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1
Parekh, Punam K. "The Photooxidation of Domoic Acid." FIU Digital Commons, 2012. http://digitalcommons.fiu.edu/etd/770.
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Domoic acid (DA) is a naturally occurring cyanotoxin, which upon ingestion, is responsible for amnesic shellfish poisoning (ASP) in both humans and animals. Produced by the marine diatom, Pseudonitzschia, DA is accumulated by a number of marine organisms including shellfish, clams and mussels which upon consumption can lead to headaches, nausea and seizures. Possessing a variety of functional groups the structure of DA contains three carboxyl groups, a pyrrole ring and a potent conjugated diene region allowing for binding to glutamate receptors in the dorsal hippocampus of the brain causing the described detrimental effects. Although limitations have been placed regarding the amount of DA that may be contained in seafood no limitations have been placed on the amount present in drinking water. Natural degradation of the toxin may occur through reactive oxygen species such as the hydroxyl radical and singlet oxygen at the conjugated diene region. In this work the photooxidation of DA via singlet oxygen has been studied using sorbic acid as a model compound. The three major reaction pathways observed during the photooxdiation process for both acids include 2 + 4 cycloaddition to produce endoperoxides , 2 + 2 reaction to afford aldehydes and ketones or an ene reaction to generate hydroperoxides. Under similar reaction conditions for SA and DA, the endoperoxide has been seen to be the major product for photoxidation of SA while the hydroperoxide has been seen to be the dominant product during photooxidation of DA.
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Fleary-Roberts, Nadia. "Towards the total synthesis of domoic acid and the isodomoic acids." Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/towards-the-total-synthesis-of-domoic-acid-and-the-isodomoic-acids(a32fd085-8e09-47b5-b533-ef259b1ae8a2).html.
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Sedehizadeh, Simon. "Towards the total synthesis of domoic acid and the isodomoic acids." Thesis, University of Manchester, 2010. https://www.research.manchester.ac.uk/portal/en/theses/towards-the-total-synthesis-of-domoic-acid-and-the-isodomoic-acids(6a1a5ea8-f5a5-4185-8222-5d3486165ac9).html.
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Silvagni, Paul Anthony. "Comparative pathology and diagnosis of domoic acid toxicity /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2003. http://uclibs.org/PID/11984.
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Lail, Erin M. "Biogeochemical cycling of domoic acid and its isomers in the ocean /." Electronic version (PDF), 2006. http://dl.uncw.edu/etd/2006/laile/erinlail.pdf.
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Knierim, Tika L. "The photodegradation of domoic acid and the effects of metal chelation /." Electronic version (PDF), 2005. http://dl.uncw.edu/etd/2005/knierimt/tikaknierim.html.
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Wittmaack, Christiana. "Behavioral Criteria for the Diagnosis of Domoic Acid Toxicosis in Zalophus californianus." NSUWorks, 2014. http://nsuworks.nova.edu/occ_stuetd/143.
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Introduction
California sea lion (Zalophus californianus) health is severely compromised by domoic acid toxicosis, which occurs in high levels during harmful algal blooms of Pseudonitzschia australis along the coast of California. Current diagnostic protocols are often inconclusive due to a 2-48 hour window of detectability within the urinary, circulatory, and gastric systems (Cook, et al. 2011 and Monte, Pers Comm, 2012). Past studies suggest that Z. californianus, with domoic acid toxicosis, commonly display abnormal behaviors (Goldstein, et al. 2008). However, many of these abnormal behaviors are also associated with other diagnoses and are therefore unreliable as diagnostic indicators. This study fills in a knowledge gap relating to abnormal behavior types and their correlation to domoic acid toxicosis and helps solve the problem of current, inconclusive, diagnostic protocols. In this study, my objectives were to identify abnormal behaviors correlated to domoic acid toxicosis, create a diagnostic ethogram, determine the applicability of the method in the field, and determine the applicability of triage based on the relationship between abnormal behaviors and domoic acid levels.
Methods
I conducted focal animal continuous scans (continuous observation of a single animal at a time, for a set period) with continuous data entry, on animals admitted to the Marine Mammal Center (main study location during 2011-2013) and the Marine Mammal Care Center (comparison location, 2013). I conducted my observations from behind a blind to prevent both human habituation and behavioral influence of the observer. Observations lasted between 10-15 minutes (10 minutes per pen in 2011, 15 minutes per animal in 2012-2013). Subjects were selected based on an admit date no later than 7 days from the observation date.
I conducted focal animal continuous scans at Pier 39, a haul out location, in the San Francisco Bay. Animals included in the study had identifying marks or were isolated from other animals (making them easy to identify). I observed animals once per observation day with a total observation period not exceeding 15 minutes per animal.
I logged domoic acid levels in feces, urine, and serum (collected by veterinary staff and analyzed with liquid chromatography and bioassays for the presence of domoic acid). I then compared these results to the types and severity of abnormal behaviors displayed by the domoic acid toxicosis sample.
Results
Results from data collected at the Marine Mammal Center suggest that head weaving (Wilcoxon, p
Results from the Pier 39 study suggest that behavioral criteria may be applicable for ruling out domoic acid toxicosis in groups of animals. However, I did not test the method during times of harmful algal blooms. Therefore, the applicability of the method for use as a diagnostic tool in the field is unknown and further research is required.
Results for the triage study were inconclusive. The number of animals that tested positive for domoic acid was small and not suitable for statistical analysis. I suggest further research into triage abilities.
Conclusion
Based on the results of these studies, I can conclude that behavioral analysis offers a reliable diagnostic tool for rescued Z. californianus. Practitioners can use behavioral diagnostic criteria with confidence for the diagnosis of domoic acid toxicosis in Z. californianus.
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Vranyac-Tramoundanas, Alexandra, and n/a. "Domoic acid-induced cardiac damage : an in vitro and in vivo investigation." University of Otago. Department of Pharmacology & Toxicology, 2007. http://adt.otago.ac.nz./public/adt-NZDU20071012.143651.
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Cardiovascular pathology is seen in both animals and humans after domoic acid intoxication. Whether this damage is direct (i.e., cardiotoxic) or indirect (i.e., CNS/autonomic seizures) is not known. We have previously shown that acute in vitro domoic acid (0.05-0.25[mu]M; 10 min) treatment of isolated cardiac mitochondria compromises mitochondrial FADH and NAD⁺-linked respiratory control and mitochondrial energetics. Domoic acid was shown to traverse and bind the cellular membrane of H9c2 cardiac myoblasts. However it did not compromise cellular viability as assessed using cell quantification or lactate dehydrogenase leakage assays. Exposure of intact H9c2 cells to domoic acid only resulted in complex II-III activity impairment and assessment of reactive oxygen species (superoxide and hydrogen peroxide) production in both isolated cardiac mitochondria and H9c2 cardiomyocytes failed to show any significant differences following exposure to domoic acid. Acute ex vivo domoic acid treatment of an isolated myocardium in Langendorff perfusion mode failed to result in cardiac haemodynamic dysfunction, however there appeared to be small but significant decrease in mitochondrial oxygen utilization. The absence of any substantial damage to intact cardiomyocytes and isolated myocardium suggested that domoic acid does not have a direct toxicological effect on cardiac energetics. We therefore investigated the possibility that cardiovascular pathology is an indirect consequence of autonomic seizure activity. Domoic acid was administered intraperitoneally or intrahippocampally and the development of cardiac pathologies was assessed and compared. Sprague-Dawley rats receiving either i.p. or i.h. domoic acid were assessed behaviourally and shown to reach similar levels in their cumulative seizure scores.
Assessment of the cardiac haemodynamics (LVDP, dP/dt, heart rate and coronary flow) revealed a significant time-dependent decrease in function at 1, 3, 7 & 14-days post-i.p. and 7 & 14-days post-i.h. domoic acid administration. Measurement of ventricular mitochondrial oxygen utilization revealed a similar time-dependent decrease in respiratory control, which appeared to be associated with increased proton leakage, shown by an increase in state-4 respiration rate (P<0.01). Assessment of the mitochondrial electron transport chain (complexes I-V) and the mitochondrial marker of integrity, citrate synthase, showed marked time-dependent impairment in both models of domoic acid -induced seizures. Oxidative stress did play a small role in the myocardial damage as indicated by the small decrease in aconitase activity (P<0.05). Plasma IL-1α, IL-1β and TNF-α levels were significantly increased from 3-days post seizures. Haematoxylin & Eosin staining of ventricular sections revealed the formation of contraction bands, inflammation and oedema, confirming a structural pathology. Cardiac damage did not differ between i.p. and i.h. animals, suggesting cardiac damage following domoic acid results from CNS autonomic seizures and resultant sympathetic storm.
This thesis has demonstrated, for the first time, that the cardiac pathology seen following domoic acid exposure is most likely to be a result of CNS activation and resultant seizure episodes, and is not a consequence of the direct interaction between domoic acid and the myocardium. We have also demonstrated for the first time, that seizure episodes result in chronic cardiac dysfunction and a structural pathology which is similar, but not identical to that seen following isoprotenerol administration in vivo.
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Hesp, Blair, and n/a. "In vitro and In vivo investigations of tolerance induction and the role of G-protein coupled kainate receptors." University of Otago. Department of Pharmacology & Toxicology, 2005. http://adt.otago.ac.nz./public/adt-NZDU20070503.150053.
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The excitotoxin domoic acid (DOM) acts at both kainic acid (KA)- and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-sensitive glutamate receptors. Clinical reports suggest that elderly people are hypersensitive to the neurological effects of DOM intoxication. Young, but not aged hippocampal slices which have been preconditioned with low concentrations of DOM or KA exhibit an acute �tolerance� to subsequent high doses of DOM or KA; application of the selective AMPA agonist fluorowillardiine (FW) fails to induce tolerance to excitotoxins. The aim of this study was to further investigate the molecular mechanism of tolerance induction in vitro, and to examine the ability of compounds to cross-condition against excitotoxic insult. In addition, in vivo techniques were used to explore the age-related susceptibility to the neurological effects of DOM and acute in vivo tolerance induction. Here we show that low doses of �classical� ionotropic kainate receptor agonists and AMPA/kainate receptor antagonists act as net inverse agonists at G-protein coupled receptors, reducing constitutive GTPase activity by up to 73% in the young hippocampus. Further evidence that inverse agonist activity at G-protein coupled receptors is responsible for acute in vitro tolerance induction by kainate receptor agonists and antagonists was also identified because preconditioning with the AMPA receptor antagonist GYKI-52466 significantly inhibited KA-induced population spike suppression in in vitro hippocampal brain slices from both Sprague-Dawley and Wistar rats. The broad-spectrum protein kinase inhibitor H-7 partially blocked tolerance induction when preconditioning occurs in the presence of suggesting that protein kinases are one of the downstream effectors of this phenomenon. Tolerance-inducing compounds are also capable of cross-conditioning against the effects of other excitotoxins; with 250 nM FW suppressed population spike area by only 62.8 � 10.0% at 30 minutes following a 500 nM KA preconditioning dose, compared to almost complete spike suppression within twenty minutes in naive hippocampal brain slices. In vivo experiments indicated that despite aged animals exhibiting significantly higher cumulative behavioural scores in response to i.p. DOM (1 mg kg⁻�; young = 102 � 9, aged = 179 � 19; P < 0.01) in response to DOM after two hours), and that this age-related supersensitivity is due to impaired renal clearance (young serum DOM = 41.5 � 30.3 ng ml⁻�, aged = 813.3 � 804.4 ng ml⁻� following administration of 1 mg kg⁻� DOM after 2.5 hours earlier). Tolerance to high doses of DOM was induced within a matter of minutes following i.p. preconditioning by low dose DOM in vivo. This was evidenced by severe seizure manifestations being almost absent in both young and aged animals, despite occurring frequently in naive animals. Therefore, this study concludes that tolerance is induced by kainate receptor ligands in vitro and in vivo within a matter of minutes, and is the result of a reduction in the turnover of G-protein coupled receptors and protein kinase activation. In addition, the increased sensitivity of aged rats to in vivo DOM is a result of elevated serum DOM concentrations most likely resulting from impaired renal clearance.
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Fehling, Johanna. "Diversity, ecology and domoic acid production of Pseudo-nitzschia spp. in Scottish waters." Thesis, Open University, 2004. https://pure.uhi.ac.uk/portal/en/studentthesis/diversity-ecology-and-domoic-acid-production-of-pseudonitzschia-spp-in-scottish-waters(4b83d442-d9f6-4b9b-bc2f-666623b42d0b).html.
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Some diatoms of the genus Pseudo-nitzschia produce the toxin domoic acid (DA). Accumulation of DA in shellfish has led to harvesting closures in western Scottish waters since 1999. This thesis investigated the diversity, ecology and distribution of toxic and non-toxic Pseudo-nitzschia species in western Scottish waters and physiological aspects of growth and toxin production dynamics of P. seriata. The temporal and spatial distribution of phytoplankton was analysed in two separate field studies. 1) Temporal changes were followed by sampling a site in coastal Scottish waters weekly to fortnightly over a period of three years. 2) The spatial distribution of the phytoplankton community was investigated by sampling a transect across-the shelf. Within both studies, physical, biological and chemical parameters were measured and correlated to temporal and spatial distribution patterns in the phytoplankton community, indicating seasonality, and differences in the distribution of toxic and non-toxic Pseudo-nitzschia species between coastal and offshore waters. From those samplings 59 clonal cultures of Pseudo-nitzschia, comprising 7 species (2 of them toxic), were established. Strains were identified via classic morphological and genetic techniques. Phylogenetic relationships were established between Scottish Pseudo-nitzschia strains. P. seriata was identified for the first time in Scottish waters as a DA producer. Laboratory experiments with cultured strains showed a) enhanced toxin production by P. seriata under silicate (Si) and phosphate (P) limitation, with higher DA production under Si than under P limitation b) similar cell yields of P. seriata, when grown in nitrate or ammonia based media c) a preference for spring light conditions (short day length) in a non-toxic P. delicatissima strain and summer light conditions (long day length) for a toxic P. seriata strain, expressed by enhanced biomass yield under the respective light condition. It was also shown that the presence of bacteria enhanced the growth of single P. seriata cells.
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McCollough, Bianca. "Toxic algae and other marine biota: detection, mitigation, prevention and effects on the food industry." Kansas State University, 2016. http://hdl.handle.net/2097/32490.
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Master of Science<br>Food Science Institute<br>Curtis Kastner<br>Harmful Algal Blooms (HABs) including Cyanobacteria and other toxic marine biota are responsible for similar harmful effects on human health, food safety, ecosystem maintenance, economic losses and liability issues for aquaculture farms as well as the food industry. Detection, monitoring and mitigation are all key factors in decreasing the deleterious effects of these toxic algal blooms. Harmful algal blooms can manifest toxic effects on a number of facets of animal physiology, elicit noxious taste and odor events and cause mass fish as well as animal kills. Such blooms can adversely impact the perception of the efficacy and safety of the food industry, water utilities, the quality of aquaculture and land farming products, as well as cause ripple effects experienced by coastal communities. HABs can adversely impact coastal areas and other areas reliant on local aquatic ecosystems through the loss of revenues experienced by local restaurants, food manufacturers as well as seafood harvesting/processing plants; loss of tourism revenue, decreased property values and a fundamental shift in the lives of those that are reliant upon those industries for their quality of life. This paper discusses Cyanobacteria, macroalgae, HABs, Cyanobacteria toxins, mitigation of HAB populations and their products as well as the ramifications this burgeoning threat to aquatic/ landlocked communities including challenges these toxic algae pose to the field of food science and the economy.
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Armstrong, Howard Meredith Dana Armstrong Howard Meredith Dana. "Harmful algal blooms on the U.S. west coast : new insights into domoic acid production and identification of yessotoxin, a new marine toxin detected in California coastal waters /." Digital Dissertations Database. Restricted to UC campuses, 2007. http://uclibs.org/PID/11984.
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GIULIETTI, SONIA. "Unveiling the hidden biodiversity of potentially toxic diatoms in the NW Adriatic Sea through integrated taxonomy approach." Doctoral thesis, Università Politecnica delle Marche, 2021. http://hdl.handle.net/11566/289742.
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Pseudo-nitzschia è tra i generi di diatomee più noti e studiati, poiché tra tutte le 56 specie accettate, 26 sono in grado di produrre una neurotossina (acido domoico) responsabile di una biointossicazione (Amnesic Shellfish Poisoning) nell’uomo. La diversità e l’andamento stagionale delle specie di Pseudo-nitzschia sono stati analizzati per la prima volta nella stazione costiera del transetto Senigallia-Susak (LTER) nel NW Adriatico dal 1988 al 2020, attraverso un approccio tassonomico integrato basato su analisi ultrastrutturale e molecolare. Almeno 6 morfotipi sono stati identificati tramite osservazione all'ottico: P. cf. delicatissima, P. cf. pseudodelicatissima, P. multistriata, P. cf. fraudulenta, P. pungens and P. cf. galaxiae. L’analisi IndVal ha rivelato che P. cf. delicatissima e P. cf. pseudodelicatissima sono taxa indicatori in primavera ed estate rispettivamente. P. cf. fraudulenta e P. pungens erano taxa indicatori della stagione invernale ed erano correlate negativamente con la temperatura e positivamente con le concentrazioni del DIN. P. multistriata proliferava in autunno ed era l’unica specie positivamente correlata con le concentrazioni di silicati. Negli anni 2018-2020 sono stati isolati, con frequenza mensile, 138 ceppi monoclonali di Pseudo-nitzschia. Combinando le analisi al microscopio elettronico a trasmissione con i risultati dell'albero filogenetico ottenuto con le sequenze LSU e ITS rDNA, sono state confermate le identità di sei specie di Pseudo-nitzschia che formano 6 distinti cluster: i.e. P. delicatissima, P. cf. arenysensis, P. calliantha, P. mannii, P. fraudulenta, P. pungens. Le analisi sul contenuto delle tossine non hanno rilevato la presenza di acido domoico in nessun ceppo. Inoltre, le analisi morfologiche di P. pungens hanno rivelato una sorprendente variabilità (sovrapposizione delle cellule in colonia, forma delle valve, morfologia dei poroidi in vista cingolare) che devia dalla forma nominale P. pungens. Infine, tramite l'osservazione sia dell'ultrastruttura del frustulo che delle sequenze molecolari (regione D1-D3 del LSU rDNA), è stata descritta Nitzschia gobbii sp. nov., una nuova diatomea planctonica con una porzione espansa al centro del frustulo e due lunghi rostri in vista valvare. Nei 30 anni di monitoraggio questa specie è stata frequentemente registrata ma forse anche erroneamente identificata come Cylindrotheca closterium o Pseudo-nitzschia galaxiae.<br>Pseudo-nitzschia is among the most known and studied diatom genus, since among the total 56 accepted species, 26 produce a neurotoxin (i.e. domoic acid) responsible for human illness (i.e. Amnesic Shellfish Poisoning).
The diversity and seasonality of Pseudo-nitzschia species were analyzed for the first time in the coastal station of the LTER-Senigallia-Susak transect in the northwestern Adriatic Sea from 1988 to 2020, by an integrated taxonomy approach, based on the ultrastructural and molecular analysis. At least six morphotypes have been detected by LM observations on field samples P. cf. delicatissima, P. cf. pseudodelicatissima, P. multistriata, P. cf. fraudulenta, P. pungens and P. cf. galaxiae. The IndVal analysis revealed that P. cf. delicatissima and P. cf. pseudodelicatissima were relevant taxa in spring and summer, respectively. P. cf. fraudulenta and P. pungens were relevant winter taxa and were negatively correlated with water temperature and positively with DIN. P. multistriata appeared in autumn and was the only species positively correlated with silicate concentrations.
Monthly isolation of the highest possible number of strains were performed from 2018 to 2020, leading to isolate 138 strains. Transmission Electron Microscopy analysis and LSU rDNA phylogenetic tree confirmed the identity of six Pseudo-nitzschia species also clustering in six distinct lineages, i.e. P. delicatissima, P. cf. arenysensis, P. calliantha, P. mannii, P. fraudulenta, P. pungens. Domoic acid was not detected in any strain. P. pungens revealed a surprisingly wide morphological variability (e.g. overlap of cells in colonies, shape of valve, poroids’ morphology in girdle view) deviating from the nominal P. pungens.
Finally, ultrastructural analysis and molecular evidence (D1-D3 region of LSU rDNA), allowed the description of Nitzschia gobbii sp. nov. a new planktonic diatom with an expanded central part of the frustule and two long rostra in valve view. This species commonly occurred during the investigated 30 years but possibly misidentified with Cylindrotheca closterium or with Pseudo-nitzschia galaxiae.
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Polischuk, Trevor M. "Intrinsic optical signals characterizing acute excitotoxicity in the hippocampal slice evoked by the marine toxin domoic acid." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq20581.pdf.
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Panlilio, Jennifer Martinez. "Impacts of developmental exposures to the harmful algal bloom toxin domoic acid on neural development and behavior." Thesis, Massachusetts Institute of Technology, 2019.
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This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.<br>Thesis: Ph. D., Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Department of Biology; and the Woods Hole Oceanographic Institution), 2019<br>Cataloged from student-submitted PDF version of thesis.<br>Includes bibliographical references.<br>Harmful algal blooms (HABs) can produce potent neurotoxins that accumulate in seafood and affect human health. One HAB toxin of concern is domoic acid (DomA), a glutamate analog produced by the marine diatom Pseudo-nitzschia spp. Current regulatory limits are designed to prevent acute neurotoxicity in adult humans. However, research shows that low-level exposure during early life can lead to long-term changes in behavior, neural connectivity, and brain morphology. To determine the underlying mechanisms of developmental toxicity, this dissertation used zebrafish as a tool to: i) Establish the developmental window of susceptibility for DomA toxicity, ii) Characterize the behavioral consequences of exposures, and iii) Identify the cellular targets and processes perturbed by DomA. I found that DomA exposure particularly at 2 days post fertilization (dpf) led to altered startle response behavior, myelination defects, and the downregulation of axonal and myelin structural genes.<br>Using vital dyes and immunolabeling, I assessed DomA-induced alterations in cells required for the startle response. I found no differences in the number of sensory neuromasts or in the sensory cranial ganglia structures that detect the acoustic stimuli. However, the majority of DomA-treated larvae lacked one or both Mauthner cells - hindbrain neurons critical for fast startle responses. DomA-treated larvae also had oligodendrocytes with fewer and shorter myelin sheaths, and appeared to aberrantly myelinate neuronal cell bodies. The loss of the Mauthner neurons and their axons may lead to a cellular environment where oligodendrocytes myelinate neuronal cell bodies in the absence of adequate axonal targets. Indeed, pharmacological treatment that reduced the oligodendrocyte number also led to the reduction in the number of these aberrant, myelinated cell bodies.<br>These results indicate that exposure to DomA at a particular period in neural development targets specific cell types, disrupts myelination in the spinal cord, and leads to prolonged behavioral deficits. These mechanistic insights support hazard assessments of DomA exposures in humans during critical periods in early development.<br>"Funding for my research came from the Ocean Ventures Fund, Hill family foundation, Woods Hole Sea grant NA14OAR4170074, and the Woods Hole Center for Oceans and Human Health (COHH), which is jointly funded by the National Institutes of Health (P01ES02192, P01ES028938), and the National Science Foundation (OCE-1314642, OCE-1840381). My funding came from the National Institutes of Health (NIH) P01ES021923-04S1, the Ocean Ridge Initiative Fellowship, the Von Damm Fellowship, and the MIT/WHOI Joint Program Academic Programs Office"--Page 5<br>by Jennifer Martinez Panlilio.<br>Ph. D.<br>Ph.D. Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Department of Biology; and the Woods Hole Oceanographic Institution)
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Vigilant, Veronica Lynn. "The occurrence of the harmful algae bloom toxin, domoic acid, in nearshore and offshore benthic communities of Monterey Bay, California /." Diss., Digital Dissertations Database. Restricted to UC campuses, 2007. http://uclibs.org/PID/11984.
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Downes-Tettmar, Naomi. "Factors that impact Pseudo-nitzschia spp. occurrence, growth, and toxin production." Thesis, University of Plymouth, 2013. http://hdl.handle.net/10026.1/1588.
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This work investigates, for the first time, the Pseudo-nitzschia (PN) dynamics in the western English Channel (L4) and the environmental factors impacting on domoic acid (DA) production in these waters. This is combined with laboratory studies examining key environmental factors and the multifactorial impact of multiple macronutrient and micronutrient availability on PN growth and DA production. An LC-MS method was established, optimised, and compared with ELISA for the accurate and reproducible extraction and determination of particulate and dissolved DA. The method was used to measure the seasonal variation in DA at L4 during 2009 and this was compared to PN seasonal abundance and diversity. Three groups a P. delicatissima-group, a P. seriata-group, and a P. pungens/multiseries-group were identified and were found to have different ecological distributions with the latter two groups significantly correlating with DA concentration. Macronutrients, in combination with other environmental factors, were found to influence PN populations at L4. Multifactorial laboratory culture experiments investigating the availability of nitrate, phosphate, and silicate, confirmed that the interrelatedness of all these nutrients significantly affected the growth, decline, and DA production of P. multiseries, and highlight the importance of both phosphate and silicate availability for DA production. When the impacts of both macronutrient (phosphate and silicate) and micronutrient (iron and copper) availability were investigated, limited growth and DA production was observed in P. multiseries cultures. Results revealed the complexity and interrelationship of factors affecting both PN growth and DA production. Furthermore, molecular methods were developed to elucidate the PN species present from 2009 Lugol’s-preserved L4 samples. DNA was successfully extracted and amplified from these samples which had been stored for up to 2 years. Initial sequence analysis identified the rbcL DNA marker as an informative site for future work with a number of L4 sequences closely relating to different Pseudo-nitzschia spp.
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Rieseberg, Ashley. "Holistic approach to the evaluation of the anthropocentric influence on domoic acid production and the corresponding impact on the California Sea Lion (Zalophus californianus) population." Thesis, Uppsala universitet, Institutionen för geovetenskaper, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-179182.
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Domoic acid (DA) is a neurotoxin produced by the harmful algae Pseudo-nitzschia that has been directly linked to mass stranding events of the California Sea Lion (CSL). The purpose of this paper is to review the anthropogenic influence on the production of this neurotoxin and examine how human activities are impacting this marine mammal species. A comprehensive and interdisciplinary literature review was conducted to evaluate the future sustainability of the CSL population. It was found that while Pseudo-nitzschia bloom developments are vulnerable to anthropogenic influences, the incontestable existence of natural contributing factors adds a certain complexity to the determination of causalities and the development of solutions. Strong evidence exists to show that DA can cause major and irreversible neurological damage in CSLs. Rehabilitation of DA-impacted CSLs is a polarizing issue in the U.S. and presents interesting implications for sustainable development. While the CSL population is currently healthy and plenteous, the strong abundance of future uncertainties warrants concern. A balance must be found between the involving social, economic, and environmental factors to ensure a promising future for the CSL species.
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Deléglise, Margot. "Suivi de la contamination des coquilles Saint-Jacques (Pecten maximus) par l'acide domoïque et exploration du rôle du microbiote dans sa décontamination." Electronic Thesis or Diss., Brest, 2024. http://www.theses.fr/2024BRES0022.
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La coquille Saint-Jacques {Pecten maximus) fait figure d’exception parmi les organismes contaminés par l’acide domoïque du fait de sa longue rétention au sein de sa glande digestive. Bien que les interdictions de pêche en cas de contamination aient un impact économique important, le mécanisme sous-jacent à cette lente dépuration reste méconnu. Les objectifs de cette thèse ont donc été de i) examiner in situ la contamination de P. maximus en corrélation avec la présence de Pseudo-nitzschia spp. et d’acide domoïque dans l'eau afin d'identifier les diverses sources de contamination possibles, ii) étudier le lien entre la dépuration de l'acide domoïque et la taille de P. maximus, iii) explorer la piste des microorganismes pour accélérer la dépuration de l'acide domoïque dans la glande digestive de P. maximus. Une surveillance menée depuis 2011 dans la rade de Brest a mis en évidence l'importance des eaux de surface et de fond ainsi que la présence de Pseudo-nitzschia spp. et d'acide domoïque dans la contamination de P. maximus, identifiant ainsi trois scénarios de contamination. Une expérience de décontamination sur deux mois a révélé que les petites coquilles semblaint dépurer l'acide domoïque plus rapidement que les grandes. Des isolements bactériens effectués sur des individus contaminés ont révélés des différences avec d'autres espèces de bivalves considérés comme dépurateurs rapides. Bien que des souches bactériennes candidates aient été identifiées, aucune réduction de l'acide domoïque n'a été observée après exposition à toxine. Un transfert de microbiote de M. edulis a été réalisé vers P. maximus, montrant un transfert de souches bactériennes dans la glande digestive de P. maximus. En conclusion, ces travaux enrichissent la compréhension des diverses sources de contamination de P. maximus par l'acide domoïque, ainsi que des réponses de ce bivalve en fonction de sa taille face à cette toxine. Cette thèse offre des pistes qui pourraient permettre d'accélérer la dépuration de l'acide domoïque chez P. maximus<br>The King scallop {Pecten maximus) is an exception among domoic acid contaminated organisms, due to its long retention within the digestive gland. Although fishing bans in the event of contamination have a significant economie impact, the mechanism behind this slow depuration remains poorly understood. The aims of this thesis were therefore to i) examine in situ the contamination of P. maximus in correlation with the presence of Pseudo-nitzschia spp. and domoic acid in the water, to identify the various sources of contamination, ii) study the link between domoic acid depuration and P. maximus size, iii) explore the possibility of microorganisms accelerating the depuration of domoic acid in the digestive gland of P. maximus. Monitoring carried out since 2011 in the Bay of Brest has shown the importance of surface and bottom waters, as well as the presence of Pseudo-nitzschia spp. and domoic acid in P. maximus contamination, thus identifying three contamination scenarios. A two-month decontamination experiment revealed that smaller scallops seemed to depurate domoic acid faster than larger ones. Bacterial isolations from contaminated individuals revealed differences with other bivalve species considered to be rapid depurators. Although candidate bacterial strains were identified, no toxin reduction was observed after exposure to domoic acid. A transfer of microbiota from M. edulis to P. maximus was performed, showing a transfer of bacterial strains into the digestive gland of P. maximus. In conclusion, this work enriches our understanding of the various sources of domoic acid contamination of scallops, as well as the response of this bivalve to domoic acid according to its size. This thesis offers new insight for accelerating domoic acid depuration in Pecten maximus
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Howard, Meredith Dana Armstrong. "Harmful algal blooms on the U.S. west coast : new insights into domoic acid production and identification of yessotoxin, a new marine toxin detected in California coastal waters /." Diss., Digital Dissertations Database. Restricted to UC campuses, 2007. http://uclibs.org/PID/11984.
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Sauvey, Aurore. "Variabilité interspécifique et intraspécifique des indices physiologiques chez les diatomées toxiques du genre Pseudo-nitzschia : influence du cycle de vie." Thesis, Normandie, 2018. http://www.theses.fr/2018NORMC266/document.
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Chez les diatomées du genre Pseudo-nitzschia, certaines espèces produisent une neurotoxine, l’acide domoïque responsable chez l’homme d’intoxications amnésiantes suite à la consommation de mollusques contaminés. En Baie de Seine, la variabilité interannuelle de ces efflorescences toxiques, qui provoquent certaines années des fermetures de zones de pêche à la coquille Saint-Jacques, est liée à des changements de diversité spécifique au sein des communautés de Pseudo-nitzschia. Cette thèse avait pour objectif de caractériser la diversité écophysiologique au sein du genre Pseudo-nitzschia afin de mieux comprendre les facteurs qui influencent les différences de toxicité et de phénologies des efflorescences des principales espèces de Pseudo-nitzschia de Baie de Seine. Différents paramètres physiologiques, tels que la croissance, la production d’acide domoïque, les quotas élémentaires, les paramètres photosynthétiques, ainsi que les paramètres de cinétiques d’absorption du nitrate, du silicate et du phosphate, ont été étudiés chez quatre espèces : P. australis, P. pungens, P. fraudulenta et P. delicatissima. De plus, la variabilité intraspécifique a été évalué en étudiant de nombreuses souches par espèce et en estimant l’influence des variations de la taille cellulaire liées au cycle de vie de Pseudo-nitzschia sur les paramètres physiologiques. Les résultats montrent une importante diversité intraspécifique de la physiologie des quatre espèces, en partie liée à la taille cellulaire et parfois au cycle de vie. La connaissance de cette diversité intraspécifique a permis de caractériser différents traits physiologiques pour chaque espèce. Les résultats apportent des éléments de compréhension sur les conditions physiologiques favorables à la production d’acide domoïque et le rôle des éléments nutritifs dans le déterminisme des efflorescences des espèces de Pseudo-nitzschia<br>In the diatom Pseudo-nitzschia, some species produce a neurotoxin called domoic acid responsible for amnesic shellfish poisoning in humans following the consumption of contaminated molluscs. In the Bay of Seine (English Channel), the variability of bloom toxicity is linked to differences in the Pseudo-nitzschia species diversity. The aim of this thesis was to characterize the ecophysiological diversity within the genus Pseudo-nitzschia in order to better understand the physiological parameters influencing differences in toxicity and bloom phenology among the main Pseudo-nitzschia species of the Bay of Seine. Growth, domoic acid production, elemental intracellular quotas, photosynthetic parameters and kinetic parameters for nitrate, phosphate, and silicic acid uptake were studied in four Pseudo-nitzschia species: P. australis, P. pungens, P. fraudulenta et P. delicatissima. The intraspecific variability was characterized with a multi-strain approach and also considering the effect of cell size on the studied physiological parameters. The results highlight the importance of the intraspecific variability in the physiology of the four species, in part due to cell size reduction linked to Pseudo-nitzschia life cycle. Delineate the intraspecific diversity allowed to characterize different physiological traits for each species. This work brings new information on the physiological conditions favorable to domoic acid production and on the role of nutrients in controlling Pseudo-nitzschia species diversity during blooms
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Deschler, Marie. "Ιntéractiοns et cοmmunicatiοn chimique entre les diatοmées tοxiques du genre Ρseudο-nitzschia et les cοnsοmmateurs zοοplanctοniques". Electronic Thesis or Diss., Normandie, 2024. http://www.theses.fr/2024NORMLH13.
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Les efflorescences d’algues toxiques (HAB), potentiellement favorisées par le réchauffement climatique et l’eutrophisation des écosystèmes côtiers, est un sujet de préoccupation majeur. C’est le cas des diatomées du genre Pseudo-nitzschia, dont certaines espèces produisent une neurotoxine, l’acide domoïque (AD), responsable d'intoxication amnésiante par les mollusques (ASP). Ces efflorescences ont des conséquences à la fois sanitaires et socio-économiques, les phénomènes ASP entrainant des interdictions de pêche, en particulier à la coquille Saint Jacques sur les côtes normandes. Les copépodes peuvent également véhiculer l’AD dans le réseau trophique. La plupart des études sur l’interaction entre Pseudo-nitzschia et les copépodes concernent des espèces arctiques. Cette thèse visait à approfondir cette interaction pour les espèces présentes sur les côtes normandes (France). Les résultats montrent des mécanismes de défense chez Pseudo-nitzschia en réponse aux prédateurs zooplanctoniques, dont l’induction de la production d’AD en présence de copépodes, observée pour la première fois chez P. australis, connue pour son fort potentiel ASP. En revanche, P. pungens et P. fraudulenta n’augmentaient pas leur production en présence de prédateurs, soulignant l’impossibilité d’extrapoler les données d’une espèce à l’autre ou d’un écosystème à l’autre. Une réponse morphologique, telle que la réduction de la taille des chaînes, a également été démontrée pour la première fois chez P. australis. Les réponses physiologique et morphologique chez P. australis étaient déclenchées à la fois par la présence directe de prédateurs et mais aussi uniquement par leurs signaux chimiques, présents dans le milieu. Ces réponses n’ont pas été associées à une sur-mortalité des copépodes. Par contre, une réduction de la nage basale et des modifications du transcriptome ont pu être mises en évidence<br>Harmful algal blooms (HAB), potentially favored by global warming and eutrophication of coastal ecosystems, are a major concern. This is the case for diatoms of the genus Pseudo-nitzschia, some species of which produce a neurotoxin, domoic acid (DA), responsible for amnesic shellfish poisoning (ASP). These efflorescences have both health and socio-economic consequences, with ASP phenomena leading to shellfish fishing bans, particularly for scallops on the Normandy coast. Zooplankton copepods can also carry DA through the food web. Most studies on the interaction between Pseudo-nitzschia and copepods have focused on Arctic species. The aim of this thesis was to investigate this interaction in greater depth for species present on the Normandy coast (France). The results show defense mechanisms in Pseudo-nitzschia in response to zooplanktonic predators, including induction of DA production, observed for the first time in P. australis, known for its high ASP potential. On the other hand, P. pungens and P. fraudulenta did not increase their production in the presence of predators, underlining the impossibility of extrapolating data from one species to another or from one ecosystem to another. A morphological response, i.e. chain size reduction, was also demonstrated for the first time in P. australis. Physiological and morphological responses in P. australis were triggered both by the direct presence of predators and solely by their cues, present in the environment. These responses were not associated with excess copepod mortality. However, a reduction in basal swimming and changes in the transcriptome were observed
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Delegrange, Alice. "Impact du phytoplancton sur les juvéniles de bars (Dicentrarchus labrax) en milieu aquacole : approches in situ et expérimentales." Thesis, Littoral, 2015. http://www.theses.fr/2015DUNK0518.
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Dans une ferme d'élevage de bar (Dicentrarchus labrax) du sud de la mer du Nord, de fortes mortalités de bar coïncident régulièrement avec l'efflorescence phytoplanctonique printanière. Le rôle du phytoplancton dans ces mortalités a donc été étudié : un suivi saisonnier (février-novembre 2012) a permis de définir les communautés phytoplanctoniques en présence et, la diversité et la toxicité du genre Pseudo-nitzschia. Ainsi, trois espèces potentiellement toxiques ont été identifiées (P. delicatissima, P. pungens, P. fraudulenta) en association avec des concentrations élevées d'acide domoïque (jusqu'à 229 pg. mL-¹). Au cours d'une expérience d'exposition (45 jours), les effets délétères de P. delicatissima sur les juvéniles de bar ont été étudiés. Si un stress d'exposition a été observé via la surproduction de mucus par l'épithélium branchial, cela n'a pas eu d'incidence sur la condition ni la physiologie des poissons. Les mortalités seraient donc davantage liées à un effet de communautés. Cette hypothèse a été testée en utilisant le pouvoir de filtration des moules (Mytilis edulis) en amont des bassins d'élevage. Cela a permis de limiter l'ampleur de l'efflorescence phytoplanctonique printanière. En conséquence, les poissons élevés dans l'eau filtrée avaient de meilleures conditions, croissance et rapport ARN:ADN que ceux élevés dans l'eau de mer non filtrée. Ce travail souligne la nécessité de généraliser le suivi des communautés phytoplanctoniques afin d'identifier les espèces délétères et leur dynamique et de développer des outils de mitigation permettant d'atténuer l'impact des efflorescences phytoplanctoniques sur l'aquaculture<br>For several years, mass mortalities of farmed sea bass (Dicentrarchus labrax) have coincided with phytoplankton spring blooms in the southern North Sea. Since these mortalities could not be explained by classical finfish diseases, phytoplankton noxious effects have been suspected and investigated. A seasonal survey allowed the identification of potentially deleterious phytoplankton species giving particular attention to the Pseudo-nitzschia genus. Three potentially toxic Pseudo-nitzschia species were identified (P. delicatissima, P. pungens, P. fraudulenta) and their presence was related to both domoic acid concentrations and phytoplankton communities. P. delicatissima being dominant over spring and presenting toxic and physical features compatible with fish mortality, a laboratory exposure experiment was carried out. Although gills irritations (mucus overproduction) revealed an exposure stress, no effect on sea bass condition nor on physiological performances was demonstrated. This suggest that phytoplankton community as a whole rather than single species should be involved in fish mortalities. This third hypothesis was tested using mussels (Mytilus edulis) as seawater filters upstream from the rearing tanks to dampen the phytoplankton spring bloom and estimate its impact on fish. Indeed, fish had better condition , growth and RNA:DNA ratio when reared in filtered seawater than in natural seawater. This work highlights the need to develop phytoplankton monitoring in fish farms so as to identify potentially deleterious species and understand their dynamics. It also demonstrates that new mitigation tools should be developed to prevent phytoplankton impacts on farmed fish
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O'dea, Sheila. "Occurrence, Toxicity, and Diversity of Pseudo-nitzschia in Florida Coastal Waters." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4187.
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Domoic acid (DA), a potent neurotoxin that has the potential to cause amnesic shellfish poisoning (ASP), is produced by members of the marine diatom genus Pseudo-nitzschia. Outbreaks of ASP in humans and of DA poisoning in birds and marine mammals have been reported across the United States and Canada since the late 1980's. Pseudo-nitzschia species can be extremely abundant in Florida waters, with densities often exceeding 106 cells/L, and sometimes exceeding 107 cells/L. Based on preliminary data, it is evident that at least nine species of Pseudo-nitzschia are found in Florida coastal waters. At least six of these species are known to produce DA in other parts of the world, and some are morphologically identical to some of the major toxin-producing species in Californian and Canadian waters. Despite the strong presence of Pseudo-nitzschia, there has never been a report of ASP or a DA-related animal mortality event from Florida.
Data collected from 2004 to 2011 show maximum Pseudo-nitzschia abundances exceeded 4 x 107 cells/L. Six species of Pseudo-nitzschia were identified from central west and southwest Florida waters via light and electron microscopy. This is the first report of P. micropora from the Gulf of Mexico. Additionally P. calliantha, P. cuspidata, and P. pungens were identified as producers of DA in Florida coastal waters; although cell quotas of DA were low. Low levels of DA were detected in about one third of the water samples analyzed and DA concentrations measured in the majority of shellfish from the study area were at least an order of magnitude below the regulatory limit of 20 µg/g, suggesting that Pseudo-nitzschia currently poses little threat to human health in Florida. However, DA production in Pseudo-nitzschia species has been shown to be variable and dependent on nutrient conditions, indicating that the potential for DA-related events to occur in Florida warrants further investigation.
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Li, Ran. "Signal peptide prediction in the space-frequency domain." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file 0.44 Mb., 68 p, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:1432421.
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Gadhavi, Paresh Laxman. "The structure of the DNA-binding domain of GAL4." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240924.
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Martinez, Sarah Jane. "Strategies for preparing segmentally isotopically labeled proteins for probing domain-domain interactions by FTIR spectroscopy by Sarah Jane Martinez." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82290.
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Fourier transform infrared (FTIR) spectroscopy is a powerful tool for probing protein structure-function relationships. With the use of isotope editing, it can also be employed to elucidate protein-nucleic acid interactions. This technique was used to study the sequence of heat-induced unfolding of the uniformly labeled 13C regulatory subunit (RSU) of E. coli aspartate transcarbamylase (ATCase) with its inhibitor CTP. The absorption of CTP in the amide I' region limits our ability to detect protein conformational changes upon binding of CTP. Therefore, by labeling the protein with 13C shifts the amide I' band ~ 40 cm -1 and clearly separates the protein bands from those of CTP. Variable-temperature (VT) FTIR spectroscopy was then employed to monitor the thermal unfolding of the labeled RSU in the presence and absence of CTP.<br>In addition, isotope editing was further explored to probe domain-domain interactions of the two domains of RSU using intein technology. Intein technology provides a novel means by which isotope editing can be performed to extract information on protein inter-domain and inter-subunit interactions by spectroscopic analysis but has not yet been exploited in Fourier transform infrared (FTIR) spectroscopy. The objective of this project is to present for the first time the feasibility of segmental labeling through intein-mediated protein ligation (IPL) for the purpose of studying conformational changes by FTIR spectroscopy, using ATCase as a model enzyme. In the first phase of this project, the RSU of ATCase, which houses a Zn-binding domain and a nucleotide binding domain, was reconstructed from its isolated domains using commercially available intein-base expression vectors. As steps towards obtaining an isotope labeled RSU, we have fused each domain to separate inteins. Following affinity purification, the intein tags were chemically cleaved and the reactive ends of the two RSU domains were ligated together to form a peptide. Although ligation was successful, improved yields are required for the FTIR spectroscopic studies.
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Sekharan, Monica R. "Structural studies of the cGMP-binding GAF domain of PDE5A /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/8502.
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Lutya, Portia Thandokazi. "Expression and purification of the novel protein domain DWNN." Thesis, University of the Western Cape, 2002. http://etd.uwc.ac.za/index.php?module=etd&.
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Proteins play an important role in cells, as the morphology, function and activities of the cell depend on the proteins they express. The key to understanding how different proteins function lies in an understanding of the molecular structure. The overall aim of this thesis was the determination of the structure of DWNN domains. This thesis described the preparation of samples of human DWNN suitable for structural analysis by nuclear magnetic resonance spectroscopy (NMR), as well as NMR analysis.
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Hufnagle, David C. "THE ANALYSIS OF BIOLOGICAL COMPOUNDS AND THREAT AGENTS WITH TERAHERTZ TIME-DOMAIN SPECTROSCOPY AND IMAGING." Miami University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=miami1354282849.
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Sirota, Leite Fernanda. "Role of the amino acid sequences in domain swapping of the B1 domain of protein G by computation analysis." Doctoral thesis, Universite Libre de Bruxelles, 2007. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210657.
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Domain swapping is a wide spread phenomenon which involves the association between two or more protein subunits such that intra-molecular interactions between domains in each subunit are replaced by equivalent inter-molecular interactions between the same domains in different subunits. This thesis is devoted to the analysis of the factors that drive proteins to undergo such association modes. The specific system analyzed is the monomer to swapped dimer formation of the B1 domain of the immunoglobulin G binding protein (GB1). The formation of this dimer was shown to be fostered by 4 amino acid substitutions (L5V, F30V, Y33F, A34F) (Byeon et al. 2003). In this work, computational protein design and molecular dynamics simulations, both with detailed atomic models, were used to gain insight into how these 4 mutations may promote the domain swapping reaction.<p>The stability of the wt and quadruple mutant GB1 monomers was assessed using the software DESIGNER, a fully automatic procedure that selects amino acid sequences likely to stabilize a given backbone structure (Wernisch et al. 2000). Results suggest that 3 of the mutations (L5V, F30V, A34F) have a destabilizing effect. The first mutation (L5V) forms destabilizing interactions with surrounding residues, while the second (F30V) is engaged in unfavorable interactions with the protein backbone, consequently causing local strain. Although the A34F substitution itself is found to contribute favorably to the stability of the monomer, this is achieved only at the expense of forcing the wild type W43 into a highly strained conformation concomitant with the formation of unfavorable interactions with both W43 and V54.<p>Finally, we also provide evidence that A34F mutation stabilizes the swapped dimer structure. Although we were unable to perform detailed protein design calculations on the dimer, due to the lower accuracy of the model, inspection of its 3D structure reveals that the 34F side chains pack against one another in the core of the swapped structure, thereby forming extensive non-native interactions that have no counterparts in the individual monomers. Their replacement by the much smaller Ala residue is suggested to be significantly destabilizing by creating a large internal cavity, a phenomenon, well known to be destabilizing in other proteins. Our analysis hence proposes that the A34F mutation plays a dual role, that of destabilizing the GB1 monomer structure while stabilizing the swapped dimer conformation.<p>In addition to the above study, molecular dynamics simulations of the wild type and modeled quadruple mutant GB1 structures were carried out at room and elevated temperatures (450 K) in order to sample the conformational landscape of the protein near its native monomeric state, and to characterize the deformations that occur during early unfolding. This part of the study was aimed at investigating the influence of the amino acid sequence on the conformational properties of the GB1 monomer and the possible link between these properties and the swapping process. Analysis of the room temperature simulations indicates that the mutant GB1 monomer fluctuates more than its wild type counter part. In addition, we find that the C-terminal beta-hairpin is pushed away from the remainder of the structure, in agreement with the fact that this hairpin is the structural element that is exchanged upon domain swapping. The simulations at 450 K reveal that the mutant protein unfolds more readily than the wt, in agreement with its decreased stability. Also, among the regions that unfold early is the alpha-helix C-terminus, where 2 out of the 4 mutations reside. NMR experiments by our collaborators have shown this region to display increased flexibility in the monomeric state of the quadruple mutant.<p>Our atomic scale investigation has thus provided insights into how sequence modifications can foster domain swapping of GB1. Our findings indicate that the role of the amino acid substitutions is to decrease the stability of individual monomers while at the same time increase the stability of the swapped dimer, through the formation of non-native interactions. Both roles cooperate to foster swapping.<br>Doctorat en sciences, Spécialisation biologie moléculaire<br>info:eu-repo/semantics/nonPublished
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Seyedhosseini, Ensieh. "Piezoelectricity and ferroelectricity in amino acid glycine." Doctoral thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/15169.
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Doutoramento em Nanociências e Nanotecnologia<br>Bioorganic ferroelectrics and piezoelectrics are becoming increasingly important in view of their intrinsic compatibility with biological environment and biofunctionality combined with strong piezoelectric effect and switchable polarization at room temperature. Here we study piezoelectricity and ferroelectricity in the smallest amino acid glycine, representing a broad class of non-centrosymmetric amino acids.
Glycine is one of the basic and important elements in biology, as it serves as a building block for proteins. Three polymorphic forms with different physical properties are possible in glycine (α, β and γ), Of special interest for various applications are non-centrosymmetric polymorphs: β-glycine and γ-glycine. The most useful β-polymorph being ferroelectric took much less attention than the other due to its instability under ambient conditions. In this work, we could grow stable microcrystals of β-glycine by the evaporation of aqueous solution on a (111)Pt/Ti/SiO2/Si substrate as a template. The effects of the solution concentration and Pt-assisted nucleation on the crystal growth and phase evolution were characterized by X-ray diffraction analysis and Raman spectroscopy.
In addition, spin-coating technique was used for the fabrication of highly aligned nano-islands of β-glycine with regular orientation of the crystallographic axes relative the underlying substrate (Pt).
Further we study both as-grown and tip-induced domain structures and polarization switching in the β-glycine molecular systems by Piezoresponse Force Microscopy (PFM) and compare the results with molecular modeling and computer simulations. We show that β-glycine is indeed a room-temperature ferroelectric and polarization can be switched by applying a bias to non-polar cuts via a conducting tip of atomic force microscope (AFM). Dynamics of these in-plane domains is studied as a function of applied voltage and pulse duration. The domain shape is dictated by both internal and external polarization screening mediated by defects and topographic features. Thermodynamic theory is applied to explain the domain propagation induced by the AFM tip. Our findings suggest that β-glycine is a uniaxial ferroelectric with the properties controlled by the charged domain walls which in turn can be manipulated by external bias.
Besides, nonlinear optical properties of β-glycine were investigated by a second harmonic generation (SHG) method. SHG method confirmed that the 2-fold symmetry is preserved in as-grown crystals, thus reflecting the expected P21 symmetry of the β-phase. Spontaneous polarization direction is found to be parallel to the monoclinic [010] axis and directed along the crystal length. These data are confirmed by computational molecular modeling. Optical measurements revealed also relatively high values of the nonlinear optical susceptibility (50% greater than in the z-cut quartz).
The potential of using stable β-glycine crystals in various applications are discussed in this work.<br>Piezo e ferroeléctricos biorgânicos são materiais que estão a atrair para si uma importância crescente por força da sua compatibilidade intrínseca com ambientes biológicos e uma biofuncionalidade aliada a um forte efeito piezoeléctrico e polarização controlada, a temperature ambiente. Aqui estudamos a piezo e ferroelectricidade no mais pequeno aminoácido, a glicina, representando uma ampla classe de aminoácidos nao-centrosimétricos.
A glicina é um elemento básico e extremamente importante em biologia, uma vez que serve de unidade base de construção para proteínas. Três formas polifórmicas com diferentes propriedades são possíveis na glicina (α, β e γ). De especial interesse para várias aplicações são as estruturas não-centrosimétricas: β-glycina e γ-glycina. A mais interessante β-polimorfa está a ser alvo de uma atenção reduzida, comparativamente às outras, por motivos de uma maior instabilidade a temperatura ambiente. Neste trabalho, Podemos crescer microcristais estáveis de glicina-β pela evaporação da solução aquosa num substrato (111)Pt/Ti/SiO2/Si que funciona como "template". Os efeitos da concentração da solução e da nucleação Pt-assistida no crescimento do cristal e evolução da fase foram estudados com recurso à difracção Raio-X e espectroscopia Raman.
Adicionalmente, a técnica de "spin-coating" foi utilizada para a fabricação de nano-ilhas de glicina-β altamente alinhadas, com a orientação dos eixos cristalográficos normalizada pelo substrato de Pt.
Estudamos a indução de domínios estruturais por meio da ponta do AFM e a variação da polarização nos sistemas moleculares da β-glicina através da técnica PFM (Microscopia de Piezo Força), comparando os resultados obtidos com modelação molecular e simulações computacionais. Mostramos que a β-glycina é de facto um piezoeléctrico à temperatura ambiente e a polarização pode ser controlada por aplicação de uma tensão a cortes não polares. A dinâmica destes domínios complanares é estudada como função da tensão aplicada e duração do pulso. A forma do domínio é ditada pela polarização interna e externa, cujo rastreio é mediado por defeitos e características topográficas. A teoria termodinâmica é aplicada para explicar a propagação dos domínios induzidos pela ponta do AFM. As nossas descobertas sugerem que a β-glycina é um ferroeléctrico uniaxial com propriedades controladas pelas fronteiras dos domínios (electronicamente carregadas), que em seu turno podem ser manipuladas por tensão externa.
Adicionalmente, propriedades ópticas não-lineares da β-glycina foram investigadas por um método de segunda geração harmonica (SHG). Este método confirmou que a simetria axial é preservada em cristais crescidos sem pós-tratamento, reflectindo a esperada simetria P21 da fase β. A direcção da polarização espontânea mostrou ser paralela ao eixo monoclínico [010] e direccionada no comprimento do cristal. Estes dados foram confirmados por modelação computacional molecular. Medições ópticas revelaram também um valor relativamente elevado para a susceptabilidade óptica não-linear (50% maior que no quartzo com corte em z).
O pontencial uso de cristais de β-glycina estáveis em diversas aplicações são também discutidos.
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Rauch, Steven Martin. "Critical Amino Acids of the Ga2 Subunit Helical Domain in Dictyostelium discoideum." Fogler Library, University of Maine, 2002. http://www.library.umaine.edu/theses/pdf/RauchSM2002.pdf.
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Parker, Maura H. "Characterization of the native and iron-replaced DNA-binding domain of the retinoic acid receptor." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1996. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ51580.pdf.
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Arpino, James. "From single amino acid deletions to whole domain insertions : engineering GFP through polypeptide backbone mutations." Thesis, Cardiff University, 2011. http://orca.cf.ac.uk/14266/.
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With an ever-expanding protein engineering toolbox different mutational techniques can be used to engineer new or altered function into protein scaffolds without the restriction of sampling just simple substitution mutagenesis. These include approaches that target backbone as well as side chain changes, such as single amino acid deletions or whole domain insertion. The problem with utilizing these mutational approaches is the difficulty in predicting both local and global structural changes on changing the backbone conformation. The aim of this thesis is to demonstrate the tolerance and beneficial influence of backbone targeted mutations using a mutant library-based screening approach, and to provide an understanding of their mechanism of action. A directed evolution transposon-based approach was used to generate libraries of single amino acid deletion and whole domain insertions into enhanced green fluorescent protein (EGFP). The later involved the insertion of cyt b562 as the donating insert domain. Library analysis revealed a wide range of sites were sampled across the backbone of EGFP. Library screening revealed widespread tolerance of EGFP to single amino acid deletions. Using the crystal structure of EGFP determined here, it was found that that loop regions where particularly tolerant. Two variants with residues G4 or A227 deleted conferred increased protein fluorescence to cell cultures with respect to EGFP. Spectral characterization and unfolding experiments identified that rather than altering the fluorescent properties of EGFP the mutations elicited their effects through altered protein folding and stability. Screening of the domain insert library revealed that sites spread along the backbone of EGFP were tolerant to cyt b562 insertion. Particularly tolerant were loops and the C-terminal end of β-strand 7, with the linker sequences playing a key role. One integral domain fusion scaffold, termed CG6, was identified in which the functions of the two individual domains were highly coupled. CG6 exhibited almost 100% fluorescence quenching upon the binding of haem to the cyt b562 domain. CG6 was also shown to potentially act as a sensor for redox state and reactive oxygen species such as H2O2 via a haem-dissociation dependent mechanism. The structure of CG6, determined by X-ray crystallography, provided the molecular basis for the functional coupling of the two domains. Critical was the side-by-side domain arrangement caused by differential linker lengths at the pivot position re-enforced by a domain-domain interface that placed the chromophores within 17-18Å of each other. Further rational design of a cyt b562-EGFP integral fusion scaffold (CG15) was performed to create novel ratiometric fluorescent redox sensors, termed CG15CC variants. The CG15CC variants have been shown to have the most reducing redox midpoint potentials of any protein based redox sensor studied to date. One of the CG15CC variants also has the fastest redox kinetics observed to date. The survey of the tolerance and influence of single amino acid deletions in EGFP conducted here has highlighted the potential beneficial nature of deletion mutagenesis and has helped provide a molecular understanding of their effect. Through domain insertion mutagenesis and retrospective structure analysis the mechanism behind the functional coupling of two domains has been described and will also help guide future work in the development of novel biomolecular switches.
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Reed, Michelle Anne Cole. "NMR studies on the acyl carrier protein domain from the type I rat fatty acid synthase." Thesis, University of Bristol, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326271.
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Poshtiban, Anahita [Verfasser]. "Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis / Anahita Poshtiban." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2020. http://d-nb.info/1218075902/34.
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Levine, Kara B. "Identification of the Human Erythrocyte Glucose Transporter (GLUT1) ATP Binding Domain: A Dissertation." eScholarship@UMMS, 1999. https://escholarship.umassmed.edu/gsbs_diss/247.
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The human erythrocyte glucose transport protein (GLUT1) interacts with, and is regulated by, cytosolic ATP. This study asks the following questions concerning ATP modulation of GLUT1 mediated sugar transport. 1) Which region(s) of GLUT1 form the adenine nucleotide-binding domain? 2) What factors influence ATP modulation of sugar transport? 3) Is ATP interaction with GLUT1 sufficient for sugar transport regulation?
The first question was addressed through peptide mapping, n-terminal sequencing, and alanine scanning mutagenesis of GLUT1 using [32P]-azidoATP, a photoactivatable ATP analog. We then used a combination of transport measurements and photolabeling strategies to examine how glycolytic intermediates, pH, and transporter oligomeric structure affect ATP regulation of sugar transport. Finally, GLUT1 was reconstituted into proteoliposomes to determine whether ATP is sufficient for the modulation of GLUT1 function in-vitro.
This thesis presents data supporting the hypothesis that residues 332-335 contribute to the efficiency of adenine nucleotide binding to GLUT1. In addition, we show that AMP, acidification, and conversion of the transporter to its dimeric form antagonize ATP regulation of sugar transport. Finally, we present results that support the proposal that ATP interaction with GLUT1 is sufficient for transport modulation.
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Phillips, Kyle. "Characterization of the role of Zea mays burp domain-containing genes in maize drought responses." Thesis, University of the Western Cape, 2016. http://hdl.handle.net/11394/5339.
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Philosophiae Doctor - PhD<br>Global climate change has resulted in altered rainfall patterns, causing annual losses in maize crop yield due to water deficit stress. Therefore, it is important to produce maize cultivars which are more drought-tolerant. This not an easily accomplished task as plants have a plethora of physical and biochemical adaptation methods. One such mechanism is the drought-induced expression of enzymatic and non-enzymatic proteins which assist plants to resist the effects of water deficit stress. The RD22-like protein subfamily is expressed in response to water deficit stress. Members of the RD22-like subfamily include AtRD22, GmRd22 and BnBDC1 which have been identified in Arabidopsis thaliana, Glycine max and Brassica napus respectively. This study aims at characterising two putative maize RD22-like proteins (designated ZmRd22A and ZmRd22B) by identifying sequence/domain features shared with characterised RD22-like proteins. Semi-quantitative and quantitative PCR techniques were used to examine the spatial and temporal expression patterns of the two putative maize Rd22-like proteins in response to, water deficit stress and exogenously applied abscisic acid in the roots and 2nd youngest leaves of maize seedlings. Using an in silico approach, sequence homology of the two putative maize Rd22- like proteins with AtRD22, GmRD22 and BnBDC1 has been analysed. Online bioinformatic tools were used to compare the characteristics of these Rd22-like proteins with those of the two maize proteins. It was shown that the putative maize RD22-like proteins share domain organisation with the characterised proteins, these common features include a N-terminal hydrophobic signal peptide, followed by a region with a conserved amino acid sequence, a region containing several TxV (x is any amino acid) repeat units and a C-terminal BURP domain-containing the conserved X₅-CH-X₁₀-CH-X₂₃-₂₇-CH-X₂₃-₂₆-CH-X₈-W motif. The putative maize Rd22-like protein appears to be
localized in the apoplast, similarly to AtRD22, GmRD22 and BnBDC1. Analysis of the gene's
promotor regions reveals cis-acting elements suggestive of induction of gene expression by water deficit stress and abscisic acid (ABA). Semi-quantitative and quantitative real time PCR analysis of the putative maize RD22-like gene revealed that the genes are not expressed in the roots. Exposure to water deficit stress resulted in an increase of ZmRD22A transcript accumulation in the 2nd youngest leaves of maize seedlings. ZmRD22A was shown to be non-responsive to exogenous ABA application. ZmRD22B was highly responsive to exogenous ABA application and responded to water deficit stress to a lesser degree. Transcript accumulation studies in three regions of the 2nd youngest leaves in response to water deficit stress showed that ZmRd22A transcripts accumulate mainly at the base and tips of the leaves. A restricted increase in ZmRD22A transcript accumulation in the middle of the leaves was observed. ZmRD22B showed a similar, but weaker transcript accumulation pattern in response to water deficit stress. However, ZmRD22B showed increased transcript accumulation in the middle region of the leaves.
In response to exogenous ABA application, ZmRd22B exhibited high transcript accumulation at the base of the 2nd youngest leaves, with the middle showing higher transcript accumulation than the tip of the leaves. It was concluded that ZmRD22A and ZmRD22B share the domain organisation of characterised RD22-like proteins as well as being responsive to water deficit stress, although only ZmRD22B was shown to be responsive to exogenous ABA application.<br>National Research Foundation (NRF)
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Taulbee, Anita R. "Terahertz Time-Domain Spectroscopy and Imaging of Biological Compounds and Tissues." Miami University / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=miami1249412289.
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Sen, Gulseren Petek. "Fabrication Of Poly (dl-lactic-co-glycolic Acid) Nanoparticles And Synthetic Peptide Drug Conjugate For Anti-cancer Drug Delivery." Master's thesis, METU, 2010. http://etd.lib.metu.edu.tr/upload/12611405/index.pdf.
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Cancer is a group of diseases in which normal cells are converted to cells capable of autonomous growth and invasion. In the chemotherapeutic control of cancer, drugs are usually given systemically so they reach toxic levels in healthy cells as well as cancer cells. This causes serious side effects. Another important problem with chemotherapy is resistance developed to cytotoxic drugs (multi drug resistance).
Doxorubicin (Dox) occupies a central position in the treatment of breast cancer. However doxorubicin induced cardiac toxicity is associated with a high incidence of morbidity and mortality. Resistance of malignant tumors to Dox is another important cause of treatment failure in patients with cancer.
One approach to overcome Dox-related toxicity is to use polymeric drug carriers, which direct the Dox away from heart tissue, and allow usage of lower dosages. In this present study two different anti-cancer drug delivery methods were evaluated. Dox was encapsulated in PLGA microparticles by single and double microemulsion solvent evaporation techniques. The highest entrapment of doxorubicin within PLGA microspheres obtained by optimization of process parameters. A sustained release of doxorubicin was obtained for 20 days.
Several protein transduction domains are known to have the ability to pass through biological membranes. One such peptide is HIV-1 TAT. In this study TAT was evaluated for its ability to carry Dox into Dox resistant MCF-7 tumor cells. Dox peptide conjugate was more potent than free drug. The concentration of drug in resistant cancer cells was increased indicating a partial reversal of drug resistance.
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Barbaro, Elena <1985>. "Tossine in matrici ambientali : quantificazione mediante metodi accoppiati HPLC/MS ad elevata sensibilità." Doctoral thesis, Università Ca' Foscari Venezia, 2013. http://hdl.handle.net/10579/2998.
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Lo scopo di questa tesi dottorato consiste nello sviluppo e nella convalida di metodi analitici HPLC-MS/MS,
volti alla determinazione di tossine algali in diverse matrici ambientali. In particolare l’attenzione si è
focalizzata sulle tossine algali d’acqua dolce, le cianotossine, e una tossina marina, l’acido domoico,
andando a sviluppare delle procedure pre-analitiche per la determinazione di queste sostanze in tre diversi
comparti ambientali: il comparto idrico, dove queste sostanze vengono emesse, i molluschi, organismi che
ne possono essere contaminati, e l’aerosol atmosferico, al fine di studiare eventuali trasferimenti dal
comparto idrico all’atmosfera.
Infine è stato studiato un metodo alternativo per la determinazione di una tossina alimentare, l’ocratossina A,
mediante la tecnica ifenata HPLC-ICP-MS.<br>The aim of this PhD thesis was to develop analytical methods using liquid chromatography coupled with a
triple quadrupole (HPLC/MS/MS) to determine algal toxins in some environmental matrices. Cianotoxins,
freshwater toxins, and domoic acid, a marine toxin, have been investigated in three different environmental
compartments: water, atmospheric aerosol and mussel tissues. Another method to determine ochratoxin A, a
potent food toxin, was studied using HPLC-ICP-MS.
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Thele, Marc. "A contribution to the modelling of the charge acceptance of lead-acid batteries - using frequency and time domain based concepts." Aachen Shaker, 2007. http://d-nb.info/991509978/04.
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Christianson, Carl Victor. "Understanding The Biosynthesis And Utilization Of Non-Proteinogenic Amino Acids For The Production Of Secondary Metabolites In Bacteria." Thesis, Boston College, 2008. http://hdl.handle.net/2345/967.
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Thesis advisor: Steven D. Bruner<br>Bacteria utilize complex enzymatic machinery to create diverse secondary metabolites. The architectural complexities of these small molecules are enhanced by nature’s ability to synthesize non-proteinogenic amino acids for incorporation into these scaffolds. Many of these natural products are utilized as therapeutic agents, and it would be advantageous to understand how the bacteria create various non-natural amino acid building blocks. With a greater understanding of these systems, engineering could be used to create libraries of potentially useful natural product analogs. The tyrosine aminomutase SgTAM from the soil bacteria Streptomyces globisporus catalyzes the formation of tyrosine to generate (S)-B-tyrosine. The precise mechanistic role of MIO in this novel family of aminomutases has not been established. We report the first X-ray crystal--> structure of an MIO based aminomutase and confirm the structural homology of SgTAM to ammonia lyases. Further work with mechanistic inhibitors provide structural evidence of the mechanism by which MIO dependent enzymes operate. We have also investigated LnmQ, an adenylation domain in the biosynthetic pathway of leinamycin. Leinamycin is an antitumor antibiotic that was isolated from soil samples in 1989. LnmQ is responsible for the specific recognition of D-alanine and subsequent activation as an aminoacyl adenylate species. We have cloned the gene into a DNA vector and expressed it in E. coli. Upon purification of the protein, crystallization conditions have been tested. Synthesis of an inhibitor that mimics the aminoacyl adenylate product catalyzed by LnmQ has been completed. Crystallization with this--> inhibitor will provide better quality crystals and a catalytically informative co-complex<br>Thesis (PhD) — Boston College, 2008<br>Submitted to: Boston College. Graduate School of Arts and Sciences<br>Discipline: Chemistry
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Lizée, Gregory Anthony. "MHC class I cytoplasmic domain, defining a role for conserved amino acid residues in class I expression, trafficking, and antigen presentation." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape4/PQDD_0020/NQ56582.pdf.
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Klyashtornyy, Vladislav. "Principles of protein nucleic acid : recognition on the examples of the ribosomal protein L1 and the cold shock domain of YB-1 protein." Thesis, Evry-Val d'Essonne, 2012. http://www.theses.fr/2011EVRY0039/document.
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Cette thèse est une étude structurale sur l’interaction entre 2 protéines modèles et des acides nucléiques : la protéine L1 (navette ribosome/ARN messagers) et le sous domaine CSD de YB-1, protéine régulatrice de la transcription et traduction. Deux méthodes sont utilisées : i) diffraction des RX par des cristaux de complexes L1:ARN ribosomal ou messager et ii) modélisation et dynamique moléculaire pour l’interaction CSD avec des homo-ribo- ou homo-deoxyribo-nucléotides simple brin. Les méthodes sont décrites avec leurs forces et limites. Les résultats sur L1-rARN/ARN éclairent les mécanismes de régulation de la traduction en montrant des différences d’affinité pour l’ARN des sous domaines I et II de L1. L’analyse de mutants de L1 dans le site de liaison à l’ARN du sous domaine I éclairent la nature des liaisons non covalentes sous tendant l’affinité de ce sous domaine pour l’ARN et souligne l’importance de la structure de L1, de sa « complémentarité » avec l’ARN et de liaisons hydrogènes non accessibles au solvant. Les travaux de modélisation et de dynamique moléculaire sur l’interaction CSD:acides nucléiques montrent que la séquence nucléotidique module l’affinité de ce complexe, l’oligonucléotide de type oligoG donnant le complexe le plus stable suivi des séquences de type oligoU et oligoA puis des oligoT et oligoC. L’orientation du brin d’ARN par rapport au CSD impacte aussi la stabilité du complexe. Une analyse des surfaces d’interaction et de la nature des liaisons intermoléculaires, montre que des principes similaires guident l’interaction L1:ARN et CSD:acides nucléiques : géométrie complémentaire des partenaires et liaisons hydrogène protégées du solvant<br>This thesis is a structural study on the interaction between two model proteins and nucleic acids: the L1 protein (shuttle ribosome/mRNA) and the CSD subdomain of YB-1, a protein that regulates transcription and translation. Two methods are used: i) X-ray diffraction by crystals of L1:ribosomal or messenger RNA complexes and ii) molecular modeling and dynamics for the CSD interaction with homo-ribo or homo-deoxyribo- single-stranded nucleotide. The methods are described with their strengths and limitations. Results on L1-rARN/ARN enlighten the mechanisms regulating translation by showing differences in affinity for RNA of the subdomains I and II of L1. Analyses of L1 mutants in the RNA binding site from the subdomain I illuminate the nature of non-covalent bonds subtending the affinity of this subdomain for RNA and stress the importance of the structure of L1, its "complementarity" with RNA and of hydrogen bonds not accessible to the solvent. Molecular modeling and dynamics of the CSD:nucleic acids interaction shows that the nucleotide sequence modulates the affinity of the complex, oligoG giving the most stable complex followed by oligoU and then oligoA or oligoT and oligoC. The orientation of the RNA strand relative to the CSD also impacts the stability of the complex. An analysis of the interaction surfaces and of the nature of intermolecular bonds, shows that similar principles guide the L1: RNA and CSD: nucleic acids interactions, i.e. a complementary geometry between partners and presence of hydrogen bonds protected from the solvent
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Thele, Marc [Verfasser]. "A contribution to the modelling of the charge acceptance of lead-acid batteries – using frequency and time domain based concepts / Marc Thele." Aachen : Shaker, 2008. http://d-nb.info/1161306323/34.
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Matthews, Sam S. "Investigation into the Effects of PEGylation on the Thermodynamic Stability of the WW Domain." BYU ScholarsArchive, 2013. https://scholarsarchive.byu.edu/etd/4280.
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The covalent attachment of poly(ethylene glycol) (PEG) to a protein surface (known as PEGylation), has been demonstrated to increase the serum half-life of therapeutic proteins by reducing kidney clearance and immunogenicity and by protecting against proteolysis. Theses beneficial effects could be further enhanced if PEGylation consistently increased protein conformational stability (i.e. the difference in free energy between the folded and unfolded states). However, the effects of PEGylation on protein conformational stability are unpredictable; PEGylation has been reported to increase, decrease, or have no effect on the conformational stability of medicinal proteins.This thesis details the results of two studies aimed at discovering the structural determinants which influence the thermodynamic impact of PEGylation on the WW domain, a small model protein. Chapter 1 is a brief introduction to protein therapeutics and protein PEGylation. Chapter 2 describes a study which demonstrates that the thermodynamic impact of PEGylation is strongly dependent on the site to which PEG is conjugated. The studies described in Chapter 3 elaborate on this site dependence, and demonstrate that PEG stabilizes the WW domain through interactions with the surface of the folded peptide, and that two factors – the orientation of the PEG chain (relative to the protein surface) and the identity of nearby side chains – play a critical role in determining the thermodynamic impact of PEGylation.
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Annoni, C. "NEW DIMENSIONS INTO PROTEIN-NUCLEIC ACIDS INTERACTIONS." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/232405.
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In the late 19th century, scientists microscopically observed the association of proteins with DNA strands. Since then, researchers have used a variety of in vitro and in vivo assays to demonstrate that proteins interact with DNA and RNA to influence the structure and function of the corresponding nucleic acid. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination and RNA processing and translocation continues to revolutionize our understanding of cell biology, normal cell development and the mechanisms of disease. Furthermore, there is the potential for constructing new molecules with excellent functionalities by assembling the very simple elements and components that are the functional subunits of these natural biopolymers. In this work of thesis we attempted making a new step forward a better knowledge of protein-nucleic acid biopolymers, focusing on two complexes of strong interest. One of the important factors for determining the expression of the function is the structure of the biopolymer. Clarification of the relationship between the structure and the function of biopolymer is of crucial importance to the development of methods for constructing tailor-made functional molecules. The first goal of my thesis focalized on the obtainment of a structural model of a well-known protein-DNA complex: Maf DNA binding domain and it DNA target (T-MARE was used in this study) with the aim of outlining a strategy for the obtainment of activity modulators. Both in silico simulations and experimental studies were carried out leading to the definition of a modus-operandi based on a disorder-order transition of the complex which is commendatory for the protein activation. On the other hand, a number of artificial enzymes have been recently constructed by using the molecular design based on structural information, screening methods that utilize libraries or by a combination of these two methods. However, the above mentioned approaches use single proteins or single nucleic acid as the structural unit, and the activity of the constructed artificial enzymes is remarkably lower in many cases, than that of the native enzymes. The construction of functional complexes (rather than a single biopolymer) as scaffold can be considered as one potential solution to these drawbacks. Further in my studies, using the HIV-Rev peptide and RRE (Rev Responsive Element) RNA complex as a scaffold, for which the tridimensional structure was fully characterized, the assemble of ribonucleopeptidic fluorescent sensors was accomplished in a stepwise manner. In this method, a randomized nucleotide sequence was introduced into the RNA subunit of RNP to construct a RNP library on which the in vitro selection method was applied. In the second step, the Rev peptide was modified with the fluorophore without altering the affinity and specificity of the RNP receptor. In the absence of a ligand for RNP, fluorescence emission was effectively quenched in the RNP complex, but recovered upon ligand binding. RNP sensors were thus created, as the ligand-binding event can be monitored by measurement of the fluorescence signals.
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Max, Klaas. "Structural and functional studies on bacterial cold shock proteins nucleic-acid binding, the architecture of a domain swap, and mechanisms contributing to their stabilization /." kostenfrei, 2007. http://www.diss.fu-berlin.de/2007/25/index.html.
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