Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

Power network in the loop : subsystem testing using a switching amplifier

“Hardware in the Loop” (HIL) testing is widely used in the automotive industry. The sophisticated electronic control units used for vehicle control are usually tested and evaluated using HIL-simulations. The HIL increases the degree of realistic testing of any system. Moreover, it helps in designing the structure and control of the system under test so that it works effectively in the situations that will be encountered in the system. Due to the size and the complexity of interaction within a power network, most research is based on pure simulation. To validate the performance of physical generator or protection system, most testing is constrained to very simple power network. This research, however, examines a method to test power system hardware within a complex virtual environment using the concept of the HIL. The HIL testing for electronic control units and power systems protection device can be easily performed at signal level. But performance of power systems equipments, such as distributed generation systems can not be evaluated at signal level using HIL testing. The HIL testing for power systems equipments is termed here as ‘Power Network in the Loop’ (PNIL). PNIL testing can only be performed at power level and requires a power amplifier that can amplify the simulation signal to the power level. A power network is divided in two parts. One part represents the Power Network Under Test (PNUT) and the other part represents the rest of the complex network. The complex network is simulated in real time simulator (RTS) while the PNUT is connected to the Voltage Source Converter (VSC) based power amplifier. Two way interaction between the simulator and amplifier is performed using analog to digital (A/D) and digital to analog (D/A) converters. The power amplifier amplifies the current or voltage signal of simulator to the power level and establishes the power level interaction between RTS and PNUT. In the first part of this thesis, design and control of a VSC based power amplifier that can amplify a broadband voltage signal is presented. A new Hybrid Discontinuous Control method is proposed for the amplifier. This amplifier can be used for several power systems applications. In the first part of the thesis, use of this amplifier in DSTATCOM and UPS applications are presented. In the later part of this thesis the solution of network in the loop testing with the help of this amplifier is reported. The experimental setup for PNIL testing is built in the laboratory of Queensland University of Technology and the feasibility of PNIL testing has been evaluated using the experimental studies. In the last section of this thesis a universal load with power regenerative capability is designed. This universal load is used to test the DG system using PNIL concepts. This thesis is composed of published/submitted papers that form the chapters in this dissertation. Each paper has been published or submitted during the period of candidature. Chapter 1 integrates all the papers to provide a coherent view of wide bandwidth switching amplifier and its used in different power systems applications specially for the solution of power systems testing using PNIL.

A multi-hypothesis topological SLAM approach for loop closing on edge-ordered graphs

We present a method for topological SLAM that specifically targets loop closing for edge-ordered graphs. Instead of using a heuristic approach to accept or reject loop closing, we propose a probabilistically grounded multi-hypothesis technique that relies on the incremental construction of a map/state hypothesis tree. Loop closing is introduced automatically within the tree expansion, and likely hypotheses are chosen based on their posterior probability after a sequence of sensor measurements. Careful pruning of the hypothesis tree keeps the growing number of hypotheses under control and a recursive formulation reduces storage and computational costs. Experiments are used to validate the approach.