Thesis (M.D.)-- University of Sydney, 1895. / Cover title. At head of title: Thesis for presentation to the examiners for the degree of M.D. at the University of Sydney, March, 1895. Bibliography: p. 65-68.

The evolutionary history of mammals, when including extinct taxa, is mainly reconstructed using tooth morphology and employs terminology based on non-monotreme mammals. Although adult monotremes are edentulous, juvenile platypuses have teeth that can be compared with extinct monotremes, but terminology can be a barrier to efficient comparison to non-monotreme mammals. Deciduous teeth and thickened epithelial plates of the extant platypus, Ornithorhynchus anatinus, are sparsely figured in the literature. New imagery of those teeth and plates from high-resolution x-ray computed tomography and scanning electron microscopy contribute to the understanding of mammal evolution and the unique morphology of platypus teeth. The teeth of the juveniles are highly variable, but early-forming features (major cusps and transverse valleys) are stable enough for comparison. Transverse lophs on monotreme teeth contain complexity not reflected in cusps alone, unlike therian mammals. These differences reinforce the need for caution when applying dental terminology that originally was produced for therian mammals. New imagery highlights potential phylogenetically informative morphology in the pulp cavity and roots. As the roots of the juvenile teeth degenerate, the epithelium below the teeth thickens into epithelial plates. Structures in the epithelial plates are broadly similar to those found in the keratin plates. New images of the epithelial plates offer insight into a series of tubes concentrated under the juvenile teeth. The tubes are a continuous conduit to the plate surface and may serve a sensory function or result from the ever-growing nature of the epithelial plate. / text

<p>Antibodies are a central part of the immune defense system, and a large variability in their specificity is needed in order to be able to react against all possible foreign substances we may encounter during our lives. In this thesis, results are presented from investigations into how an egg-laying mammal, the Australian duck-billed platypus (<i>Ornithorhynchus anatinus</i>) creates antibody variability. Our results show that despite the lack of many V gene families the antibody repertoire in the platypus seems to be well developed. A long and highly variable complementarity-determining region (CDR) 3 compensates for the limited germline diversity. Interestingly, the presence of additional cysteine residues in the CDRs may form stabilizing disulfide bridges in the antigen binding loops and thereby increasing the affinity of the antibody-antigen interaction. </p><p>Although the immune system is necessary for survival, it must be strictly controlled since it may otherwise over-react and cause more harm than benefits. Allergies and autoimmune diseases are examples of such over-reactions by the immune system. Allergies are increasing in the western world and have become one of the main medical issues of the 21^st century. IgE is the central mediator in atopic allergies such as hay fever, eczema and asthma; it is therefore a prime target in the development of allergen-independent preventative treatments. Here we present results from several studies of a novel vaccine strategy aimed at reducing the levels of IgE antibodies. The vaccine results in the induction of anti-IgE antibodies, and the skin reactivity upon allergen challenge was significantly reduced in vaccinated animals. Our results suggest that active immunization against IgE has the potential to become a therapeutic method for humans. In addition, an evaluation of possible adjuvants that could be used as immune stimulators and thus help break self-tolerance at the time of vaccination is presented.</p>

Antibodies are a central part of the immune defense system, and a large variability in their specificity is needed in order to be able to react against all possible foreign substances we may encounter during our lives. In this thesis, results are presented from investigations into how an egg-laying mammal, the Australian duck-billed platypus (Ornithorhynchus anatinus) creates antibody variability. Our results show that despite the lack of many V gene families the antibody repertoire in the platypus seems to be well developed. A long and highly variable complementarity-determining region (CDR) 3 compensates for the limited germline diversity. Interestingly, the presence of additional cysteine residues in the CDRs may form stabilizing disulfide bridges in the antigen binding loops and thereby increasing the affinity of the antibody-antigen interaction. Although the immune system is necessary for survival, it must be strictly controlled since it may otherwise over-react and cause more harm than benefits. Allergies and autoimmune diseases are examples of such over-reactions by the immune system. Allergies are increasing in the western world and have become one of the main medical issues of the 21st century. IgE is the central mediator in atopic allergies such as hay fever, eczema and asthma; it is therefore a prime target in the development of allergen-independent preventative treatments. Here we present results from several studies of a novel vaccine strategy aimed at reducing the levels of IgE antibodies. The vaccine results in the induction of anti-IgE antibodies, and the skin reactivity upon allergen challenge was significantly reduced in vaccinated animals. Our results suggest that active immunization against IgE has the potential to become a therapeutic method for humans. In addition, an evaluation of possible adjuvants that could be used as immune stimulators and thus help break self-tolerance at the time of vaccination is presented.

Vertebrate genomes exhibit global methylation of cytosine residues where they occur in a cytosine-guanine dinucleotide (CpG) context and this epigenetic mark is generally thought to be repressive to transcription. Punctuating this pervasive DNA methylation landscape are short, contiguous regions of non-methylated DNA which are found at two thirds of mammalian gene promoters. These non-methylated regions exhibit CpG content close to expected levels as they escape the depletion of CpGs observed across the methylated fraction of the genome. The unique nucleotide properties of these CpG island (CGI) regions enable their identification by computational prediction in mammalian genomes. Owing to a lack of high-resolution genome-wide DNA methylation profiles in non-mammalian species, these CGI predictions have often been used as a proxy for non-methylated DNA in these organisms. In contrast to mammals, CGI predictions in cold-blooded vertebrates rarely coincide with gene promoters, leading to the belief that CGls are significantly divergent between vertebrate species, and that unique promoter-associated features may have been acquired during warmblooded vertebrate evolution. This thesis is primarily concerned with the location, establishment and biological function of non-methylated islands of DNA in vertebrate genomes. To experimentally determine genome-wide profiles of non-methylated DNA, a novel biochemical technique was established called biotinylated ZF-CxxC affinity purification (Bio-CAP), and development of this method is discussed in Chapter 3. Experimental analysis of non-methylated DNA profiles in this thesis initially addresses two main questions: (1) 'How does the non-methylated DNA landscape compare genome-wide for seven vertebrates considering distinct tissue types and developmental stages?' (2) 'How are vertebrate non-methylated regions of DNA defined and interpreted in the nuclear environment?' To address the first question, non-methylated DNA was profiled by Bio-CAP sequencing across the genomes of seven diverse vertebrate species, representing all major branch points of vertebrate evolution, and the results are discussed in Chapters 4 and S. Contrary to previously held dogma, experimentally determined nonmethylated islands of DNA (NMls) constitute an ancient epigenetic feature of vertebrate gene regulatory elements. However, despite having numerous high-resolution maps of vertebrate non-methylated DNA, the means by which NMls are identified and maintained in the nuclear environment remains poorly understood. To address the second question and identify features which determine the methylation state of DNA, exogenous DNA sequences were introduced into mouse embryonic stem (ES) c~.II~. Non-methylated DNA was profiled by Bio-CAP sequencing to investigate how different features, such as sequence-specific binding motifs, chromatin architecture and nucleotide composition of a given DNA sequence impact local DNA methylation patterns. Interestingly, the majority of exogenous promoters were appropriately non-methylated in mouse ES cells, germline and somatic cells suggesting that gene promoters have retained strong signals for the nonmethylated state across millions of years of evolution (discussed in Chapter 6). During mouse embryogenesis, genome-scale DNA demethylation and remethylation events occur to remodel the epigenetic landscape and loss of DNA methylation during this time leads to embryonic lethality. To investigate the biological function of non-methylated DNA, the third question addressed in this thesis is (3) 'What is the developmental importance of non-methylated islands of DNA during vertebrate embryogenesis?' To investigate this, members of the ZF-CxxC domain-containing family of chromatin modifiers were ablated in zebrafish embryos to perturb the chromatin landscape at NMls, and therefore interfere with their function during early development (Chapter 7). Early embryonic development and patterning was disrupted in knockdown embryos, suggesting that interpretation of non-methylated DNA and placement of chromatin modifications at NMls is essential for normal zebrafish embryogenesis. Together this work sheds light on the evolutionary origins of NMls, the mechanisms involved in the recognition and establishment of nonmethylated loci and provides an insight into the function of non-methylated DNA during early embryonic development.