Philosophiae Doctor - PhD / Solute carrier transporters belonging to the major facilitator family of membrane transporter are increasingly being recognized as a possible mechanism to explain inter-individual variation in drug efficacy and response. Genetic factors are estimated to be responsible for approximately 15-30% of inter-individual variation in drug disposition and response. The aims of this study were to determine the minor allele frequencies of 78 previously identified single nucleotide polymorphisms in the pharmacogenomically relevant SLC22A1-3 and SLCO1B1 genes in the Admixed population of South Africa. Thereafter, to determine whether allele and genotype frequencies for these SNP were different from that reported for other African, Caucasian, and Asian populations. The inferred haplotypes from the genetic information possessed the potential to subsequently be used in future to design and interpret results of pharmacogenomic association studies involving these genes and their substrate drugs. Furthermore, to determine whether the Cape Admixed population harbour novel SNPs in the proximal promoter regions of SLC22A1- 3 and SLCO1B1-3 genes, that encodes hOCT1-3 and hOATP1 and hOATP3, respectively. SNaPshot™ multiplex single base mini-sequencing systems were developed and optimized for each of SLC22A1, SLC22A2, SLC22A3, and SLCO1B1 genes covering the previously identified 78 SNPs. These systems were then used to genotype the alleles of 130 healthy Cape Admixed subjects residing in Cape Town, South Africa. In addition, the proximal promoter regions of the SLC22A1-3 and SLCO1B1-3 genes of 96 of the participants were screened for novel SNPs by direct sequencing. The Cape Admixed subjects investigated displayed a lack of variation and were monomorphic for 78% of the SNPs screened. None of the SLC22A3 SNPs investigated was observed in this study. Sequencing of the proximal promoter regions of the SLC22 and SLCO genes did not reveal any novel SNPs in the 96 Cape Admixed subjects that were screened. This study highlights the fact that African populations do not have the same allele frequencies for SNPs in harmacogenomically relevant genes. Furthermore, the Cape Admixed and other African populations do not share all reduced-function variants of the SLC22A1-3 and SLCO1B1-3 genes with Caucasian and Asian populations. In addition, previously identified novel regulatory variants in SLC22A2 did not exhibit a significant effect on the ability of the promoter to drive transcription. However, it must be noted that these results were observed at 95% confidence interval, and that a 99% confidence interval the significance may increase theoretically. Additionally, it should be noted that more intensive studies are required to determine the potential effect these novel variants may well cause. This study lays the foundation for the design and interpretation of future pharmacogenomic association studies between the variant alleles of the SLC22A and SLCO genes in the Cape Admixed population, as well as optimizations for future expression, and more importantly, drug transport assays with respect to drug disposition and efficacy. / National Research Foundation (NRF) and the Medical Research Council (MRC)

Multidrug resistant bacteria (MDR) have the potential to push back society to the pre-antibiotic era. Although discovered before penicillin, the inexorable rise in antibiotic resistance has revitalised interest in bacteriocins as treatments for bacterial infections. Bacteriocins are protein antibiotics principal to competition amongst pathogens and commensals, but the mechanisms by which they translocate across the Gram-negative cell envelope are poorly understood. The work presented in this thesis demonstrates how the endonuclease bacteriocin pyocin S2 (pyoS2) exploits the iron transporter FpvAI to translocate across the outer membrane (OM) of Pseudomonas aeruginosa. FpvAI is a 22-strand β-barrel and virulence factor in P. aeruginosa that transports iron into the cell in the form of a small siderophore, ferripyoverdine (Fe-Pvd). Uptake of Fe-Pvd requires the proton motive force (PMF), which is transduced to the ligand-bound receptor by TonB1 and its partner proteins ExbB-ExbD in the inner membrane (IM). The crystal structure of the high affinity complex (Kd = 240 pM) formed between the N-terminal domain of pyoS2 (pyoS2^NTD) and FpvAI is presented, which shows pyoS2^NTD mimics Fe-Pvd, and induces the same conformational changes in the receptor. Fluorescently-labelled pyoS2^NTD was actively imported into P. aeruginosa PAO1 cells and this import was dependent on the PMF, TonB1 and a TonB1-box motif at the N-terminus of pyoS2^NTD. Finally, photo-activated crosslinking of stalled translocation intermediates demonstrated pyoS2^NTD translocates through the FpvAI β-barrel lumen by a process analogous to that of Fe-Pvd. Following binding to FpvAI, translocation begins by the unfolding of a force-labile portion of the plug domain, opening a narrow channel through FpvAI. This enables pyoS2 to deliver its own TonB1-box to the periplasm where contact with TonB1 activates its import through the same channel, most likely as an unfolded polypeptide. Hence, this study demonstrates that bacteria possess a rudimentary protein import system that exploits energised nutrient transporters in the OM.

The serotonergic system is involved in a broad range of functions in both vertebrates and invertebrates and is highly conserved across taxa. Serotonin is an important monoamine acting in the brains of humans and animals, and has large and varying influences on many aspects of an individual’s life. For example, in humans, serotonin modulates feelings of happiness and in fruit flies, higher levels of serotonin increase aggression. In humans, an abnormal serotonergic system can result in health issues, such as depression and obsessive compulsive disorders, for which medications have been developed, including selective serotonin reuptake inhibitors (SSRI). Because the serotonin system has a large influence on human health, understanding how it functions is of great interest to researchers. Using comparative studies to explore differences in the serotonin system across taxa can provide insight into the mechanistic details of the system. To investigate if the serotonin system is comparable between vertebrates and invertebrates, a literature study with particular focus on receptors and proteins involved was performed. In addition, this report takes part in an experimental study investigating the effect of the SSRI fluoxetine in Mediterranean field crickets.  Fluoxetine reduced exploration propensity of crickets, which was reversed, compared to what was anticipated and compared to effects seen in vertebrates. The literature review suggests that serotonin receptors are quite similar, but that proteins differ more when comparing vertebrates and invertebrates. This offers a likely explanation as to why results of studies on these different groups of animals may differ.

Abstract Patients with idiopathic Parkinson's disease (PD) may suffer from autonomic nervous system dysfunction even in the early phase of the disease. We assessed the autonomic cardiovascular and sudomotor regulation in de novo PD patients with and without medication. We also measured the dopamine (DAT) and serotonin transporter (SERT) uptake in the PD patients using 2β-carboxymethoxy-3β-(4-iodophenyl)tropane (β-CIT) SPECT and studied the clinical correlates of the uptake. Sixty PD patients were included in the study and randomised to receive levodopa, bromocriptine or selegiline (n=20 in each) as their treatment. Thirty patients were examined with β-CIT SPECT. The results of the patients were compared with those of healthy controls and within the subgroups at different time points. Cardiovascular autonomic regulation was assessed using standard cardiovascular reflex tests at baseline, after six months' medication and following a 6-week washout period. The heart rate (HR) and blood pressure (BP) regulation was impaired in PD patients at baseline, and PD medications modified the responses further. Bromocriptine and selegiline, in contrast to levodopa, increased the orthostatic BP fall and suppressed the BP response to isometric exercise. The long-term cardiovascular autonomic function was evaluated from ambulatory ECG recordings by analysis of traditional spectral and non-spectral components of HR fluctuation together with two-dimensional vector analysis and power-law relationship analysis of the HR dynamics. All spectral measures and the slope of the power-law relationship demonstrated impaired tonic cardiovascular regulation in the PD patients. Sympathetic sudomotor activity was evaluated using the sympathetic skin response (SSR). The major finding was suppression of the SSR amplitudes with an inverse correlation to clinical disability, whereas PD medication seemed to have only minor effects. The changes in amplitude and repetitiveness of the SSRs with normal adaptation suggest deficits at several levels of the SSR reflex arc. DAT uptake, assessed by β-CIT SPECT, was diminished in the striatum and especially the putamen of the PD patients, and correlated with the results of the cardiovascular reflex tests and ambulatory ECG recordings. Simultaneous measurement of SERT binding demonstrated decreased SERT availability in the thalamic and frontal areas. The results demonstrate disturbances of the reflectory and tonic cardiovascular autonomic regulation caused by PD itself. PD medications further modify the reflectory responses. The degenerative process in PD also involves the sympathetic sudomotor pathway. β-CIT SPECT provides a useful method for simultaneous assessment of DAT and SERT binding, demonstrating the deficit of serotonin metabolism in PD.

Transport av kritiskt sjuka patienter inom sjukhus innebär risker för patienten. Dessa risker är bland annat påverkan på cirkulation och syresättning. Det finns även rapporter om problem med övervakningsutrustning. Anestesisjuksköterskor arbetar under hälso- och sjukvårdslagen och ska bedriva patientsäker vård. Att säkerställa patientens ventilation och cirkulation är anestesisjuksköterskans ansvarsområde. Det saknas studier om anestesisjuksköterskors upplevelser av transporter av kritiskt sjuka patienter inom sjukhuset. Syftet var att belysa anestesisjuksköterskors erfarenheter och upplevelser av att genomföra en patientsäker transport av en kritisk sjuk patient från akutmottagningen till operationsavdelningen eller intensivvårdsavdelningen. En kvalitativ ansats valdes. Tio stycken anestesisjuksköterskor intervjuades. Alla tio intervjuer inkluderades i resultatet. Analysen av resultatet genomfördes enligt kvalitativ innehållsanalys. Detta resulterade i tre huvudkategorier och åtta subkategorier. Resultatet presenterades genom följande huvudkategorier: Att vara förberedd, Att vårda i ett team och Att vårda när livet står på spel. Det framkom i resultatet att planering inför transport har stor påverkan på hur transporten upplevdes. Det framkom även att patienten är i en utsatt situation. Upplevelsen av transporten varierade mellan anestesisjuksköterskorna, tidigare erfarenheter var en avgörande aspekt. Antalet personer som medverkade i transporten och samarbetet dem emellan kunde påverka anestesisjuksköterskans upplevelse. Det saknas riktlinjer för hur en patienttransport ska genomföras. Om riktlinjer utformas kan det vara ett hjälpmedel för anestesisjuksköterskor. Det framkommer även att patientens integritet är hotad under dessa transporter och att transportvägen upplevs vara lång. Utformning av sjukhuslokaler är en viktig aspekt för att kunna bedriva en patientsäker vård.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Kateřina Erbenová Supervisor: PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Study of interactions antiretroviral drug maraviroc with drug transporters ABCB1 and ABCG2. Maraviroc is inhibitor of CCR5 HIV virus entry into the cells representing one of the important components of antiretroviral therapy. To optimize the treatment strategies and minimize the therapeutic risks of maraviroc-containing combination antiretroviral therapy, it is important to know the interactions of this drug with other antiretrovirals. In particular, interaction on membrane transporters may affect pharmacokinetics and thereby the tissue concentrations of administered drugs, leading to insufficient efficacy of the therapy or increased toxicity. The aim of this study was to experimentally evaluate interaction of maraviroc with the two most important active drug transporters of the ABC transporter superfamily, ABCB1 (P-glycoprotein) and ABCG2 (BCRP). Using in vitro methods employing cell lines we aimed to fulfil two main goals: (1) to evaluate the inhibitory effect of maraviroc on ABCB1 and ABCG2 transporters and (2) to study if any of these transporters could transfer maraviroc as their substrate. The data...

Parkinson’s disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic (DA) neurons in the substantia nigra and motor deficits. Although the majority of PD cases are sporadic, several genetic defects in rare familial cases have been identified. Animal models of these genetic defects have been created and have provided unique insights into the molecular mechanisms of the pathogenesis of PD. However, the etiology of PD is still not well understood. Here, taking advantage of the unique features offered by zebrafish, I characterized the functions of PINK1 (PTEN-induced kinase 1) gene, which is associated with recessive familial PD, in the development and survival of DA neurons. In zebrafish, antisense morpholino knockdown of pink1 did not cause a large loss of DA neurons in the ventral diencephalon (vDC), but the patterning of these neurons and their projections were perturbed. The pink1 morphants also showed impaired response to touch stimuli and reduced swimming behaviour. Moreover, the pink1 knockdown caused a significant reduction in the number of mitochondria, as well as mitochondrial morphological defects such as smaller size or loss of cristae, thus affecting mitochondrial function. These results suggest that zebrafish pink1 plays conserved important roles in the development of DA neurons and in the mitochondrial morphology and function. To better follow DA neurons after injury or administration of toxins, I generated a transgenic zebrafish line, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of dopamine transporter (dat). In Tg(dat:EGFP) fish, all major groups of DA neurons are correctly labeled with GFP, especially the ones in the vDC, which are analogous to the ascending midbrain DA neurons in mammals. In addition, we observed that the DA neurons in the vDC could partially be replaced after severe laser cell ablation. This suggests that zebrafish may have the unique capacity of regenerating DA neurons after injury. Taken together, my studies suggested that zebrafish could be a useful alternative animal model for the study of the molecular mechanisms underlying PD and for the screening of potential therapeutic compounds for PD.

Haemophilus ducreyi is a gram-negative and heme-dependent bactreia. H. ducreyi is the responsible of causing chancroid, a sexually transmitted infection forming genital ulcers. Infection with H. ducreyi is associated with an increased risk of acquiring HIV-1 as well as increasing the risk of the HIV-1 transmission. Heme acquisition in H. ducreyi occur through a receptor mediated process in which it start with binding of hemoglobin and heme to their cognate outer membrane receptors, HgbA and TdhA, respectively. The receptors are energized by the TonB complex. Following that the deposition of heme into the periplasmic area is unclear. Profiling of the periplasmic proteome of the H. ducreyi resulted in the identification of a periplasmic- binding protein that highly expressed in heme limitation conditions, and it has been called hHbp. This protein is encoded by a gene resides in a locus of four genes displaying genetic features of an ABC transporter. The gene cluster is organized as an operon comprising an internal membrane protein (IntPro), a sulphate reductase gamma subunit (dsvC), a heme dependant periplasmic bind-ing protein (hHBP), and an ATPase. The purified periplasmic binding protein, hHbp, bind heme in a dose-dependent and saturable manner. Moreover, the binding between heme and hHbp was specifically competitively inhibited by heme. The proposal planned to cre-ate an isogenic hhbp mutant by insertional inactivation using a kanamycin cassette, to genotypically and phenotypically characterize the mutant and thereby to confirm the cru-cial role of the hhbp gene in heme transport in H. ducreyi. Several attempts to ligate a kanamycin resistance cassette into hhbp to construct such a mutant were unsuccessful de-spite the systematic alteration of the ligation conditions and the use of kanamycin re-sistant genes derived from a variety of different plasmids. The explanations for this fail-ure are uncertain. In future work, two other approaches to construct an hhbp mutant in-clude the FRT-FLP recombinase technology and the use of overlapping extension PCR with a chloramphenicol cassette.

Choline is necessary for the synthesis of phosphatidylcholine (PC), the predominant phospholipid species and an important lipid intermediate. Macrophages, critical mediators of innate immunity, have been implicated in lipid dysregulation associated with metabolic disease. Despite the importance of choline in lipid metabolism, few studies have investigated the relationship between choline metabolism and inflammation. My research revealed that macrophage polarization increased choline metabolism and the expression of the choline transporter CTL1. In addition, choline deficient macrophages showed altered cytokine secretion, suggesting choline metabolism may play an important role in regulating the immune response. This study also describes the generation of a novel CTL1-/- mouse, which showed decreased choline uptake and incorporation into lipids. As an in vivo model for choline deficiency, CTL1-/- mice represent an important model for the future study of choline metabolism. Altogether, these findings suggest an important relationship exists between choline metabolism and inflammation.

The aim of this thesis was the functional characterization of the zebrafish parl (Presenilin-Associated Rhomboid-Like) genes which code for mitochondrial proteins involved in cell survival. A mutation in PARL has been described in Parkinson’s disease patients. I investigated the role of mitochondrial PD-related proteins using a zebrafish parla and parlb deficiency model. I found that the knockdown of both parl genes is lethal. Parla plays a larger role in patterning of the DA neurons in the ventral diencephalon than Parlb. The human PARL rescued the double morphant phenotype, suggesting function conservation between zebrafish and humans. I was able to rescue the mortality and DA neuron mispatterning observed in double morphants with synthetic pink1 mRNA. This suggests that parl genes are epistatic to pink1 in zebrafish. To visualize mitochondria specifically in dopaminergic neurons of live zebrafish, I established a transgenic line Tg(dat:tom20 MLS-mCherry) where regulatory elements of the dopamine transporter (dat) were used to drive expression of a Tom20-mCherry fusion protein that is targeted to the mitochondria. I characterised the expression of Tom20-mCherry to the mitochondria of the majority of DA neuron groups. In addition, I observed a decrease in mCherry fluorescence following MPTP exposure of live fish. The PD-related mutation in PARL is located in a cleavage site of the mammalian protein, which is necessary for the production of the beta peptide; however, this site is predicted to be absent in the zebrafish Parls. To establish the cleavage patterns of the zebrafish Parls and compare them to those of human PARL, I examined the cleavage of Parl-Flag constructs in cultured cells. I detected one band for Parla-Flag and two bands representing Parlb-Flag. The parla and parlb deficiency model along with the characterization of the cleavage patterns of Parl and the Tg(dat:tom20 MLS-mCherry) transgenic line are tools which will help elucidate the role of mitochondrial proteins in PD research.