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One of the considerable strengths of this study is that I have used PET scanners with high resolution and high sensitivity that permit interrogation of the function of small volume structures.

I have also used a rigorous methodological approach to analyze PET images. In order to minimize the number of possible volumetric variations when drawing ROIs on individual PET images, I used standard ROI maps of predefined volumes of interest in MNI space that were applied to each spatially normalized parametric image. This technique has several advantages. First, the method allows the same cubic volume to be interrogated in all scans. Second, the normalization of baseline and follow-up images to the same orientation and space increases the confidence that the same anatomical structures are assessed in longitudinal studies.

Special attention was given to patients‟ selection. In chapter 4, for example the main aim of that study was to investigate brain monoaminergic innervation in parkin- patients and patients with idiopathic PD. However, I wanted to exclude that a

different extrastriatal monoaminergic pattern in the two conditions could be related to the different degrees of striatal involvement in the two groups. Therefore only parkin gene carriers underwent a PET scan for this study and patients with idiopathic PD were carefully selected from the Unit‟s database to match striatal 18

F-dopa uptake of parkin-patients.

In chapter 6, the two groups of PD patients with and without fatigue were matched for all possible confounding factors, including age, disease duration, daily intake of LEU, and UPDRSscores in both “off” and “on” conditions. Additionally, since I wanted to elucidate neurochemistry of primary fatigue, patients with dementia, depression, and EDS were excluded from the study.

However, a number of methodological weaknesses and limitations to the experimental studies reported in this thesis also need to be acknowledged. Some of these limiting issues are specific to the study design in each of the thesis‟ experiments whereas others are generally applicable to the field of brain imaging.

PET is a very expensive modality and the cost of a PET scan is quite high particularly in a research context. The impact of irradiating healthy volunteers is also relevant as it raises important ethical issues. Considering that the CSC Neurology group has

collected a large database of 18F-dopa and 11C-DASB PET images of healthy volunteers over the years, it was decided to not scan new healthy volunteers for the studies of this thesis.

Whilst striatal 18F-dopa does not appear to be age-dependent in healthy controls (Sawle et al., 1990), an age-related decline in the availability of SERT when measured with PET and SPECT has been reported (Buchert et al., 2006).

In the study in chapter 6, PD patients with and without fatigue were age-matched, whereas the healthy control subjects used for the 11C-DASB PET comparisons were 20 years younger than the PD patients. Therefore, the effect of age on the 11C-DASB reductions in the two cohorts of PD patients cannot be completely excluded. However, the reductions in 11C-DASB binding that I found in the PD patients with fatigue (about 80% of the control mean in most of the regions assessed by ROI approach) is much bigger than the maximum decline (around 21%) that could be ascribed to aging on the basis of previous studies. Additionally, I found no significant correlations between age and tracer binding in caudate, putamen, ventral striatum, and thalamus in the cohort of 15 PD patients.

Therefore, the reduction in 11C- DASB binding observed in PD patients with fatigue is unlikely to be explained by aging alone.

Probably the most notable weakness in this thesis is the lack of clinical details on the differences in non-motor symptoms between idiopathic PD and parkin-patients in Chapter 4. Unfortunately, as the idiopathic PD patients were selected from our data base to match striatal 18F-dopa uptake in parkin-patients, data on sleep problems or other non-motor symptoms in these patients were not always available. Therefore, I was not able to compare the different groups on the basis of their non-motor

symptoms. The clinical relevance of the different monoaminergic involvement in the two conditions remains to be elucidated.

In all the experiments of this thesis 18F-dopa PET has been used as a marker of monoaminergic terminals. In areas in which a definite innervation predominates, specific 18F-dopa uptake is likely to reflect the activity and density of the predominant

neuronal population (eg dopamine innervation in the striatum and serotoninergic innervation in the raphe). However, in brain structures like the hypothalamus, the red nucleus, and the pineal gland that have a mixed dopaminergic, serotoninergic and noradrenergic innervations, 18F-dopa uptake probably reflects the combined effect of activity and density of different groups of monoaminergic afferents to these structures. Therefore, the reductions of 18F-dopa uptake in these regions that I have observed in PD patients could reflect dysfunction in any or all of these pathways. Nevertheless,

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F-dopa uptake in these regions still represents a useful marker of overall

monoaminergic function and may provide the rationale for further investigations with more specific PET ligands if the involvement of a particular neurotransmitter is suspected.

Another possible limitation of this thesis is that the studies were not performed using a partial volume effect correction. In imaging, the partial volume effect is the loss of contrast between two adjacent tissues in an image caused by insufficient resolution that results in same voxels containing both tissues. As a consequence, the signal in these voxels is an average of the signal of both tissues.

Partial volume effect in my studies could have led to a decrease of the absolute Ki or BPND values. However, partial volume effect is particularly relevant in the case of

brain atrophy, such as in Alzheimer‟s disease. In PD, gross brain morphology is not altered by the disease process. Additionally, my ROIs were drawn well within the anatomical boundaries of each structure to avoid overlapping with adjacent tissues. Therefore, it is unlikely that relative regional difference in Ki and BPND values

between controls and PD patients in this thesis have been greatly altered by partial volume effects.