Detection of the Vulnerable Atherosclerotic Plaque. Pre-Clinical Evaluation of Novel PET Imaging Probes With Experimental Models

Atherosclerosis is a life-long vascular inflammatory disease and the leading cause of death in Finland and in other western societies. The development of atherosclerotic plaques is progressive and they form when lipids begin to accumulate in the vessel wall. This accumulation triggers the migration of inflammatory cells that is a hallmark of vascular inflammation. Often, this plaque will become unstable and form vulnerable plaque which may rupture causing thrombosis and in the worst case, causing myocardial infarction or stroke. Identification of these vulnerable plaques before they rupture could save lives. At present, in the clinic, there exists no appropriated, non-invasive method for their identification. The aim of this thesis was to evaluate novel positron emission tomography (PET) probes for the detection of vulnerable atherosclerotic plaques and to characterize, two mouse models of atherosclerosis. These studies were performed by using ex vivo and in vivo imaging modalities. The vulnerability of atherosclerotic plaques was evaluated as expression of active inflammatory cells, namely macrophages. Age and the duration of high-fat diet had a drastic impact on the development of atherosclerotic plaques in mice. In imaging of atherosclerosis, 6-month-old mice, kept on high-fat diet for 4 months, showed matured, metabolically active, atherosclerotic plaques. [18F]FDG and 68Ga were accumulated in the areas representative of vulnerable plaques. However, the slow clearance of 68Ga limits its use for the plaque imaging. The novel synthesized [68Ga]DOTA-RGD and [18F]EF5 tracers demonstrated efficient uptake in plaques as compared to the healthy vessel wall, but the pharmacokinetic properties of these tracers were not optimal in used models. In conclusion, these studies resulted in the identification of new strategies for the assessment of plaque stability and mouse models of atherosclerosis which could be used for plaque imaging. In the used probe panel, [18F]FDG was the best tracer for plaque imaging. However, further studies are warranted to clarify the applicability of [18F]EF5 and [68Ga]DOTA-RGD for imaging of atherosclerosis with other experimental models.

Imaging of Tumour Microenvironment for the Planning of Oncological Therapies Using Positron Emission Tomography

Tumour cells differ from normal tissue cells in several important ways. These differences, like for example changed energy metabolism, result in altered microenvironment of malignant tumours. Non-invasive imaging of tumour microenvironment has been at the centre of intense research recently due to the important role that this changed environement plays in the development of malignant tumours and due to the role it plays in the treatment of these tumours. In this respect, perhaps the most important characteristics of the tumour microenvironment from this point of view are the lack of oxygen or hypoxia and changes in blood flow (BF). The purpose of this thesis was to investigate the processes of energy metabolism, BF and oxygenation in head and neck cancer and pancreatic tumours and to explore the possibilities of improving the methods for their quantification using positron emission tomography (PET). To this end [18F]EF5, a new PET tracer for detection of tumour hypoxia was investigated. Favourable uptake properties of the tracer were observed. In addition, it was established that the uptake of this tracer does not correlate with the uptake of existing tracers for the imaging of energy metabolism and BF, so the information about the presence of tissue hypoxia cannot therefore be obtained using tracers such as [18F]FDG or [15O]H2O. These results were complemented by the results of the follow-up study in which it was shown that the uptake of [18F]EF5 in head and neck tumours prior to treatment is also associated with the overall survival of the patients, indicating that tumour hypoxia is a negative prognostic factor and might be associated with therapeutic resistance. The influences of energy metabolism and BF on the survival of patients with pancreatic cancer were investigated in the second study. The results indicate that the best predictor of survival of patients with pancreatic cancer is the relationship between energy metabolism and BF. These results suggest that the cells with high metabolic activity in a hypoperfused tissue have the most aggressive phenotype.

On Glucose Metabolism in Patients with the m.3243A>G Mutation

Background: The m.3243A>G mutation in mitochondrial DNA is the most common cause for mitochondrial diabetes. In addition, unexpected deaths related to the m.3243A>G associate with encephalopathy and cardiomyopathy. Failing mitochondrial respiratory chain in neurons, myocytes and beta cells is considered to underlie the multiorgan manifestations of the m.3243A>G. Aims: The primary aim of the study was to characterize the organ-specific glucose metabolism in patients with m.3243A>G and secondly, to study patients with or without signs of diabetes, cardiomyopathy or encephalopathy. The insulin-stimulated glucose metabolism in brain, heart, skeletal muscle, adipose tissue and liver were measured with 2-deoxy-2-[18F]fluoro-α-D-glucose in 15 patients and 14 controls. Brain oxygen metabolism was assessed with [15O]oxygen and insulin secretion was modelled based on oral glucose tolerance test. Results: The glucose oxidation in brain was globally decreased in patients with or without clinical encephalopathy. The insulin-stimulated glucose influx to skeletal muscle and adipose tissue was decreased in patients with or without diabetes as the hepatic glucose metabolism was normal. Impaired beta cell function and myocardial glucose uptake were associated with the high m.3243A>G heteroplasmy. Conclusions: This cross-sectional study suggests that: 1) The ability of insulin to stimulate glucose metabolism in skeletal muscle and adipose tissue is weakened before the beta cell failure results in mitochondrial diabetes. 2) Glucose oxidation defect is detected in otherwise unaffected cerebral regions in patients with the m.3243A>G, thus it likely precedes the clinical encephalopathy. 3) Uneconomical glucose hypometabolism during hyperinsulinemia contributes to the cardiac vulnerability in patients with high m.3243A>G heteroplasmy

Histopathological and immunohistochemical findings of swine with spontaneous influenza A infection in Brazil, 2009-2010

Swine influenza (SI) is caused by the type A swine influenza virus (SIV). It is a highly contagious disease with a rapid course and recovery. The major clinical signs and symptoms are cough, fever, anorexia and poor performance. The disease has been associated with other co-infections in many countries, but not in Brazil, where, however, the first outbreak has been reported in 2011. The main aim of this study was to characterize the histological features in association with the immunohistochemical (IHC) results for influenza A (IA), porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) in lung samples from 60 pigs submitted to Setor de Patologia Veterinária at the Universidade Federal do Rio Grande do Sul (SPV-UFRGS), Brazil, during 2009-2010. All of these lung samples had changes characterized by interstitial pneumonia with necrotizing bronchiolitis, never observed previously in the evaluation of swine lungs in our laboratory routine. Pigs in this study had showed clinical signs of a respiratory infection. Swine samples originated from Rio Grande do Sul 31 (52%), Santa Catarina 14 (23%), Paraná 11 (18%), and Mato Grosso do Sul 4 (7%). Positive anti-IA IHC labelling was observed in 45% of the cases, which were associated with necrotizing bronchiolitis, atelectasis, purulent bronchopneumonia and hyperemia. Moreover, type II pneumocyte hyperplasia, alveolar and bronchiolar polyp-like structures, bronchus-associated lymphoid tissue (BALT) hyperplasia and pleuritis were the significant features in negative anti-IA IHC, which were also associated with chronic lesions. There were only two cases with positive anti-PCV2 IHC and none to PRRSV. Therefore, SIV was the predominant infectious agent in the lung samples studied. The viral antigen is often absent due to the rapid progress of SI, which may explain the negative IHC results for IA (55%); therefore, IHC should be performed at the beginning of the disease. This study has shown how important a careful histological evaluation is for the diagnosis. Since 2009, a new histological feature of swine pneumonia in animals with respiratory clinical signs has been observed in samples from pigs with clinical respiratory disease submitted to SPV-UFRGS. In addition, the results proved the importance of histological evaluation for swine herd health management.

The influence of selected pathological states on the somatostatin-like immunoreactive (SOM-LI) endocrine cells in the mucosal layer of the porcine descending colon

This study reports on changes in the number of somatostatin-like immunoreactive (SOM-LI) endocrine cells in the porcine descending colon, caused by chemically driven inflammation, axotomy and proliferative enteropathy (PE). The distribution pattern of SOM-LI endocrine cells has been studied using the routine single-labelling immunofluorescence technique. Semi-quantitative evaluation of the number of the SOM-immunostained endocrine cells within the mucosal layer of the porcine descending colon has been based on counting of all endocrine cells immunoreactive to SOM per unit area (0,1 mm²). Under physiological conditions the number of SOM-LI endocrine cells has been shown to constitute 3,30±0,22. All applied pathological processes resulted in changes in the SOM-like immunoreactivity, which varied in particular processes studied. The number of SOM-LI endocrine cells increased to 6,28±0,31 and 4,43±0,35 during chemically driven inflammation and proliferative enteropathy, respectively, and decreased to 1,17%±0,16 after axotomy. The obtained results suggest that SOM-LI endocrine cells may participate in various pathological states within porcine descending colon and their functions probably depend on the type of pathological factor.

Immunocytochemistry of the mucilage cells of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae)

The use of monoclonal antibodies for specific pectic epitopes is an important tool in the study of the cell wall. Throughout the development of mucilage cells of Araucaria angustifolia (Bertol.) Kuntze, a gradient of distribution was observed in relation to the pectic de-esterification, as well as to the increase of galactan and arabinan epitope distribution, and to the reduction of arabinogalactans proteins (AGPs) epitope at maturity. AGP and methyl-esterified homogalacturonan (HGA) were present in the mucilage. Galactans and arabinans were also observed in the mucilage, though with weak labelling. Degradation of AGP in the maturity of mucilage cells, in cell wall, as well as in the secretion, could be involved in the programmed cell death (PCD). Different labellings found among parenchyma and mucilage cells suggested differences in the cell wall properties of the mucilage cells.

Modulation of fibronectin expression in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis

Fibronectin (FN), a large family of plasma and extracellular matrix (ECM) glycoproteins, plays an important role in leukocyte migration. In normal central nervous system (CNS), a fine and delicate mesh of FN is virtually restricted to the basal membrane of cerebral blood vessels and to the glial limitans externa. Experimental autoimmune encephalomyelitis (EAE), an inflammatory CNS demyelinating disease, was induced in Lewis rats with a spinal cord homogenate. During the preclinical phase and the onset of the disease, marked immunolabelling was observed on the endothelial luminal surface and basal lamina of spinal cord and brainstem microvasculature. In the paralytic phase, a discrete labelling was evident in blood vessels of spinal cord and brainstem associated or not with an inflammatory infiltrate. Conversely, intense immunolabelling was present in cerebral and cerebellar blood vessels, which were still free from inflammatory cuffs. Shortly after clinical recovery minimal labelling was observed in a few blood vessels. Brainstem and spinal cord returned to normal, but numerous inflammatory foci and demyelination were still evident near the ventricle walls, in the cerebral cortex and in the cerebellum. Intense expression of FN in brain vessels ascending from the spinal cord towards the encephalon preceded the appearance of inflammatory cells but faded away after the establishment of the inflammatory cuff. These results indicate an important role for FN in the pathogenesis of CNS inflammatory demyelinating events occurring during EAE.

Translational models of coronary artery disease, myocardial infarction and heart failure. Development, validation and in vivo imaging studies using positron emission tomography

Coronary artery disease is an atherosclerotic disease, which leads to narrowing of coronary arteries, deteriorated myocardial blood flow and myocardial ischaemia. In acute myocardial infarction, a prolonged period of myocardial ischaemia leads to myocardial necrosis. Necrotic myocardium is replaced with scar tissue. Myocardial infarction results in various changes in cardiac structure and function over time that results in “adverse remodelling”. This remodelling may result in a progressive worsening of cardiac function and development of chronic heart failure. In this thesis, we developed and validated three different large animal models of coronary artery disease, myocardial ischaemia and infarction for translational studies. In the first study the coronary artery disease model had both induced diabetes and hypercholesterolemia. In the second study myocardial ischaemia and infarction were caused by a surgical method and in the third study by catheterisation. For model characterisation, we used non-invasive positron emission tomography (PET) methods for measurement of myocardial perfusion, oxidative metabolism and glucose utilisation. Additionally, cardiac function was measured by echocardiography and computed tomography. To study the metabolic changes that occur during atherosclerosis, a hypercholesterolemic and diabetic model was used with [18F] fluorodeoxyglucose ([18F]FDG) PET-imaging technology. Coronary occlusion models were used to evaluate metabolic and structural changes in the heart and the cardioprotective effects of levosimendan during post-infarction cardiac remodelling. Large animal models were used in testing of novel radiopharmaceuticals for myocardial perfusion imaging. In the coronary artery disease model, we observed atherosclerotic lesions that were associated with focally increased [18F]FDG uptake. In heart failure models, chronic myocardial infarction led to the worsening of systolic function, cardiac remodelling and decreased efficiency of cardiac pumping function. Levosimendan therapy reduced post-infarction myocardial infarct size and improved cardiac function. The novel 68Ga-labeled radiopharmaceuticals tested in this study were not successful for the determination of myocardial blood flow. In conclusion, diabetes and hypercholesterolemia lead to the development of early phase atherosclerotic lesions. Coronary artery occlusion produced considerable myocardial ischaemia and later infarction following myocardial remodelling. The experimental models evaluated in these studies will enable further studies concerning disease mechanisms, new radiopharmaceuticals and interventions in coronary artery disease and heart failure.

Predictive significance of standardized uptake value parameters of FDG-PET in patients with non-small cell lung carcinoma

18F-fluoro-2-deoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) is widely used to diagnose and stage non-small cell lung cancer (NSCLC). The aim of this retrospective study was to evaluate the predictive ability of different FDG standardized uptake values (SUVs) in 74 patients with newly diagnosed NSCLC. 18F-FDG PET/CT scans were performed and different SUV parameters (SUVmax, SUVavg, SUVT/L, and SUVT/A) obtained, and their relationship with clinical characteristics were investigated. Meanwhile, correlation and multiple stepwise regression analyses were performed to determine the primary predictor of SUVs for NSCLC. Age, gender, and tumor size significantly affected SUV parameters. The mean SUVs of squamous cell carcinoma were higher than those of adenocarcinoma. Poorly differentiated tumors exhibited higher SUVs than well-differentiated ones. Further analyses based on the pathologic type revealed that the SUVmax, SUVavg, and SUVT/L of poorly differentiated adenocarcinoma tumors were higher than those of moderately or well-differentiated tumors. Among these four SUV parameters, SUVT/Lwas the primary predictor for tumor differentiation. However, in adenocarcinoma, SUVmax was the determining factor for tumor differentiation. Our results showed that these four SUV parameters had predictive significance related to NSCLC tumor differentiation; SUVT/L appeared to be most useful overall, but SUVmax was the best index for adenocarcinoma tumor differentiation.

Effects of Obesity and Weight Loss Following Bariatric Surgery on Brain Function, Structural Integrity and Metabolism

Obesity is one of the key challenges to health care system worldwide and its prevalence is estimated to rise to pandemic proportions. Numerous adverse health effects follow with increasing body weight, including increased risk of hypertension, diabetes, hypercholesterolemia, musculoskeletal pain and cancer. Current evidence suggests that obesity is associated with altered cerebral reward circuit functioning and decreased inhibitory control over appetitive food cues. Furthermore, obesity causes adverse shifts in metabolism and loss of structural integrity within the brain. Prior cross-sectional studies do not allow delineating which of these cerebral changes are recoverable after weight loss. We compared morbidly obese subjects with healthy controls to unravel brain changes associated with obesity. Bariatric surgery was used as an intervention to study which cerebral changes are recoverable after weight loss. In Study I we employed functional magnetic resonance imaging (fMRI) to detect the brain basis of volitional appetite control and its alterations in obesity. In Studies II-III we used diffusion tensor imaging (DTI) and voxel-based morphometry (VBM) to quantify the effects of obesity and the effects of weight loss on structural integrity of the brain. In study IV we used positron emission tomography (PET) with [18F]-FDG in fasting state and during euglycemic hyperinsulinemia to quantify effects of obesity and weight loss on brain glucose uptake. The fMRI experiment revealed that a fronto-parietal network is involved in volitional appetite control. Obese subjects had lower medial frontal and dorsal striatal brain activity during cognitive appetite control and increased functional connectivity within the appetite control circuit. Obese subjects had initially lower grey matter and white matter densities than healthy controls in VBM analysis and loss of integrity in white matter tracts as measured by DTI. They also had initially elevated glucose metabolism under insulin stimulation but not in fasting state. After the weight loss following bariatric surgery, obese individuals’ brain volumes recovered and the insulin-induced increase in glucose metabolism was attenuated. In conclusion, obesity is associated with altered brain function, coupled with loss of structural integrity and elevated glucose metabolism, which are likely signs of adverse health effects to the brain. These changes are reversed by weight loss after bariatric surgery, implicating that weight loss has a causal role on these adverse cerebral changes. Altogether these findings suggest that weight loss also promotes brain health.Key words: brain, obesity, bariatric surgery, appetite control, structural magnetic resonance

Anxiety and learning / response behaviour in the context of intellectual problem solving by young children

Twenty-eight grade four students were ca.tegorized as either high or low anxious subjects as per Gillis' Child Anxiety Scale (a self-report general measure). In determining impulsivity in their response tendencies, via Kagan's Ma.tching Familiar Figures Test, a significant difference between the two groups was not found to exist. Training procedures (verbal labelling plus rehearsal strategies) were introduced in modification of their learning behaviour on a visual sequential memory task. Significantly more reflective memory recall behaviour was noted by both groups as a result. Furthermore, transfer of the reflective quality of this learning strategy produced significantly less impulsive response behaviour for high and low anxious subjects with respect to response latency and for low anxious subjects with respect to response accuracy.

Factors influencing the seasonal changes in primary productivity of the photosynthetic bacteria of Crawford Lake, Ontario

A naturally occurring population of photosynthetic bacteria, located in the meromictic Crawford Lake, was examined during two field seasons (1979-1981). Primary production, biomass, light intensity, lake transparency, pH and bicarbonate concentration were all monitored during this period at selected time intervals. Analysis of the data indicated that (l4C) bacterial photosynthesis was potentially limited by the ambient bicarbonate concentration. Once a threshold value (of 270 mg/l) was reached a dramatic (2 to 10 fold) increase in the primary productivity of the bacteria was observed. Light intensity appeared to have very little effect on the primary productivity of the bacteria, even at times when analyses by Parkin and Brock (1980a) suggested that light intensity could be limiting (i.e., 3.0-5.0 ft. candles). Shifts in the absorption maxima at 430 nrn of the .bacteriochlorophyll spectrum suggested that changes in the species or strain composition of the photosynthetic bacteria had occurred during the summer months. It was speculated that these changes might reflect seasonal variation in the wavelength of light reaching the bacteria. Chemocline erosion did not have the same effect on the population size (biomass) of the photosynthetic bacteria in Crawford Lake (this thesis) as it did in Pink Lake (Dickman, 1979). In Crawford Lake the depth of the chemocline was lowered with no apparent loss in biomass (according to bacteriochlorophyll data). A reverse current was. proposed to explain the observation. The photosynthetic bacteria contributed a significant proportion (10-60%) of the lake1s primary productivitya Direct evidence was obtained with (14C) labelling of the photosynthetic bacteria, indica.ting that the zooplankton were grazing the photosynthetic bacteria. This indicated that some of the photosynthetic bacterial productivity was assimilated into the food chain of the lake. Therefore, it was concluded that the photosynthetic bacteria made a significant contribution to the total productivity of Crawford Lake.

Immuno-gluorescence study of five zygomycetous fungi with two chitin-binding probes

A polyclonal antiserum was prepared against a purified microsomal chitinase isolated from the fungus Choanephora cucurbitarum. Indirect immunofluorescence was used to localize chitinase at various developmental stages of five zygomycetous fungi and during abiotrophic mycoparasite interaction with a susceptible and resistant host. This was compared to localization of oligomers of N-acetylglucosamine with the lectin wheat germ agglutinin (WGA). Dotimmunoblot and Western blot techniques revealed that the anti-serum reacted strongly with the antigen from which it was derived. Cross reactivity of the antiserum was found with WGA and another chitin binding lectin, Phyto/acca americana agglutinin (PAA). Immuno-fluorescence results showed the direct involvement of chitinase in spore swelling, germination, sporangium development and response during mechanical injury. There appeared to be no involvement of chitinase during apical hyphal growth or new branch initiation in any of the fungi tested despite mild proteolysis and permeabilization of the cell surface prior to labelling. Binding with WGA revealed similar patterns of fluorescence to that of chitinase localization but differed by showing fluorescence and therefore chitin localization at the apex and new branch initiation when tested at different developmental stages. There was no difference between chitinase localization and binding with WGA in a susceptible host and resistant host challenged with the mycoparasite, Piptocephalis virginiana. Differences in binding ability of antichitinase and lectin WGA suggests that the latter is not a suitable indicator for indirect localization of the lytic enzyme, chitinase.

Size regulation of double and half embryos in the mice musculus

Genetic chimeras made by aggregating early mouse embryos have many uses in developmental biology and have also provided insights into embryonic growth regulation. There is an indication that the embryo can regulate for an increase in size because although aggregation chimeras are twice as big as normal embryos when made, they are born of normal size. Upward regula..... tion of size reduced embryos is also possible. Half embryos made by the isolation or destruction of one of the blastomeres of a 2-cell embryo are also born of normal size. Little is known about the timing or the mechanism of this size regulation. In this study, the timing of size regulation in double and half embryos was clearly established by comparison of cell numbers derived from serial reconstruction of light microscope sections of control and experimental embryos. It was shown that size regulation in double embryos occurred around 6dl6h and in half embryos by 7dOh. Size regulation occurred in all tissues at the same time indicating a single control mechanism for the entire embryo. More detailed examination of the growth of double embryos revealed that size regulation occurred by alteration in cell cycle length~ No excessive cell death was found in double embryos compared to the controls and continuous labelling with [3H] thymidine showed no large non-dividing cell population in double embryos. However, a comparison of the mitotic index of double and control embryos after colcemid treatment, revealed a large difference between the two around 5dl6h to 6d16h. During this period, control embryos underwent a proliferative burst not shown by the double embryos. The mechanism for cell cycle control is not clear; it may be intrinsic to the embryo or determined by the uterine environment. Evidence was found suggesting that differentiation in the postimplantation embryo was cell number dependent. The timing of differentiative events was examined in half, double and control embryos. Proamnion formation, which occurs prior to size regulation, occurs at the same cell number but at different times in the three groups of embryos. However mesoderm which appears after size regulation was seen at the same time in all grollps of embryos.

The 21-dehydroxylation of corticosteroids: evidence for an enol intermediate and the hydroxylation of steroids by fungal cyto-chrome P450: evidence for a stepwise mechanism

Two enzyme mechanisms were examined: the 21-dehydroxylation of corticosteroids by the anaerobe Eubacterium l en tum, and the hydroxylation of steroids by fungal cytochrome P450. Deuterium labelling techniques were used to study the enzymic dehydroxylation. Corticosteroids doubly labelled (2H) at the C-21 position were incubated with a culture of Eubacterium lentum. It was found that t he enzymic dehydroxylation proceeded with the loss of one 2H f rom C-21 per molecule of substrate. The kinetic isotope ef fect f or the reaction was found to be k~kD = 2. 28. These results suggest that enzyme/substr ate binding in this case may proceed via t he enol form of the substrate. Also , it appears that this binding is, at least in part, the rate determining step of t he reaction. The hydroxylation of steroids by fungal cytochrome P450 was examined by means of a product study. Steroids with a double bond at the A8 (9), ~( lO ), or ~ (ll) position were synthesized. These steroids were then incubated with fungal strains known to use a cytochrome P450 monooxygenase to hydroxylate at positions allylic to these doubl e bonds. The products formed in these incubations indicated that the double bonds had migrated during allylic hydroxylat ion. This suggests that a carbon centred radical or ion may be an intermediate i n the cytochrome P450 cat alytic cycle.