Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 126-149). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Orientador: Fernando Lopes Alberto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T09:04:32Z (GMT). No. of bitstreams: 1 Peres_TarcisiodeSouza_M.pdf: 2330056 bytes, checksum: 66c1d9241ad60e2d973cfb7361a5eb7c (MD5) Previous issue date: 2006 / Resumo: Durante todo o século XX, a pesquisa do câncer se desenvolveu de maneira sistemática, porém os últimos 25 anos foram notadamente caracterizados por rápidos avanços que geraram uma rica e complexa base de conhecimentos, evidenciando a doença dentro de um conjunto dinâmico de alterações no genoma. Desta forma, o entendimento completo dos fenômenos moleculares envolvidos na fisiopatologia das neoplasias depende do conhecimento dos diversos processos celulares e bioquímicos característicos da célula tumoral e que, porventura, a diferenciem da célula normal (GOLUB e SLONIM, 1999). Nesse trabalho buscamos o melhor entendimento das vias moleculares no processo neoplásico por meio da análise dos dados do Projeto Genoma Humano do Câncer (CAMARGO, 2001) com vistas à identificação de genes diferencialmente expressos nas neoplasias dos seguintes tecidos: mama, cólon, cabeça e pescoço, pulmão, sistema nervoso central, próstata, estômago, testículo e útero. A metodologia de geração dos transcritos utilizada pelo Projeto Genoma Humano do Câncer é conhecida como ORESTES (DIAS et al, 2000). Inicialmente, os dados de seqüenciamento (fragmentos ORESTES) foram agrupados por meio de uma técnica conhecida em Bioinformática como ¿montagem¿, utilizando o pacote de programas de computador PHRED/PHRAP (EWING e GREEN P., 1998). A comparação de cada agrupamento com seqüências conhecidas (depositadas em bases públicas) foi realizada por meio do algoritmo BLAST (ALTSCHUL et al, 1990). Um subconjunto de genes foi selecionado com base em critérios específicos e submetido à avaliação de seus níveis de expressão em diferentes tecidos com base em abordagem de inferência Bayesiana (CHEN et al, 1998), em contraposição às abordagens mais clássicas, como testes de hipótese nula (AUDIC e CLAVERIE, 1997). A inferência Bayesiana foi viabilizada pelo desenvolvimento de uma ferramenta computacional escrita em linguagem PERL (PERES et al, 2005). Com o apoio da literatura, foi criada uma lista de genes relacionados ao fenômeno neoplásico. Esta lista foi confrontada com as informações de expressão gênica, constituindo-se em um dos parâmetros de um sistema de classificação (definido para a seleção dos genes de interesse). Desta forma, parte da base de conhecimento sobre câncer foi utilizada em conjunto com os dados de expressão gênica inferidos a partir dos fragmentos ORESTES. Para contextualização biológica da informação gerada, os genes foram classificados segundo nomenclatura GO (ASHBURNER et al, 2000) e KEGG (OGATA et al, 1999). Parte dos genes apontados como diferencialmente expressos em pelo menos um tecido tumoral, em relação ao seu equivalente normal, integram vias relacionadas ao fenômeno neoplásico (HAHN e WEINBERG, 2002). Dos genes associados a estas vias, 52% deles possuíam fator de expressão diferencial (em módulo) superior a cinco. Finalmente, dez entre os genes classificados foram escolhidos para confirmação experimental dos achados. Os resultados de qPCR em amostras de tecido gástrico normal e neoplásico foram compatíveis com com os dados de expressão gênica inferidos a partir dos fragmentos ORESTES / Abstract: The XXth century showed the development in cancer research in a systematic way, most notably in the last 25 years that were characterized by rapid advances that generated a rich and complex body of knowledge, highlighting the disease within a dynamic group of changes in the genome. The complete understanding of the molecular phenomena involved in the physiopathology of neoplasia is based upon the knowledge of the varied cellular and biochemical processes which are characteristic of the tumor and which make it different from the normal cell (GOLUB e SLONIM, 1999) In this work, we investigated the molecular pathways in the neoplasic process through data analyses of the cDNA sequences generated on the Human Cancer Genome Project (CAMARGO, 2001). The following neoplasias were included: breast, colon, head and neck, lungs, central nervous system, prostate gland, stomach, testicle and womb. The methodology of generation of transcripts used by the Genome Project of Human Cancer is known as ORESTES (DIAS et al, 2000). Initially, the sequence of data (ORESTES fragments) were grouped and assembled according to similarity scores. For this purpose, we used the package of computer programs PHRED/PHRAP (EWING e GREEN P., 1998). The resulting consensus sequences, each representing a cluster, were compared to known sequences (deposited in public databanks) through the BLAST algorithm (ALTSCHUL et al, 1990). A subgroup of genes was selected based on specific criteria and their levels of expression in different tissues were evaluated by a bayesian inference approach (CHEN et al, 1998), as compared to more classical approaches such as null hypothesis tests (AUDIC e CLAVERIE, 1997). The Bayesian inference tool was represented as a PERL script developed for this work. A list of genes, putatively related to the neoplasic phenotype, was created with the support of the literature. This list was compared to the gene expression information, becoming one of the parameters of a ranking system (defined for the selection of genes of interest). Therefore, part of the knowledge related to cancer was used together with the data of gene expression inferred from ORESTES fragments. For a more accurate understanding of the molecular pathways involved in the generated information, the genes were classified according to the Gene Ontology (ASHBURNER et al, 2000) and KEGG (OGATA et al, 1999) nomenclatures. Additional global analyses by pathways related to the neoplasic phenomenon (HAHN e WEINBERG, 2002) demonstrated differential expression of the selected genes. About 52% of the genes in this pathways were differentially expressed in tumor tissue with at least a 5-fold. Finally, ten genes were selected for experimental validation (in vitro) of the findings with real-time quantitative PCR, confirming in silico results / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

The transcription efficiency of transfer RNA genes is modulated by sequences contained in their 5'-flanking region. For a tRNA val₄ gene a pentanucleotide with the sequence TCGCT was identified between positions -33 and -38. I have previously proposed that this sequence may be involved in specifically determining the rate of transcription of this gene. A general form of this sequence, TNNCT was found associated with other Drosophila tRNA genes which showed high ill vitro transcription efficiency. To further elucidate the role of TCGCT in tRNA transcription, single and double base-pair changes were created in the sequence TCGCT using site-specific mutagenesis. Mutations in the nucleotides -38T, -35C and -34T showed decreased levels of transcription whereas nucleotide changes at the nucleotides -37C and -36G did not reduce template activity. Therefore the sequence which modulates transcription of the tRNAVal₄ gene does have the general form TNNCT. Competition experiments between the Val₄ mutant -38G.-35A and a tRNASer₇ gene showed the TNNCT mutant to be a better competitor for transcription than the wild type template. Experiments analyzing the time-course of transcription, the effects of temperature and the effects of ionic strength indicated that TNNCT was not involved in determining the efficiency of stable complex formation. It is proposed that the pentanucleotide is probably responsible for influencing the rate of initiation of transcription. A sequence TGCCT contained in the anticodon stem/loop region of the Val₄ gene was also mutagenized and shown to be involved in complex stability or the elongation of Val₄ tRNAs. Using deletion analysis of the 5'-flanking sequences of a tRNASer₇ gene, a second positive transcription regulatory element was delimited. This sequence was also found in the 5'-flanks of the tRNAVal₄ and a tRNAArg gene. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The nucleotide sequence of the single Drosophila meianogaster tRNA gene contained in the recombinant plasmid, pDtl20R was determined by the Maxam and Gilbert method. This plasmid hybridizes to the 90 BC site on the Val Drosophila polytene chromosomes, a minor site of tRNA4 hybridization. The Val nucleotide sequence of the tRNA4 gene present in pDtl20R differs at four Val positions from the sequence expected from that of tRNA4 . The four differences occur at nucleotides 16, 29, 41 and 57 in the coding region. Comparison of the DNA sequence of pDtl20R to that of the plasmid pDt92R, which also hybridizes to the 90 BC site, indicates that the Drosophila fragments contained in these two plasmids are either alleles or repeats. The implications of these findings are discussed. An in vitro transcription system was developed from a Drosophila Schneider II cell line. This homologous cell-free extract support specific and accurate transcription of various Drosophila tRNA Val genes. The major product of transcription is a tRNA precursor which is processed to a tRNA sized species. Transfer RNA valine genes originating from different sites on the Drosophila chromosomes are transcribed at different rates. Comparison of the sequences in the internal promoter regions of the various genes indicates that the few differences within the coding regions may not be responsible for the observed difference in the rates of transcription. This conclusion is substantiated by studies with hybrid genes constructed during the course of this work. Preliminary evidence indicates that the Val tRNA gene which is transcribed at the highest rate may be preceded in its 5'-flanking region by a positively modulating sequence. Val The precursor RNAs directed by various tRNA genes are also processed at different rates. Transcription and processing experiments with hybrid genes suggest that nucleotide changes within the coding region, which do not affect the rate of transcription, influence the rate of processing. Time course and competition experiments demonstrate that at least two kinetic steps are required for the formation of a stable transcription complex. Studies with an in vitro constructed mutant missing in nucleotides 51-61 in the tRNA coding region suggests that this deleted region (which is highly conserved in eukaryotic tRNAs) may be involved in the primary interaction required for tRNA gene transcription. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Expression of germ-line α transcripts precedes class switching to IgA, and therefore study of the regulation of germ-line α RNA transcription is important for understanding the class switching process. Transforming growth factor β1 (TGFβ1) increases the transcription of the Ig constant region a gene and class switching to IgA in normal B cells and in the I.29μ B lymphoma cell line. The structure of germ-line α transcripts in I.29μ cells was analyzed by RNase protection and primer extension assays. Two initiation sites for germ-line α transcripts were identified 2 kb upstream to the α switch region. No TATA or Sp1 elements are found near the RNA initiation sites. The DNA segment located 5' to the initiation sites of germ-line α RNA can drive expression of a luciferase reporter gene when transiently transfected into I.29μ (subclone 22D) and A20.3 cell lines. Full constitutive expression requires no more than 106 bp of the 5' flanking segment. In deletion and substitution mutation studies, an ATF/CRE site residing within this region is very important for constitutive expression of the germ-line α promoter, but mutation of this motif does not diminish TGFβ1 inducibility. Induction by TGFβ1 requires additional sequences residing between -128 to -106 relative to the first RNA initiation site. Two copies of a tandemlyrepeated sequence 5' CACAG(G) CCAGAC 3' (termed Igα TGFβ-RE) are located in the region from -127 to -105. An oligonucleotide containing multimers of these repeats could confer TGFβ1 inducibility to a heterologous promoter. An additional copy of the TGFβ-RE was identified at -41/-30 and its deletion reduced the TGFβ1 response. Thus, tandem repeats of a novel TGFβ-RE are the positive regulatory elements for the TGFβ1 response. Gel mobility shift assays demonstrated specific binding to the TGFβ-RE by nuclear factors but the binding activity was not enhanced by TGFβ1. This study supports previously published evidence that TGFβ1 directs class switching to IgA through induction of germ-line Cα gene transcription.

The compaction of the eukaryotic genome into a complex, highly folded chromatin structure necessitates cellular mechanisms for allowing access of regulatory proteins to the DNA template. Recent advances have led to the identification of two distinct families of chromatin remodeling enzymes--multi-subunit complexes that harbor a SWI2/SNF2 ATPase family member, and the nuclear acetyltransferases. The Saccharomyces cerevisiae SWI/SNF complex, the prototype for the ATP-dependent chromatin remodeling machines, is required for expression of a subset of genes in yeast. This 2MDa multimeric assembly is believed to facilitate transcriptional enhancement by antagonizing chromatin-mediated transcriptional repression through disruption of histone-DNA contacts. In an attempt to identify components or regulators of the SWI/SNF complex, we have cloned three previously identified genes, ADA2, ADA3, and GCN5, that encode subunits of a complex distinct from SWI/SNF. During the course of this thesis work, one of these gene products, GCN5, was identified as the first catalytic nuclear histone acetyltransferase. The goal of this thesis work was to determine the role of the ADA/GCN5 complex in transcriptional activation in Saccharomyces cerevisiae. Using in vivo functional and genetic analysis, we have found that mutations in ADA2, ADA3, and GCN5 cause phenotypes strikingly similar to those of swi/snf mutants. ADA2, ADA3, and GCN5 are required for full expression of all SWI/SNF-dependent genes tested, including HO, SUC2, INO1, and Ty elements. Furthermore, mutations in the SIN1 gene, which encodes a non-histone chromatin component, or mutations in histones H3 or H4, alleviate the transcriptional defects caused by ada/gcn5 or swi/snf mutations. We have also found that ada2 swi1, ada3 swi1, and gcn5 swi1 double mutants are inviable and that mutations in SIN1 allow viability of these double mutants. To determine the biochemical activities of the native GCN5-containing complex in yeast, we have partially purified three chromatographically distinct GCN5-dependent acetyltransferase activities. We have found that these three acetyltransferase complexes demonstrate unique substrate specificities for free histones and histones assembled into nucleosomal arrays. Additionally, we found that these enzymes not only acetylate histones, but also purified yeast Sin1 protein, a non-histone chromatin component that resembles HMG1. We have also established a functional relationship between GCN5-dependent histone acetylation and polyamine-dependent chromatin condensation. We have found that depletion of cellular polyamines alleviates transcriptional defects caused by inactivation of the GCN5 histone acetyltransferase. In contrast, polyamine depletion does not alter the transcriptional requirements for the SWI/SNF chromatin remodeling complex. We have also found that polyamines facilitate oligomerization of nucleosomal arrays in vitro. Furthermore, this polyamine-mediated condensation reaction requires intact N-terminal domains of the core histones, and is inhibited by hyperacetylation of these domains. The results presented throughout this thesis support roles for the ADA/GCN5 products in antagonizing chromatin. In vivo analysis suggests a functional relationship between the ADA/GCN5 acetyltransferase complex (or complexes) and the SWI/SNF complex. These comp1exes may operate in concert at nucleosomes within specific promoters to facilitate activated transcription. Furthermore, our studies suggest that polyamines are repressors of transcription in vivo, and that an additional role of histone hyperacetylation is to antagonize the ability of polyamines to stabilize highly condensed states of chromosomal fibers.

Lee Wing Sum. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 206-226). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese Version) --- p.iv / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xxiv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.3 / Chapter 2.1 --- Peroxisomes --- p.3 / Chapter 2.2 --- Peroxisome proliferators --- p.5 / Chapter 2.3 --- Human exposure pathways to peroxisome proliferators --- p.5 / Chapter 2.4 --- Peroxisome proliferator-induced pleiotropic effects in rodents --- p.7 / Chapter 2.4.1 --- Short-term effects --- p.7 / Chapter 2.4.1.1 --- Hepatomegaly --- p.7 / Chapter 2.4.2.1 --- Peroxisome proliferation --- p.8 / Chapter 2.4.1.3 --- Alteration of gene transcriptions --- p.8 / Chapter 2.4.2 --- Long-term effect --- p.9 / Chapter 2.5 --- Mechanisms of actions of peroxisome proliferators --- p.9 / Chapter 2.5.1 --- Substrate overload --- p.9 / Chapter 2.5.2 --- Receptor-mediated --- p.11 / Chapter 2.6 --- Peroxisome proliferator-activated receptors (PPARs) --- p.11 / Chapter 2.6.1 --- Structure of PPARs --- p.11 / Chapter 2.6.2 --- Tissue-specific expression of PPARs --- p.15 / Chapter 2.6.3 --- Physiological functions of PPARs --- p.19 / Chapter 2.6.3.1 --- PPARα --- p.19 / Chapter 2.6.3.2 --- PPARγ --- p.21 / Chapter 2.6.3.3 --- PPARδ --- p.23 / Chapter 2.7 --- Role of PPARα involved in peroxisome proliferator-induced pleiotropic responses --- p.24 / Chapter 2.7.1 --- Short-term effects --- p.24 / Chapter 2.7.2 --- Long-term effect --- p.24 / Chapter 2.8 --- Mechanisms of peroxisome proliferator-induced hepatocarcinogenesis --- p.25 / Chapter 2.8.1 --- Oxidative stress --- p.25 / Chapter 2.8.2 --- Suppression of apoptosis --- p.26 / Chapter 2.8.3 --- Increased cell proliferation --- p.27 / Chapter 2.9 --- Species difference to peroxisome proliferator-induced pleiotropic effects --- p.28 / Chapter 2.10 --- Fluorescent differential display (FDD) --- p.32 / Chapter Chapter 3 --- Objectives --- p.35 / Chapter Chapter 4 --- Materials and methods --- p.37 / Chapter 4.1 --- Animals and treatments --- p.37 / Chapter 4.1.1 --- Materials --- p.37 / Chapter 4.1.2 --- Methods --- p.37 / Chapter 4.2 --- Serum triglyceride and cholesterol analyses --- p.39 / Chapter 4.2.1 --- Materials --- p.41 / Chapter 4.2.2 --- Methods --- p.41 / Chapter 4.2.2.1 --- Serum preparation --- p.41 / Chapter 4.2.2.2 --- Triglyceride determination --- p.41 / Chapter 4.2.2.3 --- Cholesterol determination --- p.42 / Chapter 4.3 --- Statistical analysis --- p.42 / Chapter 4.4 --- Tail-genotyping --- p.42 / Chapter 4.4.1 --- Materials --- p.44 / Chapter 4.4.2 --- Methods. --- p.44 / Chapter 4.4.2.1 --- Preparation of genomic tail DNA --- p.44 / Chapter 4.4.2.2 --- PCR reaction --- p.45 / Chapter 4.5 --- Total RNA isolation --- p.45 / Chapter 4.5.1 --- Materials --- p.48 / Chapter 4.5.2 --- Methods --- p.48 / Chapter 4.6 --- DNase I treatment --- p.48 / Chapter 4.6.1 --- Materials --- p.49 / Chapter 4.6.2 --- Methods --- p.49 / Chapter 4.7 --- Reverse transcription of mRNA and fluorescent PCR amplification --- p.50 / Chapter 4.7.1 --- Materials --- p.50 / Chapter 4.7.2 --- Methods --- p.53 / Chapter 4.8 --- Fluorescent differential display (FDD) --- p.53 / Chapter 4.8.1 --- Materials --- p.53 / Chapter 4.8.2 --- Methods --- p.54 / Chapter 4.9 --- Excision of differentially expressed cDNA fragments --- p.54 / Chapter 4.9.1 --- Materials --- p.57 / Chapter 4.9.2 --- Methods --- p.57 / Chapter 4.10 --- Reamplification of differentially expressed fragments --- p.57 / Chapter 4.10.1 --- Materials --- p.60 / Chapter 4.10.2 --- Methods --- p.60 / Chapter 4.11 --- Subcloning of reamplified cDNA fragments --- p.62 / Chapter 4.11.1 --- PCR-TRAP® cloning system --- p.62 / Chapter 4.11.1.1 --- Materials --- p.63 / Chapter 4.11.1.2 --- Methods --- p.63 / Chapter 4.11.2 --- AdvaTage´ёØ PCR cloning system --- p.65 / Chapter 4.11.2.1 --- Materials --- p.65 / Chapter 4.11.2.2 --- Methods --- p.66 / Chapter 4.12 --- Purification of plasmid DNA from recombinant clones --- p.69 / Chapter 4.12.1 --- Materials --- p.69 / Chapter 4.12.2 --- Methods --- p.69 / Chapter 4.13 --- DNA sequencing of differentially expressed cDNA fragments --- p.70 / Chapter 4.13.1 --- CEQ 2000 Dye Terminator Cycle Sequence system --- p.71 / Chapter 4.13.1.1 --- Materials --- p.71 / Chapter 4.13.1.2 --- Methods --- p.71 / Chapter 4.13.2 --- ABI PRISM´ёØ dRhodamine Terminator Cycle Sequencing system --- p.72 / Chapter 4.13.2.1 --- Materials --- p.72 / Chapter 4.13.2.2 --- Methods --- p.72 / Chapter 4.13.3 --- Homology search against computer databases --- p.73 / Chapter 4.14 --- Northern analysis of differentially expressed cDNA fragments --- p.73 / Chapter 4.14.1 --- Formaldehyde gel electrophoresis of total RNA --- p.74 / Chapter 4.14.1.1 --- Materials --- p.74 / Chapter 4.14.1.2 --- Methods --- p.74 / Chapter 4.14.2 --- Preparation of cDNA probes for hybridization --- p.74 / Chapter 4.14.2.1 --- PCR DIG labeling --- p.75 / Chapter 4.14.2.1.1 --- Materials --- p.75 / Chapter 4.14.2.1.2 --- Methods --- p.75 / Chapter 4.14.2.2 --- Random Prime cDNA DIG labeling --- p.75 / Chapter 4.14.2.2.1 --- Materials --- p.75 / Chapter 4.14.2.2.2 --- Methods --- p.76 / Chapter 4.14.3 --- Purification of DNA from agarose gel --- p.77 / Chapter 4.14.3.1 --- Materials --- p.77 / Chapter 4.14.3.2 --- Methods --- p.78 / Chapter 4.14.4 --- Hybridization --- p.78 / Chapter 4.14.4.1 --- Materials --- p.78 / Chapter 4.14.4.2 --- Methods --- p.73 / Chapter 4.14.5 --- Synthesis of mouse GAPDH probe from normalization --- p.80 / Chapter 4.14.5.1 --- Materials --- p.80 / Chapter 4.14.5.2 --- Methods --- p.80 / Chapter Chapter 5 --- Results --- p.82 / Chapter 5.1 --- Liver morphology --- p.82 / Chapter 5.2 --- Liver weight --- p.82 / Chapter 5.3 --- Serum triglyceride and cholesterol levels --- p.88 / Chapter 5.4 --- Confirmation of genotypes --- p.91 / Chapter 5.5 --- DNase I treatment --- p.91 / Chapter 5.6 --- FDD RT-PCR and band excision --- p.98 / Chapter 5.7 --- Reamplification of excised cDNA fragments --- p.111 / Chapter 5.8 --- Subcloning of reamplified cDNA fragments --- p.121 / Chapter 5.9 --- DNA sequencing of subcloned cDNA fragments --- p.124 / Chapter 5.10 --- Confirmation of the differentially expressed cDNA fragments by Northern blot analysis --- p.132 / Chapter 5.11 --- Temporal expression pattern of differentially expressed genes --- p.157 / Chapter 5.12 --- Tissue distribution pattern of differentially expressed genes --- p.171 / Chapter Chapter 6 --- Discussions --- p.183 / Chapter 6.1 --- "Lack of hepatomegaly, hypotriglyceridemia and hepatic nodule formation in PPARα (-/-) mice" --- p.184 / Chapter 6.2 --- "Identification of PPARα-dependent and Wy-14,643 responsive genes" --- p.185 / Chapter 6.3 --- Functional roles of the isolated cDNA fragments --- p.186 / Chapter 6.3.1 --- Fragments B14 and H4 --- p.187 / Chapter 6.3.2 --- Fragment H1 --- p.189 / Chapter 6.3.3 --- Fragment H5 --- p.192 / Chapter 6.3.4 --- Fragment H8 --- p.194 / Chapter 6.4 --- Temporal expression patterns of the isolated cDNA fragments --- p.196 / Chapter 6.5 --- Tissue distribution patterns of the isolated cDNA fragments --- p.197 / Chapter Chapter 7 --- Conclusions --- p.200 / Chapter Chapter 8 --- Future studies --- p.204 / Chapter 8.1 --- Subcloning and characterization of the other differentially expressed genes --- p.204 / Chapter 8.2 --- Overexpression and inhibition expression of specific genes --- p.204 / Chapter 8.3 --- Generating transgenic mice with target disruption of specific gene --- p.205 / References --- p.206

by Lee Wing-cheung. / "November 2001." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 152-169). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 106-143).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.