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Radiation therapy to treat head and neck cancer damages salivary glands, leading to decreased salivary flow which can cause xerostomia (perception of dry mouth), mucositis and dysphagia. These symptoms of salivary gland hypofunction are a major cause of patients stopping their treatment and greatly decrease their quality of life after treatment. Protecting from damage is essential to avoid these problems so this thesis examined some of the damage mechanisms present in salivary glands. Two intracellular processes seem to be particularly relevant. Mammalian Target of Rapamycin (mTOR) is a growth pathway often activated in cancers, repair and regeneration whereas autophagy is a self-digestion of cellular contents often associated with damage that helps to protect cells from apoptosis. Normally the two processes are linked by an enzyme UNC-like kinase (ULK) in a mutually exclusive way so that growth and degradation do not occur at the same time. However, both mTOR and autophagy have been shown to have beneficial effects after irradiation. Therefore, we decided to study the interactions between mTOR and autophagy in order to find an efficient way to uncouple mTOR and autophagy to protect irradiated salivary glands. Since ULK was an important link between mTOR and autophagy, an interesting new ULK inhibitor MRT67307 was used on salivary glands. In the first part of this study, we evaluated the effects of MRT67307 on cell cultures in order to collect enough data before trying the drug in vivo. Initially NIH 3T3 cells, a well-studied cell model, were cultured to verify the effects of MRT67307. As previously reported, the drug blocked starvation- and Torin 1 (an mTOR inhibitor)- induced autophagy. The next step was to test the effects of the drug on salivary acinar cells which were known to be very sensitive to irradiation. SMG-C6 cells were chosen since they were previously derived from rat submandibular acinar cells. In these cells, in contrast to NIH 3T3 cells, Torin 1 failed to upregulate autophagy, suggesting that mTOR and autophagy were not linked by ULK. This finding was interesting and novel and was further tested in primary-cultured cells (ie in vitro) from mouse submandibular glands. Again, administration of Torin 1 inhibited mTOR but did not activate autophagy and MRT67307 had no effect on marker of autophagy (LC3-I/LC3-II ratios). It could be inferred from these experiments that MRT67307 is a useful tool in examining mTOR/autophagy interactions through ULK1 and that in salivary glands autophagy and mTOR could be activated simultaneously. In the second part of this study, we carried out whole body irradiation of mice to study damage, mTOR-autophagy interactions and saliva flow variation in irradiated salivary glands. A dose escalation study appeared to cause minimal damage to salivary glands when the maximum dose of 11 Gy was given. To determine if salivary hypofunction had occurred whole mouth saliva was collected under temporary gaseous anaesthesia by the administration of pilocarpine (I.P.). Surprisingly, despite minimal histological indications of damage an increase in salivary function occurred. Biochemical analyses of the salivary glands indicated autophagy was transiently and weakly activated a few hours after irradiation whereas mTOR activity occurred a few days later. The use of a whole body irradiator limited the dose of irradiation to the salivary glands. Thus as a model system, transient mTOR activation probably had a beneficial effect, since pilocarpine stimulated saliva flow experiment showed a transient increase of saliva flow. However, this model system did not yield the expected salivary gland damage seen in other studies so instead another model of salivary gland damage, ductal ligation was studied. The third part of this study attempted to use autophagy-inhibitors in vivo, using the ligated salivary gland since autophagy activation was weak and did not last in irradiated salivary gland. Whole body injection of autophagy inhibitors chloroquine and MRT67307 (at two different doses and injection intervals) did not appear to have any beneficial effect on submandibular glands, except a slight delay of atrophy in both chloroquine and MRT67307 treated glands. Autophagy appeared to be mainly mTOR independent since MRT67307 failed to inhibit autophagy. This thesis contains novel data to indicate that autophagy and mTOR are independent of each in mouse submandibular glands. To the best of our knowledge, this is the first time that MRT67307 was used in vivo and no paper demonstrates an mTOR independent activation of autophagy in salivary glands. It provides the basis for further studies to protect salivary glands from irradiation damage by upregulating both mTOR and autophagy simultaneously, something that has not, so far, been tested.

This thesis seeks to examine and explain changes in the triangular relationship between Christian missionaries, an Indian princely state and the British colonial authorities. It argues that, in the second half of the nineteenth century, the missionaries and the self-declared Hindu state of Travancore maintained a largely favourable relationship despite the clear differences in their respective religious positions. For its vigorous efforts towards 'modernization', Travancore needed the educational and medical activities of the missionaries. At the same time, the Madras government demanded the abolition of caste disabilities and supported similar demands from the missionaries. However, the situation changed significantly with the emergence of Hindu revivalism and communal movements in the late nineteenth century. The state as well as the higher castes became alarmed at the conversion of large number of the lower castes to Christianity. The British authorities, faced with the rise of Indian nationalism, became much more sensitive to religious feelings in Travancore and sought to avoid intervening in social and religious matters as far as possible, though, by contrast, intervention in Cochin remained, for various reasons, more active. In this way, the missionaries practically lost support from the British government. Instead, they were obliged to pay more attention to the Maharaja and his government, which adopted various anti-missionary policies in the 1890s and 1900s. Nevertheless, the Travancore government still needed missionary educational and medical activities as well as their strong influence over the low-caste Christians who became increasingly assertive. But in the 1930s, their relationship became strained once more. The missionaries, who could not expect any substantial help from the British authorities, had almost no choice but to accept the situation. In this way, the Travancore state, while utilizing missionary activities, largely succeeded in preventing Christian influence from expanding further.

In the fifteen years since the tumor suppressor phosphatase PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) was initially discovered in the search for the enzyme that dephosphorylates Akt at the hydrophobic motif (Ser473), a number of new substrates of PHLPP1 have been identified, suggesting a broad and varied role for this newly classified serine/threonine phosphatase. Not surprisingly, deregulation of PHLPP1 signaling has been implicated in a diverse set of disease states, ranging from cancer to diabetes to inflammation. However, there was still progress to be made to better understand how PHLPP1 itself is regulated. This thesis expands on the knowledge of how PHLPP1 localization, expression, protein interactions, and function are all dynamically regulated in the cell. First, mechanistic studies reveal that the previously unstudied PHLPP1 N-terminal extension, which accounts for the first roughly 30% of the protein sequence, is dynamically regulated by phosphorylation during mitosis and is required for a mitotic “switch” in the PHLPP1 interactome. Second, biochemical analysis of this N-terminal extension revealed that it houses a nuclear localization signal, allowing it to control PHLPP1 localization and function in the context of inflammatory signaling pathways. Finally, these studies exposed that PHLPP1 expression levels are dramatically reduced in the absence of extracellular glutamine, suggesting that PHLPP1 expression is sensitive to environmental nutrient levels. Taken together, these studies not only provide novel insight into how PHLPP1 functions in a normal physiological context but also how its regulation can go awry in various disease states.

Using the budding yeast, Saccharomyces cerevisiae as our model system we set out to investigate the role of nonsense-mediated mRNA decay (NMD) pathway in the quality control of premature translation termination codon (PTC) containing transcripts to include those resulting from splicing factor mutations as well as unproductive alternative splicing events. The function of many splicing factors in pre-mRNA splicing and their involvement in the processing of a specific subset of transcripts has often been defined through loss of function analysis. We show that NMD can mask some of the effects of splicing factor mutations, and the full role of the splicing factor cannot be understood unless the RNA degradation system that degrades unspliced precursors are also inactivated. Tiling microarrays showed that inactivation of the NMD factor Upf1p in combination with splicing factor mutants prp17Δ and prp18Δ resulted in a larger spectrum of splicing defects than in the single mutants. Analysis of these double mutants also hinted to the possibility of non-productive alternative splicing. This lead us to seek out and identify splicing defects arising from alternative splice site selection through RNA-Sequencing analysis of wild-type and NMD mutant (upf1Δ, upf2Δ, and upf3Δ) strains. We found that a large fraction of intron containing genes exhibit alternative splicing, but are masked by NMD because they generate PTCs. Analysis of splicing factor mutations combined with upf1Δ revealed the role of specific splicing factors in governing the use of these alternative splice sites. Furthermore, we show the use of a non-productive alternative 5' splice site in RPL22B to be regulated during conditions of stress. These studies bring to light an unexpected flexibility of the spliceosome in splice site selection and the role of NMD in limiting the accumulation of these erroneous transcripts. Finally, we identified a small subset of two intron containing genes whose erroneous transcripts are not targeted to NMD as expected, but rather degraded by the nuclear turnover system. While most S. cerevisiae intron containing genes have only one intron, there are a few numbers of genes that contain two small introns; amongst these are the genes MATa1, DYN2, SUS1, and YOS1. The degradation of the exon2 skipped forms of these transcripts was found to be dependent on the nuclear RNA turnover pathway consisting of the 5' to 3' exonuclease Rat1p and the nuclear exosome. MATa1 was additionally found to be cleaved by the nuclear RNase III endonuclease Rnt1p. These findings show that nuclear degradation mechanisms have also evolved to complement the role of NMD to limit the accumulation of mRNAs that are erroneously spliced by the splicing machinery.