h of the extraction process, exhibited a substitution degree of the galactan backbone higher than those extracted during the 1st 2h. The extracts from P. tridentatum dried inflorescences contained higher proportions of mannans, and also of xyloglucans, both presenting a substitution degree higher than those, which were detected in lower proportion in the extracts of F. angustifolia and M. suaveolens. Through ESI-MS and ESI-MS/MS it was possible to evidence that the mannans present in the extracts of P. tridentatum presented acetyl groups on the O-2 of the mannosyl residues. It was also evidenced that the P. tridentatum mannans were more extensively acetylated than the mannans detected in the coffee infusion, LBG, and other non-conventional mannan sources. Moreover, it was detected the presence of oligosaccharides comprising hexose residues linked to non acetylated pentose residues, suggesting the possible presence of arabinose residues in the mannans from P. tridentatum extracts. The immunostimulatory activity of three fractions isolated from the extracts of F. angustifolia, M. suaveolens, and P. tridentatum, was tested and an increase in the NO production by macrophages, without compromising their cellular viability, was observed. The type I, and type II arabinogalactans detected in the extracts from F. angustifolia, and M. suaveolens seem to have contributed for the observed immunostimulatory activity. For the fraction from P. tridentatum, the mannans acetylation, and the presence of type I, and type II arabinogalactans seemed to contribute for the macrophage immunostimulatory activity observed. The possible presence of storage xyloglucans from the inflorescences seeds, also seems to have contributed for the immunostimulatory activity registered when the macrophages were stimulated with higher extract concentrations. The results obtained allow to conclude that the extracts of F. angustifolia dried leaves, M. suaveolens dried shoots, and P. tridentatum dried inflorescences contained high proportions of pectic polysaccharides, exhibiting type I arabinogalactans, together with other polysaccharides, such as type II arabinogalactans, mannans, and xyloglucans. This polysaccharide mixture seems to have contributed to the immunostimulatory activity of fractions isolated from the extracts of the studied plants. Therefore, as the same type of polysaccharides seem to be present in the decoctions and in the infusions, it seems possible that the polysaccharides might contribute for the therapeutic properties frequently associated by the popular tradition to the infusions of these plants.