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Many recent studies established a potential role for p53 in translational control of many transcripts through the modulation of the expression of several microRNAs, RNA binding proteins (RBPs), translation factors, or of ribosome biogenesis factors. To establish the relevance of translation control in p53-dependent apoptosis our group recently compared by polysome profiling two cell lines, SJSA1 and HCT116, undergoing respectively p53-dependent apoptosis and cell cycle arrest in response to the treatment with Nutlin-3, a selective activator of p53. Examining the markedly different, treatment-induced translation landscapes, the RNA helicase DHX30 was identified as a protein able to bind only in HCT116 cells to a 3’-UTR sequence motif, labeled as CGPD-motif. The motif had been identified as highly over-represented among translationally enhanced mRNAs in nutlin-treated SJSA1 cells, among which apoptosis effectors were enriched. Since the binding of DHX30 to the CGPD-motif transcripts is observed in cell cycle arresting HCT116, where those mRNA are not translationally enhanced, we reasoned that DHX30 could be part of an RBP complex responsible for reduced translation of specific pro-apoptotic mRNAs. To pursue this hypothesis, HCT116-shDHX30 clones were obtained and I studied the functions of this relatively unknown RNA helicase in this and other cell models. A translatome analysis by RNA-seq of HCT116-shDHX30 cells showed higher activation of ribosome biogenesis pathways, according to GSEA. Consistently, eCLIP data from ENCODE indicate that DHX30 can bind to most ribosomal protein transcripts, and those showed higher relative translation efficiency in HCT116-shDHX30 cells. In fact, the cells exhibited increased rRNA synthesis and higher global translation as well as a large expansion of the number of transcripts showing higher polysome-association in response to Nutlin. The DHX30 gene features two promoters, one of which can produce a transcript containing a mitochondrial localization signal. I confirmed that DHX30 has both a cytoplasmic and a mitochondrial localization by both cell fractionation and immunofluorescence. HCT116_shDHX30 cells have reduced mitochondrial metabolism, based on oxygen consumption rates but do not show evidence of compensatory glycolytic activity. Impaired mitochondrial functions could be related also to decreased expression of mitochondrial oxidative phosphorylation (OXPHOS) components as well as reduced expression of nuclear encoded mitoribosome proteins. Based on RIP assays DHX30 appears to target directly mitoribosome transcripts. Depletion of DHX30 showed reduced proliferation in various assays both in 2D and 3D culture conditions and sensitized cells to the treatment with the mitochondrial uncoupler FCCP. A similar role was observed also on mitochondrially encoded OXPHOS compomenents as well as mitoribosome components in MCF7 breast cancer cell line silenced for DHX30 with a consistent reduction in cell proliferation. In contrast, U2OS osteosarcoma cells did not show a change in the expression in mitochondrial components as well as an impact on proliferation after transient DHX30 silencing. Hence, it appears that DHX30 can exert a broad general control on cell metabolism and translation as well as a cis-element mediated control of the translation of specific mRNAs impacting both on cell fitness and apoptosis.

[Truncated abstract] The mitochondrion is the energy powerhouse that provide energy to many metabolic functions in the form of ATP. Mitochondria in plants are also known to carry out a variety of other important biochemical processes within the cell, including the anaplerotic function of tricarboxylic acid (TCA) cycle, one-carbon metabolism and portions of photorespiration. Dynamics of the mitochondrial proteome in plants underlies fundamental differences in the roles of these organelles under different developmental and environmental conditions. A quantitative comparative proteomic approach was carried out to analyze mitochondria isolated from non-photosynthetic models, cell culture and root, and compared them to mitochondria isolated from photosynthetic shoots. The glycinedependent respiration rate and the protein abundance of the photorespiratory apparatus was found to be higher in shoot than cell culture and root mitochondria. Also, there were major differences in the abundance and/or activities of enzymes in the TCA cycle between the three systems examined. The metabolic pathways that relied on the supply of intermediates from TCA cycle and photorespiration were also altered, namely cysteine, formate and one-carbon metabolism, as well as amino acid metabolism focused on 2-oxoglutarate generation, and branched-chain amino acids degradation. To further provide insight into the extent of mitochondrial heterogeneity in plants, mitochondria isolated from six organ/cell types, leaf, root, cell culture, flower, stem and silique were analyzed. Of the 251 protein spots on a 2D-gel of the mitochondrial soluble/matrix fraction, the abundance of 213 spots were significantly varied between different samples. Identification of these spots revealed a non-redundant set of 79 proteins which were differentially expressed between organ/cell types. ... Importantly, posttranslational modifications played a significant role in the dynamics of the leaf mitochondrial proteome during the diurnal cycle. Overall, these findings indicated that the mitochondrial proteome is dynamic in order to fulfil different functional and physiological requirements in response to organspecific growth and changes in the external environments. These results also indicated that the majority of the changes in the mitochondrial proteome occurred in the matrix and suggested differences in substrate choice/availability in various plant organs and during the diurnal cycle. Further, these analyses demonstrate that, while mitochondrial proteins are regulated transcriptionally by the nucleus, post-transcriptional regulation and/or post-translational modifications play a vital role in modulating the activation state and/or regulation of proteins in key biochemical pathways in plant mitochondria. The integration of proteomics data with respiratory measurements, enzyme assays and transcript datasets will allow the identification of organ-enhanced and/or light/darkresponsive metabolic pathways as well as providing potential targets for reverse genetic approaches for further functional analysis of plant mitochondria.

Late-onset Alzheimer’s disease (LOAD) is the most common form of dementia, with an incidence of up to 50% in western populations over the age of 85 and a high heritability (up to 80%). The identification of risk factors for the development of LOAD is imperative for improving our understanding of this disease and for identifying therapeutic targets for treatment or prevention. Currently, the major known risk factors for the development of LOAD are age and the ApoE ε4 genotype. Previous studies have implicated plasma levels of the amyloid beta (Aß) peptide as a LOAD-associated quantitative trait and identification of loci influencing this trait could provide new insights into LOAD. In this thesis, plasma levels of the Aß peptides Aß40 and Aß42 have been measured in two isolated populations and genome-wide linkage and association analyses were performed. The genome-wide association analyses identified a number of promising quantitative trait loci; highlighting both novel and previously reported LOAD genes for further study, whilst also providing an excellent resource for genetic convergence studies with other LOAD related traits. Several studies have reported an association between levels of oxidative stress and levels of Aß such that increasing levels of Aß appear to increase markers for oxidative stress and vice versa. The role of oxidative stress in LOAD and aging was therefore also investigated through analysis of mitochondrial mutational burden and DNA damage respectively, using DNA isolated from both blood and the brain and by carrying out a candidate gene association study of loci involved in mitochondrial function in LOAD cases and controls. Approaches to the investigation of mitochondrial genetics for the study of LOAD are comprehensively reviewed, adapted and tested and the results indicate a need for additional research in this aspect of the disease. This thesis therefore presents a focus on two aspects of genetic research into LOAD, a complex disease with multiple environmental and genetic influences which aims to advance our understanding of the disease and bring us closer to treatment and prevention strategies.