Doctor of Philosophy / Department of Biochemistry / Subbarat Muthukrishnan / The extracellular matrices of cuticle and peritrophic matrix of insects are composed mainly of chitin complexed with proteins, some of which contain chitin-binding domains. This study is focused on the identification and functional characterization of genes encoding proteins that possess one or more copies of the six-cysteine-containing ChtBD2 domain (Peritrophin A motif =CBM_14 =Pfam 01607) in the red flour beetle, Tribolium castaneum. A bioinformatics search of T. castaneum genome yielded previously characterized chitin metabolic enzymes and several additional proteins. Using phylogenetic analyses, the exon-intron organization of the corresponding genes, domain organization of proteins, and temporal and tissue-specificity of expression patterns, these proteins were classified into three large families. The first family includes 11 proteins essentially made up of 1 to 14 repeats of the peritrophin A domain. Transcripts for these proteins are expressed only in the midgut and only during feeding stages of development. We therefore denote these proteins as “Peritrophic Matrix Proteins” or PMPs. The genes of the second and third families are expressed in cuticle-forming tissues throughout all stages of development but not in the midgut. These two families have been denoted as “Cuticular Proteins Analogous to Peritrophins 3” or CPAP3s and “Cuticular Proteins Analogous to Peritophins 1” or CPAP1s based on the number of ChtBD2 domains that they contain. Unlike other cuticular proteins studied so far, TcCPAP1-C protein is localized predominantly in the exocuticle and could contribute to the unique properties of this cuticular layer. RNA interference (RNAi), which down-regulates transcripts for any targeted gene, results in lethal and/or abnormal phenotypes for some, but not all, of these genes. Phenotypes are often unique and are manifested at different developmental stages, including embryonic, pupal and/or adult stages. The experiments presented in this dissertation reveal that while the vast majority of the CPAP3 genes serve distinct and essential functions affecting survival, molting or normal cuticle development. However, a minority of the CPAP1 and PMP family genes are indispensable for survival under laboratory conditions. Some of the non-essential genes may have functional redundancy or may be needed only under special circumstances such as exposure to stress or pathogens.

Some species of Oomycetes are well studied pathogens that cause considerable economical losses in the agriculture and aquaculture industries. Currently, there are no chemicals available that are environmentally friendly and at the same time efficient Oomycete inhibitors. The cell wall of Oomycetes consists of b-(1à3) and b-(1à6)-glucans, cellulose and in some species minute amounts of chitin. The biosynthesis of cellulose and chitin in Oomycetes is poorly understood. However, cell wall synthesis represents a potential target for new Oomycete inhibitors. In this work, cellulose and chitin synthase genes and gene products were analyzed in the plant pathogen Phytophthora infestans and in the fish pathogen Saprolegnia monoica.   A new Oomycete CesA gene family was identified, containing four subclasses of genes designated as CesA1 to 4. The gene products of CesA1, 2 and 4 contain pleckstrin homology (PH) domains located at the N-terminus, which is unique to the Oomycete CesAs. Our results show that the SmCesA2 PH domain binds to phosphoinositides, F-actin and microtubules in vitro and can co-localize with F-actin in vivo. Functional characterization of the CesA genes by gene silencing in P. infestans led to decreased cellulose content in the cell wall. The cellulose synthase inhibitors DCB and Congo Red inhibited the growth of the mycelium of S. monoica and had an up-regulating effect on SmCesA gene expression. Zoospores from P. infestans treated with DCB were unable to infect potato leaves. In addition, two full-length chitin synthase genes (Chs) were analyzed from S. monoica.  Expression of SmChs2 in yeast yielded an active recombinant protein. The biochemical characterization of the in vitro product of SmChs2 confirmed that the protein is responsible for chitin formation. The chitin synthase inhibitor nikkomycin Z inhibited the SmChs2 both in vivo and in vitro.   Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that synthesis of cellulose is crucial for infection of potato by P. infestans. The PH domain is involved in the interaction of CesA with the cytoskeleton. In addition, we firmly demonstrate that the SmChs2 gene encodes a catalytically active chitin synthase. / QC 20110531

Doctor of Philosophy / Department of Entomology / Kun Yan Zhu / Chitin metabolism represents an attractive target site for combating insect pests as insect growth and development are strictly dependent on precisely toned chitin synthesis and degradation and this process is absent in humans and other vertebrates. However, current understanding on this process and the involved enzymes is rather limited in insects. In this study, two chitin synthase genes (AgCHS1 and AgCHS2 or AgCHSA and AgCHSB), and 20 chitinase and chitinase-like genes (groups I-VIII) presumably encoding the enzymes for chitin biosynthesis and degradation, respectively, were identified and characterized in African malaria mosquito, Anopheles gambiae. Immunohistochemistry analysis and developmental stage- and tissue-dependent transcript profiling by using reverse transcription PCR, real-time quantitative PCR, and in situ hybridization revealed new information on these genes. Current understanding on chitin synthases is extended by the expression profiles such as the localization of AgCHS1 and AgCHS2 transcripts in eggs, AgCHS2 transcripts in the posterior larval midgut, AgCHS1 and AgCHS2 proteins in the compound eyes, and AgCHS2 enzyme in pupal inter-segments. Chitinase and chitinase-like genes are highly diverse in their gene structure, domain organization, and stage- and tissue-specific expression patterns. Most of these genes were expressed in several stages. However, some genes are stage- and tissue-specific such as AgCht8 mainly in pupal and adult stages, AgCht2 and AgCht12 specifically in foregut, AgCht13 exclusively in midgut. Functional analysis of each chitin synthase gene was conducted by using the chitosan/dsRNA nanoparticle-based RNA interference (RNAi) through larval feeding. The repression of the AgCHS1 transcripts which are predominantly expressed in carcass initiated from the mosquito larval feeding of dsRNA suggests the systemic nature of RNAi in mosquito larvae. In addition, silencing of AgCHS1 increased larval susceptibilities to diflubenzuron, whereas silencing of AgCHS2 enhanced the peritrophic matrix disruption and thus increased larval susceptibilities to calcofluor white or dithiothreitol. Furthermore, a non-radioactive method was adapted and optimized to examine the chitin synthase activity in mosquitoes. By using this method, diflubenzuron and nikkomycin Z show limited in vitro inhibition on chitin synthase at high concentration in cell free system, whereas no in vivo inhibition was observed.

Chitin ist ein Polysaccharid, welches aus N-Acetylglucosamin (GlcNAc) aufgebaut ist. Das Biopolymer kommt in der Natur in Invertebraten, Algen, Pilzen und Einzellern vor. Chitinasen hydrolysieren Chitin. Diese Enzyme sind essentiell für die Regulierung und Entwicklung von Arthropoden und Mikroorganismen. Sie fungieren in Pflanzen als Phytoalexine. <br> Das verstärkt das Interesse am Design neuer natürlicher und synthetischer Chitinase-inhibitoren, die als potentielle Insektizide, Fungizide, Antimalaria bzw. Antiasthmatika eingesetzt werden können. <br><br> Ziel dieser Arbeit war die Synthese von neuen Thioglycosidanaloga von N-Acetyl-chitooligosacchariden und deren Untersuchung als Enzyminhibitoren. <br><br> Die geschützten 4-O-Tf-galactopyranosylthioglycoside wurden aus den korrespondierenden p-Methoxyphenylglycosiden von GlcNAc nach einer neuen Methode in einer Stufe hergestellt. Die Reaktion der galacto-Triflate mit 2-Acetamido-3,4,6-tri-O-acetyl-2-desoxy-1-thio-ß-D-glucopyranose in Gegenwart von Natriumhydrid und 15-Krone-5 in THF lieferte die Pseudo-Trisaccharide.<br><br> Die Herstellung der Glycosyl-Thiazoline aus den entsprechenden p-Methoxyphenyl-glycosiden mit Lawesson's Reagenz ist nach einem neuen Reaktionsweg beschrieben wie auch die Synthese der Thioacetamide von GlcNAc-Oligomeren.<br><br> Die Pseudo-Oligosaccharide wurden als Inhibitoren der Chitinasen von Serratia marcescens, Chironomus tentans, Acanthocheilonema viteae und Hevaea brasiliensis sowie der N-Acetyl-glucosaminidase von Chironomus tentans getestet. In einigen Fällen ergab sich eine Hemmung (IC50) im µM-Bereich. / Chitin is a polysaccharide composed of N-acetylglucosamine (GlcNAc). The biopolymer is distributed in Invertebrates, algae, fungi and Protozoa. Chitinases hydrolyse chitin. These enzymes are essential for the regulation and development of Arthropoda and micro organisms. In bacteria chitinase degrade chitin as source of carbon and act in plants as phytoalexins. Currently, there is a great interest in the design of novel natural and synthetic chitinase inhibitors, which may act as potential insecticides, fungicides, antimalaria or antiasthmatics.<br><br> Thio-oligosaccharides and related thio-glycosides are rarely described in the literature. Thio-analogues of oligosaccharides derived from N-acetylglucosamine are of interest as potential enzyme inhibitors. The synthesis and enzymatic investigations of thio-glycoside analogues of N-acetyl-chitooligosaccharides are described in this thesis.<br><br> The protected 4-O-Tf-galactopyranosyl thioglycosides were prepared by a new method in one step from the corresponding p-methoxyphenyl glycosides of N-acetylglucosamine. Coupling of galacto-triflates with 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-ß-D-glucopyranose in the presence of sodium hydride and 15-crown-5 in THF, followed by deprotection, afforded the pseudo-trisaccharides. <br><br> The preparation of glycosyl thiazolines by a new manner from the analogues p-methoxyphenyl glycosides with Lawesson's reagent is also described however the synthesis of thioacetamides of N-acetylglucosamine.<br><br> The pseudo-oligosaccharides were tested for inhibition of chitinases from Serratia marcescens, Chironomus tentans, Acanthocheilonema viteae, and Hevaea brasiliensis, and also with N-acetyl-glucosaminidase from Chironomus tentans. Inhibition with IC50 values in the µM range was found in a few cases.

Some oomycetes species are severe pathogens of fish or crops. As such, they are responsible for important losses in the aquaculture industry as well as in agriculture. Saprolegnia parasitica is a major concern in aquaculture as there is currently no method available for controlling the diseases caused by this microorganism. The cell wall is an extracellular matrix composed essentially of polysaccharides, whose integrity is required for oomycete viability. Thus, the enzymes involved in the biosynthesis of cell wall components, such as cellulose and chitin synthases, represent ideal targets for disease control. However, the biochemical properties of these enzymes are poorly understood, which limits our capacity to develop specific inhibitors that can be used for blocking the growth of pathogenic oomycetes. In our work, we have used Saprolegnia monoica as a model species for oomycetes to characterize two types of domains that occur specifically in oomycete carbohydrate synthases: the Pleckstrin Homology (PH) domain of a cellulose synthase and the so-called ‘Microtubule Interacting and Trafficking’ (MIT) domain of chitin synthases. In addition, the chitin synthase activity of the oomycete phytopathogen Aphanomyces euteiches was characterized in vitro using biochemical approaches. The results from our in vitro investigations revealed that the PH domain of the oomycete cellulose synthase binds to phosphoinositides, microtubules and F-actin. In addition, cell biology approaches were used to demonstrate that the PH domain co-localize with F-actin in vivo. The structure of the MIT domain of chitin synthase (CHS) 1 was solved by NMR. In vitro binding assays performed on recombinant MIT domains from CHS 1 and CHS 2 demonstrated that both proteins strongly interact with phosphatidic acid in vitro. These results were further supported by in silico data where biomimetic membranes composed of different phospholipids were designed for interaction studies. The use of a yeast-two-hybrid approach suggested that the MIT domain of CHS 2 interacts with the delta subunit of Adaptor Protein 3, which is involved in protein trafficking. These data support a role of the MIT domains in the cellular targeting of CHS proteins. Our biochemical data on the characterization of the chitin synthase activity of A. euteiches suggest the existence of two distinct enzymes responsible for the formation of water soluble and insoluble chitosaccharides, which is consistent with the existence of two putative CHS genes in the genome of this species. Altogether our data support a role of the PH domain of cellulose synthase and MIT domains of CHS in membrane trafficking and cellular location. / <p>QC 20151014</p>

La demande actuelle croissante d’excipients multifonctionnels par les laboratoires pharmaceutiques, amène les fabricants de matières premières à développer de nouveaux matériaux pouvant répondre à ces critères de performance. Parmi les différents procédés disponibles pour de telles productions, les techniques de co-traitement sont les plus sollicitées. Considérant l’évaluation de la fonctionnalité de différents excipients désintégrants, l’objectif de cette thèse a été de développer un tel excipient, original, économiquement intéressant et pouvant être utilisé dans la fabrication de comprimés par compression directe.Dans ce travail, nous nous sommes tout d’abord intéressés à étudier et à comparer la fonctionnalité de différents désintégrants commerciaux, en évaluant notamment l’influence des milieux réactionnels sur cette fonctionnalité.La chitine, deuxième polysaccharide naturel le plus abondant au monde, et présentant des propriétés physico-chimiques et pharmacotechniques pertinentes, a retenu notre intérêt. Un mélange co-processé, de chitine et de carbonate de calcium (CC) a été développée, et sa préparation optimisée afin de pouvoir disposer d’un matériau aux propriétés maîtrisées. Par des études conduites sur simulateur de compression, nous avons pu, d’une part, établir un profil complet de compression du CC, d’autre part, évaluer ses performances en compression directe en le formulant avec des proportions variables de composants actifs pharmaceutiques retenus comme traceurs modèles. Des études de stabilité ont enfin été réalisées, afin de déterminer ses meilleures conditions d’utilisation.Les résultats les plus marquants ont montré que la chitine présente une comprimabilité satisfaisante et des propriétés de désintégration qui ne sont pas influencées par l’environnement physico-chimique de la formulation. L’excipient co-processé CC a révélé des propriétés très intéressantes et notamment, une densité vraie et une coulabilité améliorées, par rapport à la chitine seule. Ses propriétés désintégrantes sont également particulièrement notables (comprises entre 2 et 5 s.). Son comportement en compression est par ailleurs très satisfaisant avec, une comprimabilité, une compressibilité et une compactibilité performantes; il est également à noter, que dans nos conditions de formulations, il n’a pas nécessité l'addition de lubrifiant. Les comprimés élaborés avec les composants actifs modèles ont fourni des profils de compression également performants et des aptitudes à la désintégration très satisfaisantes. Quant aux profils de dissolution pharmaceutique, ils ont révélé que, avec une teneur en CC égale à 30% m/m, la libération des actifs était rapide. Les études de stabilité conduites afin de définir les conditions optimales de conservation, ont montré que des précautions de température et d'humidité doivent être prises pour les produits formulés avec CC.L’excipient CC co-traité développé dans cette Etude présente toutes les caractéristiques d’un excellent excipient multifonctionnel (diluant, désintégrant, liant). Valorisant l’usage d’une matière première de base naturelle et très abondante (la chitine), facile à produire et économiquement rentable, il devrait pouvoir trouver un intérêt dans la formulation et la production de comprimés à dissolution rapide par compression directe. / Nowadays, there is an increasing demand for multifunctional excipients that replaces the need and use of multiple excipients. Pharmaceutical excipients coprocessing techniques represent important methods for new excipients development with enhanced functionalities. The objective of this thesis is to develop a cost-effective multifunctional excipient (filler-disintegrant-binder) used in tablet manufacturing by direct compression.In this thesis, we’ve studied and compared disintegrants functionality for different materials and evaluated the effect of media on their disintegrant’s functionality. In addition, chitin was chosen as a base to develop a multifunctional excipient used in direct compression as it showed a good and promising physicochemical and pharmaceutical properties. Chitin-Calcium carbonate (CC) coprocessed excipient was developed and its production was further optimized to ensure better powder properties and functionality. In addition, CC compression profile was established by studying its compression behavior under different conditions and formulating with active pharmaceutical ingredients to determine how it affects the formulation at different percentages. Finally, stability study was carried out to determine best conditions for the excipient handling.Results showed that chitin has good tabletability and disintegration properties that were not influenced by the physicochemical environment of the formulation. CC showed an enhancement in true density and flowability (that are considered as drawbacks for raw chitin use as an excipient) and fast disintegration (2-5s). The excipient had good tabletability, compressibility, compactibility, and it doesn’t need the use of a lubricant. CC showed a good compression profile at different manufacturing conditions (multiple lubrication levels, compression speeds and dwell times) while maintaining fast disintegration. It causes rapid disintegration and dissolution when formulated with active pharmaceutical ingredients starting from 30% w/w, and its inclusion was reflected positively on tablets strength. Stability studies showed that precautions on temperature and humidity conditions would need to be taken on CC formulated products. The results showed that the excipient serves as an excellent multifunctional excipient (filler, disintegrant, binder) used for fast disintegrating tablets produced by direct compression. It represents a cost-effective product that is efficient and easily produced at pilot plant and upon scale-up.

Antifungal proteins were isolated from the crude bacterial supernatant using ammonium sulfate salt precipitation followed by passage over DEAE -cellulose and Biogel P100 columns. The purified protein had an apparent molecular mass of 14 kDa. Its antifungal activity was retained even at 100°C, for 60 min. The results of protein identification using MALDI -TOF/TOF mass spectrometer suggested that the purified protein is indeed a chitin binding protein that has 206 acid amine containing chitin -bind -3 region with a relative molecular mass of 22230 Da. / Protein có hoạt tính kháng nấm được tinh sạch từ dịch ngoại bào chủng vi khuẩn Bacillus subtilis sau khi qua ba bước tinh sạch: tủa muối ammonium sulphate 30-70%, qua cột sắc ký trao đổi ion DEAE – cellulose và cột săc ký lọc gel Biogel P100. Protein tinh sạch có khối lượng phân tử đạt 22 kDa trên điện di SDS-PAGE. Hoạt tính kháng nấm của protein tinh sạch vẫn còn duy trì khi ủ ở 100°C trong 60 phút. Kết quả nhận dạng bằng khối phổ MALDI -TOF/TOF đã chỉ ra rằng protein bền nhiệt này là chitin binding protein được mã hóa bởi 206 acid amin cùng với khối lượng phân tử là 22230 Da. trị an toàn đối với Al và Atrazie trong môi trường nước tự nhiên về khía cạnh bảo vệ sức khỏe sinh thái.

Chitin in shrimp waste is tightly associated with proteins, lipids, pigments and mineral deposits. Therefore, these source materials have to be pretreated to remove these components. For a long time, chemical process has been used widely for extraction of chitin from shrimp waste. The chemical process however led to severe environmental damage and low chitin quality. The biological process has been shown promising to replace the harsh chemical process to reduce the environment impact. In our previous study chitin recovery from sterilized shrimp waste by Lactobacillus plantarum NCDN4 was investigated. However in large scale it is uneconomical to sterilize the shrimp waste. For that reason, in this study the microbial process using Lactobacillus plantarum NCDN4 for chitin recovery from unsterilezed shrimp waste has been investigated. Factors affecting the demineralization by this strain such as inoculum size, glucose concentration, initial pH, NaCl concentration and fermentation time were investigated. It was found that when unsterilized shrimp waste fermented with 20% L. plantarum inoculum, 12,5% glucose, and pH 6 for 4 days at 30oC, 99. 28% emineralization and 48.65% deproteination could be achieved. The ash and protein content of fermented residues were 1.33% and 22.46% respectively. Compared to sterilized condition the efficiency of demineralization and deproteination was similar. / Chitin trong phế liệu tôm liên kết chặt chẽ với protein, sắc tố và khoáng. Do vậy để thu được chitin cần có các bước tiền xử lí để loại các thành phần không phải chitin ra. Phương pháp hóa học được sử dụng rộng rãi từ lâu để tiền xử lí chitin. Tuy nhiên do phương pháp hóa học gây hại cho môi trường và tạo ra chitin chất lượng thấp, các nhà khoa học nỗ lực nghiên cứu tìm ra các phương pháp thay thế. Phương pháp sinh học được xem là rất khả quan để thay thế phương pháp hóa học. Trong nghiên cứu trước của chúng tôi, quá trình lên men phế liệu tôm thanh trùng bằng Lactobacillus plantarum NCDN4 đã được nghiên cứu. Tuy nhiên việc thanh trùng phế liệu tôm không kinh tế. Trong nghiên cứu này quá trình lên men phế liệu tôm không thanh trùng bằng Lactobacillus plantarum NCDN4 đã được khảo sát. Các yếu tố như tỷ lệ giống, nồng độ đường glucose, nồng độ NaCl, pH ban đầu của môi trường lên men và thời gian lên men đã được nghiên cứu. Kết quả cho thấy ở điều kiện 20% giống theo thể tích, 12,5% dịch đường glucose, 2% muối theo khối lượng, pH ban đầu 6, sau 5 ngày lên men lượng khoáng và protein trong nguyên liệu giảm tương ứng 99.28% và 48.65%. Lượng khoáng và protein còn lại tương ứng 1.33% và 22.46% (tính theo trọng lượng khô). So với phế liệu tôm không thanh trùng, hiệu quả loại khoáng và protein là tương đương.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Polymer composites have been gaining more importance in our daily lives because of the favorable properties that can be provided by these types of material. A polymer composite consists of improved properties when compared to the individual polymers that it is compiled of. The reason that composites are better than the individual polymers is mainly because composites are a combination of all the bene cial properties from the individual materials that was used to make the polymer composite. High impact polypropylene (HiPP) is a complex copolymer that was developed to overcome the restrictions of polypropylene (PP). Although PP have excellent properties at lower temperatures, it loses these advantages at elevated temperatures. High impact polypropylene has a much better impact strength and is processable at high temperatures. High impact polypropylene has been studied in depth for its applications and its superior properties such as an improved impact strength. The tensile properties, after the incorporation of a nano ller, have however not been investigated to our knowledge. Nano llers have reinforcing abilities due to the nano-scale diameters. Particles that have sizes on a nanometer range are mostly devoid of defects. Nano llers that are biopolymers have additional advantages such that can consist of antimicrobial abilities, renewability, biocompatibility and biodegradability. Composites reinforced with chitin nanowhiskers (chnw) have shown to have valuable applications in the latest medical, industrial and environmental developments. Di erent loadings of chnw were incorporated into a HiPP matrix in order to investigate the e ects that this nano ller will have on the tensile properties of HiPP. There were two challenges that required attention during the incorporation of chnw into HiPP. The rst major challenge was the poor interaction that exist between chnw and HiPP due to the hydrophobic nature of the HiPP matrix and the hydrophilic nature of chnw. The second problem was the agglomeration that can occur because of the hydrogen bonding between the chnw that is caused by the structure of the chnw chains. In order to gain the best dispersion of chnw within the HiPP matrix it was necessary to use compatibilizers and di erent methods of incorporation. The two types of compatibilizers that were chosen to improve the compatibility between the HiPP matrix and chnw were polypropylenegraft- maleic anhydride (PPgMA) and poly(ethylene-co-vinyl alcohol)(EVOH). Injection molding is typically used to process HiPP and was chosen as one of the methods for incorporating chnw into the HiPP matrix. A second method of incorporation was used speci cally for the nancomposites containing EVOH known as electrospinning combined with meltpressing. Tensile testing, DSC, TGA and FTIR were used to investigate the changes in the mechanical and thermal properties of the nanocomposites. SEM and TEM were employed to investigate the morphology of the electrospun ber mats and to characterize the chnw. FTIR as well as TGA were used to characterize the chitin nanowhiskers and to identify the individual components within the nanocomposites after incorporation took place. The incorporation of chnw along with the compatibilizer did show improvement in some mechanical properties of the polymer matrix. However, the in uence that each type of compatiblizer had on this e ect varied depending on the content of the chnw and compatibilizer with regards to the polymer matrix.

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>