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.

The proximal first exon architecture of the murine ghrelin gene is highly similar to its human orthologue

BACKGROUND: The murine ghrelin gene (Ghrl), originally sequenced from stomach tissue, contains five exons and a single transcription start site in a short, 19 bp first exon (exon 0). We recently isolated several novel first exons of the human ghrelin gene and found evidence of a complex transcriptional repertoire. In this report, we examined the 5' exons of the murine ghrelin orthologue in a range of tissues using 5' RACE. -----FINDINGS: 5' RACE revealed two transcription start sites (TSSs) in exon 0 and four TSSs in intron 0, which correspond to 5' extensions of exon 1. Using quantitative, real-time RT-PCR (qRT-PCR), we demonstrated that extended exon 1 containing Ghrl transcripts are largely confined to the spleen, adrenal gland, stomach, and skin. -----CONCLUSION: We demonstrate that multiple transcription start sites are present in exon 0 and an extended exon 1 of the murine ghrelin gene, similar to the proximal first exon organisation of its human orthologue. The identification of several transcription start sites in intron 0 of mouse ghrelin (resulting in an extension of exon 1) raises the possibility that developmental-, cell- and tissue-specific Ghrl mRNA species are created by employing alternative promoters and further studies of the murine ghrelin gene are warranted.

Using ontology for the representational analysis of process modelling techniques

Selecting an appropriate business process modelling technique forms an important task within the methodological challenges of a business process management project. While a plethora of available techniques has been developed over the last decades, there is an obvious shortage of well-accepted reference frameworks that can be used to evaluate and compare the capabilities of the different techniques. Academic progress has been made at least in the area of representational analyses that use ontology as a benchmark for such evaluations. This paper reflects on the comprehensive experiences with the application of a model based on the Bunge ontology in this context. A brief overview of the underlying research model characterizes the different steps in such a research project. A comparative summary of previous representational analyses of process modelling techniques over time gives insights into the relative maturity of selected process modelling techniques. Based on these experiences suggestions are made as to where ontology-based representational analyses could be further developed and what limitations are inherent to such analyses.

Building an ontology and process architecture for engineering asset management

Historically, asset management focused primarily on the reliability and maintainability of assets; organisations have since then accepted the notion that a much larger array of processes govern the life and use of an asset. With this, asset management’s new paradigm seeks a holistic, multi-disciplinary approach to the management of physical assets. A growing number of organisations now seek to develop integrated asset management frameworks and bodies of knowledge. This research seeks to complement existing outputs of the mentioned organisations through the development of an asset management ontology. Ontologies define a common vocabulary for both researchers and practitioners who need to share information in a chosen domain. A by-product of ontology development is the realisation of a process architecture, of which there is also no evidence in published literature. To develop the ontology and subsequent asset management process architecture, a standard knowledge-engineering methodology is followed. This involves text analysis, definition and classification of terms and visualisation through an appropriate tool (in this case, the Protégé application was used). The result of this research is the first attempt at developing an asset management ontology and process architecture.

High-level concept annotation using ontology and probabilistic inference

Image annotation is a significant step towards semantic based image retrieval. Ontology is a popular approach for semantic representation and has been intensively studied for multimedia analysis. However, relations among concepts are seldom used to extract higher-level semantics. Moreover, the ontology inference is often crisp. This paper aims to enable sophisticated semantic querying of images, and thus contributes to 1) an ontology framework to contain both visual and contextual knowledge, and 2) a probabilistic inference approach to reason the high-level concepts based on different sources of information. The experiment on a natural scene database from LabelMe database shows encouraging results.

Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation

Introduction During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. Methods In this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Results Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. Conclusions This study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signalling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration

Effect of female sex hormones on Chlamydia trachomatis growth and gene expression

Transmissible diseases are re-emerging as a global problem, with Sexually Transmitted Diseases (STDs) becoming endemic. Chlamydia trachomatis is the leading cause of bacterially-acquired STD worldwide, with the Australian cost of infection estimated at $90 - $160 million annually. Studies using animal models of genital tract Chlamydia infection suggested that the hormonal status of the genital tract epithelium at the time of exposure may influence the outcome of infection. Oral contraceptive use also increased the risk of contracting chlamydial infections compared to women not using contraception. Generally it was suggested that the outcome of chlamydial infection is determined in part by the hormonal status of the epithelium at the time of exposure. Using the human endolmetrial cell line ECC-1 this study investigated the effects of C. trachomatis serovar D infection, in conjunction with the female sex hormones, 17β-estradiol and progesterone, on chlamydial gene expression. While previous studies have examined the host response, this is the first study to examine C.trachomatis gene expression under different hormonal conditions. We have highlighted a basic model of C. trachomatis gene regulation in the presence of steroid hormones by identifying 60 genes that were regulated by addition of estradiol and/or progesterone. In addition, the third chapter of this thesis discussed and compared the significance of the current findings in the context of data from other research groups to improve our understanding of the molecular basis of chlamydial persistence under hormonal different conditions. In addition, this study analysed the effects of these female sex hormones and C. trachomatis Serovar D infection, on host susceptibility and bacterial growth. Our results clearly demonstrated that addition of steroid hormones not only had a great impact on the level of infectivity of epithelial cells with C.trachomatis serovar D, but also the morphology of chlamydial inclusions was affected by hormone supplementation.

Toward a fuzzy domain ontology extraction method for adaptive e-learning

With the widespread applications of electronic learning (e-Learning) technologies to education at all levels, increasing number of online educational resources and messages are generated from the corresponding e-Learning environments. Nevertheless, it is quite difficult, if not totally impossible, for instructors to read through and analyze the online messages to predict the progress of their students on the fly. The main contribution of this paper is the illustration of a novel concept map generation mechanism which is underpinned by a fuzzy domain ontology extraction algorithm. The proposed mechanism can automatically construct concept maps based on the messages posted to online discussion forums. By browsing the concept maps, instructors can quickly identify the progress of their students and adjust the pedagogical sequence on the fly. Our initial experimental results reveal that the accuracy and the quality of the automatically generated concept maps are promising. Our research work opens the door to the development and application of intelligent software tools to enhance e-Learning.

Ghrelin gene-related peptides : multifunctional endocrine/autocrine modulators in health and disease

Ghrelin is a multi-functional peptide hormone which affects various processes including growth hormone and insulin release, appetite regulation, gut motility, metabolism and cancer cell proliferation. Ghrelin is produced in the stomach and in other normal and pathological cell types. It may act as an endocrine or autocrine/paracrine factor. The ghrelin gene encodes a precursor protein, preproghrelin, from which ghrelin and other potentially active peptides are derived by alternative mRNA splicing and/or proteolytic processing. The metabolic role of the peptide obestatin, derived from the preproghrelin C-terminal region, is controversial. However, it has direct effects on cancer cell proliferation. The regulation of ghrelin expression and the mechanisms through which the peptide products arise are unclear. We have recently re-examined the organisation of the ghrelin gene and identified several novel exons and transcripts. One transcript, which lacks the ghrelin-coding region of preproghrelin, contains the coding sequence of obestatin. Furthermore, we have identified an overlapping gene on the antisense strand of ghrelin, GHRLOS, which generates transcripts that may function as non-coding regulatory RNAs or code for novel, short bioactive peptides. The identification of these novel ghrelin-gene related transcripts and peptides raises critical questions regarding their physiological function and their role in obesity, diabetes and cancer.

Personalised ontology learning and mining for web information gathering

Over the last decade, the rapid growth and adoption of the World Wide Web has further exacerbated user needs for e±cient mechanisms for information and knowledge location, selection, and retrieval. How to gather useful and meaningful information from the Web becomes challenging to users. The capture of user information needs is key to delivering users' desired information, and user pro¯les can help to capture information needs. However, e®ectively acquiring user pro¯les is di±cult. It is argued that if user background knowledge can be speci¯ed by ontolo- gies, more accurate user pro¯les can be acquired and thus information needs can be captured e®ectively. Web users implicitly possess concept models that are obtained from their experience and education, and use the concept models in information gathering. Prior to this work, much research has attempted to use ontologies to specify user background knowledge and user concept models. However, these works have a drawback in that they cannot move beyond the subsumption of super - and sub-class structure to emphasising the speci¯c se- mantic relations in a single computational model. This has also been a challenge for years in the knowledge engineering community. Thus, using ontologies to represent user concept models and to acquire user pro¯les remains an unsolved problem in personalised Web information gathering and knowledge engineering. In this thesis, an ontology learning and mining model is proposed to acquire user pro¯les for personalised Web information gathering. The proposed compu- tational model emphasises the speci¯c is-a and part-of semantic relations in one computational model. The world knowledge and users' Local Instance Reposito- ries are used to attempt to discover and specify user background knowledge. From a world knowledge base, personalised ontologies are constructed by adopting au- tomatic or semi-automatic techniques to extract user interest concepts, focusing on user information needs. A multidimensional ontology mining method, Speci- ¯city and Exhaustivity, is also introduced in this thesis for analysing the user background knowledge discovered and speci¯ed in user personalised ontologies. The ontology learning and mining model is evaluated by comparing with human- based and state-of-the-art computational models in experiments, using a large, standard data set. The experimental results are promising for evaluation. The proposed ontology learning and mining model in this thesis helps to develop a better understanding of user pro¯le acquisition, thus providing better design of personalised Web information gathering systems. The contributions are increasingly signi¯cant, given both the rapid explosion of Web information in recent years and today's accessibility to the Internet and the full text world.