Transcriptional regulation during appressorium formation and function in Glomerella cingulata : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biology at Massey University, Palmerston North, New Zealand

Glomerella cingulata, anamorph Colletotrichum gloeosporioides, causes bitter rot in apples and fruit rot in other subtropical fruits. In response to environmental cues such as contact with the host, Glomerella cingulata forms a special structure called an appressorium, which accumulates glycerol and thereby generates a sufficiently high turgor pressure to push an infection peg into the host tissue. It is known that the cAMP and MAPK signalling transduction pathways control appressorium formation and function in Colletotrichum species and other appressorium-forming fungi. This process is accompanied by a global change in gene expression. Little is known of transcriptional regulation during this process. The aim of this project was to study the transcriptional regulation of appressorium formation and function in G. cingulata. The G cingulata SAP gene had previously been shown to be expressed as a longer transcript during the early stage of appressorium differentiation. It was considered possible that the transcription factors that regulated expression of the longer transcript may be also involved in the regulation of appressorium differentiation. Identification of the transcription factor involved may help to understand the mechanisms that regulate appressorium differentiation. The plan was to use the yeast one-hybrid system to isolate the transcription factor. This required identification of the promoter regions responsible for expression of the longer SAP transcript. Therefore, the G. cingulata SAP promoter was characterized by mapping the transcription start point. Three transcription start points were determined by RLM-RACE. To further characterise the promoters, SAP-GFP reporter plasmids were constructed and transformed into G. cingulata. Even though a reasonable level of GFP expression was observed in RT-PCR experiments, however, no differences in fluorescence intensity were seen between the wild type and GFP reporter transformants. Therefore, no further attempts to study the sap promoter were made. The candidate gene approach was chosen as an alternative way to study the transcriptional regulation of appressorium formation and function in G. cingulata. The G. cingulata StuA gene was cloned using degenerate PCR, single specific primer PCR, subgenomic library screening and plasmid rescue from a disruption mutant. Targeted gene deletion of the G. cingulata StuA gene was successful. Deletion mutants display many phenotypic changes. Complementation mutants were constructed to confirm the function of this gene. A full length copy of this gene together with a second selection marker was reintroduced into the deletion mutant and the wild type phenotype was restored. Deletion mutants form appressoria at the normal rate and with unaltered morphology. In comparison with the wild type, these appressoria did not generate high turgor pressure as shown by a cytorrhysis assay. This resulted in a defect in appressorium penetration of onion epidermal cells. Nor were these mutants able to invade unwounded apples. Therefore, the G. cingulata StuA gene is required for appressorium function. In addition, deletion mutants displayed stunted aerial hyphae, "wettable" mycelium, reduced conidia production, and a defect in conidiophore and perithecium formation. These results suggested that the G. cingulata StuA gene has multiple roles in fungal development

Isolation and characterisation of the 5' region sequence for the bovine ATP-citrate lyase gene : a thesis presented in fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

ATP-citrate lyase (ACL) is one of the major lipogenic enzymes. It catalyzes the synthesis of acetyl-CoA from citrate in the cytosol. This is the first committed step towards the conversion of carbohydrate precursors into fatty acids. Acetyl-CoA serves as the major precursor for lipogenesis and cholestogenesis. Examination of this pathway shows that the rate of fatty acid synthesis from glucose is dependent on the activity of ACL. In rats the activity of this enzyme can be increased by feeding high carbohydrate diet and reduced to low levels by fasting. These changes are regulated at the transcriptional level. The ruminant provides a good model to study the regulation of expression of ACL. The levels of this enzyme are high in young ruminants, but fall to very low levels once a functional rumen is developed. In adult ruminants, acetyl-CoA for fatty acid synthesis is produced directly from acetate formed by microbial fermentation in the rumen and carried to the peripheral tissues. The down-regulation of this enzyme can be reversed by the administration of glucogenic precursors by a route that bypasses their fermentation to volatile fatty acids in the rumen. An understanding of the regulation of expression of ACL in the adult ruminant and a comparison with monogastric animals will provide significant new information about the regulation of the conversion of carbohydrate into fat. A probe containing exon 2 to exon 3 of the rat ACL gene was prepared. Its specificity to bovine genomic DNA was verified and the probe was then used to screen a bovine λ genomic library. A 17 kb clone was isolated. The restriction map of this clone was determined with several enzymes. A part of this clone (9490 base pairs) was sequenced and shown to consist of a 3 kb promoter region and doenstream seqence as far as intron 3 of bovine ACL. The transcription start sites were determined by 5'RACE. Several important features of this gene were discovered by computer analysis of the sequence. Two key transcription factor binding sites were found in the promoter region. This work provided a solid basis for further investigation towards elucidating the mechanism of the transcriptional regulation of bovine ACL and the process of lipogenesis

Frequency-Aware Contrastive Learning for Neural Machine Translation

Low-frequency word prediction remains a challenge in modern neural machine translation (NMT) systems. Recent adaptive training methods promote the output of infrequent words by emphasizing their weights in the overall training objectives. Despite the improved recall of low-frequency words, their prediction precision is unexpectedly hindered by the adaptive objectives. Inspired by the observation that low-frequency words form a more compact embedding space, we tackle this challenge from a representation learning perspective. Specifically, we propose a frequency-aware token-level contrastive learning method, in which the hidden state of each decoding step is pushed away from the counterparts of other target words, in a soft contrastive way based on the corresponding word frequencies. We conduct experiments on widely used NIST Chinese-English and WMT14 English-German translation tasks. Empirical results show that our proposed methods can not only significantly improve the translation quality but also enhance lexical diversity and optimize word representation space. Further investigation reveals that, comparing with related adaptive training strategies, the superiority of our method on low-frequency word prediction lies in the robustness of token-level recall across different frequencies without sacrificing precision