This project initiated with the aim for improving the ultrahigh resolution optical coherence tomography (UHR-OCT) system performance by considering the limitations to the axial OCT resolution for in vivo imaging of human and animal retina. To this end, a computational model was developed to simulate the effect of wavelength-dependant water absorption on the detected spectral shape of the broad-bandwidth light source used in UHR-OCT at 1060nm wavelength region, which effectively determines the axial OCT resolution in the retina. For experimental verification of the computational model, a custom built light source with a re-shaped spectrum (Superlum Inc.) was interfaced to the state-of-the-art UHR-OCT system. About 30% improvement of the axial OCT resolution in the rat retina and ~12% improvement of the axial OCT resolution in the human retina was achieved compared to the case of the almost Gaussian shaped spectrum of the standard, commercially available SLD. Although water absorption in the 1060nm spectral region strongly affects the sample beam, selecting a suitable light source with specific spectral shape can compensate for the undesired water absorption effect and thus result in significantly improved axial resolution in in vivo OCT retinal images. To demonstrate the advantages of the state-of-the-art OCT technology for non invasive retinal imaging, an established animal model of outer retina degeneration (sodium iodate (NaIO3)-induced retina degeneration) was employed for longitudinal monitoring of the degeneration and investigation of possible early and dynamic signs of damage undetected by other imaging modalities. The long-term (up to 3 months) and short-term (up to 12 hours) effect of sodium iodate toxicity on the layered structure of retina was monitored longitudinally and in vivo for the first time using OCT. An initial acute swelling of the retina, followed by progressive disruption and degeneration of outer retina was observed as a result of sodium iodate-induced damage. Changes in the thickness and optical reflectivity of individual retinal layers were extracted from the OCT images to quantify the changes occurring at different stages of the disease model. Results from this project present the theoretical and practical limits to the highest axial OCT resolution achievable for retina imaging in the 1060nm spectral range both in small animals and humans, and provided a framework for future development of novel light sources. Furthermore, UHR-OCT imaging was shown to be an effective and valuable modality for in vivo, non invasive investigation of retina degenerative disease.

El objetivo de este estudio es la calibración de diodos, para ser utilizados en dosimetria in vivo en haces de fotones de alta energía, en diversas situaciones, la mayoría de ellas no descritas en la literatura: En primer lugar se calibra un conjunto de diodos para determinar la dosis entrada y salida en la técnica específica de ICT (capítulo 3). Se hace especial hincapié en la calibración de los diodos detrás de protecciones a pulmón de transmisión parcial. En el capítulo 4 se desarrolla un algoritmo de cálculo para calcular las dosis a plano medio a partir de las dosis in vivo en ICT. Se presentan resultados en una serie de pacientes. Se comprueba la aplicabilidad de este algoritmo en presencia de heterogeneidades (pulmón) y detrás de protecciones de transmisión parcial. Se compara este algoritmo con el algoritmo propuesto por Rizzoti et alt. [41]. La calibración de diodos para altas energías de RX en técnicas estándar de tratamiento se trata en los capítulos 5 y 6. En el capítulo 5 se comparan dos tipos de diodos diseñados para trabajar con RX de 16-25 MV. En el capítulo 6 se amplía esta comparación a 4 tipos de diodos comercializados y también se presenta la calibración de un diodo para trabajar con haces de RX de energías comprendidas entre 4-8 MV. Este capítulo corresponde a la recopilación de la experiencia del Hospital de la Santa Creu i Sant Pau en la calibración de diodos para medir la dosis in vivo a la entrada en técnicas estándar así como en la implementación de la dosimetría in vivo en la rutina clínica. Esta recopilación hace inevitable que se repitan, aunque con más detalle, algunos de los procedimientos de calibración citados en capítulos anteriores. Ha sido también objetivo del capítulo 5 matizar una tesis propuesta por G. Rikner y E. Russell [11, 13, 14, 16] que hace referencia a la inferioridad desde el punto de vista dosimétrico de los diodos tipo n, en un nuevo diodo n comercializado por Pecitron en 1997.

Este estudio evalúa el efecto en la pérdida de brackets de la contaminación por saliva, introducida en distintos momentos del proceso de adhesión, usando Transbond Plus® (3M Unitek) y la resina Transbond XT® (3M). Asimismo se analiza cómo se produce el patrón de pérdidas. Material y métodos. Se llevó a cabo un “Split mouth desing” según el cual se introducía únicamente contaminación en el primer y tercer cuadrante, mientras que el segundo y el cuarto se cementaban sin contaminación. Se tomaron 46 pacientes. De forma aleatoria se asignó a cada paciente el tipo de contaminación a aplicar: grupo de saliva aplicada antes del adhesivo en el primer y tercer cuadrante; grupo de saliva aplicada después del adhesivo en el primer y tercer cuadrante. En ambos grupos, el segundo y el cuarto cuadrante, sin contaminación, ejercían de grupo control. Se cementaron un total de 531 dientes, 153 del grupo de contaminación antes, 115 del grupo de contaminación después y 263 sin contaminar o grupo control. El periodo de observación y de análisis fue, como mínimo, de seis meses para todos los dientes cementados. Resultados. No se encontraron diferencias significativas en el porcentaje de pérdidas de los grupos comparados. En la localización de las pérdidas, el estudio de los residuos tipificados mostró un número mayor de pérdidas esperadas para los segundos premolares, en los dientes contaminados y un número menor de lo esperado para los incisivos superiores contaminados y sin contaminar. La mayoría de las pérdidas se produjeron en los tres primeros meses. La forma de perderse fue predominantemente con los valores 0 y 1 del índice visual de adhesivo remanente y, al respecto, no se encontraron diferencias significativas entre los grupos.

Despite chronic dialysis treatment, patients with end stage renal disease undergoing maintenance haemodialysis (HD) remain at a substantially increased risk of morbidity. Previous reports using Doppler ultrasound (DU) during HD have revealed microembolic signals (ME) in the venous circulation. In vitro studies confirm the emergence of microbubbles of air that may pass the security system of the HD circuit without triggering the alarm. The aim of this thesis was to elucidate the presence of ME during HD and examine methods that might reduce exposure to ME in vivo. The first study utilized DU to verify the presence of ME in 40 patients during standard HD. Investigation within 30 minutes after the start of HD and just before the end of session revealed the presence of ME in the venous blood line during both phases. The air trap did not alert for the presence of ME. This indicated that ME may pass into the patient during the entire HD run. Study 2 analyzed the presence of ME prior to start and during HD when measured at the AV-access and also carotid artery. A total of 54 patients were examined using DU as the investigative technique. ME increased significantly after start of HD in the AV-access, but also at the carotid artery site. These data indicated that ME can enter the body and even pass the lung barrier. The question arose if microbubbles of air are resorbed or may cause ischemic lesions in organs such as the brain. Study 3 examined whether the amount of ME detected in the AV-access would change by using either a high or a low blood level in the venous air trap/chamber. This was a prospective, randomized and double-blind study of 20 HD patients who were their own controls. After 30 min of standard HD, measurement of ME with DU was performed for two minutes. The chamber setting was changed and after another 30 minutes a new recording was carried out for two minutes. Data showed that setting a high blood level significantly reduced the extent of ME that entered the patient. The results also indicated that ME consisted mainly of microbubbles. In study 4, twenty patients were randomized in a cross-over setting of HD. Three options were used: a wet-stored dialyzer with high blood level (WH) and a dry-stored dialyzer using either a high (DH) or a low (DL) blood level in the venous chamber. The exposure of ME, detected by DU, was least when using mode WF, more with mode DH, and most with mode DL. There was a correlation between higher blood flow and more extensive exposure to ME. Study 5 was an autopsy study of a chronic HD patient with the aim of searching for microbubbles deposited in organs. Microbubbles of gas were verified in the vessels of the lungs, brain and heart. By using a fluorescent stain of anti-fibrinogen it was verified that the microbubbles were covered by clots that had to be preformed before death occurred. This indicated that air microbubbles are not completely absorbed and could result in embolic deposition in the organs of HD patients. In conclusion, these in vivo studies showed that ME pass the air trap without inducing an alarm and enter the venous blood line of the patient. The data confirmed the presence of ME in the AV-access and also in the carotid artery. Autopsy data of a deceased HD patient demonstrated the presence of microbubbles in the capillaries of the lungs, but also in the systemic circulation such as in the brain and the heart. A high blood level in the venous chamber and wet-stored dialyzer can reduce, but not eliminate the exposure to microbubbles for patients undergoing HD.

Transcriptional regulation largely determines which proteins and the protein levels that are found in a cell, and this is crucial in development, differentiation and responses to environmental stimuli. The major effectors of transcriptional regulation are a group of proteins known as transcription factors, which importance is supported by their frequent involvement in mendelian and complex diseases. In paper I, we attempted to establish the importance of DNA sequence variation in transcriptional control, by analyzing the potential functionality of polymorphic short repetitive elements as cis-regulatory elements. However, the relevance of this study was constrained by the limited number of analyzed sequences and the in vitro nature of the experiments. To overcome these limitations, (paper II) we optimized an in vivo large-scale technology named ChIP-chip, which couples chromatin immunoprecipitation and microarray hybridization. We successfully identified the binding profiles of metabolic-disease associated transcription factors in 1% of the human genome, using a liver cellular model, and inferred the binding sites at base pair resolution. Another important characteristic of transcriptional regulation is its plasticity, which allows adjusting the cellular transcriptome to cellular and environmental stimuli. In paper III, we investigated such plasticity by treating HepG2 cells with butyrate, a histone deacetylase inhibitor (HDACi) and interrogating the changes in histone H3 and H4 acetylation levels in 1% of the genome. Observation of frequent deacetylation around transcription start sites and hyperacetylation at the nuclear periphery challenges pre-assumed HDACi mechanisms of action. Finally, in paper IV we extended the DNA binding profiles of the medically relevant transcription factors, USF1 and USF2, and H3 acetylation to the whole non-repetitive fraction of the human genome. Using motif finding tools and chromatin profiling, we uncovered the major determinants of USF-DNA interactions. Furthermore, USFs and H3ac were clearly localized around transcription start sites, frequently in the context of bidirectional promoters.

Background: Hip fractures are a major health problem in Canada, and two main contributors to hip fracture are weak bone strength and fall. Weak muscles also negatively affect bone strength and increase the likelihood of falling. Advanced imaging techniques, such as magnetic resonance imaging (MRI), offer in vivo measurement of bone strength and muscle area at the proximal femur. However, it is not known if MRI-based measurements of bone and muscle properties are repeatable (i.e. precise). Methods: The femoral neck and shaft of 14 healthy participants were scanned three times, using a 1.5T MRI with repositioning between scans. Boundaries of the femoral neck, shaft and four muscle groups were delineated semi-automatically. Geometrical and strength properties of bone and area of muscle groups were determined based on segmented images. The short-term precision errors (root mean square coefficient of variation; CVrms%) between the repeated measures were calculated accordingly. Results: MRI-based measures of bone geometry and strength and muscle area at the proximal femur demonstrated in vivo precision errors < 7.6%. The average CVrms% for bone measures and muscle area were less than 4% and 2.5% respectively. Higher CVrms% (e.g. average: 4.8%) was obtained for bone strength properties. Conclusion: This is the first study to evaluate the in vivo performance of MRI on application to the proximal femur and surrounding muscles. Results demonstrate that MRI is a promising non-ionizing technique that offers precise measures of bone and muscle at the proximal femur.

<p>Several variables have been found to affect the Th1/Th2 differentiation of newly activated CD4 T cells. This phenotype can be critical in determining effectiveness of immune responses. Experiments in this thesis were undertaken to better define the in-vivo cellular interactions involved in determining the Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>Lethally irradiated BALB/c mice reconstituted with a constant number of syngeneic, naive spleen cells were challenged with xenogeneic red blood cells (XRBC) conjugated to ovalbumin (OVA) and the Th1/Th2 phenotype of the anti-XRBC response assessed. Antigen-specific interferon-gamma (IFN-g) and interleukin-4 (IL-4) secreting cells obtained from spleens of immunized mice were enumerated by an ELISPOT assay; the relative number of IFN-g- and IL-4-producing cells is taken as a relative measure of Th1 and Th2 components of the response. When challenged with a standard dose of XRBC-OVA, predominant Th1 responses are generated; when challenged with a ten-fold lower dose, such reconstituted mice do not generate significant responses. This adoptive transfer system was employed to explore further the relationships between quantitative changes in the dose of immunizing antigen and the number of responding antigen-specific CD4 T cells, and the Th1/Th2 phenotype of immune responses generated. Unprimed transgenic CD4 T cells specific for OVA can modulate the Th1/Th2 phenotype of the anti-XRBC response upon immunization with XRBC-OVA. Addition of a small number of naive transgenic spleen cells to the standard reconstituting population of normal spleen cells results in the generation of significant numbers of SRBC-specific Th2 cells when mice are challenged with a standard dose, or can generate predominant Th1 responses when mice are challenged with a ten-fold lower dose. Transgenic cells only impact the Th1/Th2 phenotype of CD4 T cells specific for XRBC when OVA is linked to the XRBC. That CD4 T cells specific for different antigens cooperate only through the recognition of linked antigenic determinants has important implications for many aspects of immune regulation. Observations further show that thymocytes from transgenic mice can influence the XRBC-specific response phenotype in an identical manner as transgenic spleen cells, suggesting that previously polarized pro-Th1/Th2 cells are not required in the cooperative events influencing Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>These observations lead to a quantitative description, whereby antigen-mediated CD4 T cell cooperation can affect the Th1/Th2 phenotype of a primary antigen-specific immune response, and provide a context for further analysis at the molecular level.

Mycobacterium tuberculosis, the bacterium that causes tuberculosis, has an incubation period ranging from a few months to several years following infection via inhalation into the lungs. Whole body fluorescence scanners are used to image and monitor the growth of fluorescent protein expressing strains of M. tuberculosis in the lungs of animal models. Accurate quantitative analysis of bacterial growth during the early stages of infection inside lungs remains elusive, due to tissue absorption and scattering of photons emitted by the low numbers of bacteria deep in tissue. Fiber optic micro-endoscopy is uniquely suited to provide a novel solution to this problem by delivering light excitation directly to and collecting fluorescence from the infection site located in the lungs of an animal model, thereby enabling detection of fluorescent bacteria during the early stages of infection. In this thesis, I present a contact probe fiber bundle fluorescence micro-endoscope with a range of LED based excitation wavelengths, 4 μm resolution, a 750 μm field of view, and a 1 mm outer diameter. This system has detected tdTomato and GFP expressing Bacillus Calmette-Guérin (BCG) bacteria in vitro. Additionally, images of bacterial regions of infection obtained in mice subcutaneously infected with tdTomato expressing bacteria at concentrations ranging from 106 to 101 Colony Forming Units (CFU) and intra-tracheally infected mice at 106 CFU demonstrate the micro-endoscope’s capability to detect and resolve regions of bacterial infection in vivo. By relaying the bacterial fluorescence image from the infection site to an external detector, we are able to increase the sensitivity to early stages of infection.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2009. / Härtill 4 uppsatser.

Although nucleic acids primarily function as carriers of the genetic information in biology, their chemical versatility, replicability and programmability render them much more functions inside and outside of cells. Numerous nucleic acid catalysts (known as ribozymes and deoxyribozyme) and binding agents (known as aptamers) have been engineered through the combination of directed evolution and rational design. However, new technologies and theoretical frameworks are still in need to better engineer and utilize these functional nucleic acids in diagnostics and therapeutics. Aiming at engineering more powerful aptazyme-based genetic regulators, we first devised a scheme for direct selection of physiologically active ribozymes in mammalian cells. Model-driven analysis of the selection process showed that the stringency of the selection was strongly influenced by system variables such as degradation rate of un-reacted ribozymes. This analysis led to models that can be exploited to understand and predict the performance of aptazyme-based biosensors and genetic regulators. Several fundamental limitations of aptazymes-based systems were identified from the analyses of these models. As it became apparent that the signals generated by aptazymes need to be processed and amplified at molecular level to have satisfactory effects on the final readouts, we turned our focus to engineering nucleic acid-based signal processors using several newly invented schemes such as ‘entropy-driven DNA amplifier’ and ‘catalyzed DNA self-assembly.’ We first demonstrated a method to couple entropy-driven DNA amplifiers to allosteric deoxyribozymes, and then proved that the concept of catalyzed DNA self-assembly can be used to design efficient and versatile signal amplifiers for analytical applications on various platforms. These developments may potentially lead to sensitive, low-cost, and point-of-care diagnostic devices. Taken together, these works not only addressed several important issues regarding the engineering and application of nucleic acid catalysts, but also revealed a new theme in molecular engineering: In order to better engineer and utilize a part, one needs to characterize, model, and modify the system surrounding the part so that the potential of the part can be maximized. / text