The development of a non-invasive method of visualizing gene expression in larger animals could revolutionize some aspects of gene research by opening up a wider variety of animal systems to explore; some of which may be better models of human systems. Presently, most gene expression studies employ Green Fluorescent Protein (GFP) transfected into the genome of the animal system. For larger animals, an x-ray equivalent of GFP would be desirable due to the high penetrating power of x-rays. A model gene modification system is to use the Sodium (Na) Iodide Symporter (NIS) which will cause the accumulation of iodine in cells which express the NIS. To non-invasively observe the dilute iodine accumulated by the cancer cells transfected with NIS in the head of small animals, such as a rat, two synchrotron-based imaging methods were studied: K-Edge Subtraction (KES) imaging and Fluorescence Subtraction Imaging (FSI).<p> KES needs wide monochromatic x-ray beams at two energies bracketing the K-edge of the contrast agent existing or injected in the tissues. The monochromatic beam in the synchrotron facility normally is prepared by a double crystal monochromator. The appearance of the azimuthal angle (tilt error) in the double crystal monochromator creates intensity variations across the imaging field. This misalignment was studied through another two synchrotron-based imaging methods, Diffraction Enhanced Imaging (DEI) and Multi-Image Radiography (MIR), which show this problem clearly in their processed images. The detailed analysis of the effect of the tilt error, how it affects the resulting images, and how to quantify such an error were presented in the thesis. A post processing method was implemented and the artifacts caused by the improper experimental settings were discussed.<p> With the wide monochromatic beam prepared by the double crystal monochromator, a sequence of KES experiments were done and the detection limit of KES was quantified at a projected amount of 17.5mM-cm iodine in a physical model of a rat head with a radiation dose of 2.65mGy. With the raster scan of the object relative to the monochromatic pencil beam, FSI was studied to obtain higher Signal to Noise Ratio (SNR) for local area and better detection limit compared to KES. The detection limit of FSI was measured as a projected amount of 2.5mM-cm iodine in the same physical rat head with a tolerable radiation dose of 24mGy. According to the comparison of these two imaging techniques with references to imaging time and area, radiation dose, spatial resolution, and SNR, it was concluded that these two imaging techniques can be used complementarily in imaging dilute contrast material. Due to the short imaging time and large imaging area, KES is used first to provide a global view of the object, locate the area of interest, do the preliminary diagnosis, and decide whether the further FSI is necessary. Due to its high SNR for the dilute sample, FSI can be used when the area of interest is known. The combination of these two imaging techniques will be very promising and powerful. To facilitate the comparison of KES and FSI, a quality factor was developed to evaluate the performance of the imaging system.<p> The measured detection limits in our experiments are far beyond the thyroidal iodine concentration of a rat (around 1mM). To further improve the performance of KES, a bent Laue crystal monochromator was designed to do the simultaneous iodine KES imaging which overcomes the artifacts in the iodine image caused by the temporal difference for a single set of images. The designed monochromator can provide two separated x-ray beams bracketing the K-edge of iodine at the same time with a very high spatial resolution which is only depends on the source size, a very high energy resolution which can almost compete with that of the double crystal monochromator, and an acceptable photon flux.

Acute lung injury is marked by profound neutrophil influx along with fluid accumulation that impairs lung function at the cost of high mortality (up to 40%). Neutrophils are activated and their constitutive apoptosis is inhibited during this phase in order to be competent phagocytes over the next few hours. Activated neutrophils release copious amounts of toxic mediators that cause tissue damage leading to impaired barrier function and finally, impaired lung function. Therefore, one of the critical needs is to identify molecules that regulate neutrophil migration and silence activated neutrophils to prevent exuberant tissue damage. Angiostatin is an anti-angiogenic molecule highly expressed in lavage fluid of patients with acute respiratory distress syndrome. Angiostatin has recently been shown to inhibit neutrophil infiltration in mice peritonitis. However, the role of angiostatin in modulating neutrophil physiology and lung inflammation remains unknown. I studied the role of angiostatin, an anti-angiogenic molecule, in neutrophil activation and recruitment <i>in vivo</i> and <i>in vitro</i>. Angiostatin was endocytosed only by activated neutrophils, inhibited neutrophil polarity in fMLP-activated neutrophils probably through integrin &alpha;_V&beta;₃, and inhibited MAPK signalling in LPS-activated neutrophils. Angiostatin suppressed formation of reactive oxygen species and activated caspase-3 in neutrophils in both pre-and post-LPS treatments. Finally, angiostatin reduced adhesion and emigration of neutrophils in post-capillary venules of TNF&alpha;-treated cremaster muscle. The next study was designed to investigate the role of angiostatin in acute lung injury. I used <i>E. coli</i> lipopolysaccharide induced acute lung injury mouse model to test the effects of angiostatin through analyses of bronchoalveolar lavage and lung tissues. In addition, I made novel use of synchrotron diffraction enhanced imaging of mouse lungs to assess lung area and contrast ratios over 9 hours as surrogates for lung inflammation. Subcutaneous treatment with angiostatin reduced neutrophil influx, protein accumulation, lung Gr1+ neutrophils and myeloperoxidase activity, phosphorylated p38 MAPK without affecting the levels of MIP-1&alpha;, IL-1&beta;, KC and MCP-1 in lavage and lung homogenates. Diffraction enhanced imaging showed that angiostatin causes a time-dependent improvement in lung area and lung contrast ratios that reflect improvement in lung edema. Overall, the study shows that angiostatin is a novel inhibitor of acute lung injury in mice. Moreover, DEI offers a highly useful technique in evaluating dynamics of lung inflammation and to investigate the therapeutic impact of new drugs on lung inflammation. I conclude that angiostatin is a novel inhibitor of neutrophil migration, activation and acute lung injury.

Acute lung injury is marked by profound neutrophil influx along with fluid accumulation that impairs lung function at the cost of high mortality (up to 40%). Neutrophils are activated and their constitutive apoptosis is inhibited during this phase in order to be competent phagocytes over the next few hours. Activated neutrophils release copious amounts of toxic mediators that cause tissue damage leading to impaired barrier function and finally, impaired lung function. Therefore, one of the critical needs is to identify molecules that regulate neutrophil migration and silence activated neutrophils to prevent exuberant tissue damage. Angiostatin is an anti-angiogenic molecule highly expressed in lavage fluid of patients with acute respiratory distress syndrome. Angiostatin has recently been shown to inhibit neutrophil infiltration in mice peritonitis. However, the role of angiostatin in modulating neutrophil physiology and lung inflammation remains unknown. I studied the role of angiostatin, an anti-angiogenic molecule, in neutrophil activation and recruitment <i>in vivo</i> and <i>in vitro</i>. Angiostatin was endocytosed only by activated neutrophils, inhibited neutrophil polarity in fMLP-activated neutrophils probably through integrin &alpha;_V&beta;₃, and inhibited MAPK signalling in LPS-activated neutrophils. Angiostatin suppressed formation of reactive oxygen species and activated caspase-3 in neutrophils in both pre-and post-LPS treatments. Finally, angiostatin reduced adhesion and emigration of neutrophils in post-capillary venules of TNF&alpha;-treated cremaster muscle. The next study was designed to investigate the role of angiostatin in acute lung injury. I used <i>E. coli</i> lipopolysaccharide induced acute lung injury mouse model to test the effects of angiostatin through analyses of bronchoalveolar lavage and lung tissues. In addition, I made novel use of synchrotron diffraction enhanced imaging of mouse lungs to assess lung area and contrast ratios over 9 hours as surrogates for lung inflammation. Subcutaneous treatment with angiostatin reduced neutrophil influx, protein accumulation, lung Gr1+ neutrophils and myeloperoxidase activity, phosphorylated p38 MAPK without affecting the levels of MIP-1&alpha;, IL-1&beta;, KC and MCP-1 in lavage and lung homogenates. Diffraction enhanced imaging showed that angiostatin causes a time-dependent improvement in lung area and lung contrast ratios that reflect improvement in lung edema. Overall, the study shows that angiostatin is a novel inhibitor of acute lung injury in mice. Moreover, DEI offers a highly useful technique in evaluating dynamics of lung inflammation and to investigate the therapeutic impact of new drugs on lung inflammation. I conclude that angiostatin is a novel inhibitor of neutrophil migration, activation and acute lung injury.

The development of a non-invasive method of visualizing gene expression in larger animals could revolutionize some aspects of gene research by opening up a wider variety of animal systems to explore; some of which may be better models of human systems. Presently, most gene expression studies employ Green Fluorescent Protein (GFP) transfected into the genome of the animal system. For larger animals, an x-ray equivalent of GFP would be desirable due to the high penetrating power of x-rays. A model gene modification system is to use the Sodium (Na) Iodide Symporter (NIS) which will cause the accumulation of iodine in cells which express the NIS. To non-invasively observe the dilute iodine accumulated by the cancer cells transfected with NIS in the head of small animals, such as a rat, two synchrotron-based imaging methods were studied: K-Edge Subtraction (KES) imaging and Fluorescence Subtraction Imaging (FSI).<p> KES needs wide monochromatic x-ray beams at two energies bracketing the K-edge of the contrast agent existing or injected in the tissues. The monochromatic beam in the synchrotron facility normally is prepared by a double crystal monochromator. The appearance of the azimuthal angle (tilt error) in the double crystal monochromator creates intensity variations across the imaging field. This misalignment was studied through another two synchrotron-based imaging methods, Diffraction Enhanced Imaging (DEI) and Multi-Image Radiography (MIR), which show this problem clearly in their processed images. The detailed analysis of the effect of the tilt error, how it affects the resulting images, and how to quantify such an error were presented in the thesis. A post processing method was implemented and the artifacts caused by the improper experimental settings were discussed.<p> With the wide monochromatic beam prepared by the double crystal monochromator, a sequence of KES experiments were done and the detection limit of KES was quantified at a projected amount of 17.5mM-cm iodine in a physical model of a rat head with a radiation dose of 2.65mGy. With the raster scan of the object relative to the monochromatic pencil beam, FSI was studied to obtain higher Signal to Noise Ratio (SNR) for local area and better detection limit compared to KES. The detection limit of FSI was measured as a projected amount of 2.5mM-cm iodine in the same physical rat head with a tolerable radiation dose of 24mGy. According to the comparison of these two imaging techniques with references to imaging time and area, radiation dose, spatial resolution, and SNR, it was concluded that these two imaging techniques can be used complementarily in imaging dilute contrast material. Due to the short imaging time and large imaging area, KES is used first to provide a global view of the object, locate the area of interest, do the preliminary diagnosis, and decide whether the further FSI is necessary. Due to its high SNR for the dilute sample, FSI can be used when the area of interest is known. The combination of these two imaging techniques will be very promising and powerful. To facilitate the comparison of KES and FSI, a quality factor was developed to evaluate the performance of the imaging system.<p> The measured detection limits in our experiments are far beyond the thyroidal iodine concentration of a rat (around 1mM). To further improve the performance of KES, a bent Laue crystal monochromator was designed to do the simultaneous iodine KES imaging which overcomes the artifacts in the iodine image caused by the temporal difference for a single set of images. The designed monochromator can provide two separated x-ray beams bracketing the K-edge of iodine at the same time with a very high spatial resolution which is only depends on the source size, a very high energy resolution which can almost compete with that of the double crystal monochromator, and an acceptable photon flux.

Background and Rationale: Reproductive dysfunction affects more than 15% of Canadian women; however, the underlying causes remain largely unknown. Ultrasonography is the most commonly used research and diagnostic tool for imaging the ovaries and uterus. However, current ultrasonographic techniques allow the detection of ovarian structures (eg. follicles, corpora lutea) at diameters of only ≥2 mm. The increased effectiveness of synchrotron technology for imaging ovaries in comparison to conventional imaging methods is currently unknown. Overall Objective: The overall objective of this research was to determine the effectiveness of synchrotron techniques for imaging ovaries. We hypothesized that synchrotron techniques would provide greater contrast for visualizing structural details of follicles, corpora lutea (CL), and cumulus oocyte complexes (COC), compared to conventional ultrasonography. Materials and Methods: Three studies were conducted to evaluate phase-contrast based synchrotron imaging methods. The first study involved Diffraction Enhanced Imaging (DEI) of bovine ovaries (n=6). The second study involved Propagation-Based Computed Tomography (PB-CT) imaging of bovine (n=4) and human ovaries (n=4). A third, preliminary study was conducted to explore the use of Talbot Grating Interferometry (TGI-CT) imaging of bovine (n=1) and human ovaries (n=1). Fresh and formalin-fixed bovine and human ovaries were imaged without or with contrast injection into the ovarian artery. Following synchrotron imaging, all ovarian samples were evaluated using diagnostic ultrasonography and histology. Images obtained using synchrotron techniques, ultrasonography and histology were qualitative and quantitatively compared. Results: DEI allowed the identification of 71% of follicles ≥2 mm and 67% of CL detected using ultrasonography. Mean follicle diameter was similar between DEI (9.6 ± 2.4 mm), ultrasonography (9.0 ± 2.6 mm), and histology (6.9 ± 1.9 mm) for fresh ovaries without contrast (P = 0.70). Likewise, no difference in CL diameter was detected between DEI (11.64 ± 1.67 mm), ultrasonography (9.34 ± 0.35 mm), and histology (9.6 ± 0.4 mm), (P = 0.34). Antral Follicle Count (AFC; ≥2mm) was similar between ultrasonography (6.5 ± 0.7 mm, fresh with no contrast; 6.5 ± 2.5 mm, preserved with no contrast) and DEI ( 4.5 ± 0.5 mm, fresh with no contrast; 6.5 ± 0.50 mm, preserved with no contrast) (P > 0.05). However, the contrast resolution for differentiating follicles and CL was inferior with DEI compared to ultrasonography. Small antral follicles <2mm, cell layers comprising the follicle wall and COC were not detected using either DEI or ultrasonography. PB-CT imaging enabled the visualization of 100% of follicles ≥2 mm and 100% of CL that were detected with ultrasonography. CL containing a central cystic cavity were identified using PB-CT; however, CL without a central cystic cavity were not well-visualized. Mean follicle and luteal diameters did not differ among PB-CT, ultrasonography and histology (P>0.05). PB-CT was superior to ultrasonography for detecting small antral follicles <2 mm in bovine ovaries (P = 0.04), and the granulosa and theca cell layers of the follicle wall in bovine and human ovaries (P < 0.0001). However, TGI-CT images exhibited greater contrast resolution for visualizing small and large antral follicles, CL, and the cell layers of the follicle wall compared to both PB-CT and ultrasonography. High contrast structures resembling COC were detected with both PB-CT and TGI-CT, but not with ultrasonography. Only TGI-CT permitted the visualization of the oocyte within the COC in fresh and preserved ovaries. Conclusions: DEI was inferior to ultrasonography for detecting ovarian follicles and CL. PB-CT was superior to ultrasonography for visualizing follicles <2 mm, COC, and the cell layers of the follicle wall. However, PB-CT was as effective as ultrasonography for detecting and measuring follicles ≥2 mm and cystic CL. Preliminary findings suggest that TGI-CT provides the greatest contrast for imaging both ovarian macro- and microanatomy compared to PB-CT, DEI, and ultrasonography.

Diffraction Enhanced Imaging (DEI) is an X-ray imaging technique providing specific information about the molecular structure of a tissue by means of coherently scattered photons. A Controlled Drift Detector (CDD) is a novel 2D silicon imager developed to be used in X-ray imaging techniques. In this work a final (complete and detailed) analysis of DEI data taken with the CDD in the ELETTRA synchrotron light source facility in Trieste (Italy) in 2005, is presented and the applicability of both this new technique and the novel detector are discussed.

This research was inspired by a need to discover more refined technologies for imaging growing joints to facilitate research in childhood arthritis, which is among the most common chronic conditions of childhood. The objective of this project was to develop and test a new technology for imaging growing joints using diffraction enhanced imaging (DEI) combined with computed tomography (CT) using a synchrotron radiation source. DEI is a modality that derives contrast from x-ray refraction, extinction (an extreme form of scatter rejection), and absorption (as in conventional radiography). The ability to add to an image’s contrast from the refraction of x-rays, rather than that solely from absorption, generates more detailed visualization of soft tissue and of interfaces between tissues. Additionally, refraction-based imaging allows reduction of absorbed radiation dose by the sample tissue. For this research, stifle joints from four-week piglet joints were imaged by DEI-CT using the BioMedical Imaging and Therapy (BMIT) beamline at the Canadian Light Source (CLS) synchrotron facility. This new modality for imaging growing joints incorporated a novel feedback control to maintain precise alignment of the analyzer crystal, which is used to re- diffract the beam that passes through the object, throughout the scanning procedure. Results showed that high-resolution DEI-CT provided three-dimensional images of the bone and soft tissue of growing joints at a resolution on the order of microns. Fine detail within and between all joint structures and tissues, including striking detail of cartilage vasculature, a iii characteristic of growing but not mature joints, was demonstrated. This report documents for the first time that DEI combined with CT and using a synchrotron radiation source can generate more detailed images of intact, growing joints than is currently available from conventional imaging modalities. The development of this high resolution imaging system, which provides excellent contrast for both hard and soft tissues, fills an important gap in the suite of imaging modalities available for joint research, particularly during growth.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Os desenvolvimentos teóricos e experimentais na área de biomateriais têm sido aplicados diretamente a distintos campos da Medicina (odontologia, medicina regenerativa e radioterapia). Esses avanços foram concentrados tanto para diagnosticar doenças como para a quantificação de seus graus de progressão. Na perspectiva desses estudos, biomateriais estão sendo projetados e confeccionados para aplicação em diversas áreas da ciência, proporcionado avanços no radiodiagnóstico, na dosimetria para radioterapia e na calibração de equipamentos radioterápicos. Desenvolver um phantom a partir de um biomaterial se tornou um grande aliado da Medicina no tratamento de pacientes com doenças oncológicas, possibilitando melhor desempenho dos equipamentos, com a finalidade de redução dos danos causados ao tecido sadio devido ao excesso de exposição à radiação. Este trabalho utilizou polímeros: quitosana e gelatina para confecção das estruturas poliméricas e foi possível controlar as diferentes formas de produção e processamento, caracterizar e avaliar o biopolímero por técnicas físicas (ELT,MEV, e DEI) e, por consequência, analisar a aplicabilidade como phantom pulmonar de camundongo. Foi possível avaliar a morfologia dos biomateriais quantitativamente por microscopia eletrônica de varredura associada a técnica de imagem. A relevância deste trabalho se concentra em desenvolver um phantom a partir de biomateriais poliméricos que possa atuar como objeto simulador fornecendo alto contraste de imagem quando submetido a análise. Dessa forma, a escolha da técnica DEI foi satisfatória uma vez que trata-se de uma técnica de imagem de raios X de alta resolução. As imagens obtidas por DEI têm mostrado os detalhes da microestrutura interna dos biomateriais produzidos as quais possuem ≈ 10 μm de dimensão. Os phantoms confeccionados apresentaram densidade variando de 0,08 a 0,13 g/cm3. / The theoretical and experimental developments in the biomaterials area have been directly applied to different fields of Medicine (odontology, regenerative medicine and radiotherapy). These advances have focused both for diagnosing diseases such as for quantifying degrees of progression. From the perspective of these studies, biomaterials are being designed and manufactured for application in various areas of science, provided advances in diagnostic radiology, radiotherapy dosimetry and calibration of radiotherapy equipment. Develop a phantom from a biomaterial has become a great ally of medicine in the treat patients with oncological diseases, allowing better performance of the equipment in order to reduce damage to healthy tissue due to excessive exposure to radiation. This work used polymers: chitosan and gelatin, for making the polymeric structures and controlled for different types of production and processing, characterizing and evaluating the biopolymer by physical techniques (STL, SEM and DEI) and therefore analyze applicability as phantom mouse lung. It was possible to evaluate the morphology of biomaterials quantitatively by scanning electron microscopy associated with imaging technique. The relevance of this work focuses on developing a phantom from polymeric biomaterials that can act as phantom providing high image contrast when subjected to analysis. Thus, the choice of DEI technique is satisfactory since it is an imaging technique of X-ray high resolution. The images obtained by DEI have shown the details of the internal microstructure of the biomaterial produced which have ≈ 10 μm dimension. The phantoms had made density ranging from 0.08 a 0.13 g/cm3. / Tese (Doutorado)