Capsid modified replicating adenovirus to selectively target colon cancer

Résumé: Pratiquement tous les cancers du colon contiennent des mutations dans la voie de signalisation de Wnt qui active constitutivement cette voie. Cette activation mène à la stabilisation de la β-catenine. La β-catenin est transportée dans le noyau ou elle active des gènes cible en interagissant avec le facteur de transcription de TCF/LEF. Des adénovirus qui peuvent sélectivement se répliquer dans les cellules tumorales sont les agents qui peuvent permettre la déstruction de la tumeur mais pas le tissu normal. In vitro, les adénovirus avec des sites d'attachement du facteur de transcription TCF dans les promoteurs de l'adénovirus montrent une sélectivité et une activité dans une large sélection de lignées cellulaires de cancer du colon. Au contraire, in vivo, quand les adénovirus modifiés sont injectés dans la circulation, ils sont moins efficaces à cause de leur fixation par le foie et à cause de l'absence d'expression du récepteur du Coxsackie-Adénovirus (CAR). Le but de ma thèse était de modifier la protéine principale de capside de l'adénovirus, fibre, pour augmenter l'infection des tumeurs du cancer du colon. La fibre de l'adénovirus est responsable de l'attachement aux cellules et de l'entrée virale. J'ai inséré un peptide RGD dans la boucle HI de la fibre qui dirige sélectivement le virus aux récepteurs des integrines. Les integrines sont surexprimées par les cellules du cancer du colon et l'endothélium des vesseaux de la tumeur. Le virus re-ciblé, vKH6, a montré une activité accrue dans toutes les lignées cellulaires de cancer du colon, tandis que la sélectivité était maintenue. In vivo, vKH6 était supérieur au virus avec une capside de type sauvage en retardant la croissance de la tumeur. Le virus s'est répliqué plus vite et dispersé graduellement dans la tumeur. Cet effet a été montré par hybridation in situ et par PCR quantitative. Cependant, la monothérapie avec le virus n'a pu retarder la croissance des cellules tumorales SW620 greffées que de 2 semaines, mais à cause des régions non infectées la tumeur n'a pas pu être éliminée. Bien que la combinaison avec les chimiothérapies conventionnelles soit d'intérêt potentiel, presque toutes interfèrent avec la réplication virale. Les drogues antiangiogéniques sont des agents anti-tumoraux efficaces et prometteurs. Ces drogues n'interfèrent pas avec le cycle de vie de l'adénovirus. RAD001 est un dérivé de la rapamycine et il inhibe mTOR, une protéine kinase de la voie de PI3K. RAD001 empêche la croissance des cellules et il a aussi des effets anti-angiogénique et immunosuppressifs. RAD001 in vitro n'affecte pas l'expression des gènes viraux et la production virale. La combinaison de VKH6 et RAD001 in vivo a un effet additif en retardant la croissance de la tumeur. Des nouveaux peptides plus efficaces dans le ciblage de l'adénovirus sont nécessaires pour augmenter l'infection des tumeurs. J'ai créé un système de recombinaison qui permettra la sélection de nouveaux peptides dans le contexte du génome de l'adénovirus. Summary Virtually all colon cancers have mutations in the Wnt signalling pathway which result in the constitutive activation of the pathway. This activation leads to stabilization of β-catenin. β-catenin enters the nucleus and activates its target genes through interaction with the TCF transcription factor. Selectively replicating adenoviruses are promising novel agents that can destroy the tumour but not the surrounding normal tissue. In vitro, adenoviruses with TCF binding sites in the early viral promoters show selectivity and activity in a broad panel of viruses but in vivo they are less effective due to the lack of expression of the Coxsackie-Adenovirus receptor (CAR). The aim of my thesis was to modify the major capsid protein of the adenovirus, fibre, to increase the infection of colon tumours. Fibre of adenovirus is responsible for the binding to cells and for the viral uptake. I inserted an RGD binding peptide into the HI loop of fibre that selectively targets the virus to integrins that are overexpressed on tumour cells and on tumour endothelium. The retargeted virus, vKH6, showed increased activity in all colon cancer cell lines while selectivity was maintained. In vivo, vKH6 is superior to a matched virus with a wild type capsid in delaying tumour growth. vKH6 replicates and gradually spreads within the tumour as shown by in situ hybridization and Q-PCR. The virus alone can delay the growth of SW620 xenografts by 2 weeks but due to uninfected tumour regions the tumour cannot be cured. Although combination with conventional chemotherapeutics is of potential interest, almost all of them interfere with the viral replication. Growing evidence supports that anti-angiogenic drugs are effective and promising anti-tumour agents. These drugs interfere less with the viral life cycle. RAD001 is a rapamycin derivative and it blocks mTOR, a protein kinase in the PI3K pathway. RAD001 inhibits cell growth and has strong anti-angiogenic and immunosuppressive effects. RAD001 in vitro does not affect viral gene expression and viral burst size. In vivo vKH6 and RAD001 have an additive effect in delaying tumour growth, but tumour growth is still not completely inhibited. To further increase tumour infection new tumour specific targeting peptides are needed. I created an adenovirus display library that will allow the selection of targeting peptides. This system may also facilitate the production of fibre modified viruses.

Theoretical Analysis and Simulations of Vertically Vibrated Granular Materials

The dynamical properties ofshaken granular materials are important in many industrial applications where the shaking is used to mix, segregate and transport them. In this work asystematic, large scale simulation study has been performed to investigate the rheology of dense granular media, in the presence of gas, in a three dimensional vertical cylinder filled with glass balls. The base wall of the cylinder is subjected to sinusoidal oscillation in the vertical direction. The viscoelastic behavior of glass balls during a collision, have been studied experimentally using a modified Newton's Cradle device. By analyzing the results of the measurements, using numerical model based on finite element method, the viscous damping coefficient was determinedfor the glass balls. To obtain detailed information about the interparticle interactions in a shaker, a simplified model for collision between particles of a granular material was proposed. In order to simulate the flow of surrounding gas, a formulation of the equations for fluid flow in a porous medium including particle forces was proposed. These equations are solved with Large Eddy Simulation (LES) technique using a subgrid-model originally proposed for compressible turbulent flows. For a pentagonal prism-shaped container under vertical vibrations, the results show that oscillon type structures were formed. Oscillons are highly localized particle-like excitations of the granular layer. This self-sustaining state was named by analogy with its closest large-scale analogy, the soliton, which was first documented by J.S. Russell in 1834. The results which has been reportedbyBordbar and Zamankhan(2005b)also show that slightly revised fluctuation-dissipation theorem might apply to shaken sand, which appears to be asystem far from equilibrium and could exhibit strong spatial and temporal variations in quantities such as density and local particle velocity. In this light, hydrodynamic type continuum equations were presented for describing the deformation and flow of dense gas-particle mixtures. The constitutive equation used for the stress tensor provides an effective viscosity with a liquid-like character at low shear rates and a gaseous-like behavior at high shear rates. The numerical solutions were obtained for the aforementioned hydrodynamic equations for predicting the flow dynamics ofdense mixture of gas and particles in vertical cylindrical containers. For a heptagonal prism shaped container under vertical vibrations, the model results were found to predict bubbling behavior analogous to those observed experimentally. This bubbling behavior may be explained by the unusual gas pressure distribution found in the bed. In addition, oscillon type structures were found to be formed using a vertically vibrated, pentagonal prism shaped container in agreement with computer simulation results. These observations suggest that the pressure distribution plays a key rolein deformation and flow of dense mixtures of gas and particles under vertical vibrations. The present models provide greater insight toward the explanation of poorly understood hydrodynamic phenomena in the field of granular flows and dense gas-particle mixtures. The models can be generalized to investigate the granular material-container wall interactions which would be an issue of high interests in the industrial applications. By following this approach ideal processing conditions and powder transport can be created in industrial systems.

Local and Global Feature Extraction for Invariant Object Recognition

Perceiving the world visually is a basic act for humans, but for computers it is still an unsolved problem. The variability present innatural environments is an obstacle for effective computer vision. The goal of invariant object recognition is to recognise objects in a digital image despite variations in, for example, pose, lighting or occlusion. In this study, invariant object recognition is considered from the viewpoint of feature extraction. Thedifferences between local and global features are studied with emphasis on Hough transform and Gabor filtering based feature extraction. The methods are examined with respect to four capabilities: generality, invariance, stability, and efficiency. Invariant features are presented using both Hough transform and Gabor filtering. A modified Hough transform technique is also presented where the distortion tolerance is increased by incorporating local information. In addition, methods for decreasing the computational costs of the Hough transform employing parallel processing and local information are introduced.

A modified structural stress method for fatigue assessment of welded structures

Fatigue life assessment of weldedstructures is commonly based on the nominal stress method, but more flexible and accurate methods have been introduced. In general, the assessment accuracy is improved as more localized information about the weld is incorporated. The structural hot spot stress method includes the influence of macro geometric effects and structural discontinuities on the design stress but excludes the local features of the weld. In this thesis, the limitations of the structural hot spot stress method are discussed and a modified structural stress method with improved accuracy is developed and verified for selected welded details. The fatigue life of structures in the as-welded state consists mainly of crack growth from pre-existing cracks or defects. Crack growth rate depends on crack geometry and the stress state on the crack face plane. This means that the stress level and shape of the stress distribution in the assumed crack path governs thetotal fatigue life. In many structural details the stress distribution is similar and adequate fatigue life estimates can be obtained just by adjusting the stress level based on a single stress value, i.e., the structural hot spot stress. There are, however, cases for which the structural stress approach is less appropriate because the stress distribution differs significantly from the more common cases. Plate edge attachments and plates on elastic foundations are some examples of structures with this type of stress distribution. The importance of fillet weld size and weld load variation on the stress distribution is another central topic in this thesis. Structural hot spot stress determination is generally based on a procedure that involves extrapolation of plate surface stresses. Other possibilities for determining the structural hot spot stress is to extrapolate stresses through the thickness at the weld toe or to use Dong's method which includes through-thickness extrapolation at some distance from the weld toe. Both of these latter methods are less sensitive to the FE mesh used. Structural stress based on surface extrapolation is sensitive to the extrapolation points selected and to the FE mesh used near these points. Rules for proper meshing, however, are well defined and not difficult to apply. To improve the accuracy of the traditional structural hot spot stress, a multi-linear stress distribution is introduced. The magnitude of the weld toe stress after linearization is dependent on the weld size, weld load and plate thickness. Simple equations have been derived by comparing assessment results based on the local linear stress distribution and LEFM based calculations. The proposed method is called the modified structural stress method (MSHS) since the structural hot spot stress (SHS) value is corrected using information on weld size andweld load. The correction procedure is verified using fatigue test results found in the literature. Also, a test case was conducted comparing the proposed method with other local fatigue assessment methods.

Spray coating technique asa surface treatment for woodcontaining paper grades

The objective of this work was to introduce the emerging non-contacting spray coating process and compare it to the existing coating techniques. Particular emphasis was given to the details of the spraying process of paper coating colour and the base paper requirements set by the new coating method. Spraying technology itself is nothing new, but the atomisation process of paper coating colour is quite unknown to the paper industry. The differences between the rheology of painting and coating colours make it very difficult to utilise the existing information from spray painting research. Based on the trials, some basic conclusion can be made:The results of this study suggest that the Brookfield viscosity of spray coating colour should be as low as possible, presently a 50 mPas level is regarded as an optimum. For the paper quality and coater runnability, the solids level should be as high as possible. However, the graininess of coated paper surface and the nozzle wear limits the maximum solids level to 60 % at the moment. Most likelydue to the low solids and low viscosity of the coating colour the low shear Brookfield viscosity correlates very well with the paper and spray fan qualities. High shear viscosity is also important, but yet less significant than the low shear viscosity. Droplet size should be minimized and besides keeping the brrokfield viscosity low that can be helped by using a surfactant or dispersing agent in the coating colour formula. Increasing the spraying pressure in the nozzle can also reduce the droplet size. The small droplet size also improves the coating coverage, since there is hardly any levelling taking place after the impact with the base paper. Because of the lack of shear forces after the application, the pigment particles do not orientate along the paper surface. Therefore the study indicates that based on the present know-how, no quality improvements can be obtained by the use of platy type of pigments. The other disadvantage of them is the rapid deterioration of the nozzle lifetime. Further research in both coating colour rheology and nozzle design may change this in the future, but so far only round shape pigments, like typically calcium carbonate is, can be used with spray coating. The low water retention characteristics of spray coating, enhanced by the low solids and low viscosity, challenge the base paper absorption properties.Filler level has to be low not to increase the number of small pores, which have a great influence on the absorption properties of the base paper. Hydrophobic sizing reduces this absorption and prevents binder migration efficiently. High surface roughness and especially poor formation of the base paper deteriorate thespray coated paper properties. However, pre-calendering of the base paper does not contribute anything to the finished paper quality, at least at the coating colour solids level below 60 %. When targeting a standard offset LWC grade, spraycoating produces similar quality to film coating, but yet blade coating being on a slightly better level. However, because of the savings in both investment and production costs, spray coating may have an excellent future ahead. The porousnature of the spray coated surface offers an optimum substrate for the coldset printing industry to utilise the potential of high quality papers in their business.

A Study on Fractionation and Ultrafiltration of Proteins with Characterized Modified and Unmodified Membranes

Membrane filtration has become increasingly attractive in the processing of both foodand biotechnological products. However, the poor selectivity of the membranes and fouling are the critical factors limiting the development of UF systems for the specific fractionation of protein mixtures. This thesis gives an overview on fractionation of proteins from model protein solutions or from biological solutions. An attempt was made to improve the selectivity of the available membranes by modifying the membranes and by exploiting the different electrostatic interactions between the proteins and the membrane pore surfaces. Fractionation and UF behavior of proteins in the model solutions and in the corresponding biological solutions were compared. Characterization of the membranes and protein adsorptionto the membrane were investigated with combined flux and streaming potential studies. It has been shown that fouling of the membranes can be reduced using "self-rejecting" membranes at pH values where electrostatic repulsion is achieved between the membrane and the proteins in solution. This effect is best shown in UF of dilute single protein solutions at low ionic strengths and low pressures. Fractionation of model proteins in single, binary, and ternary solutionshas been carried out. The results have been compared to the results obtained from fractination of biological solutions. It was generally observed that fractination of proteins from biological solutions are more difficult to carry out owingto the presence of non studied protein components with different properties. Itcan be generally concluded that it is easier to enrich the smaller protein in the permeate but it is also possible to enrich the larger protein in the permeateat pH values close to the isoelectric point of the protein. It should be possible to find an optimal flux and modification to effectively improve the fractination of proteins even with very similar molar masses.

On the origin of the inertia: the modified Newtonian dynamics theory

The sameness between the inertial mass and the gravitational mass is an assumption and not a consequence of the equivalent principle is shown. In the context of the Sciama’s inertia theory, the sameness between the inertial mass and the gravitational mass is discussed and a certain condition which must be experimentally satisfied is given. The inertial force proposed by Sciama, in a simple case, is derived from the Assis’ inertia theory based in the introduction of a Weber type force. The origin of the inertial force is totally justified taking into account that the Weber force is, in fact, an approximation of a simple retarded potential, see [18, 19]. The way how the inertial forces are also derived from some solutions of the general relativistic equations is presented. We wonder if the theory of inertia of Assis is included in the framework of the General Relativity. In the context of the inertia developed in the present paper we establish the relation between the constant acceleration a0 , that appears in the classical Modified Newtonian Dynamics (M0ND) theory, with the Hubble constant H0 , i.e. a0 ≈ cH0 .

Recurrent Macular Edema in Central Retinal Vein Occlusion Treated with Intravitreal Ranibizumab using a Modified Treat and Extend Regimen.

Background: The aim of this study was to evaluate the stability over time of the individually defined interval of intravitreal ranibizumab injection (IVR) for the treatment of recurrent macular edema (ME) in central retinal vein occlusion (CRVO). Patients and Methods: A case series of treatment naïve patients followed in the Jules Gonin Eye Hospital for macular edema due to central retinal vein occlusion is presented. Patients were treated monthly with IVR until complete absence of fluid on qualitative SD-OCT with a minimum of 5 monthly IVR. Thereafter, they were followed according to a modified treat and extend regimen (mTER). Results: Twelve eyes (12 patients) with ME due to CRVO were included. The mean follow-up period was 31.3 months. Analysis showed that best corrected visual acuity (BCVA), central macular thickness and qualitative spectral domain optical coherence tomography (SD-OCT) showed comparable results under monthly interval, after titration of an individualized interval and when performed in a series. 78 % of treating intervals were within ± 2 weeks of the first individually adjusted interval. The mean first defined interval was 4.3 weeks and the mean interval over time was 5.5 weeks (p = 0.003). There was a trend towards longer interval over time. Conclusion: The adjusted interval of retreatment of patients with ME due to CRVO showed a high stability with a trend toward longer duration over time. An mTER regimen seems to be valuable to follow patients with ME with good stabilization of VA.