Ribosome synthesis is a very complex and energy consuming process in which pre-ribosomal RNA (pre-rRNA) processing and folding events, sequential binding of ribosomal proteins and the input of approximately 200 trans-acting ribosome assembly factors need to be tightly coordinated. In the yeast Saccharomyces cerevisiae, ribosome assembly starts in the nucleolus with the formation of a very large 90S-sized complex. This ~2.2MDa pre-ribosomal complex is subsequently processed into the 40S and 60S assembly intermediates (pre-40S and pre-60S), which subsequently mature largely independently. Although we have a fairly complete picture of the protein composition of these pre-ribosomes, still very little is known about the rRNA structural rearrangements that take place during the assembly of the 40S and 60S subunits and the role of the ribosome assembly factors in this process. To address this, the Granneman lab developed a method called ChemModSeq, which made it possible to generate nucleotide resolution maps of RNA flexibility in ribonucleoprotein complexes by combining SHAPE chemical probing, high-throughput sequencing and statistical modelling. By applying ChemModSeq to ribosome assembly intermediates, we were able to obtain nucleotide resolution insights into rRNA structural rearrangements during late (cytoplasmic) stages of 40S assembly and for the early (nucleolar) stages of 60S assembly. The results revealed structurally distinct cytoplasmic pre-40S particles in which rRNA restructuring events coincide with the hierarchical dissociation of assembly factors. These rearrangements are required to trigger stable incorporation of a number of ribosomal proteins and the completion of the head domain. Rps17, one of the ribosomal proteins that fully assembled into pre-40S complexes only at a later assembly stage, was further characterized. Surprisingly, my ChemModSeq analyses of nucleolar pre-60S complexes indicated that most of the rRNA folding steps take place at a very specific stage of maturation. One of the most striking observations was the stabilization of 5.8S pre-rRNA region, which coincided with the dissociation of the assembly factor Rrp5 and stable incorporation of a number of ribosomal proteins.

Obesity is associated with greater rates of cardiovascular disease, dyslipidemia and dysfunctional lipid metabolism. Exercise may provide an effective therapeutic tool to ameliorate dyslipidemia. However, how exercise attenuates dyslipidemia with obesity is not fully understood. Additionally, whether acute exercise or exercise training is the primary driver of such changes in this population is unknown. Furthermore, mechanisms mediating these exercise responses are not elucidated. The peroxisome proliferator-activated receptors (PPARs) provide a likely mechanism through enhanced expression of oxidative metabolism and cholesterol transport proteins augmenting fatty acid oxidation and cholesterol transport. Study one describes blood lipid and lipoprotein responses to acute aerobic exercise and exercise training in obese men and women. The primary measured effects include: increased HDL-C in men following 12 wks exercise training, and a shift from HDL3-C to HDL2-C, with concomitantly reduced HDL-C mean density and LDL3-C in women. Acute exercise of 400 kcal duration performed before and after training, yielded a decreased TC: HDL-C ratio in men, which was unaffected by training. Thus, the primary exercise-based treatment for dyslipidemia with obesity appears to be exercise training. In study two, PPARδ and PGC-1α content were significantly enhanced after acute exercise, whereas PPARα and AMPKα content were augmented only after training. These effects were seen with concomitantly increased content of target proteins involved in oxidative and lipoprotein metabolism including lipoprotein lipase, CPT-I, COX-IV, and FAT/CD36. PPARδ expression was correlated with total and LDL-cholesterol concentrations. AMPKα expression was correlated with the concentration of HDL-C and its subfractions, suggesting regulation of blood cholesterols by PPARδ and AMPKα. Study three demonstrates comparative responses to high volume resistance exercise (RE) in lean and obese Zucker rats. RE enhanced PPARδ expression regardless of phenotype, but PGC-1α in obese only. Mitochondrial biogenesis was enhanced in lean animals only, indicating PPARδ and PGC-1α content is disconnected from mitochondrial biogenesis with obesity. These studies enhance our understanding of exercise as a therapeutic tool in treating dyslipidemia and dysregulated lipid metabolism often associated with obesity. They further demonstrate the necessity for exercise training to attenuate dyslipidemia, while illustrating PPAR-mediated augmentations in oxidative and lipoprotein metabolism following exercise with obesity.

Ribosomes are universally conserved macromolecular machines that translate cellular genetic information into proteins. All ribosomes are com- posed of two ribonucleoprotein subunits. In eukaryotes these are called 40S (small) and 60S (large) subunits. Biogenesis of both subunits begins from a common precursor ribosomal RNA (rRNA) transcript in the nucleolus. The 18S rRNA of the small subunit is encoded in the 5ʹ end of the precursor transcript. U3 snoRNA and about 70 accessory factors associate with the 50 end of the pre-rRNA, to form the SSU processome or 90S pre-ribosome, which can be observed as terminal knobs in electron micrographs. After the initial processing and folding, the pre-rRNA is cleaved at site A2 to release the pre--40S. This event is dependent on the formation of the central pseudoknot, a structure that maintains the integrity of 40S architecture. Bud23 is the methyltransferase responsible for modification of the base G1575 in the P-site of the small subunit. Work presented here demonstrates that the in vivo stability, and thus function, of Bud23 is dependent on the presence of Trm112, a novel ribosome biogenesis factor identified in this work. Analysis of rRNA processing and strong negative genetic interactions with RNaseMRP mutants, provide strong evidence for that BUD23 is required for A2 cleavage. Extragenic suppressors of bud23 [delta] were identified in UTP14, UTP2, IMP4 and ECM16, coding for SSU processome components. Bud23 and the RNA helicase Ecm16 interact physically as well as genetically. Most fascinatingly, using ecm16 enzymatic mutants, this work provides compelling evidence that Ecm16 facilitates removal of U3 snoRNA from pre-rRNA, a prerequisite for central pseudoknot formation and 90S to pre--40S transition. These findings suggest a model in which binding of Bud23 monitors the status of 40S assembly, triggering Ecm16 activity to promote release of the pre--40S from 90S only after the critical folding of the small subunit rRNA. / text

The thesis focuses on the role of Psb28 proteins, namely the Psb28-1 and its homolog Psb28-2, in the biogenesis of the Photosystem II complex (PSII) in the cyanobacterium Synechocystis PCC 6803. The aims of this work were to localize the proteins within the cells, and to determine their function. A fraction of both Psb28 proteins was identified in the monomeric PSII core complexes but most proteins were found in the unassembled protein fraction associated with thylakoid membranes. Psb28-1 was mostly detected as a dimer while Psb28-2 as a monomer. Psb28-1 also differed from Psb28-2 by its higher affinity to the PSII core complex lacking CP43 antenna. Characterization of Psb28-less mutants suggested regulatory function of the proteins in PSII biogenesis in connection with chlorophyll biosynthetic pathway. Analysis of preparations isolated using FLAG-tagged versions of Psb28 proteins showed their association with Photosystem II - Photosystem I supercomplexes, especially under increased irradiance, and supported a role of Photosystem I in the PSII biogenesis.

Mitochondria not only supply the energy for cell function, but also take part in cell signaling. This review describes the dysfunctions of mitochondria in aging and neurodegenerative diseases, and the signaling pathways leading to mitochondrial biogenesis (including PGC-1 family proteins, SIRT1, AMPK) and mitophagy (parkin-Pink1 pathway). Understanding the regulation of these mitochondrial pathways may be beneficial in finding pharmacological approaches or lifestyle changes (caloric restrict or exercise) to modulate mitochondrial biogenesis and/or to activate mitophagy for the removal of damaged mitochondria, thus reducing the onset and/or severity of neurodegenerative diseases.

The widespread emergence of antibiotic resistance determinants for nearly all drug classes threatens human health on a global scale. It is therefore essential to discover antibiotics with novel functions that are less likely to be influenced by pre-existing resistance mechanisms. An emerging approach to identify inhibitors of investigator-defined cellular processes involves screening compounds for antimicrobial activity under non-standard growth conditions. Indeed, by growing cells under conditions of stress, inhibitors of specific cellular targets can be enriched, thereby allowing for the identification of molecules with predictable activities in the complex environment of the cell. Here, I exploit cold stress in Escherichia coli to identify molecules targeting ribosome biogenesis and outer membrane biosynthesis. First, through a screen of 30,000 small molecules for growth inhibition exclusively at 15°C, I was able to identify the first small molecule inhibitor of bacterial ribosome biogenesis, lamotrigine. Second, by leveraging the idiosyncratic cold sensitivity of E. coli to vancomycin, I developed a novel screening technology designed to enrich for non-lethal inhibitors of Gram- negative outer membrane biosynthesis. From this platform, I identified pentamidine as an efficient outer membrane perturbant that was able to potentiate Gram-positive antibiotics against Gram-negative pathogens, similar to the polymyxins. Remarkably, however, this compound was able to overcome mcr-1 mediated polymyxin resistance. Together, this thesis highlights the utility of exploiting the bacterial cold stress response in antibiotic discovery. / Thesis / Doctor of Philosophy (PhD)

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Lysosomes are membrane-bound organelles that have diverse functions in eukaryotic cells. Malfunctions in lysosomes result in a range of diseases known as Lysosomal Storage Disorders. After fusing with late endosomes to form hybrid organelles, lysosomes bud off and are reformed in a poorly characterized process known as lysosome formation or reformation. Only one mammalian regulator of lysosome formation has been identified, the non-selective cation channel TRPML1. In the highly similar process of Autophagic Lysosome Reformation (ALR), three known regulators have also been identified, the vesicle-coating protein clathrin and two phosphatidylinositol kinases that catalyze the formation of the membrane phospholipid PI(4,5)P₂. Here, we use an inhibitor screen coupled with a live imaging assay to identify the actin microfilament as a novel regulator of lysosome formation.

A major step in the biosynthesis of many membrane proteins is their insertion into the membrane of the endoplasmic reticulum (ER). The insertion of a multi-spanning membrane protein is a complex process since several transmembrane (TM) domains have to be correctly integrated in order to enable its correct assembly. At present it is unclear how the integration of multiple TM domains is co-ordinated by the ER translocon. The aim of this study was to analyse the molecular environment of the TM domains of a model seven TM domain protein, opsin, so as to better understand the mechanism by which integration occurs. For this purpose, stable 'integration intermediates' of defined lengths representing distinct stages of opsin biosynthesis were generated by in vitro translation of truncated mRNA in the presence of semi-permeabilised cells. Cysteine-mediated, site-specific cross-linking and immunoprecipitation were employed to examine the environment of these integration intermediates. In addition, cysteine-specific modification reagents with different physical properties were used to investigate the environment of opsin TM3 during its insertion at the ER membrane. Opsin TM domains exhibit unique patterns of adduct formation with the ER translocon components, Sec61α and Sec61β. TM1 associates with the Sec61 complex at two distinct stages during nascent chain extension, and this behaviour is dependent on the presence of subsequent TM domains. The re-association of TM1 with the transloconmay well facilitate the co-ordinated integration of TMs 1-3 into the lipid bilayer. Opsin TM4 exits the Sec61 complex as soon as the subsequent TM domain is synthesised, while TM5, TM6 and TM7 remain associated with the ER translocon throughout protein synthesis, suggesting their concerted release upon chain termination. Evidence is provided that opsin is integrated via a single Sec61 heterotrimer, despite the fact that the ER translocon appears to consist of multiple copies of the Sec61 complex. On the basis of this work, a model is presented describing the complete integration of opsin at the ER membrane.