<p>Antisense oligonucleotides (ODN) may interfere in gene expression on the basis of hybridising to its complementary messenger RNA (mRNA) sequence in the cell thereby preventing the synthesis of the peptide. Therefore, these ODNs may be potential drugs to treat human diseases by “knocking down” the expression of responsible genes or correcting the maturation process of mRNA in the field called antisense therapy. Moreover, antisense ODNs upon labelling are also potential imaging agents to monitor gene expression <i>in vivo</i>, i.e. to accomplish <i>in vivo</i> hybridisation. This would provide a non-invasive tool compared to present methods, which require tissue samples. </p><p>This goal may be reached using positron emission tomography (PET) methodology. PET is a most advanced <i>in vivo</i> imaging technology, which would allow exploring the fate of radionuclide-labelled antisense ODNs in the body; thereby providing information about biodistribution and quantitative accumulation in tissues to assess pharmacokinetic properties of ODNs. This kind of evaluation is important as part of the characterisation of antisense therapeutics but also as part of the development of antisense imaging agents.</p><p>The present study aimed to investigate ⁷⁶Br- and ⁶⁸Ga-labelled ODNs of five different modifications: phosphodiester, phosphorothioate, 2'-<i>O</i>-methyl phosphodiester, locked nucleic acid (LNA), and peptide nucleic acid. The study included exploration of the hybridisation abilities of these ODNs after labelling; furthermore, the biodistribution, metabolite analysis and uptake of the ODNs in rats regarding non-hybridisation and hybridisation specific uptake was conducted. Among the ODNs studied, LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence) displayed the most promising characteristics considering a higher retention in tissues, stability and longer plasma residence. However, biodistribution data demonstrated a non-hybridisation specific distribution in rat tissues with kidney, liver, spleen and bone marrow being the organs of high uptake. Scavenger receptors or other saturable processes unrelated to hybridisation may play a role in tissue uptake and in clearance of antisense ODNs through these organs. These processes may be sequence dependent suggesting that proof of <i>in vivo</i> hybridisation through imaging needs much more elaborate evaluations than just comparison of sense and antisense sequences and proving dose-dependency.</p>

Social phobia is a disabling anxiety disorder characterized by an excessive fear of negative evaluation in social situations. The present thesis explored the epidemiology and neurobiology of the disorder. By means of a mailed questionnaire, the point prevalence of social phobia in the Swedish general population was estimated at 15.6%. However, prevalence rates varied between 1.9 and 20.4% across the different levels of distress and impairment used to define cases. Thus, although social anxiety is widespread within the community, the precise diagnostic boundaries for social phobia are difficult to determine. Social phobia was associated with female gender, low educational attainment, psychoactive medication use, and lack of social support. A cluster analysis revealed that subtypes of social phobia mainly differed dimensionally on a mild-moderate-severe continuum, with number of cases declining with increasing severity. Public speaking was the most common social fear in all groups of social phobics and in the population at large. In the neurobiological studies, positron emission tomography was used to examine brain serotonin metabolism and changes in the regional cerebral blood flow (rCBF) response to public speaking stress following treatment with a selective serotonin reuptake inhibitor (SSRI) or cognitive-behavioral group therapy. Social phobics exhibited lowered serotonin turnover, relative to non-phobics, mainly in the medial temporal cortex including the bilateral rhinal and periamygdaloid regions. Symptom improvement with cognitive-behavioral- as well as SSRI-treatment was accompanied by a reduced rCBF-response to public speaking in the amygdala, hippocampus and adjacent temporal cortex, i.e. regions that serve important functions in anxiety. Thorough suppression of rCBF in limbic brain regions was associated with favorable long-term treatment outcome. These results provide neuroimaging evidence for a presynaptic serotonergic dysfunction in social phobia and for a common neural mechanism whereby psychological and pharmacological anti-anxiety treatments act.

Wilson’s disease is a rare inborn error of metabolism caused by a defect in ATP7B, a protein necessary for proper copper excretion into bile. It is characterised by copper accumulation with hepatic and central nervous system dysfunction. We investigated 24 Swedish families with Wilson’s disease by sequencing the entire coding sequence using a new technique called manifold sequencing. Disease causing mutations were found in 44 out of 48 alleles. From data obtained in the first study, the two most common mutations (C3207A and C2930T) were sought in 2640 anonymous DNA samples from a Swedish population, using a pooling strategy and solid-phase minisequencing. Four C3207A and one C2930T were found. From the number of C3207A, a prevalence of Wilson’s disease in Sweden of about 1 in 110,000 could be estimated. Four groups with three patients each had four different genotypes concerning mutations in ATP7B. The patients’ psychopathological symptoms were investigated, using the Karolinska Scales of Personality rating (KSP) and Comprehensive Psychopathological Rating Scale (CPRS). A trend towards lower CPRS scores was seen in the groups with mutations known to render ATP7B completely without activity. Using 61Cu liver PET in patients homozygous for mutations in ATP7B, heterozygotes, normal individuals and two patients with alcoholic liver cirrhosis, significantly slower uptake was seen in the homozygotes as compared to the heterozygotes and normal individuals. The patients with cirrhosis had values in between. This implies that 61Cu liver PET might be used as an additional rapid and little invasive diagnostic tool in Wilson’s disease. In a retrospectively studied cohort consisting of 363 patients followed in Sweden and the UK, nine cases of aggressive intra-abdominal malignancies were seen, which is more than expected. Caution should be taken in the follow-up of Wilson’s disease patients.

Antisense oligonucleotides (ODN) may interfere in gene expression on the basis of hybridising to its complementary messenger RNA (mRNA) sequence in the cell thereby preventing the synthesis of the peptide. Therefore, these ODNs may be potential drugs to treat human diseases by “knocking down” the expression of responsible genes or correcting the maturation process of mRNA in the field called antisense therapy. Moreover, antisense ODNs upon labelling are also potential imaging agents to monitor gene expression in vivo, i.e. to accomplish in vivo hybridisation. This would provide a non-invasive tool compared to present methods, which require tissue samples. This goal may be reached using positron emission tomography (PET) methodology. PET is a most advanced in vivo imaging technology, which would allow exploring the fate of radionuclide-labelled antisense ODNs in the body; thereby providing information about biodistribution and quantitative accumulation in tissues to assess pharmacokinetic properties of ODNs. This kind of evaluation is important as part of the characterisation of antisense therapeutics but also as part of the development of antisense imaging agents. The present study aimed to investigate 76Br- and 68Ga-labelled ODNs of five different modifications: phosphodiester, phosphorothioate, 2'-O-methyl phosphodiester, locked nucleic acid (LNA), and peptide nucleic acid. The study included exploration of the hybridisation abilities of these ODNs after labelling; furthermore, the biodistribution, metabolite analysis and uptake of the ODNs in rats regarding non-hybridisation and hybridisation specific uptake was conducted. Among the ODNs studied, LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence) displayed the most promising characteristics considering a higher retention in tissues, stability and longer plasma residence. However, biodistribution data demonstrated a non-hybridisation specific distribution in rat tissues with kidney, liver, spleen and bone marrow being the organs of high uptake. Scavenger receptors or other saturable processes unrelated to hybridisation may play a role in tissue uptake and in clearance of antisense ODNs through these organs. These processes may be sequence dependent suggesting that proof of in vivo hybridisation through imaging needs much more elaborate evaluations than just comparison of sense and antisense sequences and proving dose-dependency.

Chronic muscle pain is a significant medical and social problem and better understanding of the pathophysiological mechanisms involved is an important requirement for further development of diagnostics, treatment and rehabilitation methods. Experimental imaging studies have investigated functional neuroanatomy of different pain components. However, several aspects of brain mechanisms underlying brain processing of muscle pain remain unclear. The general goal of the present thesis was to study functional brain anatomy of systems underlying perception of muscle pain, processing of proprioceptive information and maintenance of fatiguing muscle contractions with an emphasize on their possible interrelations. Four series of experiment were carried out. Using an injection of hypertonic saline (HS) into the m. triceps to induce pain comparable with clinical muscle pain a significant activation of insula and putamen as well as decrease of activity in the temporal and occipital cortex in comparison with control stimulation were revealed. An advanced model of prolonged muscle pain were provided by the infusion of the HS during 20 minutes into m. erector spinae A complex dynamics of brain activity during the habituation to nociceptive stimulation was shown: initial activation of insula changed to decrease of activity in this and several other cortical areas. A conjunction analysis identified activations jointly significant in both experiments (despite localization of HS nociceptive stimulation) in the right insula, occipital and left parietal cortical areas. The study of brain activity in response to different modalities of prorioceptive inputs – passive movements, kinesthetic illusions and muscle vibration showed corresponding different patterns of activation in motor and somatosenory areas and temporal areas. Finally, the study of sustained isometric muscle contractions of various force levels and durations revealed that muscle fatigue is associated with contralateral activation of the motor and somatosensory areas and temporal areas and bilateral activation in the supplementary motor areas and cingular cortex, indicating that increased efforts needed to maintain required force and its eventual breakdown with fatigue might induce activation of additional cortical areas. Analysis of data obtained in all experimental series revealed that insula, secondary somatosensory and auditory areas are activated during both perception of muscle pain and processing of somatosensory afferentation. In conclusion, this thesis has elucidated brain processing of muscle pain showing distributed, bilateral patterns comprised of activated structures predominantly attributed to the medial pain system and deactivated structures. Furthermore, initial and late phases of tonic muscle pain are associated with different brain reactions, namely initial activation of the insula followed by a significant bilateral decrease of activity at the late stage. Area of brain cortex located near lateral sulcus and comprised of secondary somatosensory cortex, posterior part of the insula and adjacent auditory cortex is engaged in the perception of muscle pain and processing of somatosensory afferentation as well as maintenance of fatiguing muscle contractions.

This thesis investigates the potential of using positron emission tomography (PET) to study the biosynthesis and release of serotonin (5HT) at the brain serotonergic neuron. As PET requires probe compounds with specific attributes to enable imaging and quantification of biological processes, emphasis was placed on the evaluation of these attributes. The experiments established that the 5HT transporter radioligand [11C]-3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)-benzonitrile, [11C]DASB, is suitable for imaging and quantification of transporters in rats and rhesus monkeys. In addition, the binding of [11C]DASB in brain tissue is decreased when 5HT concentrations are increased by tranylcypromine administration. The sensitivity of [11C]DASB binding, under these experimental conditions, to increased endogenous 5HT concentrations demonstrates the potential of in vivo monitoring of 5HT release in rat and monkey models. The irreversible binding of 5-hydroxy-L-[β-11C]tryptophan, [11C]HTP, in the monkey brain was lower in the presence of NSD1015, which was used to inhibit the decarboxylase step in 5HT synthesis. [11C]HTP seems thus to have potential for tracking changes in the activity of this biosynthesis enzyme. In contrast, the accumulation of [11C]HTP was unaffected by clorgyline, which was used to inhibit metabolism of the probe in the brain. This appears to indicate that elimination of the main metabolite from the brain could be negligible and thus will not alter [11C]HTP quantification. The extent and distribution of the irreversible binding of a substrate for the first enzyme in 5HT formation, α-[11C]methyl-L-tryptophan, [11C]AMT, was different from those for [11C]HTP. This suggests that the two studied probe compounds provide estimates related to the enzyme activity of different steps in the 5HT biosynthesis pathway. A reference tissue version of the Patlak method for the analysis of data obtained by PET was also developed. This approach takes into account irreversible binding in the reference region and appears, therefore, to yield more reliable parameter estimates than the conventional reference Patlak analysis. The method is recommended for parameter estimation of [11C]HTP data when no metabolite-corrected plasma curve is available. Knowledge of altered 5HT synthesis and release in disease states and the consequences for effective pharmacotherapy can improve our knowledge of the aetiology of certain psychiatric and neurological diseases and enhance our ability to design more effective drugs.

<p>The use of positron emission tomography (PET) in radiation therapy has continued to grow, especially since the development of combined computed tomography (CT) and PET imaging system in the early 1990s. Today, the biggest use of PET-CT is in oncology, where a glucose analog radiotracer is rapidly incorporated into the metabolic pathways of a variety of cancers. Images representing the in-vivo distribution of this radiotracer are used for the staging, delineation and assessment of treatment response of patients undergoing chemotherapy or radiation therapy. While PET offers the ability to provide functional information, the imaging quality of PET is adversely affected by its lower spatial resolution. It also has unfavorable image noise characteristics due to radiation dose concerns and patient compliance. These factors result in PET images having less detail and lower signal-to-noise (SNR) properties compared to images produced by CT. This complicates the use of PET within many areas of radiation oncology, but particularly the delineation of targets for radiation therapy and the assessment of patient response to therapy. The development of segmentation methods that can provide accurate object identification in PET images under a variety of imaging conditions has been a goal of the imaging community for years. The goal of this thesis are to: (1) investigate the effect of filtering on segmentation methods; (2) investigate whether combining individual segmentation methods can improve segmentation accuracy; (3) investigate whether the consensus volumes can be useful in aiding physicians of different experience in defining gross tumor volumes (GTV) for head-and-neck cancer patients; and (4) to investigate whether consensus volumes can be useful in assessing early treatment response in head-and-neck cancer patients.</p><p>For this dissertation work, standard spherical objects of volumes ranging from 1.15 cc to 37 cc and two irregularly shaped objects of volume 16 cc and 32 cc formed by deforming high density plastic bottles were placed in a standardized image quality phantom and imaged at two contrasts (4:1 or 8:1 for spheres, and 4.5:1 and 9:1 for irregular) and three scan durations (1, 2 and 5 minutes). For the work carried out into the comparison of images filters, Gaussian and bilateral filters matched to produce similar image signal to noise (SNR) in background regions were applied to raw unfiltered images. Objects were segmented using thresholding at 40% of the maximum intensity within a region-of-interest (ROI), an adaptive thresholding method which accounts for the signal of the object as well as background, k-means clustering, and a seeded region-growing method adapted from the literature. Quality of the segmentations was assessed using the Dice Similarity Coefficient (DSC) and symmetric mean absolute surface distance (SMASD). Further, models describing how DSC varies with object size, contrast, scan duration, filter choice and segmentation method were fitted using generalized estimating equations (GEEs) and standard regression for comparison. GEEs accounted for the bounded, correlated and heteroscedastic nature of the DSC metric. Our analysis revealed that object size had the largest effect on DSC for spheres, followed by contrast and scan duration. In addition, compared to filtering images with a 5 mm full-width at half maximum (FWHM) Gaussian filter, a 7 mm bilateral filter with moderate pre-smoothing (3 mm Gaussian (G3B7)) produced significant improvements in 3 out of the 4 segmentation methods for spheres. For the irregular objects, time had the biggest effect on DSC values, followed by contrast. </p><p>For the study of applying consensus methods to PET segmentation, an additional gradient based method was included into the collection individual segmentation methods used for the filtering study. Objects in images acquired for 5 minute scan durations were filtered with a 5 mm FWHM Gaussian before being segmented by all individual methods. Two approaches of creating a volume reflecting the agreement between the individual methods were investigated. First, a simple majority voting scheme (MJV), where individual voxels segmented by three or more of the individual methods are included in the consensus volume, and second, the Simultaneous Truth and Performance Level Estimation (STAPLE) method which is a maximum likelihood methodology previously presented in the literature but never applied to PET segmentation. Improvements in accuracy to match or exceed the best performing individual method were observed, and importantly, both consensus methods provided robustness against poorly performing individual methods. In fact, the distributions of DSC and SMASD values for the MJV and STAPLE closely match the distribution that would result if the best individual method result were selected for all objects (the best individual method varies by objects). Given that the best individual method is dependent on object type, size, contrast, and image noise and the best individual method is not able to be known before segmentation, consensus methods offer a marked improvement over the current standard of using just one of the individual segmentation methods used in this dissertation. </p><p>To explore the potential application of consensus volumes to radiation therapy, the MJV consensus method was used to produce GTVs in a population of head and neck cancer patients. This GTV and one created using simple 40% thresholding were then available to be used as a guidance volume for an attending head and neck radiation oncologist and a resident who had completed their head and neck rotation. The task for each physician was to manually delineate GTVs using the CT and PET images. Each patient was contoured three times by each physician- without guidance and with guidance using either the MJV consensus volume or 40% thresholding. Differences in GTV volumes between physicians were not significant, nor were differences between the GTV volumes regardless of the guidance volume available to the physicians. However, on average, 15-20% of the provided guidance volume lay outside the final physician-defined contour.</p><p>In the final study, the MJV and STAPLE consensus volumes were used to extract maximum, peak and mean SUV measurements in two baseline PET scans and one PET scan taken during patients' prescribed radiation therapy treatments. Mean SUV values derived from consensus volumes showed smaller variability compared to maximum SUV values. Baseline and intratreatment variability was assessed using a Bland-Altman analysis which showed that baseline variability in SUV was lower than intratreatment changes in SUV.</p><p>The techniques developed and reported in this thesis demonstrate how filter choice affects segmentation accuracy, how the use of GEEs more appropriately account for the properties of a common segmentation quality metric, and how consensus volumes not only provide an accuracy on par with the single best performing individual method in a given activity distribution, but also exhibit a robustness against variable performance of individual segmentation methods that make up the consensus volume. These properties make the use of consensus volumes appealing for a variety of tasks in radiation oncology.</p> / Dissertation

Small intestinal neuroendocrine tumor (SI-NET) is the most common small bowel tumor in Europe and USA, with an annual incidence of around 0.3-1.3/100000 persons. SI-NETs are the most common type of gastroenteropancreatic NETs (GEP-NETs), and they are known for their ability to produce hormones such as tachykinins and serotonin, as well as for their favorable long-term prognosis in comparison to gastrointestinal adenocarcinoma. The overall aim of the thesis was to investigate unknown or unclear aspects of SI-NET disease, in connection with prognosis, treatment and follow-up. Paper I confirmed several known negative prognostic factors and also showed, for the first time, that para-aortal lymph node metastases and peritoneal carcinomatosis were associated with worse survival by multivariable analyses. Locoregional surgery was associated with a low post-operative mortality, and a prolonged long-term survival by multivariable analysis. In Paper II we continued to investigate peritoneal carcinomatosis and found it be a risk factor not only for death, but also for emergency re-surgery. Furthermore, genetic analyses of samples from primary tumors in patients with and without peritoneal carcinomatosis showed a difference in the DNA between these two groups. In Paper III the outcome after liver surgery and/or radiofrequency ablation of liver metastases was investigated. To summarize, no difference in survival was seen in patients treated with surgery/radiofrequency ablation in comparison with matched controls. However, a superior radiological response of liver metasases and lower U-5-HIAA values were seen in patients subjected to liver surgery and/or radiofrequency ablation compared to matched controls. Paper IV compared ultrasonography, computed tomography and 11C-5HTP-PET in the follow-up after radiofrequency ablation of NET liver metastases. The study concluded that 11C-5HTP-PET depicted all residual tumors after RFA and that it, if used, should be combined with computed tomography for easier interpretation, as RFA areas are not clearly distinguishable with 11C-5HTP-PET alone. Paper V studied gallstone complications after somatostatin analog treatment in SI-NET patients, and concluded that there was a rather high risk to be subjected to a cholecystectomy due to biliary colic, cholecystitis, cholangitis or pancreatitis after primary surgery in somatostatin analog treated patients.

Correlations between neuropsychiatric symptoms and beta-amyloid (Aβ) burden in specific brain regions in living Alzheimer’s disease (AD) patients remain to be elucidated. Ten mild AD patients underwent MR and [11C]SB-13 PET imaging. Neuropsychiatric symptoms were quantified with the Neuropsychiatric Inventory (NPI). NPI-depression/dysphoria, -apathy, -agitation/aggression, -anxiety, and -appetite/eating disorders scores were hypothesized to correlate with Aβ burden in particular brain regions. Pearson’s correlation coefficient revealed that depression/dysphoria scores positively correlated (p<0.05) with standardized uptake values (SUVs) from left medial temporal lobe (r=0.67), and agitation/aggression correlated with SUVs from bilateral anterior cingulate (right, r=0.71; left, r=0.78), temporal (right, r=0.71; left, r=0.75), parietal (right, r=0.77; left, r=0.81), and dorsolateral prefrontal cortex (right, r=0.74; left, r=0.73). However, NPI scores did not significantly correlate with better estimates of Aβ burden that use the cerebellum as reference region. Overall, our results confirm the lack of association between Aβ burden and neuropsychiatric symptoms reported in autopsy studies.

Correlations between neuropsychiatric symptoms and beta-amyloid (Aβ) burden in specific brain regions in living Alzheimer’s disease (AD) patients remain to be elucidated. Ten mild AD patients underwent MR and [11C]SB-13 PET imaging. Neuropsychiatric symptoms were quantified with the Neuropsychiatric Inventory (NPI). NPI-depression/dysphoria, -apathy, -agitation/aggression, -anxiety, and -appetite/eating disorders scores were hypothesized to correlate with Aβ burden in particular brain regions. Pearson’s correlation coefficient revealed that depression/dysphoria scores positively correlated (p<0.05) with standardized uptake values (SUVs) from left medial temporal lobe (r=0.67), and agitation/aggression correlated with SUVs from bilateral anterior cingulate (right, r=0.71; left, r=0.78), temporal (right, r=0.71; left, r=0.75), parietal (right, r=0.77; left, r=0.81), and dorsolateral prefrontal cortex (right, r=0.74; left, r=0.73). However, NPI scores did not significantly correlate with better estimates of Aβ burden that use the cerebellum as reference region. Overall, our results confirm the lack of association between Aβ burden and neuropsychiatric symptoms reported in autopsy studies.