Comme nous l’indiquions dans le But du Travail, le mélanome figure parmi les cancers associés aux pronostics les plus sombres, et ce en raison de son taux de réponse très faible à la radiothérapie et à la chimiothérapie. Cette résistance à la radiothérapie et à la chimiothérapie provient essentiellement du fait que les cellules de mélanomes sont résistantes à l’apoptose, et que la radiothérapie ainsi que bon nombre d’agents chimiothérapiques induisent la mort des cellules cancéreuses en y induisant l’apoptose. Nous avons voulu investiguer les rôles de la gastrine et de la galectine 1 sur le comportement biologique des cellules de mélanomes afin de voir s’il était possible de proposer la gastrine et/ou la galectine 1 comme nouvelles cibles thérapeutiques potentielles dans le cas du mélanome. <p>Notre stratégie de recherche est basée sur le principe (démontré sur le plan expérimental par de nombreuses études) selon lequel les cellules cancéreuses migrantes résistent à l’apoptose, et sont dès lors protégées contre les effets pro-apoptotiques de la chimiothérapie et de la radiothérapie qui représentent la quasi totalité de l’arsenal thérapeutique dont disposent les oncologues pour combattre les cancers. Diverses études expérimentales ont démontré que le fait de réduire le taux de migration de cellules cancéreuses résistantes à l’apoptose conférait à celles-ci une sensibilité accrue aux agents pro-apoptotiques. Nos résultats démontrent que la gastrine modifie de manière très significative les propriétés migratoires des cellules de mélanomes, sans toutefois modifier leur sensibilité à des agents pro-apoptotiques. Au contraire, la gastrine protègerait les cellules de mélanomes contre l’apoptose. Nous démontrons également dans notre travail, in vivo, un rôle pro-angiogénique pour la gastrine au sein de xénogreffes de mélanomes humains. Signalons que notre travail est le premier à démontrer un rôle potentiel de la gastrine au niveau de la biologie des mélanomes, tout au moins sur le plan expérimental. <p>Tout comme nous l’avons observé pour la gastrine, la galectine 1 semble également conférer aux cellules de mélanomes un certain degré de résistance aux agressions chimiothérapiques. Cette fois, le fait de diminuer le taux d’expression de la galectine 1 au sein de cellules du mélanome murin expérimental B16F10 (qui exprime des quantités importantes de galectine 1) renforce l’effet thérapeutique du témozolomide qui est une molécule cytotoxique. Cet effet semble survenir, tout au moins partiellement, suite à une diminution du taux d’expression de la protéine Hsp70 (suite à la diminution du taux d’expression de la galectine 1), avec pour conséquence une augmentation de la mort cellulaire par perméabilisation de la membrane des lysosomes.<p>Nous proposons une nouvelle approche thérapeutique pour combattre les mélanomes en faisant appel à la technique des petits ARN interférants (siRNA), dirigés dans le cas présent contre la galectine 1.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Virtually all extracellular proteins in humans are glycoproteins and likewise are many biopharmaceuticals. The glycosylation is directly correlated to biological function and stability of these proteins. The ability to isolate glycoproteins is thus of great importance in many applications. The most common isolation method for glycoproteins is affinity chromatography using lectins, a ubiquitous and versatile group of carbohydrate-binding proteins. The lectin Concanavalin A (ConA) has long been used for this purpose but suffers from undesired leakage into the eluate, causing an inquiry of alternative chromatography ligands or optimization of the ConA resin.In this study, a total of 20 different lectins, including ConA, were evaluated and compared in terms of suitability as ligands in affinity chromatography for glycoprotein isolation. The lectins’ binding to glycoproteins were studied, mainly through microtiter plate binding assays using a monoclonal IgG1 antibody and Conalbumin (Ovotransferrin). Further, sugar-specificities and potential eluting sugars for the lectins were examined through inhibition with eight different carbohydrates. Additionally, the glycoprotein binding and leakage of ConA columns were examined, and a potential leakagereducing treatment of ConA resin evaluated.ConA was found to be superior in binding to the investigated glycoproteins but exhibited a limited binding when immobilized to an agarose resin. This discrepancy is likely a consequence of structurally hidden glycans on the used glycoproteins and requirements of long residence time when used in a chromatographic setting. Binding competition with several sugars were investigated with a similar microtiter plate binding assay. This method displayed potential to predict the behaviour of sugars and their suitability as eluting agents in a chromatography column. The best eluting sugar for ConA was showed to be methylmannoside, ideally in combination with methylglucoside. Lastly, evaluation of ConA columns with a crosslinking glutaraldehyde-treatment showed that the ConA ligand leakage may be significantly reduced, although further studies and optimizations are needed.This study thus presents a repertoire of lectins and their differences in terms of glycoprotein-binding and sugar-specificity, as well as evaluations of ConA columns’ efficiency and potential leakage-prevention.

Congenital disorders of glycosylation (CDG) are rare multisystem metabolic diseases and their number has rapidly grown in recent years. The clinical manifestation includes very broad spectrum of symptoms. In most of all cases CDG are caused by mutations in genes encoding the enzymes of glycosylation pathway. Based on the type of defect, CDG are divided into the following groups: disorders of N-glycosylation or O-glycosylation of proteins, defects in modification of proteins by GPI anchor, disorders of lipid glycosylation and defects that impact multiple glycosylation pathways. The aim of the thesis was to find new biochemical analyses suitable for diagnostics and characterization of CDG patients. The experimental conditions were optimized for selected markers (Sambucus Nigra (SNA) lectin, proaerolysin (FLAER), antibodies to proteins CD55 and CD59) and the staining was applied to cultivated skin fibroblasts from controls and patients diagnosed with CDG by whole-exome sequencing (ATP6AP1-CDG, PIGN-CDG, SLC10A7-CDG, PISD deficiency). The experiments were performed using flow cytometry (FACS) and fluorescent microscopy (FM). The detection of sialylation by SNA lectin and analysis of the mitochondrial membrane potential changes by a fluorescent labelled probe JC-1 with FCCP simulation of mitochondrial...

En dépit d’importants progrès dans le domaine de la thérapie anti-cancéreuse, les traitements actuels restent controversés, notamment en raison de la quantité importante d'effets secondaires induits. L'immunothérapie ciblée a récemment émergée en tant qu'alternative, afin d'améliorer les modalités de traitement des patients atteints du cancer. Malgré tout, seul un nombre limité d’approches sont aujourd’hui disponibles, et une grande partie des problèmes demeurent actuellement sans solution. C'est dans ce contexte que nous nous sommes intéressés à la conception de structures biomoléculaires innovantes et bifonctionnelles, capables de rediriger des anticorps endogènes, présents naturellement dans la circulation sanguine de l'homme, contre les tumeurs et, ce, sans immunisation préalable. Les anticorps naturels circulant étant polyspécifiques et ayant la capacité d’interagir avec des antigènes glycosylés, nous nous sommes plus particulièrement concentrés sur la conception de glycoconjugués multivalents, ligands d’anticorps endogènes. Une première partie de notre étude a consisté à synthétiser différents glycodendrimères multivalents, reposant sur des châssis peptidiques et obtenus par ligations chimiosélectives, tout en variant la nature du motif glycosylé et des plateformes, ainsi que la valence du conjugué. Puis, dans un second temps, des tests d’interaction par biopuce ont été mis en place avec une lectine modèle, la lectine Helix Pomatia Agglutinin (HPA). Des protocoles expérimentaux visant à calculer des constantes de dissociation de surface, ainsi que des IC50 ont été mis en place, permettant d’identifier de bons ligands de HPA avec des affinités de l’ordre du nanomolaire. Les tests par biopuce ont ensuite été confirmés avec d’autres méthodes d’analyses (BLI, ELLA). Finalement, afin d'identifier des architectures tri-dimensionnelles permettant une affinité optimale avec des anticorps, les tests d’interaction ont été adaptés au criblage de séra humains. Un large panel de glycoconjugués a alors été criblé par biopuce avec une vingtaine de séra, permettant la détermination de structures glycosylés prometteuses, qui pourront par la suite être utilisées dans le cadre de notre approche anti-cancéreuse. / Despite significant progress in anti-cancer therapy, current treatments are still controversial due to numerous side effects. Targeted immunotherapy recently emerged as an ideal alternative to improve treatment modalities for cancer patients. However, very limited approaches are available today and major issues remain to be addressed. In this context, we are interested in the design of biomolecular structures, innovative and bifunctional, able to hijack endogenous antibodies - which are naturally present in the human blood stream - toward cancer cells without pre-immunisation. Since natural circulating antibodies are polyspecific and have the ability to interact with multiple carbohydrate antigens, we focused on the design of multivalent glycodendrimers, as ligands for endogenous antibodies. The first part of our study consisted in synthesizing several multivalent glycoconjugates, based on peptide scaffolds and obtained by chemoselective ligations. To evaluate their influence on antibodies, the nature of both the carbohydrate and the scaffold, and the valency were varied. Then, in a second part of the study, microarray assays were developed with a model lectin, the Helix Pomatia Agglutinin (HPA). Experimental procedures were designed to determine surface dissociation constant and IC50 values, leading to the identification of high affinity ligands for HPA in the nanomolar range. Microarray assays were confirmed by other analytical methods (BLI, ELLA). Finally, the assays on slides were adapted to human sera screening, in order to identify tridimensional architectures highly affine to sera antibodies. A large panel of glycoconjugates were screened by microarray with around twenty sera, leading to the determination of promising glycosylated structures, as antibody ligands. The latter could be subsequently used for our anti-cancer approach.

Comme décrit dans la partie But du Travail, les tumeurs gliales sont particulièrement agressives d’un point de vue clinique. Le glioblastome, qui correspond au grade de malignité le plus élevé des gliomes, est associé à un pronostic très sombre car aucun patient atteint de ce cancer n’a pu être guéri à ce jour. Ces tumeurs envahissent de manière diffuse (par essaimage de cellules tumorales isolées) le parenchyme cérébral, ce qui empêche une résection chirurgicale complète de la tumeur. De plus, les cellules tumorales gliales d’origine astrocytaire sont souvent résistantes à l’apoptose et donc aux thérapies adjuvantes telles que la chimiothérapie et la radiothérapie. La galectine-1 est une petite protéine intervenant directement dans les processus migratoires des cellules gliales tumorales. Nous avons donc poursuivi la caractérisation des rôles biologiques que pourrait exercer la galectine-1 au sein des gliomes.<p>Nous avons tout d’abord montré que la galectine-1 est impliquée dans la chimiorésistance des gliomes. En effet, nous avons démontré que la diminution du taux d’expression de la galectine-1, au moyen d’un siRNA au sein d’un modèle de gliome expérimental, permet d’augmenter le bénéfice thérapeutique du témozolomide in vivo sans toutefois induire d’apoptose, d’autophagie ou de perméabilisation de la membrane des lysosomes. Nous avons également montré que la diminution du taux d’expression de la galectine-1 au sein de ce modèle de gliome expérimental affecte les processus d’angiogenèse in vivo et de « vasculogenic mimicry » in vitro. Nous avons identifié la protéine ORP150 comme l’une des principales cibles de l’effet pro-angiogénique de la galectine-1, sachant que la protéine ORP150 contrôle la maturation du facteur VEGF. Nous avons ensuite montré que le rôle de la galectine-1 dans la chimiorésistance des gliomes et dans l’angiogenèse est directement lié à l’implication de la galectine-1 dans le processus de réponse au stress du réticulum endoplasmique. Via ce processus, la galectine-1 modulerait l’expression d’un certain nombre de gènes tels que ATF3, DUSP5 et HERP, qui sont impliqués dans la chimiorésistance et des gènes tels que ORP150 et MDG1 qui sont impliqués dans l’angiogenèse. <p>Enfin, nous avons également montré que la galectine-1 régule l’expression du gène BEX2 et que celui-ci joue un rôle important dans la biologie des gliomes, notamment dans les processus d’angiogenèse et de migration cellulaire. <p>En conclusion, notre travail suggère que l’étiquette « biomarqueur » pourrait être attribuée à la galectine-1 pour qualifier l’agressivité biologique des gliomes malins et que la galectine-1 pourrait représenter une nouvelle cible thérapeutique dans le combat contre les gliomes malins en général, et le glioblastome en particulier.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Recent advances in the field of host immunity against parasitic nematodes have revealed the importance of macrophages in trapping tissue migratory larvae. Protective immune mechanisms against the rodent hookworm Nippostrongylus brasiliensis (Nb) are mediated, at least in part, by IL-4-activated macrophages that bind and trap larvae in the lung. However, it is still not clear how host macrophages recognize the parasite. An in vitro co-culture system of bone marrow-derived macrophages and Nb infective larvae was utilized to screen for the possible ligand-receptor pair involved in macrophage attack of larvae. Competitive binding assays revealed an important role for β-glucan recognition in the process. We further identified a role for CD11b and the non-classical pattern recognition receptor ephrin-A2 (EphA2), but not the highly expressed β-glucan dectin-1 receptor, in this process of recognition. This work raises the possibility that parasitic nematodes synthesize β-glucans and it identifies CD11b and ephrin-A2 as important pattern recognition receptors involved in the host recognition of these evolutionary old pathogens. To our knowledge, this is the first time that EphA2 has been implicated in immune responses to a helminth.

No description available.

Glykane aus exokrinen Drüsen stellen ein Schutzsystem der Schleimhautoberflächen dar, indem sie an mikrobielle Adhäsine binden und dadurch Einfluß auf die mikrobielle Besiedelung und Invasion des Wirtes nehmen. 11 Patienten mit rasch fortschreitender Parodontitis (RPP) wurde über jeweils 20 Minuten in Ruhe und unter adrenerger Belastung Speichel aus den großen Speicheldrüsen entnommen und in einem kompetitiven Lektinbindungsinhibitionstest auf die Bindungsfähigkeit an 8 verschiedene Planzenlektine untersucht. Patienten mit RPP zeigen ein anderes Verteilungsmuster der antiadhäsiven Glycane als die Kontrollgruppe. Sie zernieren u.a. aus den Unterkieferdrüsen konstant signifikant mehr Glykane mit endständigen Mannosegruppen, aus der Parotis dagegen nur unter adrenerger Stimulation. Dies zeigt die unterschiedliche Funktion der Drüsen bei der Beeinflussung des Milieus in der Mundhöhle als auch einen unterschiedlichen Erregungszustand des Schleimhautschutzsystems bei Erkrankten und Kontrollen. Aus den Ergebnissen können sich diagnostische Aspekte für das Risiko und den Aktivitätszustand einer parodontalen Erkrankung ergeben. / Glycans from exocrine glands create a defense system of the mucosal surfaces by binding to microbial adhesins and interfering with colonisation and invasion of the host. Saliva from 11 patients with rapidly progressive periodontitis (RPP) was collected over a period of 20 minutes each in rest and during adrenergic stimulation. The samples were tested for binding properties to 8 different plant lectins by a competitive lectin binding inhibition test. A pattern of antiadhesive glycans different from the control group is secreted in patients with RPP. The latter constantly secrete significantly more glycans with terminal mannose from the mandibular glands. In the parotis this is only the case during adrenergic stimulation. This demonstrates the different purpose of the glands in maintaining the oral milieu as well as different states of activity of the mucosal defense system in RPP patients and controls. Diagnostic aspects for risk and activity of periodontal diseases can be drawn from these findings.

by Wai Ching Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 154-159). / Abstracts in English and Chinese. / Statement --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / 摘要 --- p.vii / List of Abbreviations --- p.viii / Table of contents --- p.x / List of Tables --- p.xiv / List of Figures --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.4 / Chapter 2.1 --- Plant lectins --- p.4 / Chapter 2.1.1 --- Introduction --- p.4 / Chapter 2.1.2 --- Definition and subdivision of plant lectins --- p.4 / Chapter 2.2 --- Monocot mannose-binding lectins --- p.6 / Chapter 2.2.1 --- Occurrence and carbohydrate binding specificity --- p.6 / Chapter 2.2.2 --- Molecular structure and amino acid sequence --- p.7 / Chapter 2.2.3 --- "Molecular cloning, biosynthesis and post-translational modification" --- p.10 / Chapter 2.2.4 --- Mannose-binding lectins of Family Liliaceae --- p.11 / Chapter 2.2.4.1 --- Tulipa gesneriana lectins (TGL) --- p.12 / Chapter 2.2.4.2 --- Aloe arborescens lectins (AAL) --- p.13 / Chapter 2.2.4.3 --- Polygonatum multiflorum agglutinin (PMA) and lectin-related protein --- p.14 / Chapter 2.3 --- Polygonatum odoratum lectins (POL) --- p.15 / Chapter 2.3.1 --- Isolation and purification of POL from Yu Chu --- p.15 / Chapter 2.3.2 --- Agglutinating activity and anti-viral activities of POL --- p.17 / Chapter 2.3.3 --- Bacterial expression of POL in Escherichia coli --- p.18 / Chapter 2.4 --- Plant-based production of recombinant proteins --- p.20 / Chapter 2.4.1 --- Advantages of using plants as expression system --- p.20 / Chapter 2.4.2 --- Plant-derived recombinant proteins --- p.22 / Chapter 2.5 --- Expression of heterologous proteins in rice --- p.24 / Chapter 2.5.1 --- The facts of rice --- p.24 / Chapter 2.5.2 --- Rice storage proteins --- p.25 / Chapter 2.5.2 --- Expression of lysine-rich protein (LRP)/glutelin fusion proteinin rice seeds --- p.28 / Chapter 2.5.3 --- Expression of Galanthus nivalis agglutinin in rice --- p.29 / Chapter 2.6 --- Protein trafficking in plants --- p.30 / Chapter 2.6.1 --- Golgi-dependent pathways --- p.30 / Chapter 2.6.2 --- Golgi-independent pathway --- p.32 / Chapter 2.6.3 --- Expression of protein targeting determinants in tobacco plants and suspension cells --- p.33 / Chapter Chapter 3 --- Materials and Methods --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Chemcials --- p.35 / Chapter 3.3 --- Bacterial strains --- p.35 / Chapter 3.4 --- Cloning of POL cDNA --- p.36 / Chapter 3.4.1 --- Plant materials --- p.36 / Chapter 3.4.2 --- RNA extraction --- p.36 / Chapter 3.4.3 --- RT-PCR amplification of POL cDNA --- p.36 / Chapter 3.4.4 --- 5'RACE and 3'RACE --- p.38 / Chapter 3.4.5 --- Sequencing of POL cDNA --- p.39 / Chapter 3.5 --- Analysis of POL protein --- p.40 / Chapter 3.5.1 --- Protein extraction and Tricine-SDS PAGE --- p.40 / Chapter 3.5.2 --- Western blot analysis --- p.41 / Chapter 3.6 --- Chimeric gene construction --- p.42 / Chapter 3.6.1 --- Construction of the Cauliflower mosaic virus (CaMV)35S promoter/POL constructs --- p.44 / Chapter 3.6.2 --- Construction of the glutelin-1 promoter/POL constructs --- p.48 / Chapter 3.6.3 --- Sequence fidelity of chimeric genes --- p.55 / Chapter 3.7 --- Expression of transgenes in rice --- p.55 / Chapter 3.7.1 --- Plant materials --- p.55 / Chapter 3.7.2 --- Agrobacterium transformation --- p.55 / Chapter 3.7.3 --- Callus induction --- p.56 / Chapter 3.7.4 --- Agrobacterium culture and rice transformation --- p.56 / Chapter 3.7.5 --- Selection and regeneration of rice callus --- p.56 / Chapter 3.7.6 --- Isolation of genomic DNA --- p.58 / Chapter 3.7.7 --- Southern blot analysis --- p.58 / Chapter 3.7.8 --- Extraction of leaf total RNA --- p.59 / Chapter 3.7.9 --- Extraction of seed total RNA --- p.59 / Chapter 3.7.10 --- Northern blot analysis --- p.60 / Chapter 3.7.11 --- Protein extraction and Tricine SDS-PAGE --- p.60 / Chapter 3.7.12 --- Western blot analysis --- p.61 / Chapter 3.8 --- Cytopathic effect (CPE) reduction assay --- p.61 / Chapter 3.8.1 --- Protein extraction --- p.61 / Chapter 3.8.2 --- CPE reduction assay --- p.62 / Chapter 3.9 --- Confocal immunofluorescence --- p.63 / Chapter 3.9.1 --- Preparation of sections --- p.63 / Chapter 3.9.2 --- Labelling of fluorescence probes --- p.63 / Chapter 3.9.3 --- Image collection --- p.64 / Chapter Chapter 4 --- Results --- p.65 / Chapter 4.1 --- Cloning of POL cDNA from Yu Chu --- p.65 / Chapter 4.1.1 --- RNA extraction and partial POL cDNA amplification --- p.65 / Chapter 4.1.2 --- 5'RACE and 3'RACE --- p.67 / Chapter 4.1.3 --- Sequencing of POL cDNA --- p.68 / Chapter 4.1.4 --- Sequences comparison of POL and Liliaceae lectins --- p.75 / Chapter 4.2 --- Occurence of POL protein in Yu Chu plant --- p.77 / Chapter 4.3 --- Constitutional expression of POL in rice --- p.79 / Chapter 4.3.1 --- Construction of Cauliflower mosaic virus 35S promoter constructs --- p.80 / Chapter 4.3.2 --- Southern blot analysis --- p.82 / Chapter 4.3.3 --- Northern blot analysis --- p.84 / Chapter 4.3.4 --- Western blot analysis --- p.85 / Chapter 4.3.5 --- Western blot analysis of 35S/POL T1 plant --- p.87 / Chapter 4.4 --- Seed-specific expression of POL in rice --- p.88 / Chapter 4.4.1 --- Construction of the glutelin-1 promoter constructs --- p.89 / Chapter 4.4.2 --- Southern blot analysis --- p.92 / Chapter 4.4.3 --- Northern blot analysis --- p.96 / Chapter 4.4.4 --- Western blot analysis --- p.101 / Chapter 4.4.5 --- Western blot analysis of POL-BP-8O and POL-α-TIP T1 transgenic plants --- p.117 / Chapter 4.5 --- Cytopathic effect (CPE) reduction assay --- p.122 / Chapter 4.6 --- Confocal immunofluorescence studies --- p.125 / Chapter Chapter 5 --- Discussion --- p.134 / Chapter 5.1 --- Cloning of POL cDNA --- p.134 / Chapter 5.2 --- Analysis of constitutional expression of POL in rice --- p.136 / Chapter 5.3 --- Analysis of seed-specific expression of POL in rice --- p.138 / Chapter 5.4 --- Localization of POL in POL-BP-8O and POL-α-TIP transgenic rice seeds --- p.146 / Chapter 5.5 --- Cytopathic effect (CPE) reduction assay --- p.148 / Chapter 5.6 --- Future prospects --- p.151 / Chapter Chapter 6 --- Conclusion --- p.153

Proteins that recognise the sugar surface structures on cells have an enormous potential to be used as tools in the characterisation of these structures. A group of proteins, called lectins, have been identified that can bind to carbohydrate complexes on the receptors of cells. The crude extract from Grevillea robusta seeds was found to contain lectin-like proteins that were different from most other lectins, as they would specifically target the receptors of white blood cells and not those found on red blood cells. Therefore, the lectin isolated from G.robusta could be used as a tool to identify the specific surface structures on white blood cells. The lectin was isolated using affinity chromatography where a complex (oligosaccharide) matrix was used. Agglutination, binding and sugar inhibition assays confirmed the isolated protein was a lectin. The lectin was found in low amounts (up to 5% of the total protein content) within the seeds of G.robusta. As a result of this low yield, the identification of the lectin by PAGE was difficult because the levels of protein were beyond the detection limit of the commercial staining reagents. The lectin was called the GR2 protein and was characterised as a monocot mannose binding lectin based on its sugar specificity for only mannose. A serine protease inhibitor was isolated from the seeds of G.robusta using two different chromatography methods, reverse phase HPLC (GR1.HPLC) and gel filtration chromatography (GR1.GF). Ion exchange chromatography was used to initially separate the proteins in the crude extract and the fraction containing the GR1 protein was further purified using reverse phase HPLC (GR1.HPLC). N-terminal sequencing results of the GR1.HPLC protein, showed evidence of proteolytic cleavage during the extraction process, which lead to the second purification method being established. Protease inhibitors were added to the buffers prior to being purified by gel filtration chromatography, which resulted in the GR1 protein being isolated from the crude extract without the presence of the contaminating protein. Mass spectroscopy identified the molecular weight of the GR1 protein to be 6669Da and the full amino acid sequence was derived by cDNA techniques. Sequence alignment studies of the GR1 protein showed significant similarities with the Bowman-Birk inhibitor. The positioning of the cysteine residues were conserved throughout the Bowman-Birk superfamily, however these residues were not conserved within the GR1 protein. Competitive inhibition assays on the GR1 protein revealed the protein could inhibit both trypsin and chymotrypsin at similar levels to that seen for the Bowman-Birk inhibitor. Therefore, the GR1 protein was characterised as a member of the Bowman-Birk superfamily of serine protease inhibitors. The three-dimensional structure of the GR1 protein was determined using two-dimensional NMR spectroscopy. Computer programs such as XEASY, DYANA and SYBYL® were used to tabulate the information taken from the 2D experiments, generate structures and minimise these structures respectively. The solution structure of the GR1 protein was found to contain a region of antiparallel β-sheet structure that corresponded to the trypsin binding site and the remainder of the protein consisted of loops and turns that were held together by disulfide bridges (the chymotrypsin-binding region). Structural similarities between the GR1 protein and the Bowman-Birk inhibitor existed only in the trypsin-binding site of the Bowman-Birk inhibitor. The GR1 protein is the first member of the Proteaceae family to be characterised as a Bowman-Birk inhibitor. This thesis outlines the isolation and biochemical characterisation of the two proteins found within Grevillea robusta and also describes the steps involved and results obtained in determining the three-dimensional structure of the GR1 protein.