Typescript. Includes vita and abstract. Bibliography: Includes bibliographical references (leaves 172-183). / Cancer magister, the Dungeness crab, occurs in different habitats during its life cycle, habitats which vary widely in the magnitude of salinity and temperature changes. Cancer magister hemocyanin also changes in structure and oxygenation properties during development. The following question was considered in this thesis: what are the effects of environmental salinity and temperature on metabolic rates, ionic and osmotic regulation and hemocyanin oxygen affinity in Q. magister during development. Metabolic rates and hemolYmph ionic and osmotic concentrations were measured in the megalopa, 1st juvenile, 5th juvenile and adult crab eight hours after acute exposure to 100% seawater (=32 ppt), 75% seawater and 50% seawater at both 10°C and 20°C. The oxygen binding properties of the whole hemolymph from these stages in 100% seawater at 10°C was determined. The effects of calcium and magnesium on the oxygen affinity of purified hemocyanin from different stages were also determined. In 100% seawater, routine metabolic rates of the four stages scale with body mass over the size range, 0.05 gm to 500 gm. The Q10 (10°C to 20°C) for the megalopa is higher in 75% seawater and 50% seawater than in 100% seawater. For the 1st juvenile, 5th juvenile and adult the Q10 values (10°C to 20°C) are independent of salinity. The megalopa, 1st juvenile and 5th juvenile are weaker regulators of hemolymph chloride, sodium and osmotic concentrations than the adult. The megalopa and adult, unlike the 1st juvenile and 5th juvenile, strongly regulate hemolymph calcium in reduced salinity. In 100% seawater hemolymph magnesium is significantly higher in the megalopa, 1st juvenile and 5th juvenile than in the adult. The oxygen affinities of whole hemolymph from the four stages are indistinguishable when adjusted for endogenous L-lactate concentrations; the Bohr coefficients are not significantly different among stages. The effect of magnesium on oxygen affinity of purified adult hemocyanin is influenced by proton concentration; the effect of calcium is independent of proton concentration. In 100% seawater, endogenous inorganic ion concentrations in the whole hemolymph of the various stages reduce the intrinsic stage specific differences in hemocyanin oxygen affinity.

Typescript. Includes vita and abstract. Bibliography: Includes bibliographical references (leaves 54-56). Description: 56 leaves : photos. ; 29 cm.

Hemocyanins (Hcs) and phenoloxidases (POs) are both members of the type-3 copper protein family, possessing di-cupric active sites which facilitate the binding of dioxygen. While Hcs and POs share a high degree of sequence homology, Hcs have been associated traditionally with oxygen transport whereas POs are catalytic proteins with a role in innate immunity. Evidence gathered in recent years details numerous immune functions for Hc, including an inducible PO activity. Unlike the pro-phenoloxidase activation cascade in arthropods, the endogenous mechanism(s) involved in the conversion of Hc into an immune enzyme is lacking in detail. The overall aim of this research was to characterise the physiological circumstances in which Hc is converted into a PO-like enzyme during immune challenge. A series of biochemical, biophysical and cellular techniques were used to assess the ability of phospholipid liposomes to mimic the well-characterised induction of PO activity in Hc by SDS micelles. Incubation of Hc purified from Limulus polyphemus, in the presence of phosphatidylserine (PS) liposomes, yielded ~ 90% of the PO activity observed upon incubation of Hc with the non-physiological activator, SDS. Phospholipid–induced PO activity in Hc was accompanied by secondary and tertiary structural changes similar to those observed in the presence of SDS. Subsequent analysis revealed that electrostatic interactions appear to be important in the PS-Hc activation complex. In vivo, PS-Hc interactions are assumed to be limited in quiescent cells. However, amebocytes undergoing apoptosis redistribute PS onto the outer leaflet of the plasma membrane, resulting in the potential for increased Hc-PS interactions. In the absence of a reliable culturing technique for L. polyphemus amebocytes, in vitro conditions were optimised for the short term maintenance of this labile cell type. Amebocytes retained viability and functionality in a medium that mimicked most-closely, the biochemical properties of L. polyphemus hemolymph. When presented with a fungal, bacterial or synthetic challenge, ~9% of amebocytes in vitro were found to be phagocytically active. Target internalisation was confirmed via the use of fluorescent quenchers and membrane probes. Within 4 hours of target internalisation, amebocytes underwent apoptosis, characterised by the loss of plasma and mitochondrial membrane potential, increased caspase-3 activity and extracellularisation of PS. Phagocytosis-induced cell death led to a proportional increase in the level of Hc-derived PO activity, suggesting that Hc may be interacting with PS present on terminal amebocyte membranes. The PO activity of Hc was investigated further in order to address an economically important issue; hyperpigmentation in commercial shellfish. While PO enzymes are thought to be the cause of hyperpigmentation in Nephrops norvegicus, evidence presented here suggests that cellular PO is inactivated after freeze-thawing, while extracellular Hc retains stability and displays a heightened level of inducible PO activity under similar treatments. Known PO inhibitors were used successfully to reduce Hc-derived PO activity, with inhibitors assumed to bind Hc in a manner similar to PO-inhibitor complexes. Structural and functional studies of hemocyanins and immune cells presented here provide new insights into the interactions of hemocyanin-activator complexes in invertebrates.

Le bruissement enzymatiques, appelé mélanose post mortem chez les crustacés est un phénomène enzymatique catalysé par des protéines à activités phénoloxydases (tyronase, catécholase, laccase et hémocyanine). L'utilisation de conservateurs de type sulfites (E220 à E228 et E539) reste à l'heure actuelle la solution la plus répandue pour éviter le développement de cette coloration peu attrayante pour le consommateur. Mais une partie de la population développe des réactions d'hypersensibilité en consommant des aliments sulfités. Dans l'onbjectif de rechercher une alternative à ces conservateurs, deux axes de recherche ont été développés durant ces travaux de thèse : la caractérisation biochimique des protéines responsables de la mélanose post mortem chez la crevette P. monodon, puis la recherche de molécules inhibitrices. Un fractionnement sur résine Phenyl Sepahrose™ CL-4B (HIC) suivie d'une séparation par électrophorèse SDS-PAGE ont montré la présence de trois protéines de 46, 82 et 89 kDa à activité principalement laccase. Une identification par RP-HPLC-Q/TOF a mis en évidence la présence d'hémocyanine uniquement. Un pH de 7,0 et une température comprise entre 37 et 50 °C ont mis en évidence les activités les plus importantes, en utilisant le dosage enzymatique dit "test au MBTH". Par ailleurs, un criblage à haut débit de 45 molécules potentiellement inhibitrices a été réalisé dans des conditions d'analyses standardisées grâce à l'outil de robotique de la plateforme Realcat. Une inhibition a été mise en évidence pour 23 composés, certains étant suffisamment efficaces pour être utilisés seuls. D'autres pourraient être introduits dans un cocktail de molécules inhibitrices aux fonctionnalités complémentaires. Les résultats des tests de trempage réalisés sur des crevettes entières ont montré qu'il était indispensable de compléter les études in vitro avec des essais à l'échelle de la matrice alimentaire dans son intégralité. / Enzymatic browning, called post mortem melanosis in crustaceans, is an enzymatic phenomenon catalyzed by proteins with phenoloxidase activities (tyronase, catecholase, laccase and hemocyanin). The use of sulfite preservatives (E220 to E228 and E539) remains at present the most widespread solution to avoid the development of this unattractive color towards consumers. But, a part of the population develops hypersensitivity reactions by consuming sulfited foods. With the objective to find an alternative to these conversators, two research axes have been planned : the biochemical characterization of the proteins responsible for post mortem melanosis in the P. monodon shrimp, then the search for inhibitory molecules. Fractionation on Phenyl Sepahrose™ CL-4B resin (HIC) followed by SDS-PAGE electrophoresis separation showed the presence of three proteins of 46, 82 and 89 kDa with mainly laccase activity. Identification by RP-HPLC-Q / TOF revealed the presence of hemocyanin only. A pH of 7.0 and a temperature between 37 and 50 °C showed the most important activities, using the enzymatic assay called "MBTH test". On the other hand, a high throughput screening of 45 potentially inhibitory molecules could be performed under standardized analysis conditions thanks to the robotic tools of the Realcat platform

Typescript. Includes vita and abstract. Bibliography: Includes bibliographical references (leaves 54-60). Description: xi, 60 leaves : ill. ; 29 cm.

Arthropod hemocyanin and phenoloxidase are members of a group of proteins called the Type-3 copper oxygen-binding proteins, both possessing a highly conserved oxygen-binding site containing two copper atoms each coordinated by three histidine residues (Decker and Tuczek, 2000). Despite similarities in their active site, these proteins have very different physiological functions. Phenoloxidase possesses both tyrosinase and o-diphenoloxidase activity, and is predominantly involved in reactions which protect insects from infection (Kopàcek et al., 1995). Hemocyanin is a large multi-subunit protein with a primary function as a respiratory protein, reversibly binding and transporting molecular O2 (Decker and Rimke, 1998; Decker and Tuczek, 2000). Recently, it has been demonstrated in vitro that arthropod hemocyanin possesses an inducible phenoloxidase activity when incubated with denaturants, detergents, phospholipids or proteolytic enzymes. This activity appears to be restricted to only a few subunit types, and it has been hypothesised that it may be accompanied by conformational change which opens the active site increasing access for larger phenolic substrates (Decker and Jaenicke, 2004; Decker et al., 2001; Decker and Tuczek, 2000). This possibly suggests a dual role of hemocyanin in arthropods. The presented thesis deals with two distinct aims. The first was to isolate and sequence a phenoloxidase gene from the insect Spodoptera littoralis (Egyptian Cottonleaf Worm). Despite efforts, progress was hindered by a number of experimental problems which are outlined within the relevant chapters. The second aim was to characterise the mode of SDS induced phenoloxidase activity in arthropod hemocyanin from the ancient chelicerates Limulus polyphemus (horseshoe crab) and Eurypelma californicum (tarantula) and the more modern chelicerate Pandinus imperator (scorpion), using a number of biophysical techniques. The results indicated that the SDS induced phenoloxidase activity is associated with localised tertiary and secondary conformational changes in hemocyanin, most likely in the vicinity of the dicopper centre, thus enhancing access for larger phenolic substrates. Experiments indicate that copper remains associated with the protein during these structural changes; however the nature of the association is unclear. SDS concentrations approximating the CMC appeared critical in causing the necessary structural changes required for a significant increase in the detectable phenoloxidase activity to be exhibited.