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The aim of this thesis is to describe how the use of pharmacokinetic and pharmacodynamic techniques can provide a better understanding of the optimal use of antimicrobial agents. In vivo animal experimental models and clinical PK-PD datasets for three systemically administered antifungal agents, isavuconazonium sulfate (active moiety isavuconazole), micafungin, and VL-2397, are used to accomplish this goal.

Background Epilepsy has a high prevalence of 1%, which makes it the most common serious neurological disorder. The most difficult to treat type of epilepsy is temporal lobe epilepsy (TLE) with its most commonly associated lesion being hippocampal sclerosis (HS). About 30-50% of all patients undergoing resective surgery of epileptogenic tissue continue to have seizures postoperatively. Indication for this type of surgery is only given when lesions are clearly visible on magnetic resonance images (MRI). About 30% of all patients with focal epilepsy do not show an underlying structural lesion upon qualitative neuroradiological MRI assessment (MRI-negative). Objectives The work presented in this thesis uses MRI data to quantitatively investigate structural differences between brains of patients with focal epilepsy and healthy controls using automated imaging preprocessing and analysis methods. Methods All patients studied in this thesis had electrophysiological evidence of focal epilepsy, and underwent routine clinical MRI prior to participation in this study. There were two datasets and both included a cohort of age-matched controls: (i) Patients with TLE and associated HS who later underwent selective amygdalahippocampectomy (cohort 1) and (ii) MRI-negative patients with medically refractory focal epilepsy (cohort 2). The participants received high- resolution routine clinical MRI as well as additional sequences for gray and white matter (GM/WM) structural imaging. A neuroradiologist reviewed all images prior to analysis. Hippocampal subfield volume and automated tractography analysis was performed in patients with TLE and HS and related to post-surgical outcomes, while images of MRI- negative patients were analyzed using voxel-based morphometry (VBM) and manual/automated tractography. All studies were designed to detect quantitative differences between patients and controls, except for the hippocampal subfield analysis as control data was not available and comparisons were limited to patients with persistent postoperative seizures and those without. Results 1. Automated hippocampal subfield analysis (cohort 1): The high-resolution hippocampal subfield segmentation technique cannot establish a link between hippocampal subfield volume loss and post-surgical outcome. Ipsilateral and contralateral hippocampal subfield volumes did not correlate with clinical variables such as duration of epilepsy and age of onset of epilepsy. 2. Automated WM diffusivity analysis (cohort 1): Along-the-tract analysis showed that ipsilateral tracts of patients with right/left TLE and HS were more extensively affected than contralateral tracts and the affected regions within tracts could be specified. The extent of hippocampal atrophy (HA) was not related to (i) the diffusion alterations of temporal lobe tracts or (ii) clinical characteristics of patients, whereas diffusion alterations of ipsilateral temporal lobe tracts were significantly related to age at onset of epilepsy, duration of epilepsy and epilepsy burden. Patients without any postoperative seizure symptoms (excellent outcomes) had more ipsilaterally distributed WM tract diffusion alterations than patients with persistent postoperative seizures (poorer outcomes), who were affected bilaterally. 3. Automated epileptogenic lesion detection (cohort 2): Comparison of individual patients against the controls revealed that focal cortical dysplasia (FCD) can be detected automatically using statistical thresholds. All sites of dysplasia reported at the start of the study were detected using this technique. Two additional sites in two different patients, which had previously escaped neuroradiological assessment, could be identified. When taking these statistical results into account during re-assessment of the dedicated epilepsy research MRI, the expert neuroradiologist was able to confirm these as lesions. 4. Manual and automated WM diffusion tensor imaging (DTI) analysis (cohort 2): The analysis of consistency across approaches revealed a moderate to good agreement between extracted tract shape, morphology and space and a strong correlation between diffusion values extracted with both methods. While whole-tract DTI-metrics determined using Automated Fiber Quantification (AFQ) revealed correlations with clinical variables such as age of onset and duration of epilepsy, these correlations were not found using the manual technique. The manual approach revealed more differences than AFQ in group comparisons of whole-tract DTI-metrics. Along-the-tract analysis provided within AFQ gave a more detailed description of localized diffusivity changes along tracts, which correlated with clinical variables such as age of onset and epilepsy duration. Conclusions While hippocampal subfield volume loss in patients with TLE and HS was not related with any clinical variables or to post-surgical outcomes, WM tract diffusion alterations were more bilaterally distributed in patients with persistent postoperative seizures, compared to patients with excellent outcomes. This may indicate that HS as an initial precipitating injury is not affected by clinical features of the disorder and automated hippocampal subfield mapping based on MRI is not sufficient to stratify patients according to outcome. Presence of persisting seizures may depend on other pathological processes such as seizure propagation through WM tracts and WM integrity. Automated and time-efficient three-dimensional voxel-based analysis may complement conventional visual assessments in patients with MRI-negative focal epilepsy and help to identify FCDs escaping routine neuroradiological assessment. Furthermore, automated along-the-tract analysis may identify widespread abnormal diffusivity and correlations between WM integrity loss and clinical variables in patients with MRI-negative epilepsy. However, automated WM tract analysis may differ from results obtained with manual methods and therefore caution should be exercised when using automated techniques.

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Atypical antipsychotics are commonly prescribed for the treatment of psychiatric disorders such as schizophrenia. However, long term use of atypical antipsychotics lead to metabolic adverse effects such as weight gain, insulin resistance, and dyslipidemia, all of which are independent risk factors for cardiometabolic disease. Adipose tissue plays an important role in glucose and lipid homeostasis and has been linked to atypical antipsychotic-induced metabolic toxicity. The main aim of this thesis was to characterise the effect of atypical antipsychotics in vitro using murine 3T3-F442A preadipocyte cell lines and primary human adipocytes. Cells were incubated with clozapine (1-20μM), olanzapine (0.2-20μM) and aripiprazole (0.2-20μM) every 48 hours for 10 (3T3-F442A) or 13 (primary human adipocytes) days. Lipid accumulation was measured by Oil Red O assay. Protein and gene expression of peroxisome proliferator-activated receptor gamma and lipin 1 were measured by Western Blot and RT-PCR respectively. Adipokine secretion was measured by ELISA. Global lipidomic profiling of drug-treated adipocytes was undertaken using LC-MS and selected lipid species were validated by RT-PCR of enzymes that mediate the ceramide metabolism pathway. In differentiating 3T3-F442A cells clozapine (20μM:1.56 absorbance units±0.097; p=0.001) and olanzapine (20μM:1.57±0.14; p=0.07) but not aripiprazole showed an increase in lipid accumulation as compared to the vehicle. Clozapine but not olanzapine or aripiprazole upregulated the protein expression of peroxisome proliferator-activated receptor gamma (Mean fold change±SD; 307.34±26.30; p=0.0001) and lipin1 (213.46±26.43; p=0.02), and increased the secretion of adiponectin (736.77ng/ml±66.06; p=0.001; Vehicle: 323.26 ng/ml±53.07) and tumour necrosis factor alpha (45.24pg/ml±0.16; p=0.0001; Vehicle: 32.89pg/ml±0.34). In primary human adipocytes clozapine significantly increased lipid accumulation (1μM: 1.10±0.08; p=0.04) and leptin secretion (1μM: 1.06±0.10; p=0.04) but both clozapine and olanzapine led to a reduction in adiponectin secretion (Clozapine: 0.31±0.18; p=0.03, olanzapine: 0.39±0.21; p=0.01). Aripiprazole showed opposite effects to that of clozapine and olanzapine on lipid accumulation, gene expression and adipokine release. Drug uptake experiments showed that there was a 20-fold increase in clozapine uptake by differentiating primary human adipocytes (1293.4 pmol/million cells±170.56; p=0.006) in comparison to the murine adipocytes (64.25pmol/million cells±26.96). Co-incubation of clozapine with aripiprazole showed that aripiprazole significantly reversed clozapine-mediated reduction in secretion of adiponectin (1.23±0.09; p=0.04). Higher doses of both clozapine and olanzapine but not aripiprazole resulted in significant changes in the global lipidome profile of primary human adipocytes. Clozapine and olanzapine but not aripiprazole significantly decreased the ceramide 18 species and resulted in significant downregulation in the gene expression of ceramide biosynthesis enzymes, serine palmitoyltransferase long chain base subunit 2 (Clozapine: 0.60±0.13; p=0.003; Olanzapine: 0.62±0.16; p=0.007) and Delta-4-Desaturase Sphingolipid 1 (Clozapine: 0.67±0.18; p=0.01; Olanzapine: 0.83±0.11; p=0.02). In conclusion, we have shown that atypical antipsychotics may cause metabolic toxicity by directly affecting adipocyte function and metabolism; however, this could be potentially reversed in vitro by various therapeutic strategies. Atypical antipsychotics also significantly alter the adipocyte lipidome which may contribute to metabolic adverse effects in schizophrenia patients. The mechanisms and pathways identified in this study now need to be validated in in vivo and clinical models which will aid the identification of toxicity biomarkers in atypical antipsychotic-treated individuals.

Maintaining the appropriate transcriptional balance in the cell is a complex process involving numerous mechanisms, including the action of transcription factors and non-coding regulatory elements. Such processes are key to maintaining healthy central nervous system (CNS) functioning, and can be modulated through the interaction of both genes and environment in a ‘G x E’ mechanism. If the regulation of a certain gene or gene set is altered inappropriately in the brain, this can result in neuronal dysfunction which may contribute to psychiatric or CNS conditions. This thesis primarily aimed to extend our understanding of transcriptional regulation at the MIR137 schizophrenia-associated locus, and to add to our understanding of the role of repetitive DNA and retrotransposons in the regulation and evolution of genes involved in wider CNS pathways. The chromosome 1p21.3 locus encompassing the microRNA, MIR137, has been repeatedly highlighted by GWAS as one of the most robust loci for association with schizophrenia. The evidence presented in this thesis identified multiple evolutionary conserved regions (ECRs) which act as transcriptional regulators at this locus, as well as a regulatory gene network comprising MIR137 and the transcriptional regulators REST and EZH2, which are likely to modulate the expression of multiple CNS- and schizophrenia-associated gene sets. Extending our view of the MIR137 locus identified a brain-expressed RNA, EU358092, which shared near identical expression and regulatory profiles to MIR137, suggesting potential co-expression and -regulation of RNAs across this locus. The second half of this thesis explored repetitive DNA, including variable number tandem repeats (VNTRs) and the retrotransposon subfamilies, Long Interspersed Nuclear Elements (LINEs) and SINE-VNTR-Alus (SVAs), which have been shown to act as modulators of gene expression. Common polymorphisms in the VNTR containing MIR941, a human-specific, brain expressed microRNA at chromosome 20q13.3, resulted in altered copy number of MIR941, with two genotypes being specific to a schizophrenia cohort. SVAs were implicated in the recent evolution of multiple zinc finger loci, which may have had the potential to alter the regulation of large transcriptional networks in a species-specific manner, while LINE elements were likely to have been involved in recent genomic remodelling around GABA and glutamate signalling genes. Taken together, the work contained in this thesis considered the roles of a wide range of DNA elements with relevance to CNS-expressed genes, from the oldest and most highly conserved regions of the genome, to the most recent retrotransposon insertions. This work identified roles for these elements in the evolution and regulation of genes involved in schizophrenia risk and neuroprotection, and further identified gene networks and additional transcripts which may contribute to the maintenance of healthy brain functioning. The impact of genetic variation at these regions - in the form of single nucleotide polymorphisms (SNPs), altered VNTR copy number, or polymorphic retrotransposon insertions - and their effect on CNS functioning, has been a key theme throughout this work. In conclusion, this would provide evidence to suggest that genetic polymorphisms which alter the function, size, or location of such elements at loci involved in brain-related processes could contribute to schizophrenia risk in a way that would likely be modulated through an interaction between environmental stimuli and genotype.

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