Geometric and tomographic methods of reconstructing three-dimensional cerebral blood vessels from two-dimensional digital subtraction angiograms are studied experimentally. / Three-dimensional vessel geometry is reconstructed from center-line coordinates of corresponding vessel branches in both stereo and biplane angiogram pairs. The problem associated with finding corresponding vessel branches in biplane images was shown to be reduced by re-projection of stereoscopically reconstructed vessels. Results indicate that the limiting factor in reconstruction accuracy is the degree of vessel foreshortening in biplane image pairs. / An iterative algorithm ('Clean') is adapted to tomographic reconstruction of vessel cross-sections from a small number of views. Star-pattern artifacts in images initially formed by back-projection are removed by iterative deconvolution guided by 'a priori' object knowledge. This procedure is repeated for a set of two-dimensional sections that describe the three-dimensional vascular structure. Results show that there is sufficient detail in reconstructed sections to determine the location of vascular structures.

The sensitive and specific detection of proteins is at the center of many routine analyses in fundamental research, medical diagnosis, food quality control and environmental safety. The current gold standard for these applications remains the laborious and costly microwell plate ELISA. Over the last decade, new miniaturized devices have emerged: microfluidic systems that can drastically reduce the costs and the time of analysis. Many approaches and designs have been proposed. However, some recurrent difficulties remain that prevent the achievement of a system with the necessary balance between scientific performance, cost-effectiveness and user friendliness. These limitations include the complexity to maintain a constant flow rate in a simple and repeatable fashion, to mix solutions in a laminar flow regime, to control undesired surface effects, and to connect the chip to external pumping instruments. This thesis describes a novel microfluidic immunoassay platform that addresses the aforementioned issues while also achieving highly sensitive parallel measurements for the rapid quantification of protein biomarkers. The development of this platform followed three consecutive stages: (i) the establishment of an initial design for the simple manipulation of solutions in stop-flow mode, and the elaboration of strategies for mixing and for the simultaneous detection of parallel reactions, (ii) the introduction of the concept of Dual Network system, which removes the need for channel passivation against the non-specific adsorption of proteins, and (iii) the optimization of the critical assay parameters for the quantification of the cytokine TNF-alpha. The main attributes of the developed platform are also presented: the straightforward fabrication process, the simplified flow control, the enzymatically generated fluorescent signal, and the multi-purpose use of magnetic beads. These microbeads were utilized as functionalized substrate to capture the analyte, but also to / La détection sensible et spécifique de protéines se trouve au cœur d'analyses de routine dans la recherche fondamentale, le diagnostique médical, le contrôle qualité de la nourriture et la sûreté environnementale. Le standard actuel pour ces applications est toujours le coûteux et laborieux test ELISA en micropuits. Au cours de la dernière décennie, de nouveaux dispositifs miniaturisés ont fait leur apparition : des systèmes microfluidiques pouvant réduire de manière drastique les coûts et les temps d'analyse. Plusieurs approches et designs ont été proposés. Cependant, certaines difficultés récurrentes entravent toujours l'avènement d'un système possédant l'équilibre nécessaire entre la performance scientifique, le maintient de coûts bas, et la facilité d'utilisation. Ces limitations incluent la complixité de fixer une vitesse de flot constante de façon simple et reproductible, de mixer des solutions en régime laminaire, de contrôler les effets de surfaces indésireux, et de connecter la puce à des instruments de pompage externes. Cette thèse décrit une nouvelle plateforme d'immunoessais microfluidiques, qui adresse les problèmes mentionnés tout en réalisant des mesures hautement sensibles et en parallèle pour la quantification de biomarqueurs protéiques. Son développement a suivi trois étapes consécutives : (i) l'établissement d'un design initial pour la manipulation aisée de solutions en mode stop-flow, l'élaboration de stratégies de mixage et de détection simultanée de réactions parallèles, (ii) l'introduction du concept de système Dual Network, qui supprime la nécessité de passiver les canaux contre l'adsorption non-spécifique de protéines, et (iii) l'optimisation des paramètres critiques de l'essai pour la quantification de la cytokine TNF-alpha. Les attribues principaux de la plateforme sont également présentés : le procédé de fabrication rapide, le contrôle du flot simplifié, le signal$

Myeloid cell leukemia 1 (MCL-1), an anti-apoptotic BCL-2 member, is active in the preservation of mitochondrial integrity during apoptosis. By collective data from nuclear magnetic resonance (NMR) spectroscopy and titration calorimetry, we revealed the selectivity of MCL-1 in binding BH3 ligands of interest to mammalian biology, and proved that the core domain of MCL-1 (cMCL-1) is necessary and sufficient for BH3 ligand binding. We characterized the in vitro protein-protein interaction between cMCL-1 and activated BID, which occurs in a very slow manner in solution but is otherwise similar to the interaction between cMCL-1 and BID-BH3 peptide. We also present the solution structure of complex cMCL-1:BID-BH3, which may greatly facilitate drug discovery studies of human tumor malignancies. BAK, a multi-region pro-apoptotic protein, directly mediates the mitochondrial outer membrane permeablization (MOMP). We completed a structural investigation of BAK by X-ray crystallography. We report two structures of BAK's homo-dimers, one zinc-mediated (cBAK) and one disulphide-bond-linked (cBAK-o). Their dimerizing sites locate closely at D160 and H164 in cBAK and C166 in cBAK-o, which allow them to compose a unique regulatory element to switch BAK's activity as suggested in mitochondria activity-testing assays. BAK is tightly regulated through protein-protein interactions by MCL-1. We characterize the conformational changes in BAK and MCL-1 using detergents to mimic the membrane environment, and studied their interaction in vitro. The non-ionic detergent IGEPAL and the zwitterionic detergent CHAPS have different effects on these two proteins, but both initiate the heterodimerization. The complex of MCL-1 and BAK can be disrupted by either a BID-BH3 peptide, which acts through binding to MCL-1, or a mutation in BH3 region of BAK (L78A), demonstrating the essential role of BAK's BH3 in its regulation by MCL-1. This thesis concludes with a hybrid model for BAK activation: / La protéine MCL-1 (Myeloid cell leukemia 1), qui appartient à la classe de protéines anti-apoptotiques BCL-2, joue un rôle dans le maintien de l'intégrité mitochondriale durant l'apoptose. Les résultats obtenus par résonance magnétique nucléaire (RMN) et par titrage calorimétrique, nous ont permis de mettre en évidence la sélectivité de la protéine MCL-1 pour les ligands mammifères d'interêt biologiques qui contiennent le motif BH3 et nous avons ainsi démontré que le domaine central du facteur MCL-1 (cMCL-1) est nécessaire et suffisant pour cette interaction. Nous avons caractérisé in vitro l'interaction entre le domaine cMCL-1 et le facteur activé BID; cette interaction se produit lentement en solution mais est similaire à celle observée entre le domaine cMCL-1 et le peptide BID-BH3. De plus nous avons résolu la structure du complexe cMCL-1:BID-BH3, qui est une cible potentielle qui pourrait être à la base d'un criblage d'une banque de petites molécules dans le cas de tumeurs humaines malignes. BAK, une protéine pro-apoptotic modulaire, permet la perméabilité de la membrane externe de la mitochondrie: ce mécanisme est dénommé “MOMP” pour “the mitochondrial outer membrane permeablization”. Nous avons accompli l'étude structurale de la protéine BAK par cristallographie et diffraction de rayons X. Nous présentons deux complexes de la protéine BAK: un homodimère lié par une molécule de zinc (cBAK) et une qui contient un pont disulfure (cBAK-o). Le site de dimérisation se situe proche des résidu D160 et H164 pour cBAK et C166 pour cBAK-o, ce qui leur confère un élément de régulation unique pour moduler l'activité de BAK comme suggéré dans des essais d'activité mitochondriale. La protéine BAK est finement régulée grâce à son interaction protéine-protéine avec MCL-1. Nous avons caractérisé les changements conformations des facteurs BAK et MCL-1 à l'aide de détergents pour modéliser un environnement m

The blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) signal is one of the most recently developed imaging techniques used to identify localized changes in brain activity. The BOLD signal is a function of cerebral blood flow (CBF), the cerebral metabolic rate of oxygen consumption (CMRO2), and cerebral blood volume (CBV). Normally coupled changes in these physiological parameters during regional increases in neural activity will result in BOLD signal increases. This makes the BOLD signal useful as a tool for identifying areas of increased neural activity. Moreover, it can also be used to infer changes in neurophysiology as a response to certain stimuli in the healthy brain, in individuals with certain neurological conditions affecting the cerebrovasculature and under the effect of some types of drugs. There is a major challenge to be faced when using the BOLD signal to study neurophysiological changes that occur under the effect of certain drugs or in the case of neurovascular disease. Under these circumstances, the underlying physiology and normally coupled vascular/metabolic response to a stimulus will be altered. Consequently, changes seen in the BOLD signal during regional increases in neural activity may not be associated with the same neurovascular changes that would occur in the healthy brain. Caffeine is an example of a widely used drug which has been shown to elicit such changes in the neurophysiological state. Two experiments were performed on a group of healthy volunteers in order quantify the effects of caffeine on both baseline neurophysiology and the BOLD signal changes evoked during a visual stimulus. The first involved the measurement of the oxygen extraction fraction(OEF) by performing venous blood magnetic resonance (MR) relaxometry and the second, the measurement of the BOLD and CBF response to a visual stimulus. The results obtained demonstrate that after caffeine consumption there is an increase in baseline OEF (OEF0) and a decrease in baseline CBF (CBF0), but a non-significant change in baseline CMRO2 (CMRO2,0). The caffeine-induced change in the individual BOLD and CBF response to a visual stimulus was found to correlate negatively with individual caffeine-induced change in CBF0. However, the average percent change in visually evoked BOLD and CBF signals across all subjects remained unaltered following caffeine consumption, whereas the CMRO2 response to a visual stimulus was found to increase. / L'effet BOLD (blood oxygenation level dependent) est l'une des plus récentes techniques d'imagerie par résonance magnetique (IRM) utilisée pour identifier les changements localisés d'activité cérébrale. Le signal BOLD varie en fonction de la circulation sanguine cérébrale (CBF), du taux métabolique cérébral de la consommation d'oxygène (CMRO2), et du volume sanguin cérébral (CBV). Les changements normalement-couplés de ces paramètres physiologiques lors d'une augmentation régionale d'activité neurale causeront une augmentation du signal BOLD. Cela rend le signal BOLD utile comme outil pour identifier les régions d'augmentation de l'activité neuronale. Deplus, le signal BOLD permet de déterminer quels changements neurophysiologiques suivent certains stimulus dans les cas suivants : le cerveau sain; le cerveau de personnes atteintes d'affections neurologiques affectant la cérébrovasculature; et le cerveau sous l'effet de certains types de médicaments. Un défi majeur se présente lorsque le signal BOLD est utilisé pour étudier les changements neurophysiologiques se produisant sous l'effet de certains médicaments ou en cas de maladie neuro-vasculaire. Lors de ce type d'utilisation, la physiologie sous-jacente et la réponse normalement-couplée vasculaire/métabolique à un stimulus est altérée. Par conséquent, les changements observés dans le signal BOLD au cours des augmentations régionales d'activité neuronale ne correspondent pas aux changements neurovasculaires qui se produisent normalement dans le cas du cerveau sain. La caféine est un médicament couramment utilisé qui a suscité de tels changements dans l'état neurophysiologique. Deux expériences ont été réalisées sur un groupe de volontaires sains dans le but de quantifier les effets de la caféine sur la neurophysiologie de base et les changements évoqués sur le signal BOLD lorsd'un stimulus visuel. La première expérience mesure la fraction de l'extraction d'oxygène (OEF) en effectuant de la relaxométrie par résonance magnetique sur le sang veineux. La deuxième expérience mesure la réponse du signal BOLD et CBFà un stimulus visuel. Les résultats montrent que, après la consommation de caféine il y a une augmentation de l'OEF, une augmentation de la réponse du CMRO2 au stimulus visuel, une diminution de la CBF de base (CBF0) et un changement non-significatif du CMRO2 de base (CMRO2,0). On observe une corrélation négative entre les changements générés par la consommation de caféine sur les réponses du signal BOLD et de la CBF au stimulus visuel et les changements générés par la consommation de caféine sur la CBF0. Néanmoins, en moyenne, la consommation de caféine ne génére aucun changement sur la réponse du signal BOLD et de la CBF au stimulus visuel.

The primary aim of this thesis is the reconciliation of two seemingly disparate quantitative magnetic resonance imaging (MRI) techniques proposed to characterize human brain white matter (WM) in health and disease. Quantitative magnetization transfer imaging (QMTI) and multi-component analysis of T2 relaxation (QT2) both attempt to quantify myelin content in vivo, but are based on fundamentally different models of WM. QMTI probes the macromolecular component of tissue using a two-pool model of magnetization transfer, while QT2 isolates the water signal from distinct micro-anatomical compartments. The specific objectives were to determine the interrelationship between measurements made with both techniques in the context of potential pathological changes associated with multiple sclerosis (MS), and to apply both to track WM changes in the acute phase of MS lesions. First, simulations were used to evaluate the theoretical sensitivity of each technique to the characteristics of a model of WM that incorporates four pools of magnetization, based on published in vitro measurements. Next, the experimental reproducibility of each technique was investigated, and the impact of certain basic variations in the data acquisition and analysis procedures was evaluated. In the final stage, both methods were applied longitudinally in vivo to assess the dynamic changes that occur in acute, contrast-enhancing lesions of MS. The theoretical results illustrate the sensitivity and limitations of QMTI and QT2 to specific pathology-inspired modifications of WM, and shed new light on the potential specificity of often-neglected QMTI parameters. The reproducibility of both techniques is acceptable for use in repeated clinical measurements, and QMTI has lower variability overall. The importance of corrections for magnetic field inhomogeneity in QMTI is demonstrated, and a simple optimization of the QMTI data acquisition is introduc / L'objectif principal de cette thèse est la réconciliation de deux techniques quantitatives d'imagerie par résonance magnétique, en apparence difféerentes, utilisées pour la caractérisation de la susbtance blanche du cerveau humain en santé ou affectée par la maladie. Les techniques d'imagerie quantitative par transfert de magnétisation (QTM) et d'analyse de la relaxation T2 par de multiples composantes (QT2) proposent toutes deux des mesures in vivo de la quantitée de myéline, mais à l'aide de modèles fondamentalement différents. D'un côté, l'imagerie QTM sonde la composante macro-moléculaire des tissues à l'aide d'un modèle à deux réservoirs pour le transfert de magnétisation. De l'autre, l'imagerie QT2 sépare les signaux acqueux provenant de compartiments micro-anatomiques distincts. Plus spécifiquement, cet ouvrage cherche à mieux comprendre l'interdépendance des mesures de ces deux techniques dans le contexte pathologique de la sclérose en plaques (SEP), pour ensuite les appliquer à l' étude de lésions aigues de SEP. En premier lieu, des simulations ont été effectuées pour évaluer la sensibilité de chaque technique aux caractéristiques d'un modèle plus complet de la substance blanche, qui découle de résultats in vitro publiés et incorpore quatre réservoirs de magnétisation. Ensuite, la reproductibilité de chacune des techniques a été évaluée; de plus, quelques variations élémentaires des méthodes d'acquisition et d'analyse des données examinées. En dernier lieu, les deux techniques ont été utilisées in vivo afin de mesurer les changements dynamiques des lésions aigues de SEP, présentant un hyper-signal rehaussée par un agent de contraste. Les résultats des simulations démontrent d'un point de vue théorique la sensibilité et les limites de chacune de ces technique aux changements dans la substance blanche. Ces résultats apportent égalem

The effect of shear rate on the ADP-induced aggregation of human blood platelets in flow through tubes was studied over the full physiologically significant range. The extent of single platelet aggregation at 0.2 $ mu$M ADP in citrated platelet-rich plasma, PRP, was greatest at mean tube shear rate, G = 314 s$ sp{-1}$; however, aggregate size steadily decreased from G = 39.3 to 1800 s$ sp{-1}$. At 1.0 $ mu$M ADP the rate of aggregation increased up to G = 1800 s$ sp{-1}$ where virtually no unaggregated platelets remained after 43 s of flow, although, aggregate size was still limited by shear rate. A shear-dependent delay in the onset of aggregation and an increase in collision efficiency with time suggest the existence of a time and shear-dependency in the expression of bonds mediating aggregation. Greater aggregation of platelets from female donors than male donors was due to differences in the ionized calcium concentration, (Ca$ sp{2+}$), in the plasma of donors of different hematocrit when the chelating agent citrate is used as anticoagulant. At physiological (Ca$ sp{2+}$) aggregation was much greater in heparinized and hirudinized plasma than in citrated plasma and no sex difference was present. Aggregation in whole blood was much greater than in PRP due to a shear-dependent increase in the frequency of collision between activated platelets caused by the motion of red cells.

Photodynamic therapy (PDT) is a treatment that makes use of light and a photosensitizing drug to destroy malignant cells. Current clinically approved drugs suffer from many limitations; the most prevalent of these is due to the absorption coefficient of human tissues in the wavelength regime where these drugs are excitable. Semiconductor quantum dots (QDs) can overcome this drawback since they can be synthesized to become excited at any wavelength. The goal of this thesis is to explore the possibility of using core/shell Indium Phosphide/Zinc Sulfide (InP/ZnS) quantum dots (QDs) for photodynamic therapy applications. Electron paramagnetic resonance (EPR) spectroscopy and colorimetric assays were used to identify the nature of toxic species produced. From these findings, the physical mechanism by which these particles produce toxic species is discussed. It was found that InP/ZnS QDs produced superoxide anions and hydroxyl radicals, the levels of which depended on the ZnS shell thickness. Furthermore, the level of cellular uptake was studied in different cell lines using confocal microscopy. It was found that InP localized in the perinuclear region of all cell lines and that B16 melanoma cells showed the most efficient levels of uptake (2.5 times greater than the uptake from KB cells). Lastly, conjugation of InP QDs to the chemotherapeutic drug doxorubicin (Dox) was studied using flow cytometry and colorimetric assays. It was found that conjugation of Dox to InP led to enhanced levels of cell death; it is proposed that this was due to more efficient drug delivery by the conjugate. In summary, photosensitization processes in InP/ZnS QDs can be exploited for PDT applications; these particles also prove to be promising as a drug delivery agent. Despite this, photophysical processes of these QDs must be further explored to ameliorate their design for PDT. / La thérapie photodynamique (TPD) est un traitement médical qui détruit les cellules cancéreuses en utilisant des photons de lumière, typiquement en forme de laser, afin d'activer des drogues photosensibles. Présentement, les médicaments approuvés pour usage clinique ont d'importantes limitations. Particulièrement, le coefficient d'absorption des tissus humains se retrouve dans la même gamme de longueur d'onde où les médicaments sont excitables; par conséquent, leur efficacité est compromise. Les nanoparticules de matériaux semi-conducteurs, appelées aussi points quantiques (PQs), ont l'habilité de surpasser cette limitation parce qu'ils peuvent être produits pour absorber la lumière à n'importe quelle longueur d'onde. L'objectif de cette thèse est donc d'évaluer la possibilité d'utiliser les PQs pour la TPD. Plus spécifiquement, les PQs composés d'un cœur de phosphure d'indium (InP) avec une coquille du sulfure de zinc (ZnS) ont été examinés. La spectroscopie par résonance paramagnétique électronique (RPE) et les tests colorimétriques ont été utilisés pour identifier la nature des espèces toxiques produites, ainsi que le mécanisme responsable de leur formation. Les résultats ont montré que les particules de InP/ZnS produisent des anions de superoxyde et des radicaux d'hydroxyle; la quantité des radicaux formés dépend de l'épaisseur de la coquille ZnS. En plus, la microscopie confocale a été utilisée pour évaluer l'ingestion intracellulaire des PQs par divers types de cellules. Ces images ont démontré que les PQs se concentrent dans le cytoplasme autour du noyau et que les cellules mélanomes de type B16 sont celles qui absorbent le plus (2.5 fois plus que les cellules KB). Finalement, les PQs ont été conjuguées à un agent chimiothérapeutique (doxorubicin (Dox)) et leur toxicité a été explorée par cytométrie en flux et des tests colorimétriques. La mort cellulaire a augmenté avec l'attachement de PQs, ce qui s'explique par une amélioration de la livraison intracellulaire de Dox. En conclusion, les PQs InP/Zn révèlent être des candidats prometteurs en tant que médicaments et agents de livraison pour la TPD, cependant certains éléments de leur structure restent à être améliorés.

A method is described in this study whereby dose distributions calculated by a treatment planning system (TPS) were evaluated by using dose distributions calculated with Monte Carlo (MC) simulations. The MC calculated dose data were used as a benchmark. A generic Siemens MD 2 linear accelerator was simulated with the BEAMnrc MC code to obtain beam specific dynamic variables in a phase space file (PSF) related to particle fluence in a plane at a known distance from a water phantom. Dose distributions from various field sizes were produced by simulations with the DOSXYZnrc MC code. Two datasets were produced consisting of percentage depth dose (PDD), profiles and diagonal profile data for 6 and 15MV x-ray beams. The CadPlan TPS was commissioned with these datasets for both energies. Analyses of TPS calculated dose distributions were done in a water phantom and dose distributions for various clinical cases on patient CT data. Patient CT datasets were transformed into patient CT models that were suitable for dose calculations with DOSXYZnrc. These models consisted of various media with various densities for which interaction cross section data is available. Dose distributions for a number of clinical treatment plans could be devised on both the TPS and DOSXYZnrc. These included head and neck, breast, lung, prostate, oesophagus and brain plans. Calculations on the TPS were done for the Single Pencil Beam (SPB) and in some cases the Double Pencil Beam (DPB) convolution algorithms in combination with the Batho and ETAR (Equivalent Tissue-air ratio) inhomogeneity correction algorithms. Dose distributions were normalized to the depth of maximum dose (dmax) for single fields and to the ICRU reference point in full treatment plans. The location of these points was the same for the TPS and DOSXYZnrc distributions. PDD curves, beam profiles, dose-volume histograms (DVHs) and equivalent uniform doses (EUDs) were produced to aid in the evaluation of the TPS dose calculation accuracy. Results demonstrated that the assumptions in the convolution models used to produce beam penumbra regions, especially in blocked field cases, fail to account for scattered dose contributions outside the treatment field and overestimated the dose underneath small or thin shielding blocks. The PB algorithms in combination with the inhomogeneity corrections show total disregard for lateral and longitudinal electron transport through heterogeneous media. This effect is pronounced in regions where electronic equilibrium is not found, like low density lung. This region, in combination with high density bone nearby, proved even larger discrepancies as dose absorption decreases in low density media and increases in high density media. A small 15 MV field passing through lung tissue exhibited large dose calculation errors by the PB algorithms. The dataset produced here is flexible enough to be used as a benchmark for any TPS utilizing commissioning measurements in water. This method can address commissioning results as well as any clinical situation requiring dose calculation verification.

South Africa is not excluded from the problems encountered world-wide in the treatment of nosocomial urinary tract infections, commonly caused by enzyme-producing Enterobacteriaceae. These enzymes include the Ã-lactamases and extended-spectrum Ã-lactamases (ESBLs) capable of hydrolysing the Ã-lactam agents and in particular the expanded-spectrum cephalosporins frequently used. The study was designed to determine the role of Ã-lactamases in resistance development in commonly encountered pathogens implicated in urinary tract infections and to characterise the enzymes involved. Resistance to the Ã-lactam agents amoxicillin, ceftriaxone, ceftriaxone, piperacillin and cefoxitin was suspected to involve the presence of one or more β-lactamases in the isolates from Bloemfontein hospitals. Diverse and complex β-lactamases were identified and ESBLs were detected in 80% of the isolates. These β-lactamases were characterised by isoelectric focusing (IEF) and genetic analysis (DNA amplification by PCR) to investigate the presence of possible genes responsible for resistance development. The production of blaTEM and blaSHV type genes was demonstrated. Isolates harbouring these genes were highly resistant to amoxicillin and piperacillin, with MIC90s of >128μg/ml. Resistance to these antibiotics was shown to be readily transferred between strains and there was an indication that the resistance genes are carried on plasmids and was transferred by conjugation. A plasmid of 9-10 kb was detected in 83% of the isolates and could be one of the mechanisms implicated in the transfer of ESBLs in uropathogenic bacteria. Ã-Lactam resistance could be attributed to the presence and action of Ã-lactamases such as the TEM and SHV type enzymes and this resistance can be transmitted between bacteria, causing problems specifically in the hospital environment. Further and continuous investigations are required to find a solution for this ever increasing problem.

The need to find new manners in which to combat cardiovascular disease and associated thrombotic complications, remains a high priority in industrialised countries. Even in third-world countries the implications and associated risks of these diseases are being felt more and more. The advent of the biotechnology era and employment of recombinant DNA techniques has brought about exponential advances in understanding the complex mechanisms of haemostasis, and is employed to find new ways to combat pathological thrombotic complications. The challenge is to harness the many tools and techniques produced by the ongoing biotechnology explosion, and apply them to elucidate questions still unanswered and explore areas still unknown. In this study it was illustrated that modern molecular biology techniques can be applied in many areas of thrombosis and haemostasis research. The display of cDNA libraries on the surfaces of filamentous bacteriophages was used in the search for novel antithrombotic compounds from a haematophagous insect Hippobosca rufipes. Phages displaying the cDNA libraries were panned against human a-thrombin and selected according to their binding affinity and inhibition ability. To illustrate the use of a Escherichia coli expression system, a domain of a enzyme was cloned, expressed, and the recombinant peptide isolated and refolded. ADAMTS-13 was recently identified as an important role player in the realm of von Willebrand factor activity, including primary haemostasis and pathological disorders. The second carboxy-terminal CUB domain of ADAMTS-13 was amplified from full-length cDNA, cloned into a expression vector system, and expressed as insoluble inclusion bodies in the cytoplasm of E. coli, from where it was isolated and refolded. In this study, molecular techniques were used in different phases of research into the specific activity and interactions of a particular component of the haemostatic system. This illustrated the marriage of biotechnology with fundamental medical research in an era of interdisciplinary sciences.