<p>The use of positron-emitting tracers has been extended beyond tomographic facilities in the last few years, giving rise to a general positron-emitting tracing technique. The methodological part of the present thesis involved the evaluation of the performance of storage phosphor (SP) plates, with tracers labeled with high-energy, short-lived, positron-emitting radionuclides, using homogenized tissue specimens and autoradiography with frozen brain sections. The SP plates showed superior sensitivity and a linear response over a wide radioactivity range. Autoradioradiography provided reliable results due to (a) adequate sensitivity for low radioactivity concentration, b) an excellent linear range, and (c) satisfactory resolution. Though equilibration time of receptor-ligand interaction was dependent upon section thickness, quantification was possib with thinner sections.</p><p>An initial finding using frozen section autoradiography of rat brain and spinal cord showed preferential binding of [¹¹C]4-NMPB, a muscarinic acetylcholine (mACh) receptor antagonist, to the M4 subtype of mACh receptors. Further work to ascertain this specificity, by use of binding studies on cell membranes from CHO-K1 cells expressing individual subtypes of human mACh receptors, suggested lack of subtype selectivity. With respect to the possible cliinical use in glutamatergic neuropathology, [¹¹C]cyano-dizocilpine, as a potential PET tracer for the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, was studied. The <i>in vivo</i> visualization of specific binding could not be achieved, though <i>in vitro</i> binding demonstrated good specificity and preferential binding to the activated for of the NMDA receptors.</p><p>The use of the glucose analogue [¹⁸F]fluorodeoxyglucose (FDG) to study glucose utilization was evaluated in experimental traumatic brain injury (TBI). A trauma-induced increased uptake of FDG was seen, whereas the uptake of [1-¹⁴C]glucose remained unchanged. This discrepancy might be due to the increased postraumatic affinity of FDG for the endothelial glucose transporter proteins and/or to the hexokinase enzyme. [¹¹C]Cyano-dizocilpine, [¹¹C]4-NMPB, and [¹¹C]flumazenil were utilized in autoradiography to evaluate changes in NMDA, mACh, and GABA_A receptors, espectively, in experimental TBI. Observations showed a global decrease in the binding potential BP) of (i) [¹¹C]cyano-dizocilpine acutely and 12 hrs after TBI, and (ii) of [¹¹C]4-NMPB at 12 hrs after TBI, and (iii) a decrease in the BP of [¹¹C]flumazenil in the cortex and hippocampus ipsilateral to the site of injury. The demonstrated changes in receptor binding after TBI are indicative of a widely dissipated effect of TBI on the particular neurotransmitter receptor systems as compared with what would be expected from FDG studies after TBI, i.e., a local disturbed neurotransmission.</p>

<p>The positron emitting radionuclide ⁶⁸Ga (T_1/2 = 68 min) might become of practical interest for clinical positron emission tomography (PET). The metallic cation, ⁶⁸Ga(III), is suitable for complexation with chelators, either naked or conjugated with biological macromolecules. Such labelling procedures require pure and concentrated preparations of ⁶⁸Ga(III), which cannot be sufficiently fulfilled by the presently available ⁶⁸Ge/⁶⁸Ga generator eluate. This thesis presents methods to increase the concentration and purity of ⁶⁸Ga obtained from a commercial ⁶⁸Ge/⁶⁸Ga generator. The use of the preconcentrated and purified ⁶⁸Ga eluate along with microwave heating allowed quantitative ⁶⁸Ga-labelling of peptide conjugates within 15 min. The specific radioactivity of the radiolabelled peptides was improved considerably compared to previously applied techniques using non-treated generator eluate and conventional heating. A commercial ⁶⁸Ge/⁶⁸Ga generator in combination with the method for preconcentration/purification and microwave heated labelling might result in an automated device for ⁶⁸Ga-based radiopharmaceutical kit production with quantitative incorporation of ⁶⁸Ga(III).</p><p>Macromolecules were labelled with ⁶⁸Ga(III) either directly or via a chelator. The bifunctional chelator, DOTA, was conjugated in solution to peptides, an antibody and oligonucleotides. The peptides had varied pI values, constitution, and length ranging from 8 to 53 amino acid residues. The oligonucleotides were of various sequences and length with modifications in backbone, sugar moiety and both 3' and 5' ends with a molecular weight up to 9.8 kDa. The bioconjugates were labeled with ⁶⁸Ga(III), and the resulting tracers were characterised chemically and biologically. The identity of the ⁶⁸Ga-labelled bioconjugates was verified. The tracers were found to be stable and their biological activity maintained. Specific radioactivity was shown to be an important parameter influencing the feasibility of accurate imaging data quantification.</p><p>Furthermore, ⁶⁸Ga-labelled peptide imaging was shown to be a useful tool to study peptide adsorption to microstructures in a chemical analysis device.</p>

Les hormones thyroïdiennes sont importantes à la fois pour le développement, la maturation et le fonctionnement du système nerveux central tout au long de la vie. Alors que l’association entre l’absence d’hormones thyroïdiennes dans l’hypothyroïdie congénitale et le retard mental profond est bien documentée, l’hypothyroïdie acquise à l’âge adulte peut se manifester par une variété de symptômes tant somatiques, que neuropsychologiques ou psychiatriques comme des troubles de l’attention, troubles de la concentration, troubles de mémoire, un ralentissement psychomoteur mais aussi une humeur dépressive, de l’anxiété et même parfois un délire de persécution. De plus, les processus physiopathologiques reliant l’activité thyroïdienne à ces symptômes restent peu clairs dans l’hypothyroïdie acquise à l’âge adulte. L’objectif de cette thèse est d’examiner les relations entre état hypothyroïdien et dépression. En utilisant des échelles psychométriques, dix patients thyroïdectomisés pour carcinome thyroïdien ont été évalués pour la dépression, l’anxiété et le ralentissement psychomoteur ; ils ont été examinés à la fois en eu- et hypothyroïdie après retrait des hormones thyroïdiennes. Une tomographie par émission de positrons a été utilisée, avec de l’eau marquée à l’oxygène-15 et du fluorodéoxyglucose marqué au fluor-18 comme traceurs, de manière à corréler le débit sanguin cérébral régional (rDSC) et le métabolisme cérébral au glucose (rMGC) avec l’état mental des patients. Deux techniques différentes d’analyse d’images ont été appliquées (régions d’intérêt et cartes statistiques paramétriques). En hypothyroïdie, il y avait une diminution généralisée du rDSC (23.4%, p<0.001) et du rMGC (12.1%, p<0.001) sans diminution locale spécifique. Les patients étaient aussi significativement plus déprimés (p<0.001), plus anxieux (p<0.001) et plus ralentis sur le plan psychomoteur (p<0.005) en hypothyroïdie qu’à l’état euthyroïdien. Ces résultats indiquent que l’activité cérébrale est globalement réduite en hypothyroïdie sévère de courte durée sans observation des modifications régionales habituellement décrites dans la dépression majeure. Nous avons ensuite conduit une étude cognitive qui a examiné les fonctions attentionnelles, mnésiques et exécutives de même que l’intensité des symptômes anxieux et dépressifs dans l’état hypothyroïdien et le trouble dépressif majeur et le lien possible entre ces symptômes et les perturbations cognitives. Cette étude a confirmé l’existence d’un ralentissement psychomoteur associé à des perturbations attentionnelles et exécutives mais non mnésiques dans la dépression majeure, de même que dans l’hypothyroïdie. Cependant, alors que les patients déprimés présentaient un biais conscient pour du matériel à valence émotionnelle négative, ce biais n’a pas été observé chez les patients hypothyroïdiens. Alors que l’état hypothyroïdien s’accompagnait de symptômes anxieux et dépressifs, il semble que ces derniers soient trop discrets pour qu’un biais attentionnel puisse être observé avec du matériel à valence émotionnelle négative. En conclusion, les perturbations cognitives observées en hypothyroïdie semblent être directement reliées à cette condition métabolique hypothyroïdienne plutôt que la conséquence des symptômes dépressifs associés à cette condition. / The thyroid hormone is important both for the functional development and maturation of the central nervous system and for its proper functioning throughout life. Whereas the association between the absence of thyroid hormone in congenital hypothyroidism and profound mental retardation is well documented, adult onset hypothyroidism may have a variety of somatic, neuropsychological and psychiatric symptoms such as inattentiveness, inability to concentrate, deficits in memory, psychomotor slowing but also depressive mood state, anxiety, and sometimes persecutive delusions. In addition, the pathophysiological process relating thyroid activity to these symptoms remain unclear in adult onset hypothyroidism. The objective of this thesis is to examine the relationships between hypothyroidism and depression. Using psychometric scales, ten patients that had undergone total thyroidectomy for thyroid carcinoma were evaluated for depression, anxiety and psychomotor slowing; they were examined both when euthyroid and hypothyroid after thyroid hormone withdrawal. Positron emission tomography was used, with oxygen-15-labeled water and fluorine-18 FDG as the tracers, to correlate the regional cerebral blood flow (rCBF) and cerebral glucose metabolism (rCMRGlc) with the mental state in patients. Two different image analysis techniques (regions of interest and statistical parametric maps) were applied. In hypothyroidism, there was a generalized decrease in rCBF (23.4%, p<0.001) and in rCMRGlc (12.1%, p<0.001) and there were no specific local defects. Patients were also significantly more depressed (p<0.001), anxious (p<0.001) and psychomotor slowed (p<0.005) in hypo- than in euthyroid status. These results indicate that the brain activity was globally reduced in severe hypothyroidism of short duration without the regional modifications usually observed in primary depression. We conducted then a cognitive study which examined attentional, mnemonic and executive functions as well as the intensity of anxiety and depressive symptoms in hypothyroidism and major depression and the possible link between these symptoms and cognitive disturbances. This study confirmed the existence of psychomotor slowing associated with attentional and executive disturbance but without mnemonic disturbances in major depression as well as in hypothyroidism. However, while depressed subjects manifested a conscious bias with material of negative emotional valence, no such bias was found in the hypothyroid subjects. While the hypothyroid state is accompanied by anxiety/depressive symptoms, it seems that the latter are too discrete for an attentional bias to be observed with material with a negative emotional valence. In conclusion, the cognitive disturbances observed in hypothyroidism seem to be directly related to the metabolic condition of hypothyroidism rather than the consequence of the depressive symptoms associated with this condition.

The use of positron-emitting tracers has been extended beyond tomographic facilities in the last few years, giving rise to a general positron-emitting tracing technique. The methodological part of the present thesis involved the evaluation of the performance of storage phosphor (SP) plates, with tracers labeled with high-energy, short-lived, positron-emitting radionuclides, using homogenized tissue specimens and autoradiography with frozen brain sections. The SP plates showed superior sensitivity and a linear response over a wide radioactivity range. Autoradioradiography provided reliable results due to (a) adequate sensitivity for low radioactivity concentration, b) an excellent linear range, and (c) satisfactory resolution. Though equilibration time of receptor-ligand interaction was dependent upon section thickness, quantification was possib with thinner sections. An initial finding using frozen section autoradiography of rat brain and spinal cord showed preferential binding of [11C]4-NMPB, a muscarinic acetylcholine (mACh) receptor antagonist, to the M4 subtype of mACh receptors. Further work to ascertain this specificity, by use of binding studies on cell membranes from CHO-K1 cells expressing individual subtypes of human mACh receptors, suggested lack of subtype selectivity. With respect to the possible cliinical use in glutamatergic neuropathology, [11C]cyano-dizocilpine, as a potential PET tracer for the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, was studied. The in vivo visualization of specific binding could not be achieved, though in vitro binding demonstrated good specificity and preferential binding to the activated for of the NMDA receptors. The use of the glucose analogue [18F]fluorodeoxyglucose (FDG) to study glucose utilization was evaluated in experimental traumatic brain injury (TBI). A trauma-induced increased uptake of FDG was seen, whereas the uptake of [1-14C]glucose remained unchanged. This discrepancy might be due to the increased postraumatic affinity of FDG for the endothelial glucose transporter proteins and/or to the hexokinase enzyme. [11C]Cyano-dizocilpine, [11C]4-NMPB, and [11C]flumazenil were utilized in autoradiography to evaluate changes in NMDA, mACh, and GABAA receptors, espectively, in experimental TBI. Observations showed a global decrease in the binding potential BP) of (i) [11C]cyano-dizocilpine acutely and 12 hrs after TBI, and (ii) of [11C]4-NMPB at 12 hrs after TBI, and (iii) a decrease in the BP of [11C]flumazenil in the cortex and hippocampus ipsilateral to the site of injury. The demonstrated changes in receptor binding after TBI are indicative of a widely dissipated effect of TBI on the particular neurotransmitter receptor systems as compared with what would be expected from FDG studies after TBI, i.e., a local disturbed neurotransmission.

The present thesis investigated family history and neurobiology of social phobia. Social phobia is a disabling disorder characterized by a marked fear of scrutiny in a variety of social situations. By using a validated questionnaire, study I related family history of excessive social anxiety to social phobia and avoidant personality disorder in epidemiologically identified probands in the Swedish general population. A two- to threefold increased relative risk of social anxiety was observed for both diagnostic groups. Thus, having an affected family member is associated with approximately a doubled risk for both social phobia and avoidant personality disorder. The neurobiological studies explored situational and anticipatory elicited anxiety by means of positron emission tomography and 15O-water. Study II examined the functional neuroanatomy of social anxiety provocation in social phobics and a healthy comparison group during a public speaking task. Social phobia symptomatology was associated with higher neural activity in the amygdaloid complex, i.e. "the alarm system" of the brain, and lower activity in the prefrontal cortex. Study III examined the neural correlates of anxiety elicited by the anticipation of public speaking in individuals with social phobia. Anticipatory anxiety was accompanied by enhanced regional cerebral blood flow in the dorsolateral prefrontal and inferior temporal cortices as well as in the amygdaloid-hippocampal region. Brain blood flow was lower in the temporal pole and in the cerebellum. These results suggest that social phobia has a neuroanatomical basis in a highly sensitive fear network centered in the amygdaloid-hippocampal region and encompassing the prefrontal cortex.

The present thesis explored the neurobiological basis of three aspects of defense behaviors in humans. Positron emission tomography methodology was used, and changes in regional cerebral blood flow (rCBF) were measured as an index of neural activity. Firstly, brain function was studied in a group of patients suffering from combat-related posttraumatic stress disorder, using a symptom provocation paradigm with combat sounds in order to elicit fear. Exposure to auditory trauma reminders relative to neutral sounds was associated with increased rCBF in sensorimotor areas, the cerebellar vermis, the periaqueductal gray matter, and the right amygdala, whereas decreased activity was observed in the retrosplenial area of the posterior cingulate cortex. Secondly, the neural circuitry mediating the acoustic startle response and its habituation was studied in a group of healthy subjects. During acoustic startle stimulation as compared to a resting condition, increased rCBF was found in a medial posterior area of the pons corresponding to the nucleus reticularis pontis caudalis. As a result of startle repetition, altered activity was found in the cerebellum, pointing to its involvement in startle habituation. Thirdly, neural activity associated with startle modulation by phobic fear was studied in a group of subjects with specific animal phobias during exposure to pictures of their feared and non-feared objects, paired and unpaired with acoustic startle stimuli. As a result of startle potentiation, increased rCBF was found in the left amygdaloid-hippocampal region, and medially in the affective division of the anterior cingulate cortex. In conclusion, these results provide evidence for the involvement of limbic and paralimbic brain areas during fear provocation and fear-potentiated startle and for a similar neurocircuitry underlying startle in humans and animals.

Positron Emission Tomography (PET) permits regional myocardial perfusion, fibrosis and oxidative metabolism to be non-invasively quantified with radioactive tracers such as [15O]-water and [1-11C]-acetate. PET is an established research tool in congestive heart failure (CHF), a major cause of morbidity and mortality. However, as CHF is a syndrome that eventually affects all aspects of cardiac and systemic hemodynamic function, more clinically relevant information from a single PET scan is desirable. The aim of this thesis therefore was to develop and implement some new concepts in cardiac PET. A new method for the measurement of cardiac output with any tracer was validated in animal experiments and CHF patients. The early pulmonary retention of [1-11C]-acetate was inversely related to left ventricular (LV) function in animals and was directly proportional to lung water content and severity of LV diastolic dysfunction in patients. Eight patients with acute myocardial infarction were followed with serial PET from 3 hours to 3 weeks after trombolytic treatment. PET revealed that myocardial perfusion and the extraction and utilization of fuel substrates all decreased closer to the infarct centre. The rate of oxygen utilization within the infarct at 3 h predicted degree of myocardial fibrosis, pulmonary oedema and tissue viability at 3 weeks. Seventeen patients with CHF due to chronic ischemic cardiomyopathy and severely reduced LV function were evaluated with [1-11C]-acetate PET before and after coronary artery bypass surgery. There was a dramatic improvement in physical performance and symptoms, which was not correlated to the standard LV ejection indices. PET revealed that functional improvement was associated with improved LV loading conditions, reversed remodeling and homogenization of oxidative metabolism rather than increased output.

Liquid chromatography – mass spectrometry (LC-MS) methods were developed for the analysis of positron emission tomography (PET) radiotracers in biological matrices. Additionally, radiotracer chemistry was explored as an analytical tool for supporting LC-MS method development and imaging molecular interactions in miniaturised chemical analysis systems. Conventional radiodetection methods can offer high sensitivity in the analysis of radiotracers in biological matrices, although with the short half-life of PET tracers, this mass sensitivity decreases rapidly with time. This limits the time frame for analysis, and may compromise the precision and accuracy of the later measurements. Performing LC-MS analysis of the dominant stable isotope form of the tracer removes such time restrictions. An LC-MS/MS method was developed for determination of the tracer flumazenil in human plasma, with high inter-assay precision (RSD &lt; 7%) and accuracy (95 – 104%). The method was applied in a multiple scan PET study where the plasma concentration spanned from 0.07 to 0.21 nM. The method removed the time restrictions associated with radiodetection methods and thus provided the opportunity of analysing a greater number of samples than would have been possible with radioanalysis. Furthermore, an LC-MS/MS method was developed that provided an efficient metabolic screening tool of potential PET tracers, whereby the substrates could be collected directly from 11C-labelling batches. This permitted repeated incubation experiments without the need of repeated labelling syntheses. A para-methoxy-benzamide analogue of the radiotracer WAY-100635 was thus identified as a potential tracer with improved metabolic stability. Additionally, a capillary LC-MS method was developed with rapid (0.75 min) and efficient (&gt; 99%) on-line high flow-rate extraction for determination of metabolic stability of PET radiotracers. Finally, the concept of radionuclide imaging of miniaturised chemical analysis systems was demonstrated with the direct study of interactions within capillary extraction columns and microchannels moulded in a plastic CD and poly(dimethylsiloxane).

The positron emitting radionuclide 68Ga (T1/2 = 68 min) might become of practical interest for clinical positron emission tomography (PET). The metallic cation, 68Ga(III), is suitable for complexation with chelators, either naked or conjugated with biological macromolecules. Such labelling procedures require pure and concentrated preparations of 68Ga(III), which cannot be sufficiently fulfilled by the presently available 68Ge/68Ga generator eluate. This thesis presents methods to increase the concentration and purity of 68Ga obtained from a commercial 68Ge/68Ga generator. The use of the preconcentrated and purified 68Ga eluate along with microwave heating allowed quantitative 68Ga-labelling of peptide conjugates within 15 min. The specific radioactivity of the radiolabelled peptides was improved considerably compared to previously applied techniques using non-treated generator eluate and conventional heating. A commercial 68Ge/68Ga generator in combination with the method for preconcentration/purification and microwave heated labelling might result in an automated device for 68Ga-based radiopharmaceutical kit production with quantitative incorporation of 68Ga(III). Macromolecules were labelled with 68Ga(III) either directly or via a chelator. The bifunctional chelator, DOTA, was conjugated in solution to peptides, an antibody and oligonucleotides. The peptides had varied pI values, constitution, and length ranging from 8 to 53 amino acid residues. The oligonucleotides were of various sequences and length with modifications in backbone, sugar moiety and both 3' and 5' ends with a molecular weight up to 9.8 kDa. The bioconjugates were labeled with 68Ga(III), and the resulting tracers were characterised chemically and biologically. The identity of the 68Ga-labelled bioconjugates was verified. The tracers were found to be stable and their biological activity maintained. Specific radioactivity was shown to be an important parameter influencing the feasibility of accurate imaging data quantification. Furthermore, 68Ga-labelled peptide imaging was shown to be a useful tool to study peptide adsorption to microstructures in a chemical analysis device.

The most important aspects of nuclear medicine imaging systems such as Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) are the spatial resolution and the sensitivity (detector efficiency in combination with the geometric efficiency). Considerable efforts have been spent during the last two decades in improving the resolution and the efficiency by developing new detectors. Our proposed improvement technique is focused on the readout and electronics. Instead of using traditional pulse height analysis techniques we propose using free running digital sampling by replacing the analog readout and acquisition electronics with fully digital programmable systems. This thesis describes a fully digital data acquisition system for KS/SU SPECT, new algorithms for high resolution timing for PET, and modular FPGA based decentralized data acquisition system with optimal timing and energy. The necessary signal processing algorithms for energy assessment and high resolution timing are developed and evaluated. The implementation of the algorithms in field programmable gate arrays (FPGAs) and digital signal processors (DSP) is also covered. Finally, modular decentralized digital data acquisition systems based on FPGAs and Ethernet are described.