The recent emergence of the field of proteomics has been driven by advances in mass spectrometry methods and instrumentation. Due to the large amount of data generated, success at peptide and protein identification is contingent on reliable software algorithms. The software programs in use at the time the work in this dissertation was carried out were well suited to the task of identifying unmodified peptides and proteins in complex mixtures. However, the existing programs were not able to reliably identify protein modifications, especially unpredicted modifications. This dissertation describes the development of two novel software algorithms that can be used to screen LC-MS-MS data files, and identify MS-MS spectra that correspond to peptides with either predicted or unpredicted modifications. The first program, SALSA, is highly flexible and uses user defined search criteria to screen data files for spectra the exhibit fragmentation patterns diagnostic of specific modifications or peptide sequences. SALSA facilitates exhaustive searches, but requires user expertise to both generate search criteria and to validate matched spectra. The second program, P-Mod, provides automated searches for spectra corresponding to peptides in a search list. P-Mod is able to identify spectra derived from either modified or unmodified peptides. All sequence-to-spectrum matches reported in the P-Mod output are assigned statistical confidence levels derived using extreme value statistics.

Tracing semantic change in various translations of The Bible.

In eukaryotes, the genomic double-stranded DNA (dsDNA) coils around histones to form nucleosomes. Arrays of these nucleosomes bundle together to generate chromatin. Most DNA-related processes require interactions between chromatin-protected DNA and cellular machinery. Access of cell machinery to genomic DNA is partially regulated by the position and stability of nucleosomes, which may be influenced by changes in nucleosomal DNA. DNA is composed of adenine (A), guanine (G), cytosine (C), thymine (T) nucleotides and their derivatives. It has been shown that some C derivatives participate in directing multiple biological processes, and aberrant modification patterns are often linked to diseases. It has been proposed that T derivatives exhibit similar effects. This thesis focuses on elucidating the effect of naturally occurring DNA modifications on the properties of dsDNA and nucleosomes. dsDNA sequences systematically modified with various T derivatives were characterized using classical biophysical techniques to assess the effect of these DNA modifications. The results indicate that in the sequence context studied, 5-hydroxymethyluracil modifications destabilize dsDNA, while dense symmetrical 5-formyluracil (fU) modifications alter the dsDNA structure. These effects may provide clues to the differential protein recruitment observed in previous research. In vitro studies on nucleosome occupancy and stability revealed that 5-formylcytosine (fC) modifications have positive effects on nucleosome formation and stability compared to the unmodified counterpart by influencing the intrinsic biochemical and biophysical properties of the nucleosomes. These results provide casual links for the observation in vivo between fC and the increased nucleosome occupancy and positioning. In order to further understand the positional effect of fC on the nucleosomes, a method was developed for quick and reliable incorporation of C derivatives into dsDNA at desired positions. The positive effect of fC modifications on nucleosome occupancy and stability observed here has necessitated further studies to gain deeper insights into the biological functions of fC in the nucleosome context. Cryo-EM can be used to elucidate the structural foundation for the changes fC posts to nucleosome, and protein interacting assays will identify the cellular machineries specifically recruited/repulsed by fC-modified nucleosomes. The effect of DNA modifications elucidated by the above studies advances our understanding on the role that DNA modifications play in regulating cellular processes.

Transgenic lines of sugarcane with increased total sugar content have been obtained in previous work in our lab, through the introduction of a sucrose isomerase (SI) gene designed vacuole-targeting of the SI gene product. Some of the resulting transgenic lines accumulate the high value sugar isomaltulose (IM) without decrease in stored sucrose content. Other lines show enhanced sucrose content with low levels of IM in mature storage tissues. Under containment glasshouse conditions, these “SugarBooster™” lines have shown up to two-fold increase in the total sugar concentration in the harvested juice. This remarkable step above the former ceiling in stored sugar concentration may permit new insights into the mechanisms by which plants regulate sugar accumulation, a pivotal question in plant biology. Studies conducted in this thesis are a part of the effort to understand the underlying mechanisms. Experiments were focused on the regulation of SI and endogenous genes related to sucrose metabolism, as revealed by transcript levels. Enzyme activity was also tested for sucrose phosphate synthase (SPS), a key enzyme in plant sucrose biosynthesis. Initially, the relationship between IM content and SI transcript level was analyzed. Real-time quantitative polymerase chain reaction (RT-qPCR) successfully detected SI gene transcripts in internodes of different maturities in SugarBooster™ lines, but no correlation was found between the transcript level and IM content. In contrast, northern blotting to distinguish full-length SI mRNA from degraded transcripts revealed a positive relationship between IM content and functional transcripts of SI. The results imply that stabilization of SI mRNA is important for high IM content but that high sucrose accumulation accompanied by low IM involves other mechanisms. Therefore, transcript levels of several key genes in sucrose metabolism were analyzed in internodes at various developmental stages in SugarBooster™ iv and control lines. These genes encode sucrose phosphate synthase (SPS), sucrose synthase (SuSy), soluble acid invertase (SAI), neutral invertase (NI), cell wall invertase (CWI), a putative sugar transporter type 2a (Type2a) and sucrose transporter type 6 (Type6). Sucrose content was negatively correlated to the transcript levels of SAI, but there were no significant correlations between sugar content and mRNA levels of other tested enzymes across all developmental stages. However, interesting transcript patterns that might contribute to high sucrose accumulation were observed in some lines. Examples from different lines include: SuSy transcripts increased in immature internodes but decreased in mature ones; NI expression decreased in mature internodes and sugar transporters increased in some cases. Further analysis was undertaken on transcript regulation of five SPS families, as SPS is the rate-limiting enzyme for sucrose biosynthesis in some plant systems. As recently reported from a CSIRO analysis of sugarcane progeny segregating for sucrose content, transcripts of SPS family 2 predominated in the stem for both SugarBooster™ and control lines. Transcripts of SPS families 3 and 4 were less abundant and families 1 and 5 were rare. More abundant mRNAs of SPS families 2, 3 and 4 indicate that they play an important role in sucrose accumulation in the stem. However, none of the mRNA levels of the five families had a significant correlation to the SPS enzyme activity or to the sucrose content measured in corresponding tissues. In contrast, SPS enzyme activity was found positively correlated to sucrose content. It revealed that regulation of SPS in sugarcane is mainly at the level of enzyme activity. These studies have been constrained by the availability of experimental material in early vegetative generations of SugarBooster™ lines under containment glasshouse conditions. As the opportunity emerges for more material from approved field trials, it will be important to identify the most stable lines under field conditions, for further studies on metabolites, carbon partitioning and enzyme activity to fill the gap in understanding between regulation at transcript level and the SugarBooster™ phenotypes.

Epigenetics refers to heritable changes in phenotype without alterations to the genotype. Epigenetic changes involve two main mechanisms: DNA methylation and histone modification. Methylation of DNA at cytosine bases is the best-studied epigenetic process to date. CpG methylation states are thought to be maintained throughout cell divisions. However, loss of DNA methylation or DNA demethylation has been observed in specific stages of mammalian development. Such prominent examples of developmental DNA demethylation processes occur in developing primordial germ cells and in preimplantation embryos. However, little is known about DNA methylation changes of other tissues in mammalian development. Therefore, the first aim of this PhD study was to investigate changing nuclear distributions and levels of DNA methylation during development in order to discover dynamic variations amongst developing mouse tissues. In addition, a transgenic MBD-GFP mouse was employed to visualise DNA methylation in tissues. Several hypothetical mechanisms for the enzymatic removal of 5mC have been proposed. One of the proposed candidates is Tet-mediated successive oxidation of 5mC to generate 5hmC, 5fC and 5caC. 5hmC has therefore been considered as a transient intermediate in an active cytosine demethylation pathway. Nevertheless, some studies suggest that 5hmC may also function as an epigenetic modification in its own right. Thus, the second aim of this study was to address the research question of how and where 5hmC originates during development. In order to be able to identify tissues undergoing dynamic nuclear changes in DNA methylation and hydroxymethylation states during early mouse development, new working protocols for immunodetection of 5mC and 5hmC on tissue cryosections were required. The protocol optimisation for 5mC immunodetection is discussed in greater detail in Chapter 3. It was found that DNA methylation immunostaining of cryosections required heat-mediated DNA denaturation, which was partly compatible with protein immunostaining. Next, Chapter 4 focuses on identifying tissues undergoing dynamic changes in 5mC and 5hmC patterns during development from E9.5 to E14.5 mouse embryonic stages, using optimised immunohistochemistry protocols. These protocols revealed interesting dynamic observations of 5mC and 5hmC in the developing cerebral neocortex, surface ectoderm, liver, red blood cells, diaphragm and heart. These findings suggested that dynamic changes of 5mC and 5hmC during neocortical and compact myocardial development were in good agreement with a model where the formation of 5hmC may correlate with the loss of old 5mC, but the observations were also consistent with an involvement of de novo methylation in the generation of 5hmC. In other developing tissues, including surface ectoderm, liver, red blood cells, diaphragm and cardiac trabeculae, dynamic changes in 5mC and 5hmC levels were in line with a model where the 5hmC may act as a new epigenetic mark that functions independently. The optimised protocol also confirmed DNA demethylation of the germ cells at E12.5. The presence of three Tet family enzymes (Tet1, Tet2, Tet3) and de novo methyltransferase DNMT3A in mouse E12.5 tissues is reported in the second part of Chapter 4. It was found that Tet1, Tet2, Tet3 and Dnmt3a were present at detectable levels in neocortex, liver, diaphragm and heart. Contrastingly, no apparent signals for Tet1, Tet2, Tet3 and Dnmt3a were observed in red blood cells. This result was expected due to the very low levels of 5hmC staining in E12.5 red blood cells. The third aim of the present study was to investigate the existence of crosstalk between various epigenetic mechanisms. Thus, Chapter 5 focuses on exploring the relationship between 5mC and repressive histone marks, H3K9me3 and H3K27me3. Histone methylation dynamics at H3K9 and H3K27 were observed during mouse fetal development in neocortex and heart. The overall distribution patterns of H3K9me3 and H3K27me3 demonstrated strong association with developmental changes in 5mC, suggesting that these three repressive epigenetic marks work in concert to establish a silenced state of heterochromatin. Chapter 6, on the other hand, focuses on visualising DNA methylation in tissues using mouse transgenic tools. It was found that brain, liver, heart and neural tube expressed high levels of GFP. But no apparent developmental dynamics of GFP was observed. In conclusion, this study will contribute scientific understanding of dynamic DNA methylation and nuclear heterochromatin organisation during mammalian development, and its role in the specification and maintenance of cell lineages forming tissues and organs. This knowledge will provide insight into current barriers to cell fate reprogramming, which will be of benefit to cell regenerative biomedical technologies.

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This study set out to examine the effectiveness and reliability of six modifications to the Zhang et al. (2004) CPT-based lateral spread model. A regression analysis, distribution charts, and a discriminant analysis are performed to determine how effective the modifications are on the model. From the comparisons and statistical analysis performed in this study, application of these modifications reduces over-predictions from strain-based prediction methods. Unfortunately, the tendency to under-predict displacements on average is also increased.

Lantibiotics like mutacin 1140 are receiving a considerable amount of attention because of their broad spectrum of activity, high potency, low immunogenicity, and good structural stability. Mutacin 1140 is produced by Streptococcus mutans JH1140 and is a type A (I) lantibiotic. Lantibiotics are ribosomally synthesized bacteriocins that undergo post-translational modifications to form lanthionine or β-methyllanthionine rings as well as 2, 3-didehydroalanine (Dha), 2, 3-didehydrobutyrine (Dhb), and S-amino vinyl-D-cysteine (AviCys). Their mode of action is pore formation and/or abduction of lipid II from the site of new cell wall synthesis. In order to gain further knowledge of both the structural integrity and structureunction relationship of type A (I) lantibiotics, chemical modifications or site directed mutagenesis was utilized. In the first aim of this study, two type A (I) lantibiotics were used, nisin A produced by Lactococcus lactis and gallidermin produced by Staphylococcus gallinarium. They both share homology in rings A and B, the lipid II binding domain, with rings A and B of mutacin 1140. What was discovered was that oxidation of the lanthionine rings results in the complete loss of bioactivity due to the loss of affinity for lipid II. Interestingly, the lateral assembly ability of the oxidized variants is not affected. In the second aim, the dehydrated threonine residue (Dhb) at position 14 in gallidermin underwent chemical modification using several thiol-compounds. The results showed that this residue is amendable to modification through thiol chemistry with some loss of bioactivity. However, the MICs for the chemical variants were still in the nanomolar range. From this work the first ever in vivo images of gallidermin were produced. The last aim of the study utilized site directed mutagenesis of the mutA gene of mutacin 1140 to determine the role of various residues in ring A and the hinge region of the peptide. It was determined that neither Trp4, Dha5, nor Arg13 are important for bioactivity but a set distance between rings A and B is essential. The majority of the mutants constructed showed either similar or increased bioactivity.

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