Дисертації з теми "Amphitrione"

The focus of this research was to determine how three different factors affect the kinetics of the reaction between Dehaloperoxidase ? Hemoglobin (DHP-Hb), a halogenated phenol substrate, and a peroxide co-substrate. The factors examined were the order of addition of the substrate and co-substrate, the pH of the solutions, and the difference between using hydrogen peroxide (H2O2) and m-Chloroperbenzoic acid (MCPBA) as the co-substrates. The order of addition studies were carried out on a stop-flow apparatus. The results presented here demonstrate that the substrate must bind to DHP-Hb prior to the addition of the peroxide co-substrate. The effect of pH was examined using stop-flow and photodiode array spectrometry. These studies show how the pKa of the substrate affects the mechanism of the reaction. The kinetic results actually depend on the pKa of the substrate used. Stop-flow studies and UV-Vis assays were used to compare H2O2 and MCPBA as co-substrates. These studies demonstrate that the use of MCPBA as the co-substrate yields a faster rate of product formation. The use of H2O2 as the co-substrate results in a larger amount of product formation at all pHs studied, with the exception of pH 6.0.

The enzyme dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a heme protein, which has a globin fold, but can function as both a hemoglobin and a peroxidase. As a peroxidase, DHP is capable of converting para-halogenated phenols to the corresponding quinones in the presence of hydrogen peroxide. As a hemoglobin, DHP cycles between the oxy and deoxy states as it reversibly binds oxygen for storage. Herein, we report that the distal histidine shows a large conformational flexibility in the deoxy form. Crystals of deoxy ferrous DHP were obtained by reducing the ferric wild-type DHP in sodium dithionite solution and the structure was determined at 100K to a resolution of 1.22Ã. The heme iron in the deoxy ferrous DHP is five-coordinate and has an out-of-plane displacement of 0.23 Ã for the heme iron relative to the oxy form. The distal histidine, H55 is observed in conformations, which are analogous to the open and closed forms of myoglobin. In the closed conformation, H55 is located inside the distal pocket, but does not penetrate as deeply into the distal pocket as in the metaquo ferric or oxy ferrous structures. This observation is consistent with the hypothesis that H55 interacts with heme iron ligands through hydrogen bonding in the closed conformation. There are two open or solvent-exposed conformations, in which H55 is more than 9.5 Ã away from the heme. The comparison of the deoxy structure with the other structures provides new insight into the correlation between the heme iron ligation and the conformation of distal histidine in the DHP.

Biofouling is the accumulation of sessile marine organisms, such as algae, tube worms and barnacles on man-made substrata and has negative economic and ecological implications. Ship hulls are readily fouled, which significantly increases drag while decreasing ship fuel efficiency when moving through water. Fouled hulls have also become important vectors of invasive species. These problems are minimized when hulls are painted with a toxic anti-fouling or non-toxic foul-release coating. Due to recent restrictions of anti-fouling paint use, research and development of non-toxic alternatives has increased. Novel hull coating efficiency is often quantified by the critical removal stress value of barnacles from the coating. Barnacle adhesive cement protein content is thought to be responsible for barnacles’ incredible ability to adhere underwater. The expression level and type of adhesive proteins has eluded scientists due to their extreme insolubility within cured barnacle cement. Identification of these proteins may provide insight to the adhesion of fouling species and aid coating development. Barnacles are a cosmopolitan organism, able to withstand a wide range of environmental conditions, yet foul-release coating research had not previously incorporated environmental factors as variables in determining coating performance. Temperature is known to affect protein structure and function and is also a formative factor of barnacle larvae survival and development. Even so, the interaction between temperature and barnacle adhesion to has not previously been explored. We examined the effect of temperature on barnacle adhesion to foul-release coatings. After observing differences in critical removal stress due to temperature, we attempted to attribute these differences to specific proteins within the adhesive using 2D SDS PAGE. Gel image analysis determined that there were significant differences in cement protein expression between barnacles raised within different temperatures. Preliminary protein identification with Mass Spectronomy (MALDI TOF/TOF) was performed, however further research and a larger barnacle genomic database is needed to elucidate barnacle cement protein sequences.

Despite being a model organism to study settlement in marine invertebrates, little is known about the genetics of the barnacle Balanus amphitrite. To fill this gap, cDNA libraries representative of different developmental stages were generated and sequenced. Nearly 14,000 genes were annotated, which may represent 2/3 of the species’ total protein coding regions. The database that was created to allow public access to this genetic information will profoundly benefit future research aiming to understand the molecular regulation of development and settlement in this species. Furthermore, a quantitative real-time PCR assay to study gene expression in B. amphitrite was designed and validated. Eleven genes were studied for their ability to normalize qRT-PCR data. Total RNA extracted from seven developmental stages was reverse transcribed and the expression stability of the selected genes was compared. It was found that transcripts encoding cytochrome b and NADH dehydrogenase subunit 1 were expressed most stably, and their use to normalize gene expression data is recommended. Conflicting evidence exists on the role of bacteria in B. amphitrite settlement. However, there is a paucity of information on the microbial community naturally associated with this barnacle. In order to reveal the existence of stable associations, a 16S rRNA-based, taxon-specific qPCR assay was developed to monitor the preponderance of 5 bacterial phyla and classes. Furthermore, attempts to profile these qPCR products by DGGE were made. This new method was applied to characterise the bacterial communities associated with different B. amphitrite developmental stages and body parts. It was found that the structure of these communities changed throughout the barnacle life cycle in a highly reproducible manner. Furthermore, bacteria isolated from the barnacle shell were capable of inducing settlement of conspecific larvae. The analysis of these communities at a lower taxonomic level should confirm if any of these ecologically important bacteria are vertically transmitted.

Barnacles are dominant hard–fouling organisms in marine waters. They attach to substrates by secreting a complex proteinaceous adhesive. Understanding the chemical composition of this multi–protein underwater adhesive and how it is affected by environmental variables, such as oceanic temperatures, is critical for developing nontoxic solutions to control biofouling. Previous experiments in our lab revealed an inverse relationship between critical removal stress (CRS) and temperatures at which barnacles were reared. Further investigations showed that this correlation is not attributed to differences in physical properties such as barnacle size or short–term changes in the viscosity of adhesive. Therefore, the observed effects may be influenced by a physiological response to temperature during initial growth and development. We hypothesized that rearing temperature affects the expression of proteins found in the adhesive matrix. To elucidate the underlying mechanisms responsible for the temperature effect, we analyzed uncured barnacle adhesive using two-dimensional gel electrophoresis (2DGE) and matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS). In our analysis, we 1) detected differences in protein expression at two experimental temperatures (15°C and 25°C) and 2) identified several proteins that may serve functional roles in the process of adhesion. Our data are also consistent with a model that the curing process of barnacle adhesive may be analogous to the process of wound healing in animals.

Many barnacle species are gregarious and their cypris larvae display a remarkable ability to explore surfaces before committing to permanent attachment. The cuticular tissue of adult barnacles contains an α2-macroglobulin-like glycoprotein contact cue referred to as the settlement-inducing protein complex (SIPC) which increases the attractiveness of surfaces and signals cyprids to settle. Despite decades of research into marine fouling and the development of anti-fouling systems, detailed knowledge of the biochemical and structural composition of marine pheromone cues is poor. This cue is key to barnacle gregarious settlement and represents an attractive target for custom synthesis of antagonistic surfaces. Using the tropical acorn barnacle Balanus amphitrite, this project endeavoured to characterise the structure and glycan moiety of the SIPC. The SIPC active fraction was purified by ion exchange chromatography and gel filtration and detected through SDSPAGE gel antibody immunoblotting. The carbohydrate structure was characterised using a combination of hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-fluorescence) and exoglycosidase digestions. This provided evidence of predominantly oligomannose glycans with the occurrence of monofucosylated oligomannose glycans in lower proportions. The characterisation of high mannose glycosylation is supported by observations on the effect of mannose in solution increasing settlement in B. amphitrite cypris larvae. Protocols to create surface bound carbohydrate-functionalised polymers were successfully developed and confirmed using X-ray photoelectron spectroscopy, ellipsometry and contact angle measurements. These surfaces were shown to act as SIPC mimics, cueing settlement on contact. The SIPC is known to contain seven potential N-glycosylation sites. Additional work using transmission electron microscopy and atomic force microscopy has further enhanced understanding of the glycoprotein structure. Obtaining complete structural characterisation of the SIPC remains a goal that has the potential to inspire solutions to the age-old problem of barnacle fouling.

La tesi indaga la civiltà dello spettacolo ferrarese sotto Ercole I d’Este, un sovrano che controlla, promuove e dirige la vita festiva e cerimoniale con fermezza secondo un preciso disegno politico. Con il sussidio di un’ampia gamma di fonti cronistiche, epistolari, archivistiche, letterarie e iconografiche, il lavoro tenta di ricostruire le fonti di ispirazione, la trama simbolica e allegorica del progetto teatrale erculeo e le realizzazioni materiali di alcuni spettacoli. Il focus si concentra su un aspetto ancora non indagato in profondità, perché dato per scontato o ingiustamente ritenuto estraneo all’ambito delle discipline dello spettacolo: la ricezione attiva di Plauto e Terenzio ma anche di Luciano o Ovidio e il loro specifico riuso nei volgarizzamenti commissionati dal Duca agli intellettuali della sua corte. Intrecciando le prospettive tradizionali della filologia classica con quelle teatrologiche si indagano, cioè, i modi attraverso i quali si assimila a più livelli e si attualizza espressivamente il patrimonio ritrovato della cultura classica identificando in questo passaggio la cifra costitutiva della scena erculea, l’aspetto che, in altre parole, maggiormente la definisce e la distingue da altre realtà coeve. Il lavoro è articolato in due parti precedute da un’introduzione, nella quale si fissano alcuni punti nodali relativi al contesto ferrarese tardo-quattrocentesco. Ai nastri di partenza si colloca l’illustre umanista Guarino Veronese, decisivo protagonista, con il suo magistero, nell’esegesi e nella diffusione dei capolavori comici latini, che imprime un forte impulso riformista alla vita culturale estense, arrivando a rivoluzionare il concetto stesso di princeps e di corte. Dallo Studio alla città: un Apologo di Pandolfo Collenuccio è il punto di avvio di una riflessione teorica intorno alla categoria estetica di comico e di commedia; quindi, si guarda al laboratorio teatrale erculeo nei suoi tratti ibridi e polimorfi, quale spazio privilegiato di incontro tra l’eredità romanza della cultura volgare e i classici da poco riscoperti dagli umanisti. L’indagine è condotta attraverso una campionatura di episodi festivi e di documenti estrapolati come più significativi. La prima sezione esplora la festa per le nozze di Alfonso d’Este, primogenito di Ercole ed erede al soglio ducale, e Anna Sforza, nipote di Ludovico il Moro e sorella del duca nominale di Milano Gian Galeazzo Maria Sforza, ricostruita alla luce di evidenze documentarie inedite. Il confronto tra l’ingresso trionfale della sposa a Ferrara, avvenuto il 12 febbraio 1491, e la speculare entrata processionale degli Este a Milano del 22 gennaio dello stesso anno, rivela con chiarezza le differenze tra le rispettive tattiche promozionali di Ercole I e Ludovico il Moro. Il complesso cerimoniale architettato da Ercole per la giornata conclusiva dei festeggiamenti costituisce da questo punto di vista una sorta di prototipo delle sue strategie mitopoietiche con cui l’immagine del principe e della corte vengono trasfigurate tra teatro, iconografia e letteratura. L’Amphitrione di Pandolfo Collenuccio, andato in scena con tutta probabilità proprio in quella circostanza, funge da ponte con la seconda sezione della ricerca, dedicata ai volgarizzamenti plautini. La scelta è ricaduta su quelli di sicura provenienza ferrarese: oltre al volgarizzamento collenucciano dell’Amphitruo, rientrano nel gruppo la Cassina e la Mustellaria di Girolamo Berardo e l’Asinaria finora ritenuta anonima e della quale si tenta un’attribuzione. A questo insieme si è aggiunta la Vita de Iosep andata in scena a Ferrara nel 1504, un singolare esperimento di “commedia religiosa” ascrivibile a Collenuccio che assimila il duca estense alla figura del patriarca biblico Giuseppe. L’indagine sui singoli testi si svolge lungo due principali direttrici: A. L’analisi dei motivi tematici. Il filtro culturale imposto dalla scrittura in volgare condiziona le originarie tematiche plautine, di cui naturalmente il volgarizzamento fornisce una declinazione sua propria. L’obiettivo è valutare l’originale apporto dell’autore, che non si limita a ricevere e rielaborare attivamente gli spunti plautini, ma ne aggiunge anche di nuovi, provenienti dal repertorio novellistico-popolare. B. L’interpretazione delle modifiche “strutturali” apportate dai volgarizzatori al dettato plautino. In questa fase si cercano di individuare e analizzare i moduli del testo che ricorrono spesso in corrispondenza di situazioni in cui venga alterato significativamente il testo di Plauto, come ad esempio laddove viene inserita una fine d’atto diversa da quella dell’originale (reimpostando di conseguenza la sequenza fine di atto–inizio del successivo secondo una serie di schemi che hanno tutta l’aria di essere considerati dai traduttori come “canonici”) o in corrispondenza delle sequenze testuali lacunose, mutile o molto problematiche dell’originale. Si tratta di passi che per forza di cose richiedevano un considerevole lavoro di rimaneggiamento (quando non di scrittura ex novo) per poter essere adattati alle esigenze della rappresentazione. In gran conto è tenuta l’eventuale presenza di un codice formulare che descriva il movimento e le forme di interazione dei personaggi sul palco, per noi potenzialmente utilissimo in qualità di porta di (parziale) accesso all’azione scenica.

碩士
國立中山大學
海洋生物研究所
98
Barnacles are mostly sessile, hermaphroditic, internal-fertilizing and usually non-selfing crustaceans. They have the longest penis length, relative to body length, among all organisms. Because of their immobility, barnacles have to extend penes to reach mates and transfer the sperm. The longer the penis the more mates they can reach. However, maintaining long penes may be costly. How do barnacles allocate the reproductive investment and adjust the length of penes? Previous researches indicated that wave actions influenced the penis length of barnacles. Here we explore if biological factors are also involved in determining the penis length. Our hypotheses are that the penis length of Balanus amphitrite may be determined by (1) the presence of neighbors, (2) neighbor numbers, and (3) neighbor distance. Experimental results indicate that the neighbor distance could influence the penis length of B. amphitrite, and the penis length increase with increasing distance of neighbors. The neighbor numbers were not found to influence the penis length. In the treatment of lone individuals, the penis lengths first increased and then decreased, and they are significantly shorter than those in the treatment with neighbors. When neighbors exist, B. amphitrite detects the density by distance of neighbors. The greater the distance to neighbors, the lower the densities of neighbors. Because of the mates are enough to mate, the penes length decrease with increasing density to save cost. When neighbors do not exist, B. amphitrite may save cost by decreasing or not investing pens length . Our result shows that the plasticity of the penis length of B. amphitrite is an adaptation by natural selection.

碩士
國立中山大學
海洋生物科技暨資源學系研究所
104
Biofouling is a serious global issue of our oceans. Marine fouling organisms can attach on immersed artifical surfaces thus thus cause economic loss. This study utilized the major fouling barnacle species Amphibalanus amphitrite to test anti-fouling activities of more than one hundred natural compounds, including crude extracts, pure compounds and the derivatives isolated from marine organisms. There are more than one hundred natural compounds isolated from marine organisms and the derivatives were screened. There are three steps in our experiments: (1) Preliminary test: we prepared all natural compounds and the derivatives at the concertation of 10 ppm or 20 ppm and recorded the settlement condition of barnacle cypris larvae for 48 hours. (2) Antifouling activity test: we tested antifouling activities of potential compounds from step 1 at concentrations from 1-100 ppm. Then we estimated half maximal effective concentration (EC50), half lethal concentration (LC50) and therapeutic ratio (TR) of each compound. (3) Field test: we mixed the candidate compound with marine paint, then observed actual effect of the antifouling paint in the field. Our results showed that the pure compound S28 has high antifouling activity. The EC50 of S28 is 8.824 ppm, LC50 is 101.080 ppm and the therapeutic ratio (TR) is 11.454. These results suggested S28 is effective and has low toxicity. In conclusion, S28 has great potential for future development of antifouling paint.

The present study addressed the need to understand how short-term variations in metal concentrations in the environment determine its concentrations in a biomonitor, and how this information affects the use of the biomonitor in environmental monitoring programs. As a case study, the barnacle biomonitor Balanus amphitrite present in Ross Creek (Townsville, Queensland, AU) and the heavy metal Cd were used. The research methodology for this study comprised three integrated approaches: field measurements; the performance of laboratory experiments, and the development of an ecotoxicological simulation model, in order to understand the processes controlling Cd accumulation in Balanus amphitrite in the field. Two sampling programs were carried out along Ross Creek, in the dry season of 2002 and the wet season of 2004, in which barnacles, their food sources (two class sizes of suspended particulate material, SPM, and microzooplankton) and water (dissolved phase) were sampled weekly for Cd concentrations and mass abundances. Sampling periods were selected to test whether the concentration of Cd in the biomonitor responded to any variation in the dissolved and particulate phase Cd concentrations in Ross Creek, as caused by rainfall variation. In both sampling periods, the Cd concentration in the dissolved phase increased upstream, ranging from 1.6 to 283 ngl,", The Cd concentration in the barnacle's food sources exhibited the same pattern - ranging from <0.01 to 2.10 mg kg" for the small size class of SPM (0.4550 um), from 0.07 to 1.62mg kg" for large SPM (50-200 um), and from 0.03 to 0.80 mg kg-I for microzooplankton (50-200 um). The Cd concentration in two populations of Balanus amphitrite increased upstream between two sites 2.20 km apart and ranged from 2.15 to 6.40 mg kg" and from 5.22 to 12.8mg kg-I. Even though no significant temporal variation was detected for the Cd sources to the barnacles, the biomonitor Cd concentrations varied over the three sampling months, within each sampling period, exhibiting specific patterns for this variation. These observations suggest that changes in the Cd concentrations in the food sources and the relative mass abundance of these sources may result in a specific Cd concentration in Balanus amphitrite. Similar Cd concentrations, within sites, were observed for the particles between the dry and wet seasons. Only the most contaminated site exhibited significant differences in the dissolved Cd concentration between seasons. Because more than 95% of the total Cd in the Ross Creek water <200 um) was in the dissolved phase <0.45 um), the differences in the dissolved Cd concentration resulted in the barnacles from the most Cd-contaminated site being exposed to a total Cd concentration in the wet season (45.8 ng L-I) that was a half of that in the dry season (91.6 ng L-I) . Such Cd differences were not indicated by the biomonitor whose Cd concentration did not vary significantly between dry (8.4 mg kg") and wet (7.4 mg kg") seasons. A budget analysis based on Thomann's bioenergetic kinetic model, indicated that Cd flux from food contributes >80% of the Cd concentration in Balanus amphitrite. Thus, because no significant variation was identified for the Cd concentration in the food, no variation in the Cd concentration in the biomonitor was observed at the most contaminated site between seasons. A sensitivity analysis on the model showed that physiological characteristics of the biomonitor are the key parameters controlling Cd accumulation in Balanus amphitrite, rather than the metal concentration in the dissolved or particulate phases. This, coupled with the fact that the Cd flux from food is the major source of Cd to Balanus amphitrite suggests no tight coupling between Cd in the biomonitor and its availability in the environment. A simulation model was developed based on Thomann's bioenergetic kinetic model. The daily-simulated Cd concentration in Balanus amphitrite produced by the model reproduced the general trend observed in the field. However, even though high and low patterns of Cd concentration in this organism could be reproduced by the model, it could not reproduce the short-term temporal variations accurately. A model investigation suggested that variations in the mean weight of the sampled barnacles might mask the real pattern of temporal variation of the barnacles Cd concentration; even though no size effect has been identified in the field data. Two simulation exercises indicated that Balanus amphitrite may present some weakness in indicating temporal variations in Cd concentrations in the environment. The model results suggested that this organism could not indicate a 6-month Cd-pulse in the environment that increased the Cd concentration in its main source (small SPM, 0.45-50 um) by a factor of 2.8 using a realistic sampling effort. In addition, this organism took more than a year to reach equilibrium for its Cd concentration in a simulated relocation experiment. These problems may be critical for the use of Balanus amphitrite as a biomonitor, and suggest that this organism can only provides a poor measure of current bioavailability of the metal in the environment. However, if a long-term mean Cd availability in the particulate fraction (sized <200 um) is required, Balanus amphitrite can provide such an information.