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Protein synthesis is one of the multiple levels at which gene expression is regulated. The rate-limiting steps of protein synthesis occur during initiation. The binding of the ribosome to the mRNA in translation initiation is catalyzed by the proteins of the eukaryotic initiation factor 4 (eIF4) group. In mammals, the mRNA cap-binding protein eIF4E is present in limiting levels and is regulated by several mechanisms. This thesis examines the regulation of eIF4E during the development of the genetically tractable organism, Drosophila melanogaster. A Drosophila eIF4E gene was cloned, its position was mapped cytologically, and this gene was shown to encode two cap-binding protein isoforms via alternative splicing. Antisera specific to the eIF4E isoforms were raised and purified to characterize the expression of eIF4E during development. Several mutant alleles of eIF4E were identified and demonstrate that this gene is essential for the viability of Drosophila. Furthermore, eIF4E mutants arrest in growth during early larval stages. The lethality and growth defects of eIF4E mutant alleles were rescued by a transgene containing a wild-type copy of eIF4E expressed under the control of its endogenous promoter. Ser251 of Drosophila eIF4E is in a sequence context identical to site on which eIF4E is phosphorylated in response to extracellular stimuli in other organisms. To examine the biological significance of the phosphorylation of eIF4E, transgenic flies were generated in which Ser251 was mutated. We show that eIF4E from Ser251 mutant lines cannot incorporate labeled orthophosphate. Interestingly, flies in which the only source of eIF4E is non-phosphorylatable are semi-lethal and escapers are small in size. These results are evidence that Ser251 of eIF4E is required for the normal growth of a multicellular organism.

The low cycle thermo-mechanical fatigue of laser beam welded conventionally cast Inconel 738, directionally solidified René 80 and single crystal René N5 has been evaluated. Results have been compared to gas tungsten arc and baseline alloy conditions. Metallographic examination of laser beam welds and the associated heat affected zone were conducted by scanning electron microscopy and energy dispersive spectroscopy. The impact of laser beam welding on thermo-mechanical fatigue properties of Inconel 738, René 80 and René N5 has been evaluated and recommendations for improvements and areas of further research have been presented.

All bacteria initiate translation using formylated methionine, yet directly after translation, the formyl-group is removed. This sequence of addition and removal appears futile, yet every sequenced bacterial genome encodes the enzymes for formylation and deformylation, suggesting this process is essential. Puzzlingly, the process is absent from both Archaea and Eukaryotes, and moreover, bacterial mutants lacking both the formylase and deformylase activities are viable, albeit with a diminished growth rate. We created an Escherichia coli strain devoid of formylase and deformylase activity. This strain was then allowed to evolve over 1500 generations whereupon it reached wild-type growth rate, demonstrating that formylation can be completely dispensed with. This raises an additional question: if the formylation cycle is unnecessary, how did it emerge and why has it persisted? Our results show that the formylation-deformylation cycle could have evolved as a toxin-antitoxin pair (TA) with post-segregational killing (PSK) activity. TAs ‘addict’ cells to the plasmids that carry them by inducing PSK. We measured the stability of formylase-deformylase encoding plasmids and their ability to elicit PSK in our evolved E. coli strain. We report several lines of evidence consistent with the formylation-cycle having evolved from a plasmid-borne PSK element: 1) in the absence of deformylation, formyl-methionine on proteins is cytotoxic in bacteria 2) deformylation relieves the cytotoxicity of formyl-methionine, 3) the loss of a plasmid containing formylase and deformylase genes from evolved cells results in cessation of growth – a standard PSK phenotype. In addition, we introduced the E. coli formylase and deformylase genes into yeast and demonstrate that Met-tRNA formylation is not lethal, even in the absence of deformylation. This suggests PSK would be ineffectual in yeast, accounting for the absence of formylation from eukaryotic cytoplasmic translation. We also report the presence of formylase and deformylase genes in the two representative members of the archaeal Methanocopusculum genus. Moreover, we demonstrate that these genes have been acquired by a recent horizontal gene transfer from bacteria. Our results indicate that formylmethionine use in bacteria evolved, not through a direct functional benefit to cells, but through competition between infectious genetic elements.

The Tracks rites of passage are processes that mark the adolescent transition, for the participant, the family and the community, between the two life stages of childhood and adulthood. Adolescent initiation rites offer a community led journey of separation, transition and integration as a way to work meaningfully with adolescents as they move between the life stages of childhood into adulthood. In Aotearoa/New Zealand the Tracks programme provides a five day contemporary rite of passage for adolescents and, where possible, their fathers. The rite of passage is based on the assumption that adolescents need opportunities to find their voices and make meaning if they are to become more aware of who they are and where they belong. The methodology recognises that I, as researcher and insider in the Tracks organisation, needed to develop a holistic approach to insider research so that I could call on my understandings of the organisation and also guard against bias. The holistic approach involves the four interpenetrating strategies of appreciative inquiry, narrative inquiry, a blend of approaches to self-study that include meditation and critical reflection, and most importantly organic inquiry. The four strategies are based on coherence theories that describe learning as being organic, interconnected and emergent. Data were gathered from interviews and cycles of critical self-reflection in the form of a learning journal. Data comes from interviews with the mother or fathers and young men of six families who have participated in the Tracks rite of passage programme. I have also discussed this work with a number of professionals in the field of youth work. The project found that Tracks had created conditions that empowered these young men with an increased capacity to make sense of their lives. Fathers expressed how challenging and rewarding they had found it to speak in honest terms with their sons, and that they were supported to do the inner work necessary to be able to speak in such ways. All of the family members expressed a need to have more support after the event. The findings suggest a need to explore further the nature of the work happening at Tracks. It validates Lashlie’s (2005) theory that adolescents need their fathers and other men to be involved in their lives at the time of transition. Tracks also helps fathers to get to grips with the inner work of developing emotional maturity. The work happening at Tracks invites further research into and debate on the value of emotional intelligence. The Tracks rite of passage offers an alternative perspective to understand the unacceptably high rates of adolescent morbidity and mortality happening in New Zealand.

The single crystal superalloy PWA 1484 is used in hot section turbine blade applications due to its performance at high temperatures. In practice, the turbine blades are often coated in order to protect them from environmental degradation. However, under repeated cyclic loading, the coating may serve as a site for crack initiation in the blades. Fundamental out-of-phase (OP) thermo-mechanical fatigue (TMF) studies, primarily using uncoated solid cylindrical test samples, have previously examined both crack initiation and propagation in PWA 1484. In this work, mechanical strain-controlled OP TMF tests were performed on coated and uncoated specimens of a hollow cylindrical geometry in order to study the effects of both geometry and coating on the TMF crack initiation behavior. To accomplish this, it was necessary to create and analyze a modified gripping mechanism due to the unique geometry of the test samples, and as predicted by hand calculations and finite element analysis, these modifications proved to be successful. The TMF test results for the uncoated material were compared to those from previous studies under the same testing conditions, and it was found that the differences in geometry had a minimal impact on fatigue life. Comparisons of the results for the coated and uncoated material suggested that the coating may have offered a slight improvement in life, although insufficient results were available to determine whether these differences were statistically significant. Damage mechanisms resulting from different test conditions were also observed through microscopy on failed specimens.

Nickel based alloys have been developed as a material offering superior general and localized corrosion resistance compared to the more traditionally used in chemical and oil plant in the most aggressive environment such as hydrochloric acid and ferric chloride. Hence the addition of Cr and Mo to Ni creates alloys with exceptional corrosion resistance in a diverse range of environments. This study examines the roles of Cr and Mo in the corrosion behavior of Ni alloys. The performance of three nickel-base alloys IN600, IN601 and C22 was examined in increasing saline solution severity of sodium chloride, concentrated hydrochloric acid and ferric chloride solution at pH = 0.0. The passive corrosion and breakdown behavior of these alloys suggests that Cr is the primary element influencing general corrosion resistance, while the repassivation potential is strongly dependent on the Mo content. This indicates that Cr plays a strong role in maintaining the passivity of the alloy, while Mo acts to stabilize the passive film after a localized breakdown event. Corrosion fatigue test results indicate that fatigue life of IN600, IN601 and C22 specimens tested in 3.5 % sodium chloride solution are essentially the same as for specimens tested in air. Test results also showed that for IN600, IN601 and C22 alloys, the number of cycles to failure was highest in air and sodium chloride solution, followed by specimens fatigued in hydrochloric acid, and was least in specimens fatigued in ferric chloride solution. No evidence of surface pitting was found on C22 specimens in all three solutions whereas IN600 and IN601 were both pitted. However, pits were generally larger in IN600 likely due to lower Cr content than in IN601.

Nickel based alloys have been developed as a material offering superior general and localized corrosion resistance compared to the more traditionally used in chemical and oil plant in the most aggressive environment such as hydrochloric acid and ferric chloride. Hence the addition of Cr and Mo to Ni creates alloys with exceptional corrosion resistance in a diverse range of environments. This study examines the roles of Cr and Mo in the corrosion behavior of Ni alloys. The performance of three nickel-base alloys IN600, IN601 and C22 was examined in increasing saline solution severity of sodium chloride, concentrated hydrochloric acid and ferric chloride solution at pH = 0.0. The passive corrosion and breakdown behavior of these alloys suggests that Cr is the primary element influencing general corrosion resistance, while the repassivation potential is strongly dependent on the Mo content. This indicates that Cr plays a strong role in maintaining the passivity of the alloy, while Mo acts to stabilize the passive film after a localized breakdown event. Corrosion fatigue test results indicate that fatigue life of IN600, IN601 and C22 specimens tested in 3.5 % sodium chloride solution are essentially the same as for specimens tested in air. Test results also showed that for IN600, IN601 and C22 alloys, the number of cycles to failure was highest in air and sodium chloride solution, followed by specimens fatigued in hydrochloric acid, and was least in specimens fatigued in ferric chloride solution. No evidence of surface pitting was found on C22 specimens in all three solutions whereas IN600 and IN601 were both pitted. However, pits were generally larger in IN600 likely due to lower Cr content than in IN601.

Scaffolding proteins control the localization of protein kinases. During translation, the scaffolding proteins eIF4G and DAP5 recruit the Ser/Thr kinase Mnk1 to phosphorylate the mRNA cap-binding protein eIF4E and modulate translation. Biochemical deletion analysis previously showed that the interaction between Mnk1 and eIF4G/DAP5 is mediated by N-terminal residues in Mnk1 and C-terminal residues in eIF4G/DAP5. Using X-ray crystallography I have determined the structure (1.5 A) of the C-terminal domain of DAP5 (DAP5C). This structure reveals that DAP5C contains two atypical HEAT domains similar to the ones seen previously in the structure of the C-terminal region of eIF4G (4GC). Using ITC I showed that the Kd for the interaction between the N-terminus ofMnk1 and 4GCIDAPSC is 20 muM and 10 muM, respectively. Using NMR chemical shifts we have mapped the residues on both Mnk1 and 4GC/DAP5C which are important for maintaining this interaction. Finally, using SAXS a low resolution configuration of the hMnk1-4GC complex was modeled. It is hoped that an understanding of the structural basis for the recruitment of protein kinases to their sites of action will allow the design of small-molecule compounds that can be used to modulate the location of the kinase and hence its activity.

Initiation of DNA replication requires the action of the Dbf4/Cdc7 kinase complex (DDK) which is also a phosphorylation target of Rad53 kinase in the S-phase checkpoint. DDK is thought to trigger DNA replication by phosphorylating members of the Mcm2-7 complex present at origins of replication. While DDK phosphorylation sites have been identified on Mcm2-7, the contributions made by Dbf4 and Cdc7 to the targeting of the complex have not been established. DDK has also been implicated in the S-phase checkpoint response since it is removed from chromatin in a Rad53-dependent manner. The interaction of Dbf4 and Cdc7 with each of the Mcm2-7 subunits was assessed and showed an interaction between Dbf4 and Mcm2 and Mcm6, while interactions between Cdc7 and Mcm4 and Mcm5 were observed. Mutations in Mcm2 and Mcm4 that disrupt the interactions with Dbf4 or Cdc7 showed modest growth impairment and compromised DNA replication, while simultaneous abrogation of both interactions resulted in lethality. Strains overexpressing Mcm2 or Mcm4 were sensitive to genotoxic agents, while overexpression of Mcm2 in a Mcm4Δ175-333 strain background resulted in a severe growth impairment as well as sensitivity to genotoxic stress. ChIP analysis revealed the possibility of Dbf4/Cdc7 localization to origin flanking regions through most of S-phase, which may redistribute to origins at the time of firing. Fluorescence microscopy of Mcm2 and Dbf4 in S-phase seem to show a punctate pattern of staining, consistent with these factors’ localization to ‘replication factories.’ By using a Dbf4ΔN mutant, the N-motif was shown to be required for the Rad53-mediated removal of Dbf4 from chromatin under checkpoint conditions. Initial optimization of a DNA combing protocol was also performed, which along with Dbf4ΔN mutant and the fluorescently-epitope tagged strains, will be useful tools for evaluating a role for DDK in the S-phase checkpoint response. Altered levels of DNA replication factors have been implicated in many human cancers. The data presented in this study provide novel insight into the normal process of the initiation of DNA replication which can be applied to research involving higher eukaryotes, including humans, and can serve as a benchmark for comparison with the cancer phenotype.