Thèses : « Biological Mechanism of Action »

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Deans, Bryan. « Studies on the mechanism of action on antitumour imidazotetrazinones ». Thesis, Aston University, 1994. http://publications.aston.ac.uk/11048/.

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This thesis attempts to identify the cellular targets important to the cytotoxicity of imidazotetrazinones, to elucidate the pathways by which this damage leads to cell death, and to identify mechanisms by which tumour cells may circumvent this action. The levels of the DNA repair enzymes O6-alkylguanine-DNA-alkyltransferase (O6-AGAT) and 3-methyladenine-DNA-glycosylase (3MAG) have been examined in a range of murine and human cell lines with differential sensitivity to temozolomide. All the cell lines were proficient in 3MAG despite there being 40-fold difference in sensitivity to temozolomide. This suggests that while 3-methyladenine is a major product of temozolomide alkylation of DNA it is unlikely to be a cytotoxic lesion. In contrast, there was a 20-fold variation in O6-AGAT levels and the concentration of this repair enzyme correlated with variations in cytotoxicity. Furthermore, depletion of this enzyme in a resistant, O6-AGAT proficient cell line (Raji), by pre-treatment with the free base O6-methylguanine resulted in 54% sensitisation to the effects of temozolomide. These observations have been extended to 3 glioma cell lines; results that support the view that the cytotoxicity of temozolomide is related to alkylation at the O6-position of guanine and that resistance to this drug is determined by efficient repair of this lesion. It is clear, however, the other factors may influence tumour response since temozolomide showed little differential activity towards 3 established solid murine tumours in vivo, despite different tumour O6-AGAT levels.

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Mungthin, Mathirut. « Studies on the mechanism of action and mechanism of resistance to quinoline-containing antimalarial drugs in Plasmodium falciparum ». Thesis, University of Liverpool, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263874.

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Sanders, Paul Michael. « Mechanism of action of a tumour derived lipid mobilising factor ». Thesis, Aston University, 2003. http://publications.aston.ac.uk/11005/.

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Cancer cachexia comprises unintentional and debilitating weight loss associated with certain tumour types. Fat loss in cachexia is mediated by a 43kDa Lipid Mobilising Factor (LMF) sharing homology with endogenous Zinc-a2-Glycoprotein (ZAG). LMF and ZAG induced significant lipolysis in isolated epidydimal adipose tissue. This is attenuated by co-incubation with 10mM of antagonist SR59230A and partially attenuated by 25mM PD098059 (indicating b3-AR and MAPK involvement respectively). LMF/ZAG induced in vitro lipid depletion in differentiated 3T3-L1 adipocytes that seen to comprise a significant increase in lipolysis (p<0.01), with only a modest decrease in lipid synthesis (p=0.09). ZAG significantly increased in vitro protein synthesis (p<0.01) in C2C12 myotubes (without an effect on protein degradation). This increase was activated at transcription and attenuated by co-incubation with 10mM SR59230A. Proteolytic digestion of ZAG and LMF followed by sephadex G50 chromatography yielded active fragments of 6-15kDa, indication the entire molecule was not required for bioactivity. Cachexigenic MAC16 cells demonstrated significant in vitro ZAG expression over non-cachexigenic MAC13 (p<0.001). WAT and BAT excised from MAC16 mice of varying weight loss demonstrated increased ZAG expression compared to controls. Dosing of NMRI mice with s/c ZAG failed to reproduce this up-regulation, thus another cachectic factor is responsible. 0.58nM LMF conferred significant protection against hydrogen peroxide, paraquat and bleomycin-induced oxidative stress in the non-cachexigenic MAC13 cell line. This protection was attenuated by 10mM SR59230A indicating a b3-AR mediated effect. In addition, 0.58nM LMF significantly up regulated UCP2 expression (p<0.001), (a mitochondrial protein implicated in the detoxification of ROS) implying this to be the mechanism by which survival was achieved. In vitro, LMF caused significant up-regulation of UCP1 in BAT and UCP2 and 3 in C2C12 myotubes. This increase in uncoupling protein expression further potentiates the negative energy balance and wasting observed in cachexia.

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Matkar, Smita S. « Mechanism of action of potential anticancer drugs ». Scholarly Commons, 2008. https://scholarlycommons.pacific.edu/uop_etds/2368.

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Traditionally, inoperable or metastatic cancers have been treated by causing massive DNA damage in order to induce self-destruction (apoptosis) of the rapidly multiplying cancer cells. Initially, this strategy works for many cancers, in particular those which express normal p53 tumor suppressor protein. However, most cancers eventually aquire mutations in either p53 or other signaling molecules and fail to initiate apoptosis in response to severe DNA damage. During this study three types of compounds were investigated for their DNA damaging and anticancer effects: a pair of novel metal containing compounds, a pair of natural products, and a known synthetic drug which had been used many years ago for completely different indication. It was shown that all stop the growth of cancer cells and that the latter two classes do not require functional p53 because they work equally well in cells with normal (wildtype), mutant or no p53. The two nickel complexes investigated in this dissertation, differ in their ability to cause DNA damage and cell death. The oxidized form of the nickel complex, [Ni(CR-2H)] 2+ causes DNA damage and cell death at a much lower concentration than its reduced counterpart [Ni(CR)] 2+ . The phenanthridine alkaloids, Sanguinarine and Chelerythine cause high levels of DNA strand breaks and extremely rapid apoptosis which is not due to DNA damage because the quick onset precludes extensive signaling. The effects of the phenanthridines were linked to production of large amounts of reactive oxygen species (ROS), in particular hydrogen peroxide (H 2 O 2 ). The importance of ROS for the action of anticancer drugs as well as antibiotics is increasingly being recognized. In addition we also investigated the thioxanthone Lucanthone or Miracil D (which was used for the treatment of parasitic worms more than 50 years ago). It causes DNA strand breaks and apoptosis. Apoptosis occurs on a timescale consistent with signaling. However, p53 does not seem to be involved and alternative mechanisms are being investigated. This work provides new directions for designing novel anticancer drugs that are not subject to the limitations of DNA damaging agents.

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Duan, Xuchen. « Physiological and biological mechanisms of bisphosphonate action ». Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:36b0439d-2f89-4c1e-8bb3-941b4e6ee847.

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Bisphosphonates (BPs) are stable analogues of pyrophosphate widely used for the treatment of bone diseases characterised by increased bone resorption. Studies over the years have shown that the pharmacological potencies of BPs are dependent both on their binding affinities for bone mineral and on their inhibitory actions on osteoclasts. In addition, potential effects on other cell types present locally in the environment of skeletal tissues have been reported. The present study systematically evaluated the relative mineral-binding affinities of individual BPs of clinically relevance in mixtures of these compounds and the changes with elution pH by using column chromatography with ceramic hydroxyapatite and fluoroapatite combined with mass spectrometric identification and quantitation of the individual BPs. The results indicate that pH has a profound effect on the ionisation of the phosphonate and R2 functional groups, with BPs having greater affinities at lower pH as shown by increased retention times. Moreover, two other approaches, namely using Langmuir adsorption isotherms and competition assays based on fluorescent BP, have been developed to assess the mineral-binding capacities and dissociation constants of BPs. These results suggest that there are substantial differences among BPs in their binding to hydroxyapatite. From the cellular aspect of my study, I present evidence for the anti-apoptotic effects of BPs in osteocytes and osteoblasts. However, the study of prosurvival signalling pathways involved in these cells needs to be optimised. The work described in this thesis provides novel insights into the physiological and biological mechanisms of BP action. My project has provided a better knowledge of the physicochemical properties of BPs, which are highly relevant to their differential distributions within bone, their biological potencies, and their durations of action. Additionally, the cell culture studies may provide new information on the cellular effects of BPs on osteocytes and osteoblasts.

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Moreno, González M. del Carmen. « Characterization and mechanism of action of the biological control agent Pantoea agglomerans EPS125 ». Doctoral thesis, Universitat de Girona, 2006. http://hdl.handle.net/10803/7796.

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La soca EPS125 ha mostrat ser un efectiu agent de control biològic de diferents patògens fúngics de postcollita en diferents fruits. Degut a la seva elevada eficàcia, es va plantejar desenvolupar aquesta soca comercialment i per aquest motiu en el present treball es plantejà complementar la informació necessària pel seu registre.
D'acord amb els resultats obtinguts mitjançant proves fenotípiques i genotípiques, la soca EPS125 queda inclosa dins l'espècie Pantoea agglomerans (Enterobacter agglomerans-Erwinia herbicola). En relació a la utilització de fonts de carboni, en el perfil i contingut d'àcids grassos cel·lulars i en el polimorfisme en la longitud dels fragments de macrorestricció genòmica (MRFLP), la soca EPS125 mostrà trets característics que la diferencien d'altres soques. Els dos marcadors moleculars (125.2 i 125.3) específics per la soca EPS125 dissenyats en el present treball mostraren ser semiespecífics per la seva detecció mitjançant la tècnica PCR i Real Time PCR. Quedant pendent l'anàlisi d'especificitat de l'ús combinat dels dos marcadors moleculars en una reacció PCR multiplex. P. agglomerans EPS125 ha mostrat ser molt efectiva en el control de Penicillium expansum en poma amb una dosi efectiva mitjana de 2.7x105 a 7x105 ufc/ml, i una ratio de 25-101 cèl·lules de la soca EPS125 per inactivar una espora del patogen segons el model de saturació hiperbòlica. Segons les aproximacions fenotípiques i estudis genotípics realitzats, sembla que els mecanismes de biocontrol utilitzats per la soca EPS125 contra P. expansum en poma estan directament relacionats amb la capacitat de formació de biofilm per aquesta soca.
Strain EPS125 has shown effectiveness against a wide range of fungal pathogens in a large variety of fruit. However, to develop this strain as commercial biopesticide an extensive characterization is essential. For this reason, the objective of this PhD thesis was to complete the necessary information for its future registration.
According to morphological and biochemical tests, strain EPS125 pertain to Pantoea agglomerans (Enterobacter agglomerans-Erwinia herbicola) species. This strain showed typical traits different from other bacteria in relation to the ability to use several carbon sources, the fatty acid profiles and the macrorestriction fragment length polymorphism (MRFLP) pattern. The two DNA molecular markers of P. agglomerans EPS125 (125.2 and 125.3) obtained in the present work were semispecific in the detection of strain EPS125 by means of PCR and Real Time PCR. However, the combined use of the two primer sets in a multiplex PCR reaction would be specific. P. agglomerans EPS125 was highly effective against P. expansum in apple fruit having a median effective dose from 2.7x105 to 7x105 cfu/ml and a ratio of 101 and 25 EPS125 cells to inactivate one pathogen spore according to the hyperbolic saturation model.
Biocontrol mechanisms used by P. agglomerans EPS125 against P. expansum in apple fruit may be related with the ability of biofilm formation by this strain as show phenotypic approaches and genotypic studies.

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Tatarski, Miloš. « Molecular mechanism of dBigH1 action ». Doctoral thesis, Universitat de Barcelona, 2018. http://hdl.handle.net/10803/663021.

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INTRODUCTION: For decades it was known that many species contain embryo specific linker histone H1 variants that replace the somatic H1 during early embryogenesis. This is especially important because the early embryo shows typically zero to very little activity of transcription, and the first cleavages of the embryo depend exclusively on maternally deposited factors that are important for transcription and chromatin assembly. The first species shown to contain an embryo specific histone H1 was the sea urchin. Other species like the mouse, Xenopus or the zebrafish followed. Even in humans there are embryo specific H1 variants. Drosophila seemed to be an exception to this, until in 2013 the first linker histone H1 variant was discovered that was called dBigH1. Like other embryo H1 variants, dBigH1 is expressed in the early embryo and disappears when cellularization starts and it gets replaced by the somatic H1. Likewise, to its counterparts in other species, dBigH1 is responsible for the inhibition of transcription during the early stage of fly development. OBJECTIVES: In this thesis, we addressed the questions about the mechanism of inhibition of dBigH1 as well as the factors that are responsible for its deposition into chromatin. RESULTS: To answer the first question, we used an in vitro system for chromatin reconstitution based on an extract from early Drosophila embryos (DREX) that contains dBigH1 and all other factors needed for proper chromatin assembly. We then used the reconstituted chromatin in transcription experiments using HeLa nuclear extract that contains all factors needed for transcription. We saw that transcription for chromatin reconstituted in DREX could be reduced when the extract was previously depleted from dBigH1 using specific antibodies against it. By adding back recombinant dBigH1 to the depleted extract we were able to restore the initial lever of transcription. This showed us that dBigH1 was the repressive factor, as it was already confirmed in vivo. We then used a truncated construct of dBigH1 where we depleted the N-terminal domain of the protein. This was of particular interest as the N-terminal region of dBigH1 is the one that differs most form the somatic H1. It is much longer and more importantly very enriched with acidic residues, something that is very unique amongst all embryo specific H1 variants. We saw that when using the truncated construct, transcription was inhibited to a much lesser extent than with the full length dBigH1, proposing that the N-terminal domain is indeed responsible for the inhibition of transcription. To answer the second question about the factors needed for dBigH1 deposition, we used Drosophila testis to study dBigH1 in vivo. dBigH1 shows a very similar expression pattern in testis as the chromatin remodeler ACF1. This is why we decided to investigate a possible interaction between those two proteins. Additionally, we knew that ACF1 uses NAP1 as a histone chaperone for H1 in some species, so we also asked if NAP1 could play a role in dBigH1 deposition as well. Indeed, we saw that when using flies deficient for ACF1 we see much less dBigH1 in the testis tip where the germal stem cells (GSC) reside, suggesting that ACF1 plays an important role in dBigH1 deposition. In accordance, we see more dBigH1 in the GSCs when using flies overexpressing ACF1. At the same time, we can see that when depleting NAP1 from DREX, we see more dBigH1.

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Muraro, Lucia. « Studies of Botulinum Neurotoxins Mechanism of Action ». Doctoral thesis, Università degli studi di Padova, 2009. http://hdl.handle.net/11577/3425607.

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Botulinum neurotoxins (7 serotypes of BoNTs, named from A to G) and tetanus neurotoxin (TeNT) are the most powerful clostridial toxins (CNTs). They are responsible for botulism and tetanus respectively. TeNT and BoNTs bind to peripheral nerve terminals and inhibit neurotransmitter release from presynaptic neuronal cells by proteolytic cleavage of proteins involved in the fusion of synaptic vesicles with the cell membrane. BoNTs act at the level of Peripheral Nervous System (PNS) causing flaccid paralysis whereas TeNT acts at the level of Central Nervous System (CNS) causing spastic paralysis. In particular BoNT A cleaves and disables SNAP25 (synaptosome-associated protein 25), impairing the release of acetylcholine at neuromuscular junction. Structurally, CNTs are composed of two polypeptide chains linked by a single disulphide bond: the 50 kDa Light Chain (LC), which acts in the cytosol as a metalloprotease; and the 100 kDa Heavy chain, which includes a translocation domain (HN) and a receptor binding domain (HC). The three functional domains are structurally distinct and arranged in a linear fashion, such that there is no contact between the LC and HC domain. HC is further composed of two distinct subdomains HCN and HCC. These neurotoxins act at femtomolar concentration and the high affinity binding is due to multiple binding sites, either for membrane ganglioside and neuronal specific membrane proteins. BoNT/A binds to SV2 (synaptic vesicle 2) and to the ganglioside GT1b. It was thought that these two binding sites were located one in HCN subdomain and the other in the HCC subdomain. HCN share some sequence homology with lectins so it was a good candidate to bind ganglioside. Recently by crystallographic analysis it has been shown that both the protein receptor and ganglioside sites of BoNT/B are in the HCC domain. Due to the high homology between all CNTs it is likely that also the SV2 and GT1b sites are in the BoNT A HCC domain. If this is the case the role of N-terminal subdomain of BoNT A is still unknown. The aim of this project is to investigate the role of HCN in the binding of BoNT A to the plasma membrane. It is important to notice that the sequence of this toxin portion is conserved among all CNTs. The sequence of BoNT/A coding for the HCN domain (aa from 855 to 1093) has been cloned as His-Tag fusion protein, and fused to the Enhanced Green Fluorescent Protein (EGFP) and to the monomeric cherry red fluorescent protein (mCherry). By fluorescent microscopy observations we have shown that both the fluorescent chimera were able to bind to the plasma membrane of epithelial and neuronal cells. The fluorescent HCN domain remains at the plasma membrane during incubation times that allow the internalization of whole binding domain, HC. The fluorescent staining is not homogenous on the plasma membrane but is enriched in bright spots. For TeNT binding a role of lipid raft have been establish but for BoNTs the question seems to be still open. Ours data show that the sphingomyelin binding toxin lysenin, colocalized with HCN staining and treatment with sphingomyelinase diminished the HCN binding on epithelial cells. Moreover, in dot blot analysis HCN was able to directly interact with anionic lipid in particular phosphatidylinositol 5 phosphate (PI(5)P). A role for negative charged lipid in the binding of BoNTs and TeNT to lipid bilayer, it was already suggested; our hypothesis is that the N-terminal portion of the binding domain is able to bind anionic lipid in the environment of lipid raft. We suggest that these additional interactions with the membrane surface may play the role of positioning the toxin on the membrane surface ready for membrane insertion.

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Ashraf, Sadia. « Study of mechanism of action of Scorpion neurotoxins ». Doctoral thesis, Università degli studi di Padova, 2013. http://hdl.handle.net/11577/3423586.

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Summary Scorpions are important representative of arthropods. They have been well adapted to the extremes of environmental conditions. Scorpions also play a vital role in ecological systems by maintaining balance between different populations in an ecosystem. Apart from their positive role scorpions are responsible for more than 1.2 million stings per year with almost 3000 deaths worldwide per year and thus posing serious threat to public health. Two families of scorpions i.e. Buthida and Hemiscorpiidae are dangerous for humans. Scorpion sting can result in mild effects of redness and pain to very severe and lethal effects which can result failure of multiple organs eventually leading to death. Scorpion venom is composed of many different components such as low molecular weight peptides, nucleotides, lipids and certain enzymes. The diverse detrimental effects of scorpion sting result due to presence of many different types of toxins (neurotoxin, cardiotoxin, nephrotoxin, hemolytic toxin) and different enzymes (phosphodiesterases, phospholipases and hyaluronidases) in their venom. Since scorpions have a long evolutionary history during this long time period scorpions have developed a series of venom peptides that display a diverse range of biological functions. The most widely studied components of scorpion venom are the ion channel-modulating toxins which have been studied and described in detail in literature. Antarease a new class of unique scorpion metalloproteases capable of cleaving SNARE proteins has been described only recently with no previous evidence on presence of such enzymes in scorpions. Up till now SNARE proteins have only been shown to be targets of clostridial neurotoxins. To investigate such a class of metalloproteases we have analyzed the action of different scorpion venoms both on recombinant SNARE proteins as well as in neuronal cell models. Conclusion: This study shows for the first time the presence of antarease like proteins in scorpion species: Buthus eupeus and Orthochirus scrobiculosus. Both of the scorpion species contain active components i.e. metalloproteases that are able to cleave SNARE proteins in a mechanism similar to antarease.
RIASSUNTO Gli Scorpioni sono importanti rappresentanti del phylum Artropodi. Essi si sono ben adattati a condizioni ambientali estreme e giocano un ruolo fondamentale in diversi ecosistemi. Allo stesso tempo gli scorpioni sono responsabili di più di 1.2 milioni di punture per anno con quasi 3000 morti in tutto il mondo. Sono due le famiglie di scorpioni pericolose per l’uomo: Buthida e Hemiscorpiidae. Le punture di scorpione possono causare da effetti lievi come rossore, e dolore a effetti gravi che causano danni a diversi organi ed eventualmente la morte del soggetto colpito. Il veleno di scorpione è costituito da diversi componenti come peptidi a basso peso molecolare, lipidi ed enzimi. Gli effetti patologici della puntura di scorpione sono causati dalla presenza di diverse tossine (neurotossina, cardiotossina, nefrotossina, tossina emolitica) e diversi enzimi (fosfodiesterasi, fosfolipasi, ialuronidasi) nel veleno. Dato che gli scorpioni hanno una lunga storia evolutiva, durante questo lungo periodo hanno sviluppato un serie di peptidi e proteine che possiedono diverse funzioni biologiche. Grazie alla loro abbondanza e quindi alla facilità d’isolamento, i componenti del veleno più studiati sono le tossine che esplicano la loro azione sui canali ionici i quali sono stati molto studiati e descritti in dettaglio in letteratura. Recentemente è stata descritta una nuova classe di metalloproteasi di scorpione capace di proteolizzare le proteine SNARE che è stata denominata Antarease. Fino ad ora le proteine SNARE che hanno un ruolo chiave nel processo di neuroesocitosi, sono state descritte essere il bersaglio molecolare solo di neurotossine batteriche quali le tossine del tetano e del botulismo. Per studiare questa nuova classe di metalloproteasi, abbiamo analizzato l’azione di diversi veleni di scorpioni sia su proteine SNARE ricombinanti sia su modelli di neuroni primari in coltura. Questo studio ha dimostrato la presenza di metalloproteasi simili all’Antarease in specie di scorpioni Buthus eupeus e Orthochirus scrobiculosus e che questi enzimi sono in grado di proteolizzare in maniera specifica le proteine SNARE.

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LIPARI, Elisa. « DEVELOPMENT AND QUALIFICATION OF BIOANALYTICAL METHODS FOR DEAMIDATED IFNβ-1a AND INVESTIGATION ABOUT THE MECHANISM OF ACTION ». Doctoral thesis, Università degli Studi di Palermo, 2021. http://hdl.handle.net/10447/497430.

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Interferon beta-1a (IFNβ-1a) is a recombinant IFNβ with the tradename Rebif involved in several biological activities. Recently, it has been reported that artificial deamidation of IFNb-1a increases its biological response. Given the therapeutical potential, an investigation on the deamidated variant has been carried out via different approaches to discover the mechanism underlying this biological effect. The antiviral and immunomodulatory activity of deamidated cytokine was assessed using two precise and accurate cell-based assays. As expected, deamidated IFNβ-1a showed an increase in the biological response and its canonical pathway and receptor binding affinity were thorough analysed. Deamidated interferon beta increases STAT1 phosphorylation and ISGs expression compared to its native form. A full in-depth analysis in receptor binding highlighted a change in receptor affinity in deamidated IFNβ-1a. In particular, deamidation destabilizes the interaction with IFNAR2 through a change in the H-bonds network, increasing the affinity to IFNAR1 and consequently to the whole receptor complex. The higher receptor binding and the consequent strong signaling and gene expression, explains the greater biological activity of the deamidated IFNβ-1a compared to the native fcorm. These results open new perspective on the therapeutic potential of this molecule which could have significant beneficial effects on patients with MS and beyond

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Burman, Jonas. « Mechanisms of action of β-blockers for the treatment of heart failure ». Thesis, Linköpings universitet, Biologi, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-167006.

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Heart failure is a syndrome in which the heart is unable to supply the entire body with oxygen. It is manifested in shortness of breath and exercise intolerance. One class of drugs that have proven effective in managing the progression of heart failure is β-blockers. These drugs bind to β-adrenergic receptors with high affinity, thus preventing the binding of endogenous catecholamines such as epinephrine and norepinephrine to the receptors by outcompeting them. The most common explanation of how β-blockers help manage the progression of heart failure is that by slowing the heart rate, it reduces the strain put on the heart. There may however be other ways that β-blockers help decrease morbidity and mortality of heart failure. Alternative reasons to how β-blockers aid the treatment of heart failure have been proposed based on the literature. It was found that the compensatory mechanisms intended to alleviate failure may be the main reasons that actually worsen it. Prolonged stimulation by epinephrine and norepinephrine damage the myocardium through oxidative damage, signaling for apoptosis and cardiac remodeling, as well as causing an increase in blood volume through the RAS-system. By blocking these maladaptive responses, β-blockers such as Carvedilol, Metoprolol and Nebivolol, together with other drugs such as ACE-inhibitors, and lifestyle changes help manage the progression of heart failure as well as increase the quality of life for the patients suffering from it

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Jagatia, Heena. « The biological role and mechanism of action of rbpA and carD in Streptomyces coelicolor A3 (2) and Mycobacterium tuberculosis ». Thesis, University of Sussex, 2018. http://sro.sussex.ac.uk/id/eprint/80557/.

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Salazar, Montoya Vivian Angélica. « Exploring the mechanism of action of human antimicrobial ribonucleases ». Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/310611.

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Las ribonucleases humanas son un grupo heterogéneo de proteínas pertenecientes a la superfamilia de la Ribonucleasa A. Estas proteínas se caracterizan por su capacidad de hidrolizar ácidos ribonucleicos y por la presencia de actividad antimicrobiana frente diversos organismos patógenos como bacterias, hongos, parásitos y virus. El primer objetivo del presente estudio doctoral se centra en la caracterización de la actividad antimicrobiana y en modelos de membrana de las formas nativas de la ribonucleasa 3 purificadas a partir de eosinófilos. Las distintas formas nativas presentan modificaciones postraduccionales dadas por diversos grados de glicosilación que se correlacionan con la activación de los eosinófilos durante los procesos inflamatorios. El estudio establece la capacidad antimicrobiana de las formas nativas y su actividad en modelos de membrana. Los resultados indican que las modificaciones postrasduccionales modulan la actividad biológica de la RNasa 3, sugiriendo una contribución in vivo en su función fisiológica. Como segundo objetivo de esta tesis, se evaluó por primera vez en nuestro grupo de investigación la actividad antimicótica de las ribonucleasas 3 y 7 frente al hongo Candida albicans, el cual fue elegido como modelo patógeno eucariota. Se determinó y caracterizó la presencia de actividad frente a Candida por parte de ambas ribonucleasas humanas. Por último, el tercer objetivo de esta tesis se centra en la purificación y caracterización de la ribonucleasa 8, la más reciente ribonucleasa humana descrita, identificada inicialmente en placenta. La RNasa 8 presenta un patrón inusual de enlaces disulfuro respecto a sus proteínas homólogas. Este cambio estructural modifica la estabilidad de la proteína y expone regiones que facilitan el proceso de agregación proteica. Fue necesaria la previa optimización de un protocolo alternativo de purificación. Se analizaron sus propiedades antimicrobianas, sugiriendo su posible participación en la respuesta inmunitaria innata. Los resultados del presente estudio corroboran las propiedades antimicrobianas de diversas ribonucleasas humanas miembros de la familia de la RNasa A, sugiriendo una función ancestral en el sistema de defensa innato. El estudio contribuye a la comprensión de su mecanismo de acción y plantea su potencial uso como herramientas terapéuticas.
Human ribonucleases are a heterogeneous group of proteins belonging to the superfamily of RNase A. These proteins are characterized by their ability to hydrolyse ribonucleic acids and the presence of antimicrobial activity against various pathogens including bacteria, fungi, parasites and viruses. The first objective of this doctoral study is focused on the antimicrobial characterization of native Ribonuclease 3 forms purified from eosinophils. Native forms present posttranslational modifications giving different glycosylation grades that modulate their activity during inflammatory processes. This study aims to establish the antimicrobial properties of native forms purified from eosinophils and their activity in a membrane model system. Results indicate that post-translational modifications contribute to the the protein biological activities, suggesting a related physiological role. As a second objective, we evaluated for the first time the antifungal activity of the antimicrobial RNase 3 and RNase 7 against Candida albicans, an eukaryotic pathogen selected as a simple model to test the antimicrobial mechanism of action. Both human ribonucleases displayed a high antifungal activity. Results highlighted a dual mechanism of action, where cell lysis takes place at high protein concentration, while depolarization, cell internalization and cellular RNA degradation is achieved at sublethal doses. Finally, the last objective is focused on the characterization of ribonuclease 8, also called the placental RNase, the most recent human ribonuclease described. RNase 8 has gained and lost one cysteine residue in non-conserved positions in a mechanism called "disulphide shuffling". The protein tendency to aggregate required the design of an alternative purification protocol. We analysed its antimicrobial abilities, suggesting a possible role in innate defence. The results of this study confirmed the high antimicrobial activity of several human ribonucleases from the RNase A superfamily suggesting an ancestral role in the host immune defence response. The study contributed to the understanding of their mechanism of action and set the basis for applied drug design.

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Chandranath, Swaminathan Irwin. « Comparitive activities and mechanisms of action of three novel antiulcer agents ». Thesis, University of Central Lancashire, 2000. http://clok.uclan.ac.uk/21028/.

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Antiulcer agents, notably inhibitors of gastric acid secretion, have been the most successful category of drugs to be discovered in recent years; and moreover, there are currently four such agents in the world list of top 25 best selling drugs. Histamine H2 antagonists have been the number one selling pharmaceutical product for more than a decade and inhibitors of the parietal cell HIC-ATPase, so called "proton pump inhibitors" (PPI), look set to continue this success. The proposed study was designed to establish the relative efficacy and mechanisms of action of three novel agents using both in vitro and in vivo models. The three compounds namely AG-1749 (Lansoprazole), PD-136450 and transforming growth factor alpha (TGF(x) were studied to evaluate their antisecretory and antiulccr activities. Lansoprazole, the second PPI to be developed for clinical use, is a non-competitive inhibitor of the H1C-ATPase and has recently been launched in a number of countries. PD-136450 is a competitive antagonist of central and peripheral cholecystokinin-B (CCK-B) receptors (gastrin receptor) and it under clinical development as an anxiolytic but which has actions on the stomach and pancreas. Anxiolytic drug is otherwise known as anti-anxiety drugs, which are used to treat anxiety disorders, like depression, panic disorders, phobias and many personality disorders. TOFu is a polypeptide growth factor, which acts at the EGF receptor and displays potent mitogenic and antisecretory activity. The initial study deals with the comparison of the three compounds with omeprazole and ranitidine in terms of their ability to inhibit acid secretion and their activity in a range of experimental ulcer models. Potency, duration of action and activity against a range of stimulants of acid secretion (histamine, pentagastrin and basal) was determined in anaesthetized rat models by establishing dose-response relationships. The compounds represent a spectrum of activities in as much as lansoprazole is a potent, long acting inhibitor, PD-136450 is an orally active but selective inhibitor, while TOFu has a very short duration and is only active after parenteral administration. In a view to find out the mechanism of action of these drugs on gastric acid secretion, isolated gastric glands from rabbits were employed as an in vitro technique using radiolabeled 14C-aminopyrine as a marker. The results show that lansoprazole was the most potent antisecretory agent compared to other two drugs. The second phase of the study deals with the activity of the three compounds against gastric ulcers induced by acid hypersecretion, indomethacin and stress. This study enabled us to assess the extent to which antisecretory activity per se compared with other actions such as wound healing (TGFa) or anxiolytic activity (PD-136450) contribute to ulcer healing. As other workers already established that prostaglandins and nitric oxide are involved in the cytoprotective activity, the present study investigated the influence of prostaglandin and nitric oxide by using indomethacin and L-NAME pretreatment on the cytoprotective activity of lansoprazole, PD-136450 and TGFcz. Moreover, the three drugs were tested for their activities on the mucus and bicarbonate production in the stomach. It was found that lansoprazole and TGFc increased the gastric mucus secretion while PD-136450 did not show any change. Moreover it was evidenced from this study that the protective activity of PD-136450 is associated with the influence of bicarbonate secretion from the pancreas. In conclusion, the results of this study have indicated that lansoprazole, PD- 136450 and TGFct are potent antisecretory and antiulcer agents which have great therapeutic importance.

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Kodack, David Philip. « Signaling of the pleiotrophin growth factor through the anaplastic lymphoma kinase receptor studies on mechanism of action, biological activity and inhibitors / ». Connect to Electronic Thesis (CONTENTdm), 2009. http://worldcat.org/oclc/457188845/viewonline.

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Jaksevicius, Andrius. « Elucidation of mechanisms by which culinary herbs and spices exert their inhibitory action on the growth of CRC cells in vitro ». Thesis, Kingston University, 2017. http://eprints.kingston.ac.uk/41153/.

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Colorectal cancer (CRC) is one of the most commonly diagnosed types of cancer in the developed countries and the incidence is rising in the developing regions. Chronic inflammation, which is propagated by overexpression of cyclooxygenase-2 (COX-2) and its major product prostaglandin E2 (PGE2), plays a key role in the development of CRC. Culinary herbs and spices (CHS) are rich in polyphenols, have a high anti-oxidant capacity and possess anti-inflammatory activity. It has been shown that CHS inhibit the growth of CRC cells, however, their anti-carcinogenic mechanisms are mainly unknown. Hence, the aim of this study was to identify the CHS that were most potent inhibiting the growth of CRC cells, and subsequently to elucidate their anti-carcinogenic mechanisms, in particular, focusing on COX-2, the Wnt/β-catenin signalling pathway, and proteins involved in apoptosis. Another goal was to investigate whether combining the CHS would result in synergistic effects on the above. This study demonstrated that CHS extracted in water/or ethanol and their combinations inhibited CRC cell growth. This study also revealed that the most potent CHS extracted in ethanol (turmeric (TE), bay leaf (BLE) and ginger (GE)) and combinations downregulated the expression of COX-2 and suppressed COX-2 activity by reducing PGE2 release; their effect was comparable to that of the selective COX-2 inhibitor Celecoxib (50 μM). These CHS also induced apoptosis in CRC cells by targeting several key proteins: p53, caspase-3, and PAPR. However, the CHS did not have an effect on Wnt signalling pathway, which partially could be due to insufficient treatment time. In conclusion, this study demonstrated that CHS and their combinations inhibited CRC cell growth, inhibited COX-2 expression and activity, and modulated several key molecules involved in the development of CRC. Based on these findings, CHS have the potential to be utilized for CRC chemoprevention and possibly be used as a complimentary treatment. However, in vivo studies are needed to establish the true potential of these foods.

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López, Antón Nancy. « Identification of novel mechanisms of action contributing to the biological activity of cytotoxic natural compounds ». Diss., lmu, 2006. http://nbn-resolving.de/urn:nbn:de:bvb:19-53326.

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Rawls, Eric L. « Neural Mechanisms of Action Switching Moderate the Relationship Between Effortful Control and Aggression ». ScholarWorks@UNO, 2016. http://scholarworks.uno.edu/td/2234.

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Aggression and violence are social behaviors that exact a significant toll on human societies. Individuals with aggressive tendencies display deficits in effortful control, particularly in affectively charged situations. However, not all individuals with poor effortful control are aggressive. This study uses event-related potentials (ERPs) to decompose the chronology of cognitive functions underlying the link between effortful control and aggression. Specifically, this study investigates which ERPs moderate the effortful control - aggression association. We examined three successive ERP components (P2, N2 and P3) for stimuli that required effortful control. Results indicated that N2 activation, but not P2 or P3 activation, moderated the relationship between effortful control and aggression. These effects were present in negative and neutral contexts. This moderating effect was consistent with previous studies linking neural processing efficiency with reduced activation during cognitive control tasks. Our results suggest that efficient cognitive processing moderates the association between effortful control and aggression.

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LAZZATI, ZELDA. « Speciation of particulate matter's organic fraction and its mechanis of action on human health ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2009. http://hdl.handle.net/10281/7466.

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Three main researches have been employed for the implementation of a protocol analysis for the characterization and quantification of the lignin fraction in the particulate matter at the concentration matrix level; the implementation of different methods of analysis of the toxic interesting pollutants, Oxy-PAHs; Nitro-PAHs and the Bisphenol A, that together with the large set of performed analysis, allowed the characterization of some PM fractions in relation with Indoor and Outdoor concentrations, human exposure and Urban – Rural – Remote sites composition. At last an in silica method was developed for the research of the proteins involved in the interaction with the pollutants of interest, optimized on Bisphenol A because of its history and recent interaction study with the Nuclear Receptors. From the involved pathway the Blood Serine Proteases are used to test the accuracy and reproducibility of obtained Autodock4.0 and Dock4.0 data. The method results useful for research on the biological mechanism of action in relation with both matrix concentrations and in vivo and in vitro studies. The data predicted will be confirmed by NMR analysis. The newest docking program gives more and more reproducible data, accurate and empirically shaped on the domain problem, at last the experimental data had to confirm or not confirm the predictions.

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Teixeira, Lúcia de Fátima Moreira. « Role and mechanism of action of human immunoregulatory iNKT cells in Leishmania infection : new approaches for vaccination ? » Doctoral thesis, Faculdade de Farmácia da Universidade do Porto, 2010. http://hdl.handle.net/10216/63808.

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Teixeira, Lúcia de Fátima Moreira. « Role and mechanism of action of human immunoregulatory iNKT cells in Leishmania infection : new approaches for vaccination ? » Tese, Faculdade de Farmácia da Universidade do Porto, 2010. http://hdl.handle.net/10216/63808.

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Mohaibes, Raheem J. « Efficacy and mechanism of action of novel synthetic fatty acids derivatives in a transgenic Drosophila melanogaster Model of a Alzheimer's disease ». Doctoral thesis, Universitat de les Illes Balears, 2015. http://hdl.handle.net/10803/378038.

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- Introducció Alzheimer's disease (AD) is a neurodegenerative disorder characterized by early synaptic and late neuronal loss, affecting more than 26 million people worldwide. Among patients affected with dementia, more than half suffer from Alzheimer’s disease. The biggest risk factor for developing Alzheimer's disease is age. β-amyloid (Aβ) plaques and neurofibrillary p-Tau tangles accumulates in the brains of elderly patients playing a central role in the pathogenesis of AD. During the last years the fruit fly, Drosophila melanogaster has increasingly been used as a model for neurodegenerative disease. Although the adult fly has a simpler nervous system than those of vertebrates, it is capable of higher-order brain functions, including aversive and appetitive learning, and recalling learned information from prior experiences. - Contingut de la investigació This work has been focused on modeling Alzheimer's Disease in Drosophila by expressing two human genes associated with AD (Aβ42 and Tau) in the fly central nervous system. This model displays AD-like neuropathological as well as behavioral symptoms. The main goal of developing such a model is to analyse and study the effect of new synthetic fatty acids molecules in the pathogenesis of AD. Additionally, the model organisms established in this study could provide tools that help to understand disease-specific processes resulting in neuronal loss. This study argues that Drosophila can be used to study the behavioural basis of human neurodegenerative diseases and may provide a model to identify novel therapeutic avenues for neurodegenerative diseases as Alzheimer’s disease. In this work also was studied the effect of membrane lipid therapy on cognitive decline of a transgenic model of Drosophila. This model overexpresses the human amyloid peptide of 42 amino acids (Aβ42), and human Tau protein that play a key role in the development of this disease. - Conclusió The treatment has been based on the use of DHA and its hydroxylated derivate OHDHA, ARA and its hydroxylated form OHARA and EPA and its hydroxylated form OHEPA at 1, 3, 10, 30, 100 and 250 μg/ml of standard food. After testing the transgenes expression in the F1 generation by PCR analysis and Western blot it was evaluated the toxicity of the compounds, and it was demonstrated that food supplementation with OHDHA, OHARA, OHEPA partially restored the loss of locomotor activity and increased the life-span of the flies expressing the human transgenes whereas the DHA, ARA, EPA, form had not significant effects. It has been observed that the concentrations of 30 and 100 μg/ml of hydroxylated form, including the mixtures of (OHDHA+OHARA), (OHEPA+OHARA), and 30 μg/ml of TGMs, LP183A1, LP183A2, was used, have led to cognitive improvement and have maintained or increased the lifespan with respect to the control group. In addition it was analyzed the lipid content from Drosophila heads by using gas chromatography and it was found that the food supplementation with either hydroxylated or non-hydroxylated compounds induced changes in the fatty acid profile of Drosophila. Furthermore it was discovered that the amount of short chain fatty acids (SCFA), from the heads of F1 treated with ARA, EPA and DHA was less than that from untreated F1 flies. Concerning the hydroxylated fatty acids, the reduction in the levels of short chain fatty acid (SCFA) was similar to that of the non-hydroxylated fatty acids. All food supplement tested induced an increase of long chain fatty acids (≥ 18C). ARA, EPA and DHA were present in the fatty acid profile of flies treated with the respective non-hydroxylated food supplements. This fact proves the absorption and incorporation of dietary PUFAs into the Drosophila body tissues.
- Introducció La enfermedad de Alzheimer (AD, del inglés Alzheimer's disease) es una patología neurodegenerativa caracterizada por una pérdida temprana de conexiones sinápticas y, de manera tardía, de neuronas. Esta enfermedad afecta a unos 40 millones de personas en todo el mundo. Entre las personas con demencia, más de la mitad sufren AD. El mayor riesgo para desarrollar la enfermedad de Alzheimer es la edad. De hecho, las placas β-amiloide (Aβ) y ovillos neurofibrilares de fosfo-Tau se acumulan en los cerebros de pacientes ancianos, jugando un papel central en la patogénesis de AD. Además, se han encontrado reducciones significativas en los niveles de los lípidos fosfatidiletanolamina y ácido docosahexaenoico (DHA) en el cerebro de pacientes con AD. Durante la última década, la mosca de la fruta (Drosophila melanogaster) se ha utilizado como modelo para enfermedades neurodegenerativas, debido a que puede ser utilizada para el análisis de conductas como el aprendizaje aversivo y apetitivo, así como su capacidad de utilizar la información aprendida de previas experiencias, aunque la mosca adulta presenta un sistema nervioso mucho más simple que el de vertebrados. - Contingut de la investigació La presente investigación se centra en la utilización de Drosophila como modelo de AD mediante la sobreexpresión de los genes humanos asociados con AD (Aβ42 y Tau) en el sistema nervioso central de la mosca. El principal objetivo de desarrollar este modelo es analizar y estudiar el efecto de ácidos grasos sintéticos novedosos en la terapia de la AD. Conjuntamente, los organismos modelo establecidos en este trabajo pueden constituir un sistema que permita la comprensión de los procesos específicos de la enfermedad que desencadena la pérdida neuronal. Con todo ello, el presente trabajo demuestra que se puede usar Drosophila para estudiar las bases comportamentales de las enfermedades humanas neurodegenerativas y puede suponer un modelo para identificar nuevas terapias para dichas enfermedades, tales como AD. Además, se ha estudiado el efecto de la terapia lipídica de membrana en el declive cognitivo del modelo transgénico de AD de Drosophila. - Conclusió Los tratamientos empleados se basan en el uso de DHA y su derivado hidroxilado OHDHA, ARA y su forma hidroxilada OHARA y EPA y su forma hidroxilada OHEPA, así como derivados de triacilgliceroles (triacilglicerol miméticos, TGM) a dosis crecientes y añadidos en la comida. Tras confirmar la expresión de los transgenes en la generación F1 de las moscas por PCR y western blot, se analizó la toxicidad de los distintos compuestos y se demostró que la suplementación de comida con OHDHA, OHARA, OHEPA restauró la pérdida de actividad locomotora, parcialmente, además, aumentó la vida media de las moscas expresando los transgenes humanos, mientras que DHA, ARA, EPA no presentaron efectos significativos. Se observó que las concentraciones de 30 y 100 μg/ml de las formas hidroxiladas, incluyendo las mezclas de (OHDHA+OHARA), (OHEPA+OHARA) y 30 μg/ml de TGMs, LP183A1, LP183A2, mejoraron la capacidad cognitiva y aumentaron la vida media con respecto al grupo control no tratado. También se analizó el contenido lipídico en membranas de la cabeza de moscas mediante cromatografía de gases y se observó que la suplementación de la comida, tanto con los compuestos hidroxilados como los no-hidroxilados estudiados, indujo cambios en el perfil de ácidos grasos de Drosophila melanogaster. Entre ellos, se observó una menor cantidad de ácidos grasos de cadena corta en cabezas de moscas F1 tratadas con ARA, EPA and DHA en comparación con moscas no tratadas. En cuanto a los ácidos grasos hidroxilados, presentaron un nivel similar en la reducción de los niveles de ácidos grasos de cadena corta. Además, todos los suplementos añadidos a la comida indujeron un aumento de los ácidos grasos de cadena larga (≥ 18C). Finalmente, se observó la presencia de ARA, EPA y DHA en el perfil de ácidos grasos de las moscas tratadas con el correspondiente ácido graso no-hidroxilado. Este hecho prueba la absorción e incorporación de los ácidos grasos poliinsaturados presentes en la dieta en los tejidos de la Drosophila.

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Raitano, Arthur Bartholomew. « Reciprocal modulation of tumor necrosis factor and gamma interferon receptors in human carcinoma cells : Biological significance and mechanisms of action ». Diss., The University of Arizona, 1991. http://hdl.handle.net/10150/185483.

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Human recombinant cytokines may have a role in the clinical treatment of pancreatic and colorectal cancers. In the present studies, the growth inhibitory actions of recombinant human tumor necrosis factor (rhTNF) and recombinant human gamma interferon (rhIFN-γ) were examined in several human pancreatic and colorectal carcinoma cell lines in vitro in relation to the expression TNF and IFN-γ receptors. rhTNF and rhIFN-γ exerted significant, but differential, growth inhibitory effects in five of six cell lines examined. All six cell lines exhibited high affinity binding sites for both ¹²⁵I-labeled rhTNF and ¹²⁵I-labeled rhIFN-γ. However, the basal number of binding sites in general did not correlate with the relative growth inhibitory effects induced by either rhTNF or rhIFN-γ. In contrast, in three cell lines in which the cytokines exerted synergistic effects, rhTNF increased by 2-3 fold the number of ¹²⁵I-rhIFN-γ binding sites. Further, in two of these cell lines, rhIFN-γ also upregulated ¹²⁵I-rhTNF binding. As demonstrated by anti-IFN-γ receptor antibody labeling and the use of transcriptional and translational inhibitors, the increase in ¹²⁵I-rhIFN-γ binding by rhTNF was due to enhanced synthesis and expression of IFN-γ receptor protein. Recombinant human lymphotoxin (rhLT), which binds to the TNF receptor, and recombinant human interleukin-1 alpha (rhIL-1), which binds to a distinct receptor and mimicks many of the biological actions of TNF, also increased the expression of IFN-γ receptors. Further, rhIL-1 exerted synergistic growth inhibitory effects with rhIFN-γ. Taken together, these results suggest that the synergistic effects of either TNF/LT or IL-1 and IFN-γ may involve the upregulation of IFN-γ receptors. The mechanisms by which rhTNF and rhIL-1 upregulate IFN-γ receptors are unclear. However, the upregulatory effects of both rhTNF and rhIL-1 were attenuated by the Ca²⁺ ionophore A23187 and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate, suggesting that the mechanisms involved in IFN-γ receptor upregulation by TNF and IL-1 are negatively modulated by Ca²⁺ and protein kinase C activation. The results of this dissertation suggest that immunotherapy with recombinant cytokines may be useful in that treatment of pancreatic and colorectal cancer in vivo.

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Cabrefiga, Olamendi Jordi. « Fire blight (Erwinia amylovora) of rosaceous plants. Pathogen virulence and selection and characterization of biological control agents ». Doctoral thesis, Universitat de Girona, 2004. http://hdl.handle.net/10803/7924.

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El fuego bacteriano, causado por Erwinia amylovora, es una enfermedad muy importante a nivel comercial y económico porque afecta a plantas de la familia de las rosáceas y es especialmente agresiva en manzano (Pyrus malus) y peral (Pyrus communis), así como en plantas ornamentales (Crataegus, Cotoneaster o Pyracantha). Esta enfermedad está distribuida por todo el mundo en zonas climáticas templadas de Amércia del Norte, Nueva Zelanda, Japón, Israel, Turquí y Europa. En España, el fuego bacteriano fue detectado por primera vez en 1995 en el norte del País (Euskadi) y más tarde en nuevos focos aparecidos en otras áreas. La enfermedad puede ser controlada comercialmente mediante la aplicación de pesticidas quimicos (derivados de cobre, antibioticos). Sin embargo, muchos de los productos químicos presentan baja actividad o causan fitotoxicidad, y la estreptomicina, el producto más eficaz, esta prohibido en muchos países, incluyendo España. Por tanto, en ausencia de apropiados agentes químicos, el control biológico se contempla como una buena alternativa. En el presente trabajo, un agente de control biológico, Pseudomonas fluorescens EPS62e, ha sido seleccionada de entre 600 aislados de las especies P. fluorescens y Pantoea agglomerans obtenidos de flores, frutos y hojas de plantas de la familia de las rosáceas durante una prospección llevada a cabo en varias áreas geográficas de España. La cepa ha sido seleccionada por su capacidad de suprimir la infecciones producidas por E. amylovora frutos inmaduros, flores y brotes de peral en condiciones de ambiente controlado, presentando unos niveles de control similares a los obtenidos mediante el control químico usando derivados de cobre o antibióticos. La cepa además ha mostrado la capacidad de colonizar y sobrevivir en flores y heridas producidas en frutos inmaduros en condiciones de ambiento controlado pero también en flores en condiciones de campo. La exclusión de E. amylovora medinate la colonización de la superficie, el consumo de nutrientes, y la interacción entre las células del patógeno y del agente de biocontrol es la principal causa de la inhibición del fuego bacteriano por la cepa EPS62e. Estas características constituyen aspectos interesantes para un desarrollo efectivo de la cepa EPS62e como un agente de biocontrol del fuego bacteriano en condiciones comerciales.
Fire blight, caused by Erwinia amylovora is a serious disease of rosaceous plants with great commercial and economic interest that is distributed over the world. Disease may be controlled commercially by the application of chemicals (copper compounds, antibiotics). Many chemical agents have low activity or cause phytotoxicity, and streptomycin, the most effective antibiotic, is not approved for use in many countries, including Spain. Therefore, in the absence of suitable chemical control agents, biological control could provide a useful alternative. In the present work, a biological control agent, Pseudomonas fluorescens EPS62e, has been selected among 600 isolates of P. fluorescens and Pantoea agglomerans obtained from flowers, fruits and leaves of rosaceous plants in a survey performed through several geographic areas of Spain. This strain has been selected for its capacity to suppress immature fruit, blossom and shoot infections caused by E. amylovora, under controlled environment conditions, providing control levels similar to chemical control with copper or antibiotic compounds. The strain has also shown the capacity to colonize and survive well in flowers and wounds on immature fruit under controlled environment conditions but also in flowers under natural conditions. Pre-emptive exclusion of the pathogen E. amylovora by surface colonization and nutrients depletion, and cell-to-cell interaction appear to be the main mechanisms of biocontrol. These characteristics constitute interesting traits for an effective development as a fire blight biological control agent under commercial conditions.

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Zemaryalai, Khatera. « Investigations into the roles of potassium channels in hair growth : studies confirming the presence of several ATP-sensitive potassium (K+ATP) channels in hair follicles and exploring their mechanism of action using molecular biological, cell culture, organ culture and proteomic approaches ». Thesis, University of Bradford, 2010. http://hdl.handle.net/10454/4461.

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Hair disorders cause significant distress. The main, but limited, treatment for hair loss is minoxidil, an ATP-sensitive potassium (KATP) channel opener whose mechanism of stimulation is unclear. The regulatory component of KATP channels has three forms: SUR1, SUR2A and SUR2B which all respond to different molecules. Minoxidil only opens SUR2B channels, though SUR1 and SUR2B are present in human hair follicles. To expand our understanding, the red deer hair follicle model was used initially. Deer follicles expressed the same KATP channel genes as human follicles when growing (anagen), but no channels were detected in resting follicles. This reinforces the importance of KATP channels in active hair growth and the usefulness of the deer model. To assess whether SUR1 KATP channels are actually involved in human hair growth, the effects of a selective SUR1 channel opener, NNC55-9216, on scalp follicle growth in organ culture was examined. NNC55-9216 stimulated anagen; its effect was augmented by minoxidil. This creates the potential for more effective pharmaceuticals to treat hair loss via SUR1 channels, either alone or in combination with minoxidil. The dermal papilla plays a crucial regulatory role in hair follicle activity determining the type of hair produced. Minoxidil had no effect on dermal papilla cell proliferation, but altered the profile of proteins produced when assessed by proteomics. Further research into the roles of KATP channels and greater understanding of the significance of these protein changes should enhance our knowledge of hair biology and help the development of new, improved therapies for hair pathologies.

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Martis, Prithy Caroline. « RENCA macrobeads inhibit tumor cell growth via EGFR activation and regulation of MEF2 isoform expression ». Wright State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=wright1597229612949836.

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Salvadori, Mirian Graciela da Silva Stiebbe. « Mecanismo de ação da atividade antinociceptiva e anti-inflamatória do (-) - mirtenol ». Universidade Federal da Paraíba, 2013. http://tede.biblioteca.ufpb.br:8080/handle/tede/6810.

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The essential oils of herbs have a variety of bioactive compounds, such as monoterpenes. These monoterpenes have several pharmacological activities described as analgesic, anti-inflammatory, antidepressant and anticonvulsant, among others. The (-)- myrtenol is a monoterpene alcohol monocyclic, of pleasant odor, used in the cosmetics industry. However, the absence of research on possible pharmacological activities of this monoterpene has encouraged the present research. This study investigates the effect of (-)- myrtenol, intraperitoneally in adult male Swiss mice under experimental models of pain and inflammation. Initially, the research was initiated with lethal dose 50 (LD50) of monoterpene, in order to establish safe doses for subsequent tests. To investigate the action profile of monoterpene in the central nervous system, a pharmacological screening behavior was carried out, the effect of which in animals treated with (-)- myrtenol was that of analgesia. Then methodologies were conducted to evaluate the antinociceptive activity. The (-)- myrtenol (25, 50 and 100 mg/kg, i.p) increased the latency to the onset of abdominal writhing induced by acetic acid and reduced the number of writhing when compared to the control group. In the formalin test, using the same doses, (-) myrtenol did not alter the duration of paw licking in the neurogenic phase (0-5 min), but it inhibited significantly (p <0,001) the time of paw licking along the inflammatory phase (15-30 min). In the test of nociception induced by glutamate, the three doses of monoterpene reduced time of paw licking. In the hot plate test, which is sensitive and specific to drugs that act through a central mechanism, the (-)- myrtenol did not alter the paw withdrawal latency. With these results, we propose that the antinociceptive action of (-)- myrtenol may be a peripheral and not central mechanism of action. In an attempt to elucidate the mechanism of action involved in the antinociceptive effect of (-)- myrtenol, pharmacological tools were used in the formalin test. The antinociception produced by (-)- myrtenol (100 mg/kg, i.p) was reversed by naloxone (5 mg/kg, s.c) naloxonazine (10 mg/kg, s.c), glibenclamide (10 mg/kg, s.c), L-NOARG (50mg/kg, i.p) and yohimbine (0,15 mg/kg, i.p) only in the second phase of the formalin test. In view of the outstanding action of monoterpene in the second phase of the formalin test, we have investigated its possible anti-inflammatory activity. In this evaluation, treatment with (-)- myrtenol (50 and 100 mg/kg, i.p) was effective in reducing the paw edema induced by carrageenan (500 mg / paw), prostaglandin E2 (5 nmol / paw) and bradykinin (3 nmol / paw) at all times tested. In the model of carrageenan-induced peritonitis (1%), the monoterpene decreased the influx of leukocytes and also the levels of IL-1β and TNF-α in peritoneal fluid. Therefore, this study has demonstrated that (-)- myrtenol has antinociceptive activity with the participation of opioid receptor μ1, K+ATP channels, oxidonitrergic and adrenergic α2. Furthermore, (-)- myrtenol has exhibited anti-inflammatory activity by inhibiting the formation of paw edema and reduced leukocyte influx, possibly by inhibiting the production of pro-inflammatory cytokines IL-1β and TNF-α.
Os óleos essenciais obtidos de plantas medicinais possuem uma variedade de compostos bioativos, como os monoterpenos. Estes monoterpenos possuem distintas atividades farmacológicas descritas, como analgésica, anti-inflamatória, antidepressiva e anticonvulsivante dentre outras. O (-)-mirtenol é um monoterpeno, álcool monocíclico, de odor agradável, utilizado na indústria de cosméticos. No entanto, a ausência de pesquisas sobre as possíveis atividades farmacológicas deste monoterpeno incentivou à realização deste trabalho. O presente estudo investigou o efeito do (-)- mirtenol, pela via intraperitoneal, em camundongos suíços machos adultos em modelos experimentais de dor e de inflamação. Inicialmente, foi realizada a pesquisa da dose letal 50 (DL50) do monoterpeno, no intuito de estabelecer doses seguras para os testes subsequentes. Para investigar o perfil de ação do monoterpeno no sistema nervoso central foi realizado a triagem farmacológica comportamental e o principal efeito observado nos animais tratados com (-)- mirtenol foi analgesia. Em seguida, foram realizadas metodologias para avaliar a atividade antinociceptiva. O (-)- mirtenol (25, 50 e 100 mg/kg, i.p.) aumentou a latência para o inicio das contorções abdominais induzidas por ácido acético e reduziu o número de contorções, quando comparado ao grupo controle. No teste da formalina, utilizando as mesmas doses, o (-) - mirtenol não alterou o tempo de lambida da pata na fase neurogênica (0-5 min), mas inibiu significativamente (p<0,001) o tempo de lambida da pata na fase inflamatória (15-30 min). No teste da nocicepção induzida por glutamato, as três doses do monoterpeno reduziram o tempo de lambida da pata. Já no teste da placa quente, que é sensível e específico para drogas que atuam por mecanismo central, o (-)- mirtenol não alterou a latência na retirada da pata. Com estes resultados podemos propor que a ação antinociceptiva do (-)- mirtenol pode ser por mecanismo de ação periférica e não central. Na tentativa de elucidar o mecanismo de ação envolvido no efeito antinociceptivo do (-)- mirtenol foram usadas ferramentas farmacológicas no teste da formalina. A antinocicepção produzida pelo (-)- mirtenol (100 mg/kg i.p.) foi revertida pela naloxona (5 mg/kg s.c.), naloxonazine (10 mg/kg s.c.), glibenclamida (10 mg/kg s.c.), L-NOARG (50mg/kg i.p.) e ioimbina (0,15 mg/kg i.p) somente na segunda fase do teste da formalina. Tendo em vista a destacada ação do monoterpeno na segunda fase do teste da formalina, investigamos sua possível atividade anti-inflamatória. Nessa avaliação, o tratamento com o (-)- mirtenol (50 e 100 mg/kg, i.p.) foi capaz de reduzir o edema de pata induzido por carragenina (500 μg/pata), prostaglandina E2 (5 nmol/pata) e bradicinina (3 nmol/pata) em todos os tempos testados. No modelo da peritonite induzida por carragenina (1%), o monoterpeno diminuiu o influxo de leucócitos e também os níveis das citocinas IL-1β e TNF-α no lavado peritoneal. Portanto, este trabalho demonstrou que o (-)- mirtenol possui atividade antinoceptiva com participação dos sistemas opióidérgico μ1, canais de K+ATP, óxidonitrérgico e α2-adrenérgico. Além disso, possui atividade anti-inflamatória ao inibir a formação do edema de pata e reduzir o influxo de leucócitos possivelmente pela inibição da produção das citocinas pró-inflamatórias IL-1β e TNF-α.

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Murakami, Mario Tyago. « Estratégias de inibição, mecanismos moleculares e interações intermoleculares em complexos macromoleculares / ». São José do Rio Preto : [s.n.], 2006. http://hdl.handle.net/11449/100483.

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Orientador: Raghuvir Krishnaswamy Arni
Banca: Antônio Carlos Martins Camargo
Banca: Nilson Ivo Tonin Zanchin
Banca: Beatriz Gómez Guimarães
Banca: Roberto da Silva
Abstract: This work presents some features of essential biological processes such as the haemostatic system, integrity of biological membranes and thermostability of proteins. Crystallographic, spectroscopic and in silico tools have been used to obtain information at the molecular level of macromolecular complexes, action mechanisms and inhibition pathways. Worms, snakes, ticks, leeches and spiders produce a variety of proteins, which interfere in the regulation of these systems. Different toxins isolated from these organisms were characterized providing necessary information for the development of a new anti-myonecrotic molecule and reveal a new factor Xa exosite that is important for macromolecular substrates recognition and inhibition. The first crystal structure of a member of the sphingomyelinases D family was determined by the "quick cryo-soaking" technique and the catalytic mechanism was proposed, which involves a magnesium-binding site and two catalytic histidines. An alternative activation of the protein C pathway that does not require thrombomodulin was structurally characterized and revealed the dual role of the elestrotatic surface charge around the active site and the three strategically positioned carbohydrate moieties in the approach, recognition and activation of protein C.
Doutor

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Pham, Huy Dien. « Effets de differents anti-inflammatoires sur la migration et le metabolisme oxydatif des polynucleaires neutrophiles de rat ». Paris 6, 1987. http://www.theses.fr/1987PA066194.

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Gao, Hui. « Extracting key features for analysis and recognition in computer vision ». Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1141770523.

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Ruiz, Carmona Sergio. « Virtual screening for novel mechanisms of action : applications and methodological developments ». Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/400297.

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The main motivation of this thesis has been to validate, improve and develop new methods with respect to the ones available nowadays in the area of drug discovery, in order to be able to study more challenging targets in the near future that currently are out of our reach. As the productivity of the pharmaceutical industry is decreasing year after year over the last decades, the improvement of such methods would be a step forward. As we are mostly a computational lab, this thesis has focused on different computational approaches such as docking, molecular dynamics or chemoinformatics. On the first part of the thesis (first author on the publication in PLoS Computational Biology in 2014), I worked on Docking-based Virtual Screening (VS). Particularly, in validating rDock, a little-known but very powerful program that was published and released during this thesis as open source software. In order to validate it, we performed several benchmarking experiments with DUD and ASTEX sets to compare the performance of rDock against Glide and AutoDock Vina, two commonly used docking programs. The capabilities of rDock with respect to binding mode prediction (predict how a ligand structure will be upon binding to its receptor) and virtual screening (selecting the most likely active ligands amongst thousands or millions of drug-like molecules) were compared with Glide and Vina, and we demonstrated that rDock performed as well as them. On the second project of the thesis (first author on the publication in Nature Chemistry in 2016), we wanted to develop a novel computational tool for drug discovery not only that was complementary to the existing ones, but also that improved them by adding new ways of interpreting the data. Taking advantage of the already known technique of Steered Molecular Dynamics (SMD), we proposed an approach consisting in reducing the size of the system, focusing around a key interaction point and running SMD to discriminate between active and inactive ligands. This approach, or as we call it: "Dynamic Undocking", is intended to foster drug design efforts in the lead optimization stage by improving the efficiency of the in silico assessment of protein-ligand binding affinity. After a positive retrospective assessment of the method using different systems of the DUD set, a prospective validation was required to evaluate its feasibility in a real drug discovery project. Hsp90 was selected as the test system: A fragment library was created and a subset of fragments was selected for a first stage of docking-based VS. About 300.000 ligands were docked with rDock and the top-scoring ones were subjected to Dynamic Undocking. In a collaboration with Vernalis, a pharmaceutical company in the UK, we tested tens of compounds selected with Dynamic Undocking and we were able not only to find positive and novel hits but also to improve hit-rate with respect to standard fragment screening by almost 10 fold. Finally, we had the opportunity to participate in the D3R Grand Challenge 2015 where we could apply all the methods from this thesis (first author on the publication in Journal of Computer-Aided Molecular Design in 2016). This challenge was designed as a blind public test where different groups around the world tried to predict the binding mode and the affinity of a set of ligands for their respective protein target. Our approach consisted in a combination of docking and Dynamic Undocking and our results were placed amongst the best for the two systems of the challenge. We also discussed how the level of available data and previous knowledge on each of the systems impacted on the final results.
La motivación principal de esta tesis ha sido validar, mejorar y desarrollar nuevos métodos con relación a los disponibles hoy en día en el área del desarrollo de fármacos, para en un futuro poder estudiar dianas que actualmente están fuera de nuestro alcance. Debido a que la productividad de la industria farmacéutica está disminuyendo durante los últimos años, una mejora en los métodos disponibles sería un gran paso adelante. Esta tesis se ha centrado en diferentes métodos computacionales, como el docking o la dinámica molecular. En la primera de las partes, trabajé en el cribado virtual (Virtual Screening) basado en docking. Concretamente, participé en la validación del programa de docking rDock mediante la comparación con dos programas muy usados hoy en día de su capacidad de predecir correctamente el modo de unión de un ligando con su proteína diana y de sus resultados en el cribado virtual de posibles fármacos. En la segunda parte de la tesis, participé en el desarrollo de un método computacional novedoso en el diseño de fármacos que complementase y mejorase los métodos actualmente disponibles. Éste método, bautizado en inglés como “Dynamic Undocking”, consiste en una implementación específica de dinámica molecular mediante la cual somos capaces de detectar si un ligando puede ser activo o inactivo de manera rápida y eficiente. Se validó el método de manera retrospectiva y posteriormente se aplicó en otro proyecto con el objetivo de encontrar nuevos posibles fármacos para una proteína relacionada con cáncer. Gracias a una colaboración con una empresa del Reino Unido, encontramos nuevos ligandos de manera que aumentamos la tasa de éxito con relación a un método estándar en casi 10 veces. Por último, participé en el “D3R Grand Challenge 2015”, un experimento a escala mundial donde los participantes aplicaron diferentes métodos y compararon sus resultados respecto a dos métricas distintas: la predicción del modo de unión y la capacidad de ordenar los ligandos proporcionados por la organización por su afinidad respecto a la proteína diana. En nuestro caso, aplicamos una combinación de docking y “Dynamic Undocking” con unos resultados excelentes.

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Attarzadeh, Yazdi Ghassem. « Molecular mechanism of glucocorticoid action ». Thesis, University of Edinburgh, 2006. http://hdl.handle.net/1842/26161.

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The aim of this thesis project was to investigate the mechanisms by which glucocorticoid hormones regulate the activity of BK channels in human embryonic kidney 293 (HEK293) cells as the model system for glucocorticoids-action. It was shown that glucocorticoids act via endogenously expressed type II receptors in a concentration- and time-dependent manner in these cells. Dexamethasone (100 nM) had no significant effect on Dexras1 mRNA but significantly increased serum- and glucocorticoid-induced protein kinase 1 (SGK-1) mRNA. Biochemical analysis showed that SGK-1 protein is increased by dexamethasone in a Triton X-100 insoluble fraction. Further work was directed toward analysing the possible association of SGK-1 and protein phosphatases with two BK channel α-subunit variants: ZERO-BK and STREX-BK, the latter contains the 59 amino-acid splice insert encoded by the stress hormone induced exon (STREX). HEK293 cells stably expressing the respective channel subunits were analysed. Immunoprecipitations with antisera directed against the BK α-subunits showed that protein phosphatase 2A (PP2A) but not SGK-1 is constitutively associated with the STREX as well as the ZERO variant BK channel. Furthermore, the cytoplasmic C-terminal segment of the STREX-BK channel was necessary for cell-surface expression of the channel and the association of the channel with PP2A. Dexamethasone, failed to change the apparent amount of immunoreactive PP2A co-immunoprecipitating with the channel. In conclusion: SGK-1 but not Dexras1 is a protein rapidly induced by dexamethasone in HEK293 cells. PP2A but not SGK-1 is in complex with both ZERO and STREX-BK channels, and dexamethasone does not alter this association. The cytoplasmic tail of the BK channels is essential for PP2A interaction.

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Conti, Lucio. « The molecular mechanism of TFL1 action ». Thesis, University of East Anglia, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.423575.

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During Arabidopsis development the shoot apical meristem (SAM) generates lateral primordia which display stage-specific traits. In long days, wild-type Arabidopsis generates leaves in an initial vegetative phase (V). Upon integration of environmental and endogenous signals, the SAM enters the reproductive phase. First it makes an 11 phase, which consists of 2-3 leaves (cauline) subtending secondary shoots (coflorescences). Next it enters the 12 phase and produces flowers on its flanks. The TFL 1 gene is a key component of the phase change machinery as mutations in TFL 1 affect the timing of phase switching. Also ffl1 mutants enter a novel phase whereby the SAM, after 12, is converted into a terminal flower, a phase normally absent in wild type. The molecular mechanism of how TFL 1 protein acts is unclear. In animal systems, TFL 1-like proteins have been shown to be components of signal transduction pathways. To understand the mechanism underlying TFL 1 function I aimed to identify proteins interacting with TFL 1 by introducing into Arabidopsis a functional TAP tag version of TFL 1 under the control of the 35S promoter. I set up conditions which allowed me to isolate and visualize by total protein staining TAPtag TFL 1. However, no obvious proteins appeared to co-purify with TFL 1. To understand how TFL 1 is modified, and to follow TFL 1 protein expression throughout development and in cell fractions, I developed polyclonal antibodies against TFL 1. These antibodies recognized TFL 1 in vivo and were used to characterize TFL 1 biochemically. TFL 1 detection by immunoblots in conjunction with mass-spectrometry analysis showed that TFL 1 was not subjected to obvious modifications unlike animal homologues. Moreover, from cellular fractionation experiments TFL 1 was located in the cytosol. To reveal essential downstream functions required for TFL 1 signaling, I characterized a suppressor mutant, called sof1, of plants ectopically expressing TFL 1. I mapped sof1 within a confined region on the bottom of chromosome 3. Physiological analysis of sof1 led to a model of SOT1 action in controlling phase change. TFL 1 mRNA is found in a unique expression domain which comprises a group of cells in the centre of the SAM and yet TFL 1 affects the identity of lateral primordia. By using affinity purified anti-TFL 1 antibodies I showed that TFL 1 protein moves and is distributed throughout the SAM. This might account for the effect of TFL 1 on controlling overall shoot identity and raises important questions on the role of the TFL 1 protein outside its mRNA expression domain.

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Beastall, J. C. « The mechanism of action of azone ». Thesis, University of Nottingham, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380112.

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Williams, Daffydd Griffin. « Mechanism of action of penetration enhancers ». Thesis, Cardiff University, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320625.

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Taylor, Catherine. « A mechanism of action of strigolactone ». Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709140.

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Taylor, M. R. G. « Mechanism of action of Rad51 paralogs ». Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1458671/.

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Homologous recombination (HR) is an essential DNA break repair mechanism that remains incompletely understood. HR is a complex multistep process initiated by the loading of RAD-51 recombinase as filaments onto single stranded DNA (ssDNA). This structure directly invades an intact homologous duplex, which serves as a template for repair DNA synthesis. Numerous positive regulators of HR have been described, including the Rad51 paralogs, but the mechanism of action of Rad51 paralogs in promoting HR is unknown. In this study, I have characterized the mechanism of action of a novel Rad51 paralog complex, RFS-1/RIP-1, from C. elegans. RFS-1 is a Rad51 paralog required for RAD-51 focus formation at stalled replication forks, indicating an early positive regulatory role in HR. I demonstrate that RFS-1 interacts with a nematode-specific orphan protein, RIP-1. I identify a cryptic Walker B ATPase-like motif within RIP-1, which is functionally important in establishing the RFS-1/RIP-1 interaction interface. rip-1 and rfs-1 mutant animals phenocopy for essentially all phenotypes analysed. Together these data suggest RFS-1/RIP-1 functions as a constitutive complex. I show recombinant RFS-1/RIP-1 can be purified and specifically binds ssDNA but lacks measurable ATPase activity. RFS-1/RIP-1 also strongly stimulates strand invasion activity by RAD-51, consistent with a pro-recombinogenic function in vivo. I define for the first time the mechanism of action underlying the intrinsic ability of Rad51 paralogs to stimulate HR. Using a combination of biochemical and biophysical approaches, notably electrophoretic mobility shift assays, stopped-flow reaction kinetics and nuclease protection assays, I show RFS-1/RIP-1 dramatically alters the properties of RAD-51-ssDNA filaments such that RAD-51 is more stably associated with ssDNA yet the ssDNA is more sensitive to nuclease degradation. RFS-1/RIP-1 exerts these effects primarily downstream of filament formation, ruling out a major role in RAD-51 loading. I propose RFS-1/RIP-1 remodels RAD-51-ssDNA filaments to a conformation poised for pairing with the template duplex and strand invasion.

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Phisit, Prapunwattana Yongyuth Yuthavong. « Mechanism of antimalarial action of tetracycline / ». abstract, 1986. http://mulinet3.li.mahidol.ac.th/thesis/2529/29E-Phisit-P.pdf.

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Jeganathan, Karthik Babu. « The mitotic checkpoint : mechanism and biological relevance ». [S.l. : Groningen : s.n. ; University Library Groningen] [Host], 2008. http://irs.ub.rug.nl/ppn/.

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Clark, Alice Rosemary. « The filamin A actin binding domain structure and function : implications for a gain-of-function mechanism for the otopalatodigital syndrome : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand [Ph. D] EMBARGOED ». Massey University, 2010. http://hdl.handle.net/10179/1185.

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Embargoed until 1 January 2011
The filamin family act as scaffolding proteins associating with actin filmanents, acting through a highly conserved actin binding domain (ABD). The ABD of the filamins is homologous to that found in other F-actin binding proteins such as dystrophin. Mutations in the filamin A gene cause a wide range of disease symptoms in humans reflecting the diversity of the roles that filamin A has in cell structure and signalling pathways. The diseases fall into two separate phenotypic groups. Periventricular nodular heterotopia (PVNH) generally results from the complete loss of filamin A protein, and affects the central nervous system. The clinically separate otopalatodigital disorders (OPD) spectrum disorders are skeletal disorders and were hypothesised to be gain of function phenotype diseases. At the beginning of this work, there was very little structural data available for the human filamins, and none for the crucial highly conserved actin binding domain. This lack of structural data limited the interpretation of the biochemical and genetic data and constrained our understanding of the disease associated mutations that cluster in this domain. These studies aimed to provide insights into the structure and mechanism of actin binding domains, and thus provide a better understanding of the diseases caused when this domain is mutated. A secondary structural analysis and crystal structures of the wildtype and OPD2 associated mutant ABDs were obtained. The overall fold of the three proteins was equivalent as determined by circular dichroism spectroscopy and x-ray crystallography. The ABD from filamin A E254K showed 3.7 fold increased F-actin affinity, accompanied by a reduced thermostability (of 5.6 °C). Western blotting of OPD2, frontometaphyseal dysplasia (FMD) and PVNH patient fibroblast lysates showed similar levels of filamin A compared to the control cells. In addition the OPD and PVNH patient fibroblasts were able to adhere to fibronectin and migrate with an equivalent rate to control cells. Together these results have allowed correlations to be developed between structure, protein stability, actin affinity, cellular phenotype and the overall clinical phenotype. Showing that, at least in one example, OPD2 may be due to an increased actin affinity providing further evidence for a gain of function mechanism of OPD2.

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Thomson, Robert Brent. « Cellular mechanisms of acid/base transport in an insect excretory epithelium ». Thesis, University of British Columbia, 1990. http://hdl.handle.net/2429/31306.

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The cellular mechanisms responsible for rectal acidification in the desert locust, Schistocerca gregaria, were investigated in isolated recta mounted as flat sheets in modified Ussing chambers. In the absence of exogenous CO₂, HCO₃⁻, and phosphate, the isolated rectum (under both open- and short-circuit current conditions) was capable of rates of net acid secretion (J[subscript]H+) similar to those observed in vivo, demonstrating the viability of the preparation and suggesting that rectal acidification was due to proton secretion rather than selective movements of HCO₃⁻ or phosphate. The possibility that trace levels of metabolic CO₂ might be generating sufficient HCO₃⁻ to account for the observed rates of rectal acidification (via HCO₃⁻ reabsorption) was assessed by adding exogenous CO₂/HCO₃⁻ to the contraluminal bath. The small increases in J[subscript]H+ observed after addition of 2% or 5% CO₂ were shown to be due to simple hydration of CO₂ which had diffused into the lumen (from the contraluminal bath), rather than changes in rates of HCO₃⁻ reabsorption. Since measurable quantities of luminal HCO₃⁻ did not directly affect the apical acid/base transport mechanism per se, it was concluded that metabolic CO₂ could not generate sufficient HCO₃⁻ in the lumen to account for the rates of rectal acidification observed under nominally CO₂/HCO₃⁻-free conditions and that J[subscript]H+ must be due to a proton secretory rather than bicarbonate reabsorptive mechanism. Microelectrode measurements of intracellular pH (pHi) and apical and basolateral membrane potentials (Va and Vb respectively) indicated that luminal pH was not in equilibrium with either contraluminal pH or pHi and that the mechanism responsible for active luminal acid secretion resided on the apical membrane. Preliminary measurements of bath total ammonia (ie. NH₃ + NH₄+) levels in the previous experiments suggested that the rectum was actively secreting ammonia at significant rates across the apical membrane into the lumen. If the ammonia crossed the apical membrane as NH₃ rather than NH₄+, rates of luminal ammonia secretion (J[subscript]Amm) would have to be added to J[subscript]H+ to obtain corrected values of luminal proton secretion. In the absence of exogenously added ammonia and CO₂, ammonia was preferentially secreted into the lumen under both open- and short-circuit current conditions. J[subscript]Amm was dependent on the presence of luminal amino acids and was relatively unaffected by K[superscript]+ removal or changes in luminal pH from 7.00 to 5.00. Bilateral Na+ substitution or luminal addition of ImM amiloride reduced J[subscript]Amm by 63% and 65% respectively. The data consistently demonstrate that the rectum secretes significant quantities of endogenously produced ammonia preferentially into the lumen as NH₄+ rather than NH₃ via an apical Na[superscript]+/NH₄[superscript]+ exchange mechanism. Clearly, rates of net acid secretion estimated by titratable acidity do not have to include a correction for luminal ammonia secretion. Although J[subscript]H+ was completely unaffected by changes in contraluminal pH, it could be progressively reduced (and eventually abolished) by imposition of either transepithelial pH gradients (lumen acid) or transepithelial electrical gradients (lumen positive). Under short-circuit current conditions, the bulk of J[subscript]H+ was not dependent on Na[superscript]+, K[superscript]+, CI⁻, Mg₂+, or Ca+ and was due to a primary electrogenic proton translocating mechanism located on the apical membrane. A small component (10-16%) of J[subscript]H+ measured under these conditions could be attributed to an apical amiloride-inhibitable Na[superscript]+/H[superscript]+ exchange mechanism. Inhibition of JH+ by anoxia or reduction of luminal pH unmasked a significant proton diffusional pathway on the apical membrane in parallel with the active proton pump. The fact that J[subscript]H+ was significantly inhibited (42%-66%) by contraluminal addition of ImM cAMP and relatively unaffected by changes in contraluminal pCO₂ or pH suggests that net acid secretion in the locust rectum in vivo is modulated by circulating hormonal factors rather than haemolymph pH or pCO₂ per se.
Science, Faculty of
Zoology, Department of
Graduate

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Díaz, i. Cirac Anna. « Mechanism of action of cyclic antimicrobial peptides ». Doctoral thesis, Universitat de Girona, 2011. http://hdl.handle.net/10803/38252.

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This PhD thesis is the result of the combination of experimental and computational techniques with the aim of understanding the mechanism of action of de novo cyclic decapeptides with high antimicrobial activity. By experimental techniques the influence of the replacement of the phenylalanine for tryptophan residue in their antimicrobial activity was tested and the stability in human serum was also analyzed, in order to evaluate their potential therapeutic application as antitumor agents. On the other hand, the interaction amongst the peptide BPC194 c(KKLKKFKKLQ), the best candidate from the whole library of cyclic peptides, and a model anionic membrane was simulated. The results showed a structure-function relationship derived from the stable conformation of the peptides involved in the membrane permeabilization. As a result, a rational design was performed being BPC490 the peptide with best antimicrobial activity compared with the best active peptide from the original library.
Aquesta tesi doctoral resulta de la combinació d’estudis mitjançant tècniques experimentals i computacionals amb l’objectiu d’entendre el mecanisme d’acció de "de novo" decapèptids cíclics amb elevada activitat antimicrobiana. Experimentalment, es va avaluar la influència de la substitució dels residus de fenilalanina per triptòfan en la seva activitat antimicrobiana i també la seva estabilitat sèrum humà, per tal de valorar la seva possible aplicació terapèutica envers el càncer. Per altra banda, es va simular la interacció del pèptid BPC194 c(KKLKKFKKLQ), millor candidat de la biblioteca de pèptids cíclics, amb models aniònics de bicapa lipídica. Els resultats van posar en manifest una relació estructura-funció derivada de la conformació estable dels pèptids que participen directament en la permeabilització de la membrana. Es va procedir doncs al disseny racional de nous pèptids cíclics sent el pèptid BPC490 el que va presentar una millor activitat bacteriana en comparació amb el pèptid més actiu de la llibreria original.

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Reißmann, Stefanie. « Mechanism of Action of Group II Chaperonins : ». Diss., lmu, 2007. http://nbn-resolving.de/urn:nbn:de:bvb:19-73195.

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Hirschkorn, Martin C. « Dynamic Model of a Piano Action Mechanism ». Thesis, University of Waterloo, 2004. http://hdl.handle.net/10012/877.

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While some attempts have been made to model the behaviour of the grand piano action (the mechanism that translates a key press into a hammer striking a string), most researchers have reduced the system to a simple model with little relation to the components of a real action. While such models are useful for certain applications, they are not appropriate as design tools for piano makers, since the model parameters have little physical meaning and must be calibrated from the behaviour of a real action. A new model for a piano action is proposed in this thesis. The model treats each of the five main action components (key, whippen, jack, repetition lever, and hammer) as a rigid body. The action model also incorporates a contact model to determine the normal and friction forces at 13 locations between each of the contacting bodies. All parameters in the model are directly measured from the physical properties of individual action components, allowing the model to be used as a prototyping tool for actions that have not yet been built. To test whether the model can accurately predict the behaviour of a piano action, an experimental apparatus was built. Based around a keyboard from a Boston grand piano, the apparatus uses an electric motor to actuate the key, a load cell to measure applied force, and optical encoders and a high speed video camera to measure the positions of the bodies. The apparatus was found to produce highly repeatable, reliable measurements of the action. The behaviour of the action model was compared to the measurements from the experimental apparatus for several types of key blows from a pianist. A qualitative comparison showed that the model could very accurately reproduce the behaviour of a real action for high force blows. When the forces were lower, the behaviour of the action model was still reasonable, but some discrepancy from the experimental results could be seen. In order to reduce the discrepancy, it was recommended that certain improvements could be made to the action model. Rigid bodies, most importantly the key and hammer, should be replaced with flexible bodies. The normal contact model should be modified to account for the speed-independent behaviour of felt compression. Felt bushings that are modelled as perfect revolute joints should instead be modelled as flexible contact surfaces.

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Plested, Charles Paul. « Mechanism of action of seronegative myasthenia gravis ». Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301392.

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Keane, Richard. « HPMA copolymer-aminoellipticine conjugates : mechanism of action ». Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269617.

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Beecroft, Marikka Shannon. « Antimicrobial chelators and their mechanism of action ». Thesis, Durham University, 2019. http://etheses.dur.ac.uk/12933/.

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Limiting the availability of metals in an environment is known to restrict bacterial growth and proliferation. For example, humans sequester metals to help prevent infection by pathogens, a system termed nutritional immunity. Chelators are small molecules that bind tightly to metals and thus have antibacterial properties that mimic these innate immune processes. This activity of chelators has not been studied extensively, although experiments with EDTA suggest that it disrupts bacterial membrane permeability by stripping lipopolysaccharide from the bacterial outer surface, possibly due to the stabilising Mg2+ and Ca2+. The work described here examines in detail the antibacterial effect of 11 chelators on Escherichia coli and how this relates to cellular starvation. Four distinct effects on cellular metal content were found with these chelators i) no change, ii) reduction to manganese, iii) reduction in zinc, and iv) reduction in iron combined with an increase in manganese. There was limited correlation between chelant metal affinities in solution with effects seen in cells. The chelants also exhibited variation in antibacterial efficacy, which was enhanced when used in combination, most yielding synergistic or additive effects. These chelants therefore offer significant potential as tools to probe metal homeostasis systems and as antibacterials. EDTA, DTPMP and Octopirox were studied further by screening their effects on growth using a selection of E. coli mutants. DTPMP and Octopirox have similar effects on cellular metal content, depriving cells of iron and inducing uptake of manganese; mutant data suggests that DTPMP primarily affects Fe3+ uptake, while Octopirox Fe2+. All three chelators also seem t have effects on oxidative damage and tolerance, especially EDTA which deprives cells of manganese. The E. coli transcriptional response to EDTA was also investigated by RNA-SEQ. EDTA has wide-ranging effects on cellular metabolism, upregulating genes involved in carbon utilisation, energy production, translation and transcriptional regulators, including some iron-sulphur cluster proteins. Overall, the results offer the first detailed insight into the antibacterial effect of a structurally diverse group of chelants and the first step in understanding the relationship between metal affinity and their antibacterial mechanisms of action.

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Chant, Eleanor Laura. « The mechanism of action of ColE1 Rcd ». Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613713.

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Jackson, Natalie Diane. « The mechanism of action of peroxygen biocides ». Thesis, University of York, 1999. http://etheses.whiterose.ac.uk/9825/.

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Bagnobianchi, A. « The molecular mechanism of action of Bendamustine ». Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1461456/.

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Bendamustine has demonstrated clinical efficacy in the treatment of haematological malignancies and distinguish itself from other alkylating agents. The mechanistic and clinical differences associated with Bendamustine may be related to its structural features including a benzimidazole ring, although the mechanism of action is poorly understood. Understanding the molecular mechanism of Bendamustine could explain the therapeutic efficacy and identify potential biomarkers for response. The Bendamustine-DNA interaction in naked DNA, cytotoxicity, and ICL formation and repair (unhooking) in naked DNA or in cell lines and patient multiple myeloma cells by the single cell gel electrophoresis (comet) assay, were analyzed. DNA damage response (DDR) and potential mechanisms of acquired resistance to Bendamustine were also evaluated. Bendamustine alkylated DNA at guanine-N7 positions, produced ICLs in naked DNA and in cells, and demonstrated a cytotoxic effect comparable to conventional ICL drugs (Cisplatin, Melphalan). However, ICLs were not efficiently repaired (unhooked) in A549 cells, which could repair Cisplatin or Melphalan ICLs. In plasma cells from both Non-Melphalan Treated or Melphalan Treated patients, no evidence of efficient repair of Bendamustine ICLs was observed. Bendamustine DDR compared to Cisplatin or Melphalan gave a more selective pattern of expression of genes involved in DNA damage signaling pathways, cells defective in ERCC1, XPF, and homologous recombination repair showed less sensitivity to Bendamustine, there were differences in ɣH2AX and RAD51 foci formation and cell cycle distributions. In derived acquired resistant cell lines, resistance was associated with a reduced level of ICL at an equimolar drug dose compared to the parental lines. The molecular mechanism of Bendamustine is similar to conventional alkylating agents: DNA alkylation and ICL formation. However, ICL repair inefficiency and altered DDR are the differences between Bendamustine and conventional alkylating agents due to its benzimidazole ring that influences, showed by G1/S arrest of cell cycle population, its mechanism.

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Thèses sur le sujet « Biological Mechanism of Action »

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