The Centers for Disease Control and Prevention estimated 23,000,000 cases of viral gastroenteritis caused by Norovirus in 2000, 40% of which were transmitted by food including: a variety of fresh produce, cake, deli meats, fruit salad, cheeses and ice. (CDC, 2003). An estimated 83,391 cases of Hepatitis A virus was reported in 2000, of which 5% was attributed to foodborne transmission (CDC, 2003). These figures underscore an urgent need for a method that can isolate virus from a variety of food matrices. The aim of this study was to develop an overall assessment of the inhibitory effects of a variety of food matrices on Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Additionally, to compare a sequence specific hybridization probe amplification format to a non sequence specific SYBR Green format using the Roche LightCycler. The secondary aim was to evaluate the effectiveness of a food virus concentration and isolation protocol under development at the Florida Department of Health Bureau of Laboratories, Tampa. Three food specimens consisting of prepackaged smoked ham, fresh cilantro, and Thompson's green grapes were seeded with three dilutions of poliovirus 3 (Sabin strain). A viral concentration procedure under development at the Florida Department of Health Bureau of Laboratories, Tampa was used to isolate the virus. Real Time RT-PCR was carried out on the Roche LightCycler in SYBR Green and Hybridization probe formats. Spiking the virus-negative samples of each matrix with a dilution series of poliovirus 3 created post flocculation spikes. This post-flocculation dilution series amplification allowed a standard curve to be created unique to each food matrix. The flocculation and concentrations specimens were then amplified and the standard curves from the post-flocculation seed were used to calculate the loss associated with the concentration procedure. This study reports significant differences (p<0.05) in recovery detected between the various matrices, and Real Time RT-PCR formats. The concentration protocol under development at the Florida Department of Health Bureau of Laboratories, Tampa, demonstrates a 12-78% recovery of seeded virus in a simulated “real world” virus contamination event among the various matrices.

L'étude concerne les lasers de puissance à semi-conducteurs émettant aux alentours de 1480nm dont l'utilisation principale est l'amplification Raman dans les fibres optiques pour les futurs systèmes de télécommunications. Cette amplification nécessite des puissances de pompe supérieures à 400mW. Les travaux ont porté sur la conception et la caractérisation des lasers de pompe en vue d'une augmentation de la puissance de sortie. Après avoir identifier les principaux effets limitant la puissance des pompes (effets thermiques, Longitudinal Spatial Hole Burning, pertes internes), trois solutions ont été mises en œuvre au cours de cette thèse pour réduire ces effets. Nous avons montré que l'introduction de guide large multimode (MMI) permettait à la fois une réduction des effets thermiques et une augmentation de la puissance de saturation de l'ordre de 10% en comparaison au laser à ruban droit. La cavité évasée a permis une réduction du Longitudinal Spatial Hole Burning entraînant un gain en puissance de saturation de l'ordre de 30% avec une puissance de 625mW pour un courant de 2.5A. Enfin, la diminution des pertes internes via l'utilisation d'un quaternaire faible indice coté InP dopé n (structure à "cladding asymétrique") a permis l'obtention d'un record de puissance (1 Watt couplé sous 5A) dans une fibre monomode avec un guide à ruban droit.

Le RadLV/VL₃ clone V-13 est un rétrovirus murin thymotrope, non défectif, fortement leucémogène et écotrope. Il induit des lymphomes des cellules T en s'intégrant dans le génome de l'hôte. Le virus peut causer une tumeur en modifiant le fonctionnement des gènes près desquels il s'est intégré. Ces intégrations, si retrouvées au même endroit dans plus d'une tumeur, définissent un site commun d'intégration. Ce projet a pour but de trouver un ou des nouveaux sites d'intégration du rétrovirus RadLV/VL₃ clone V-13. Pour ce faire, un oligonucléotide de séquence connue, nommé splinkerette, a été utilisé. L'ADN d'une souris tumorale est coupée puis une splinkerette y est ligasée. Ceci permet d'amplifier, par des réactions de PCR, les régions flanquantes aux intégrations rétrovirales. Une fois clonées, les produits des PCR sont séquencés, permettant de situer les intégrations dans le génome de la souris, qui est disponible dans les banques de données. Il est ainsi possible de repérer les gènes à proximité de l'intégration rétrovirale. Certains gènes d'intérêt ont déjà été retrouvés grâce à cette technique pour d'autres rétrovirus. Dans le cas du RadLV/VL₃, parmi une vingtaine de tumeurs et environ 70 bandes analysées, les gènes Notch l, Rasgrp1, Rorc, Lef1, Gfi1, Ncor2, Scarb1, Lfng, Mad, Myb, Ahi1, Supt4h, Bzrap1, Sept9, Fos, Jundm2, Myc, Pim1, Ccnd3, Bcl211 et Gpc3 ont été trouvés. Plusieurs de ces oncogènes n'ont jamais été associés au rétrovirus RadLV/VL₃. Deux régions potentiellement oncogéniques sur les chromosomes 7 et 11 ont été identifiées. La compréhension du fonctionnement des oncogènes permettra d'empêcher leur expression aberrante, ou d'utiliser des rétrovirus comme vecteurs dans la thérapie génique afin de corriger l'expression de gènes mutés. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : ADN, Clonage, Leucémie, Oncogène, PCR, RadLV/VL₃, Rétrovirus, Séquençage, Site commun d'intégration, Sonde radioactive, Splinkerette, Tumeur.

The goal of this thesis is developing a two-color Ytterbium (Yb) fiber amplifier system that can be used for generation of mid-infrared radiation. Previously, our group reported generating 20 µW of average power, at a wavelength of 18µm. This was accomplished through the amplification of a two color-seed with peaks at 1040nm and 1110nm, through a two stage amplification without any compression. The mid-infrared radiation (MIR) was generated with a 4.5 ps pulse duration by the method of difference-frequency mixing, using 300 mW of average power from the two-color Yb-fiber amplifier. Because there was no limitation by two-photon absorption, MIR output power could be scaled by increasing the amplifier power. The current project aims to increase the peak power of the laser pulses to improve the efficiency of the nonlinear mixing. The two-colour seed is generated by continuum generation in a photonic crystal fibre, pumped by 200 mW of average power from a mode-locked Yb:fibre laser. In order to efficiently increase the energy of the two wavelengths, the 4.6 mW seed pulse is now pre-amplified up to 21 mW in a 2.7 m length single mode, single core Yb:fibre . The pre-amplifier used a double-ended pumping scheme with two single mode diode lasers at 976 nm each having 150 mW maximum pump power. A notch filter was placed in the output beam to eliminate any Amplified Spontaneous Emission. After further amplification in a 7 m length of double clad, Yb-fibre, a maximum average power of 727 mW was achieved for two colours peaked at 1035 nm and 1105 nm wavelengths. The pump power for this stage was 6 W. A grating stretcher is now used to select the two-colour input along with stretching the pulses. A three grating compressor is used to compress the output pulses to 466 fs pulse duration. After compression the average power of the two colours is 350 and 110 mW for wavelengths at 1035 and 1105nm, respectively. These higher power pulses are planned to be used to increase the mid-infrared generation efficiency.

This document contains the development of an amplifier for an ECG-signal and interfacing it to wireless communication. The purpose of this project is to get a clear ECG-signal without any noise, save it and send it through wireless communication.A challenge of the wireless communication unit is to send as little information as possible to make the communication faster, without loss of information in the ECG-signal.The context for this project is the integration of wireless communication in medical applications for home healthcare. This means that, patients are no longer bound to a specific healthcare location where they are monitored by medical instruments. Wireless communication will not only provide them with safe and accurate monitoring, but also the freedom of movement.

Many of today’s methods for the detection of biomolecules suffer from a high limit ofdetection due to poor signal generation upon recognition of target. By applying andoptimizing proximity ligation assay (PLA) in Western blotting (WB), the limit of detectionhas been lowered down to the picomolar range. In this report I have optimized the differentparameters that affect the signal generation and explored possibilities to increase the ease ofuse, by merging protocol steps and performing signal generating reactions at roomtemperature.

The technique of DNA origami utilizes the specific and limited bonding properties of DNA to fold single stranded DNA sequences of various lengths to form a predesigned structure. One longer sequence is used as a scaffold and numerous shorter sequences called staples, which are all complementary to the scaffold sequence, are used to fold the scaffold into intricate shapes. The most commonly used scaffold is derived by extracting the genome of the M13 phage and the staples are usually chemically synthesized oligonucleotides. Longer single stranded sequences are difficult to synthesize with high specificity, which limits the choices of scaffold sequences available. In this project two main methods of single stranded amplification, Rolling Circle Amplification (RCA) and the usage of helper phages, were explored with the goal to produce both a 378 nt scaffold and staple sequences needed for folding a DNA origami structure. To facilitate imaging by Transmission Electron Microscopy (TEM) of this small structure, the DNA origami structure was created to form a polymer structure. Production of the scaffold sequence in high yield was unsuccessful and no well-defined polymers were found in the folded samples, though a few results showed promise for further studies and optimizations. Due to time constraints of this project, only production of the scaffold sequence was tested. Unfortunately the scaffold produced by the helper phages was of the complementary strand to that used to design the DNA origami structure, and could therefore not be used for folding. The correct strand was produced by the RCA where the yield was too low when using Phi29 DNA polymerase for proper folding to take place, though small scale RCA by Bst DNA polymerase on the other hand showed promising results. These results indicate that the scaffold production may not be far off but still more experience in producing intermediate size oligonucleotides may be necessary before succeeding in high yield production of this 378 nt long sequence. The promise given by this production is to enable high yield, high purity, low cost and also an easily scalable process set-up. This would be an important step in future DNA nanotechnology research when moving from small scale laboratory research to large scale applications such as targeted drug delivery systems.

The goal of this thesis is developing a two-color Ytterbium (Yb) fiber amplifier system that can be used for generation of mid-infrared radiation. Previously, our group reported generating 20 µW of average power, at a wavelength of 18µm. This was accomplished through the amplification of a two color-seed with peaks at 1040nm and 1110nm, through a two stage amplification without any compression. The mid-infrared radiation (MIR) was generated with a 4.5 ps pulse duration by the method of difference-frequency mixing, using 300 mW of average power from the two-color Yb-fiber amplifier. Because there was no limitation by two-photon absorption, MIR output power could be scaled by increasing the amplifier power. The current project aims to increase the peak power of the laser pulses to improve the efficiency of the nonlinear mixing. The two-colour seed is generated by continuum generation in a photonic crystal fibre, pumped by 200 mW of average power from a mode-locked Yb:fibre laser. In order to efficiently increase the energy of the two wavelengths, the 4.6 mW seed pulse is now pre-amplified up to 21 mW in a 2.7 m length single mode, single core Yb:fibre . The pre-amplifier used a double-ended pumping scheme with two single mode diode lasers at 976 nm each having 150 mW maximum pump power. A notch filter was placed in the output beam to eliminate any Amplified Spontaneous Emission. After further amplification in a 7 m length of double clad, Yb-fibre, a maximum average power of 727 mW was achieved for two colours peaked at 1035 nm and 1105 nm wavelengths. The pump power for this stage was 6 W. A grating stretcher is now used to select the two-colour input along with stretching the pulses. A three grating compressor is used to compress the output pulses to 466 fs pulse duration. After compression the average power of the two colours is 350 and 110 mW for wavelengths at 1035 and 1105nm, respectively. These higher power pulses are planned to be used to increase the mid-infrared generation efficiency.

The human epidermal growth factor receptor-2 (HER2) protein has been associated with breast cancer progression and the HER2 status can be used to determine the type of treatment for each breast cancer patient. The purpose of this study was to examine the HER2 protein and gene statuses in breast cancer tissue samples using two methods and analyze the concordance between them. Ten paraffin-embedded, formaldehyde-fixed breast cancer tissue samples from the Biobank at the Department of Pathology and Cytology at Sundsvall Hospital were analyzed in this study. All samples were from women born between 1931 and 1976. The methods used were immunohistochemistry (IHC) to visualise the HER2 protein and silver in situ hybridization (SISH) to detect gene amplification. The IHC staining method is an indirect detection of the HER2 protein using antibodies. The SISH method used in this study is a Dual ISH which detects both the HER2 gene and the centromere region of Chromosome 17 on the same tissue slide. A HER2 gene/Chromosome 17 ratio was calculated according to the manufacturer’s instructions. This ratio was used to determine HER2 gene status. Out of ten samples, seven were positive with IHC and three were negative. The results from the SISH staining exposed a gene amplification in three of the IHC positive samples, while seven samples did not contain any amplified HER2 genes. The conclusion was that the concordance between IHC and SISH for HER2 was 60 percent.

<p>This document contains the development of an amplifier for an ECG-signal and interfacing it to wireless communication. The purpose of this project is to get a clear ECG-signal without any noise, save it and send it through wireless communication.A challenge of the wireless communication unit is to send as little information as possible to make the communication faster, without loss of information in the ECG-signal.The context for this project is the integration of wireless communication in medical applications for home healthcare. This means that, patients are no longer bound to a specific healthcare location where they are monitored by medical instruments. Wireless communication will not only provide them with safe and accurate monitoring, but also the freedom of movement.</p>