The discovery of cyclic AMP (cAMP), nearly 50 years ago by Sutherland radically altered the appreciation of metabolic regulation. Since then the presence of cAMP and its tremendous physiological impact has been demonstrated in many prokaryotic systems. In fact, virulence mechanisms of several pathogens known today exploit cAMP dependent pathways. Interestingly the genome of Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis, encodes as many as 16 adenylyl cyclases (enzymes that convert ATP to 3’, 5’-cAMP) and 10 cyclic-nucleotide binding proteins. Recent reports show that bacterial-derived cAMP manipulates host signaling for bacterial survival, suggesting an important role for cAMP in the pathogenesis of M. tuberculosis. A large number of non-pathogenic species of mycobacteria also share and conserve several of these cAMP metabolism genes, suggesting that cAMP is not only important for pathogenesis but also may play a critical physiological role in the genus. The work carried out in this thesis aims at a better understanding of the role of cAMP by studying the adenylyl cyclases and cyclic AMP receptor proteins (CRPs) from Mycobacterium smegmatis, a non-pathogenic member of the genus. Intracellular cAMP levels in a cell are precisely maintained by modulating the activities of the adenylyl cyclases (cAMP synthesising enzymes), the phosphodiesterases (cAMP hydrolysing enzymes) and the secretion machinery, if any. To assess the role of cAMP in mycobacteria, cAMP levels were measured in M. smegmatis during growth and under various stress conditions. The results show that cAMP levels peak at log phase of growth and decline thereafter. Under acidic conditions or on perturbing the cell-wall, cellular cAMP levels are altered, which indicate a possible role of cAMP in stress adaptation. Earlier work in our laboratory has led to the identification of multiple adenylyl cyclases in the mycobacterial genomes. These cyclases are similar in sequence to the mammalian enzymes and several of them have been shown to be active in vitro displaying a diverse range of biochemical properties. The M. smegmatis genome encodes 10 adenylyl cyclase-like genes. In order to understand the role of cAMP in M. smegmatis, individual cyclases were analysed for their biochemical properties and physiological functions. The work presented in this thesis is concerned with the functional characterization of MSMEG_3578 and MSMEG_3780, two of the several adenylyl cyclases from M. smegmatis. MSMEG_3578 encodes for a protein that comprises two transmembrane domains, an extracellular receptor-like domain, a membrane anchoring HAMP domain and an intracellular cyclase domain. The cyclase domain is closely related to mammalian cyclases but lacks the canonical residues that are critical for the catalysis of class III cyclases. Interestingly, the stop codon of this gene overlaps with the start codon of the downstream gene, MSMEG_3579 (a putative cyclic nucleotide gated mechanosensitive ion channel), suggesting a functional link between the two genes. The conservation of this gene pair across the mycobacterial genus indicates the importance of this putative receptor-effector pair in the physiology of mycobacteria. Additionally, microarray analysis by various groups have shown that this gene pair in Mycobacterium tuberculosis is differentially regulated in conditions that mimic stress the bacteria may experience during infection. In order to ascertain the physiological role of MSMEG_3578, a knock-out M. smegmatis strain was generated and tested for growth and cAMP defects. The knock-out strain showed growth and cAMP profiles similar to the wild-type strain. Over-expression of MSMEG_3578 in M. smegmatis resulted in a significant rise in cAMP levels. Interestingly, over-expression of the MSMEG_3578 adenylyl cyclase in E. coli did not lead to an elevation in cAMP levels indicating that other mycobacterial proteins may be required for the activity of MSMEG_3578 in vivo. In agreement with this, the purified adenylyl cyclase domain of MSMEG_3578 was found to be biochemically inactive in vitro. Additionally, the over-expressing strain has altered colony morphology as compared to the wild type strain. Perturbation of cAMP levels by over-expression of other cyclases also leads to a similar colony morphology phenotype, indicating the phenotype to be controlled by cAMP in general rather than by a specific cyclase in the cell. MSMEG_3780 is a highly conserved, biochemically active adenylyl cyclase, speculated to play a house-keeping function in M. smegmatis. Its orthologs from M. tuberculosis (Rv1647) and M. leprae (ML1399) have been biochemically characterized earlier in our laboratory. To unravel the role of this gene in vivo, the ∆MSMEG_3780 strain was tested for growth and cAMP defects under various conditions. The deletion strain did not show any difference in growth rate or morphology when compared to the wild-type strain. However it showed a reduction in intracellular cAMP levels at the log-phase of growth. Reintroduction of the MSMEG_3780 gene in the deletion strain restored cAMP to wild-type levels, thus indicating a crucial role for this adenylyl cyclase in the maintenance of intracellular cAMP levels during logarithmic growth. In order to investigate the regulation of the MSMEG_3780 gene, its promoter activity was tested under various stress-conditions. Acid-stress specifically resulted in the repression of the MSMEG_3780 promoter activity, a condition which also leads to a decrease in cAMP levels in the cells. Further evidence for the susceptibility of the MSMEG_3780 gene to acid-stress was obtained when the ∆MSMEG_3780 strain failed to reduce intracellular cAMP content upon sustained acid-stress conditions. Since Rv1647 shares similar biochemical properties with MSMEG_3780 and can also heterodimerize with the MSMEG_3780 protein in vitro, it was interesting to test whether the M. tuberculosis ortholog could functionally complement MSMEG_3780. To assess this, a complement strain was generated that contained the Rv1647 gene under the control of MSMEG_3780 promoter, integrated into the genome of ∆MSMEG_3780 strain. Rv1647 protein was able to restore the cAMP phenotype seen on acid stress as well as the cAMP levels in the mutant strain at the log phase of growth. This study indicated the role of cAMP and MSMEG_3780 in acid adaptation and also suggested a non-redundancy of adenylyl cyclases in mycobacteria, where different individual cyclases may have unique functions in the cells. Since Rv1647 could complement the cAMP defective phenotype in ∆MSMEG_3780, this suggests functional parallels between the proteins from the two species. Bacterial adaptation to environmental stress is brought about by a rapid change in its gene expression profile. Cyclic AMP plays an important role by binding to and activating a transcriptional factor, cAMP receptor protein or CRP. We have identified two CRPs from M. smegmatis, viz., MSMEG_0539 and MSMEG_6189 that possess high similarity at the amino acid level (78% overall sequence identity). The CRP ortholog from M. tuberculosis, Rv3676 has been characterized structurally, biochemically and functionally earlier. Western blot and RT-PCR analyses showed that CRPs in M. smegmatis were present during all phases of growth. Both the CRPs were cloned, expressed and shown to bind cAMP. Since the DNA binding domains of Rv3676 and the two M. smegmatis CRPs are nearly identical, the CRPs from M. smegmatis were predicted to bind similar target sequences. Interestingly, a CRP site was identified in the promoter of the MSMEG_3780 gene, suggesting a possible feed-forward or feed-back loop, where the enzymatic product of the adenylyl cyclase now governs its own gene expression. We performed Electrophoretic Mobility Gel Shift Assays (EMSAs) with M. smegmatis lysates to show that CRP binds to the MSMEG_3780 promoter at the CRP site. Subsequent Chromatin Immunoprecipitation (ChIP) assays confirmed that CRP binding to the MSMEG_3780 promoter occurred in vivo. To investigate the role of CRP in the regulation of the MSMEG_3780 gene, luciferase reporter assays with the wild-type and CRP site mutant promoters were carried out. Results suggest that CRP regulates the MSMEG_3780 gene under osmotic stress. However, CRP did not play any role in basal expression of MSMEG_3780 during growth. To assess which CRP of the two is functionally linked to the MSMEG_3780 promoter, we carried out a footprint assay with purified CRPs. It was intriguing to note that both the CRPs were in fact able to bind the promoter albeit under different conditions. Whereas MSMEG_6189 bound the promoter both in the presence and absence of cAMP, MSMEG_0539 bound the promoter only in the presence of cAMP. MSMEG_6189 thus deviates from the accepted CRP paradigm that seeks an absolute requirement of cAMP for specific DNA binding. The present study identifies cAMP as an important stress signal in M. smegmatis. Using MSMEG_3780 as a model gene, the role of cAMP in mycobacteria was studied. The two divergent CRPs that were characterized may function and dictate cAMP-mediated and perhaps independent functions in cells. Taken together, our results provide compelling evidence for the important role of cAMP in the general physiology and stress adaptation in M. smegmatis.

No description available.

Tuberculosis continues to be one of the major causes of morbidity and mortality worldwide. Several mycobacterial species such as M. tuberculosis and M. africanum are responsible for causing this disease in humans. Reports of high cAMP levels in mycobacterial species (as compared to other bacteria such as E. coli) suggested that this second messenger may play an important role in the biology of mycobacteria. Further, it was reported that infection with mycobacteria led to an increase in the cAMP levels within the host macrophage. More recent studies have shown that this cAMP increase may be due to bacterially derived cAMP, hinting at a role for cAMP in mycobacterial pathogenesis. Given this background, the study of cAMP in mycobacteria proves to be an interesting field of research. Signalling through cAMP involves an interaction of this cyclic nucleotide with a cAMP-binding protein. These proteins typically contain a cyclic nucleotide-binding domain (CNB domain) linked to another (effector) domain. The CNB domain is thought to allosterically control the activity of the effector domain, thus mediating cellular responses to altered cAMP levels. For example, in the case of eukaryotic protein kinase A (PKA), binding of cAMP to the CNB domain results in relieving the inhibitory effects of the regulatory subunit on the catalytic subunit. The catalytic subunit then phosphorylates its target substrates, eliciting a variety of cellular responses. This work involves the characterisation of novel cAMP-binding proteins from mycobacteria, in an attempt to better understand cAMP signalling mechanisms in these organisms. The genome of M .tuberculosis H37Rv is predicted to code for ten CNB domain-containing proteins. One of these genes is Rv0998 (KATmt). KATmt was found to contain a GCN5 related N-acetyltransferase (GNAT) domain linked to a CNB domain. KATmt finds orthologues throughout the genus Mycobacterium, thereby suggesting its role in the basic physiology of these organisms. In addition, such a domain fusion is unique to mycobacteria and hence promises to deliver insights into the biology of this medically important genus. Presented here are the biochemical and functional characterisation of KATmt and its orthologue from M. smegmatis, MSMEG_5458 (KATms). Recombinant KATms bound cAMP with high affinity, validating the functionality of its CNB domain. Mutational and analogue-binding studies showed that the biochemical properties of the CNB domain were similar to mammalian protein kinase A and G-like CNB domains. The substrate for the GNAT acetyltransferase domain was identified to be a universal stress protein from M. smegmatis (MSMEG_4207). MSMEG_4207 was acetylated at a single lysine residue (Lys 104) by KATms in vitro. Further, cAMP binding to KATms increased the initial rate of acetylation of MSMEG_4207 by 2.5-fold, suggesting allosteric control of acetyltransferase activity by the CNB domain. To ascertain that KATms acetylated MEMEG_4207 in vivo, an in-frame deletion of the KATms gene was generated in M. smegmatis (ΔKATms). MSMEG_4207 was immunoprecipitated from wild-type M. smegmatis and the ΔKATms strains, followed by mass spectrometric analysis. Acetylated MSMEG_4207 was only present in the wild-type strain, confirming that KATms and MSMEG_4207 is an in vivo enzyme-substrate pair. Key biochemical differences were observed between KATms and KATmt. KATmt had an affinity for cAMP in the micromolar range, close to three log orders lower than that of KATms. In addition, KATmt showed strictly cAMP-dependent acetylation of MSMEG_4207. This demonstrates that orthologous proteins often evolve under varied selective pressures, resulting in divergent properties. Using a combination of bioluminescence resonance energy transfer (BRET) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS), the conformational changes that occur upon cAMP binding to the CNB domain of KATms were monitored. A BRET-based conformation sensor was constructed for KATms by inserting KATms between GFP2 (green fluorescent protein) and Rluc (Renilla luciferase). An increase in BRET upon cAMP binding to the sensor was observed. HDXMS analysis revealed that besides the CNB domain, the only other region that showed conformational changes in KATms upon cAMP-binding was the linker region. To confirm that the linker region was important in propagating the effects of cAMP-binding to the acetyltransferase domain, an additional construct for BRET analysis encompassing the CNB domain and the linker region was generated. The magnitude of the increase in BRET was similar to the full length BRET-based sensor, validating the crucial role of the linker region in propagating cAMP-mediated conformational changes. A ‘PXXP’ motif found in the linker region, showed maximum exchange in HDXMS analysis. Mutation of both these proline residues to alanine in KATms, as well as KATmt, resulted in decoupling of cAMP-binding and allosteric potentiation of acetyltransferase activity. In contrast to the intricate parallel allosteric relays observed in other CNB domain-containing proteins, the CNB domain in KATms functions as a simpler cyclic nucleotide binding-induced switch involving stabilization of the CNB and linker domain alone. Therefore, KATms is an example of a primordial CNB domain where conformational changes are a consequence of binding-induced ordering alone. Using a computational approach, putative substrate proteins of KATmt from M. tuberculosis were identified. The substrate specificity of lysine acetyltransferases is determined loosely by a consensus sequence around the lysine residue which is acetylated. Using this property of protein acetyltransferases, the genome of M. tuberculosis H37Rv was mined for proteins harboring lysine residues in a similar sequence context as seen in MSMEG_4207. In vitro biochemical analysis of some of the predicted substrates helped confirm a subset of enzymes belonging to the fatty acyl CoA synthetase (FadD) class as substrates of KATmt. The acetylation of FadDs by KATmt was cAMP-dependent. In each of the four proteins tested, acetylation was found to occur at a single conserved lysine residue. To confirm that FadDs were acetylated by KATmt in vivo, BCG_1055, the orthologue of KATmt in M. bovis BCG, was deleted using the specialised transduction method. FadD13, one of the FadDs acetylated by KATmt in vitro, was immunoprecipitated from wild-type M. bovis and the ΔBCG_1055 strains using a FadD13-specific polyclonal antibody. Acetylated FadD13 was almost completely absent in ΔBCG_1055 but substantial amounts of acetylated FadD13 were present in the wild-type strain, indicating that FadD13 was indeed an in vivo substrate of KATmt. The functional consequences of acetylation of FadDs were analysed using an in vitro fatty acyl CoA synthetase assay. The activities of FadD2 and FadD13 were inhibited on acetylation with KATmt, while acetylation of FadD5 resulted in the formation of a novel product. Therefore, modification of the highly conserved lysine residue in these enzymes by acetylation led to loss or alteration of their enzymatic activity, suggesting that acetylation may be used as a regulatory mechanism to modulate the activities of some of the FadDs by KATmt in a cAMP-dependent manner. Given the extensive role of FadDs in cell wall biosynthesis and lipid degradation in mycobacteria, it seems possible that post-translational control by KATmt in a cAMP-dependent manner constitutes a novel mechanism utilised by these bacteria to regulate these pathways. This direct regulation of protein lysine acetylation by cAMP appears to be unique to mycobacteria, as orthologues of KATmt are not found outside this genus. In addition, the biochemical differences between KATmt and its orthologue from M. smegmatis KATms, indicate species specific variation, on a common theme. This study is the first report of protein lysine acetylation in mycobacteria. In addition to the identification of several proteins subject to this post-translational modification, the effect of acetylation on the enzymatic activities of some of them has been elucidated.

No description available.

Orientador: Gilberto de Nucci / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T11:10:20Z (GMT). No. of bitstreams: 1 Arruda_AndreMoreiraMartins_M.pdf: 11172054 bytes, checksum: db7fb87cc200cb091d3be4733e8d8af5 (MD5) Previous issue date: 2013 / Resumo: A presença de um par de órgãos copuladores, os hemipênis, é a característica mais singular do grupo Squamata, que reúne as serpentes e os lagartos. Para que ocorra a ereção, o hemipênis sofre ingurgitamento dos corpos cavernosos por sangue e linfa, além de contar com o auxílio da contração do músculo propulsor do pênis e o relaxamento do músculo retrator. O coito nestes animais pode durar até 28 horas, porém, os mecanismos envolvidos, as estruturas e sua base farmacológica de funcionamento são ainda pouco conhecidas. O hemipênis consiste de dois corpos cavernosos funcionalmente concêntricos, um deles contendo feixes de fibras musculares lisas. Em mamíferos, sintases de NO neuronais e endoteliais estão presentes em estruturas neurais e no endotélio, respectivamente, enquanto a guanilato ciclase solúvel e PDE5 (fosfodiesterase tipo 5) estão expressas no músculo liso trabecular. Partindo disto, para investigar as vias presentes no tecido das cobras, foram construídas curvas concentração-resposta cumulativas de relaxamento para a acetilcolina (ACh), nitroprussiato de sódio (SNP), BAY41-2272 e tadalafil em corpos cavernosos de Crotalus (CCC) pré-contraídos com fenilefrina. Relaxamentos induzidos por estímulo elétrico (EFS) também foram feitos na ausência e presença de L-NAME (100 mm), ODQ (10 mM) e tetrodotoxina (TTX, 1 mM). Em CCC pré-contraídos, o relaxamento dependente de frequência, gerado por EFS, durou três vezes mais do que aqueles em CC mamíferos. Embora estes relaxamentos sejam praticamente abolidos por L-NAME ou ODQ, eles não foram afetados pela TTX. Em contraste, o EFS promoveu relaxamento em corpos cavernosos de sagui que haviam sido incubados com TTX / Abstract: The presence of a pair of copulatory organs, the hemipenes, is the most unique feature of the group Squamata, which includes snakes and lizards. For an erection to occur, the hemipenes suffer engorgement of the corpora cavernosa with blood and lymph, besides counting with the aid of contraction of the propellant muscle and relaxation of penis retractor muscle. Coitus in these animals can last up to 28 hours, however, the mechanisms involved, the structures and their pharmacological basis are still little known. The hemipenis consists of two concentric functionally cavernous bodies, one containing bundles of smooth muscle fibers. In mammals, neuronal NO synthases and endothelial cells are present in the endothelium and neuronal structures, respectively, whereas the soluble guanylate cyclase and PDE5 (phosphodiesterase type 5) are expressed in trabecular smooth muscle. To investigas the tissue were constructed cumulative concentration-response curves for relaxation to acetylcholine (Ach), sodium nitroprusside (SNP), BAY41-2272 and tadalafil in the corpora cavernosa of Crotalus (CCC) pre contracted with phenylephrine. Relaxations induced by electrical stimulation (EFS) was also tested in the presence and absence of L-NAME (100 mm), ODQ (10 mM) and tetrodotoxin (TTX, 1 mM). In precontracted CCC, dependent relaxation frequency generated by EFS last three-times more than those in DC mammals. Although these relaxations are virtually abolished by L-NAME or ODQ, they were not affected by TTX. In contrast, EFS caused a relaxation of the corpus cavernosum in marmosets that had been incubated with TTX / Mestrado / Farmacologia / Mestra em Farmacologia

The increased use of thermoplastics in load carrying components, especially in the automotive industry, drives the needs for a better understanding of its complex mechanical properties. In this thesis work for a master degree in solid mechanics, the mechanical properties of a PA 6/66 resin with and without reinforcement of glass fibers experimentally been investigated. Topics of interest have been the dependency of fiber orientation, residual strains at unloading and compression relative tension properties. The experimental investigation was followed by simulations implementing existing and available constitutive models in the commercial finite element code LS-DYNA. The experimental findings showed that the orientation of the fibers significantly affects the mechanical properties. The ultimate tensile strength differed approximately 50% between along and cross flow direction and the cross-flow properties are closer to the ones of the unfilled resin, i.e. the matrix material. An elastic-plastic model with Hill’s yield criterion was used to capture the anisotropy in a simulation of the tensile test. Residual strains were measured during strain recovery from different load levels and the experimental findings were implemented in an elastic-plastic damage model to predict the permanent strains after unloading. Compression tests showed that a stiffer response is obtained for strains above 3% in comparison to tension. The increased stiffness in compression is although too small to significantly influence a simulation of a 3 point bend test using a material model dependent of the hydrostatic stress.

Cette thèse est consacrée à la régulation de la guanylate cyclase soluble (sGC), le récepteur endogène du monoxyde d'azote (NO) chez les mammifères qui est impliqué dans la transduction du signal. L'enzyme sGC est activée par la fixation du NO sur son hème et catalyse alors la formation du cGMP à partir du GTP. Alors que la sGC est présente dans de nombreuses cellules de mammifères, le domaine hémique bactérien homologue (H-NOX) est impliqué dans la détection du NO et la régulation du métabolisme. Un objectif important est la découverte d'inhibiteurs de la sGC pour ralentir la progression tumorale.Le criblage de composés naturels d'une chimiothèque mesurant l'activité de la sGC purifiée a révélé six inhibiteurs actifs (Ki = 0.2 – 1 µM). Avec deux autres composés actifs en photothérapie (hypericin et hypocrellin) nous avons démontré que ces inhibiteurs sont des effecteurs allostériques qui ne se fixent ni sur l'hème, ni sur le site catalytique ou de fixation des activateurs, découvrant une nouvelle classe de composés pharmacologiques ciblant la voie de signalisation NO/cGMP.La transition structurale induite par l'activateur riociguat en synergie avec le CO a été étudiée par spectroscopie d'absorption résolue en temps pour démontrer des changements de coordination de l'hème. Deux états d'activation distincts de la sGC par le CO existent simultanément avec les coordiantions 6c-hème et 5c-hème en présence de l'activateur qui induit la rupture de la liaison Fe-His de l'hème, à l'instar de l'activateur naturel NO. De plus, nous montrons que l'isoliquiritigénine, commercialisée comme activateur de la sGC, et en réalité un inhibiteur de la sGC.La dynamique ds ligands CO, NO, and O2 a été mesurée sur 12 ordres de grandeur temporelle pour le type sauvage et un mutant du transporteur bactérien du NO (AXCP). La simple mutation Leu16Ala augmente l'afinité pour le CO 108 fois, celle du NO 106 fois et rend cette protéine réactive à O2. Dans le cas de CO et NO dont les affinités pour L16A-AXCP sont les plus grandes jamais mesurées, la recombinaison bimoléculaire n'est pas détectable. Des simulations de dynamique moléculaire ont démontré que le CO dissocié est contraint de rester à 4 Å du Fe2+ par Ala16, contrairement au type sauvage Leu16.La dynamique de O2 a été mesurée dans la protéine senseur Tt H-NOX par spectroscopie d'absorption transitoire et confirme l'hypothèse que Tt H-NOX n'est sans doute pas un senseur de NO stricto sensu mais un senseur redox. Les propriétés de Tt-H-NOX ne sont pas compatibles avec le rôle d'un simple transporteur de NO. / This thesis is devoted to the regulation of soluble guanylate cyclase (sGC), the endogenous nitric oxide (NO) receptor in mammals involved in signal transduction. The enzyme is activated by the binding of NO to its heme and catalyzes the formation of cGMP from GTP. While sGC is present in many mammalian cells, the homologous bacterial domain (H-NOX) is involved in NO detection and metabolism regulation. An important objective was to find sGC inhibitors to slow down tumor progression.The screening of natural compounds from a chemical library, tested on purified sGC activity, revealed six active inhibitors (Ki = 0.2 – 1 µM). Together with two agents for photodynamic therapy (hypericin and hypocrellin) we demonstrated that these inhibitors are allosteric modulators which bind neither to the heme nor to the catalytic and activator sites, revealing a new class of pharmacological compounds targetting the NO/cGMP signaling pathway.The structural transition induced in sGC by stimulator riociguat in synergy with CO was studied by transient absorption spectroscopy to demonstrate coordination changes of the heme. Two different activation states of sGC with CO 6c-heme and 5c-heme exist simultaneously in the presence of the stimulator which induces the breaking of the heme Fe-His bond, as does the sGC natural effector NO. In addition, the effect of isoliquiritigenin, which is sold as a sGC activator, was shown to be actually an inhibitor of sGC.The dynamics of the ligands CO, NO and O2 were measured over 12 orders of magnitude in time in wild type and mutant of a bacterial NO transporter (AXCP). The single mutation Leu16Ala increased 108-fold the CO affinity, ~106-fold the NO affinity and makes this protein reactive to O2. In the case of CO and NO, whose affinities for L16A-AXCP are the largest ever measured, the bimolecular rebinding was absolutely not detectable. Molecular dynamic simulations demonstrated that dissociated CO is constrained to stay within 4 Å from Fe2+ by Ala16, contrarily to wild-type Leu16.The dynamics of O2 in Tt-H-NOX proteins measured by transient absorption spectroscopy confirmed the hypothesis that Tt-H-NOX may not be a NO-sensor stricto sensu but a redox sensor. The properties of the Tt-H-NOX protein are not compatible with the role a mere NO-carrier.

No description available.

A series of lateral load tests were performed on a full-scale pile cap with three different backfill conditions, namely: with no backfill present, with densely compacted clean sand in place, and with loosely compacted clean sand in place. In addition to being displaced under a static loading, the pile cap was subjected to low frequency, small displacement loading cycles from load actuators and higher frequency, small displacement, dynamic loading cycles from an eccentric mass shaker. The passive earth pressure from the backfill was found to significantly increase the load capacity of the pile cap. At a displacement of about 46 mm, the loosely and densely compacted backfills increased the total resistance of the pile cap otherwise without backfill by 50% and 245%, respectively. The maximum passive earth pressure for the densely compacted backfill occurred at a displacement of approximately 50 mm, which corresponds to a displacement to pile cap height ratio of 0.03. Contrastingly passive earth pressure for the loosely compacted backfill occurred at a displacement of approximately 40 mm. Under low and high frequency cyclic loadings, the stiffness of the pile cap system increased with the presence of the backfill material. The loosely compacted backfill generally provided double the stiffness of the no backfill case. The densely compacted backfill generally provided double the stiffness of the loosely compacted sand, thus quadrupling the stiffness of the pile cap relative to the case with no backfill present. Under low frequency cyclic loadings, the damping ratio of the pile cap system decreased with cap displacement and with increasing stiffness of backfill material. After about 20 mm of pile cap displacement, the average damping ratio was about 18% with the looser backfill and about 24% for the denser backfill. Under higher frequency cyclic loadings, the damping ratio of the pile cap system was quite variable and appeared to vary with frequency. Damping ratios appear to peak in the vicinity of the natural frequency of the pile cap system for each backfill condition. On the whole, damping ratios tend to range between 10 and 30%, with an average of about 20% for the range of frequencies and displacement amplitudes occurring during the tests. The similar amount of damping for different ranges of frequency suggests that dynamic loadings do not appreciably increase the apparent resistance of the pile cap relative to slowly applied cyclic loadings.

No / Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in dyslipidemic disorders. Although oxLDL stimulates activatory signaling, it is unclear how these events drive accelerated thrombosis. Here, we describe a mechanism for oxLDL-mediated platelet hyperactivity that requires generation of reactive oxygen species (ROS). Under arterial flow, oxLDL triggered sustained generation of platelet intracellular ROS, which was blocked by CD36 inhibitors, mimicked by CD36-specific oxidized phospholipids, and ablated in CD36(-/-) murine platelets. oxLDL-induced ROS generation was blocked by the reduced NAD phosphate oxidase 2 (NOX2) inhibitor, gp91ds-tat, and absent in NOX2(-/-) mice. The synthesis of ROS by oxLDL/CD36 required Src-family kinases and protein kinase C (PKC)-dependent phosphorylation and activation of NOX2. In functional assays, oxLDL abolished guanosine 3',5'-cyclic monophosphate (cGMP)-mediated signaling and inhibited platelet aggregation and arrest under flow. This was prevented by either pharmacologic inhibition of NOX2 in human platelets or genetic ablation of NOX2 in murine platelets. Platelets from hyperlipidemic mice were also found to have a diminished sensitivity to cGMP when tested ex vivo, a phenotype that was corrected by infusion of gp91ds-tat into the mice. This study demonstrates that oxLDL and hyperlipidemia stimulate the generation of NOX2-derived ROS through a CD36-PKC pathway and may promote platelet hyperactivity through modulation of cGMP signaling. / the British Heart Foundation (PG/11/37/28884 and PG/13/90/30578) and Heart Research UK (RG2614)