The role of formula diets with different macronutrient composition in the treatment of obesity

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Identifying the molecular function of MacroD2 and TARG1

Human cells need to react to environmental stimuli in a timely manner. One way to achieve this fast reaction time is the attachment of small chemical or biological units to proteins, so-called post-translational modifications. Post-translational modifications are added, read, and removed by different sets of enzymes dependent on the type of post-translational modification. One type of post-translational modification is ADP-ribosylation, a modification where either single or multiple units of ADP-ribose are added to proteins by a family of enzymes called PARPs. ADP-ribosylation is involved in a plethora of cellular pathways and in a multitude of essential cellular functions such as DNA damage repair, transcription, and the cell cycle. Proteins modified with a single ADP-ribose moiety are called mono-ADP-ribosylated (MARylated). In MARylated proteins, where the ADP-ribose moiety is linked to the protein via acidic amino acids, the modification can be reversed by three enzymes - MacroD1, MacroD2, and TARG1. While MacroD1 is exclusively mitochondrial, both MacroD2 and TARG1 are present in the nucleus and cytoplasm. Not much is known about the function of MacroD2 and TARG1 so far. Both enzymes are connected to the response to DNA damage and to neurological defects in literature. Therefore, the aim of this thesis was to identify which functions both enzymes possess in human cells. To this end, I utilized a two-pronged approach. Firstly, I identified protein interaction partners of MacroD2 with the BioID approach. I used BioID since this system was generated to identify weak and transient interactions which is necessary since ADP-ribosylation is rapidly added and removed. With the interactors of MacroD2 identified with the BioID approach, I found that many proteins with gene ontology terms related to actin and focal adhesions were enriched. This led to the hypothesis that MacroD2 might be involved in the regulation of the actin cytoskeleton. As a second prong, I generated and validated CRISPR/Cas knockout cell lines lacking either MacroD2, TARG1 or both enzymes. With those cell lines I systematically screened for phenotypes related to the identified MacroD2 interactors. I screened all cell lines for defects in intensity or localization of the actin cytoskeleton and focal adhesions with immunofluorescence experiments. I could not identify any defects. Subsequently, I addressed if the knockout cell lines had defects in actin regulated processes such as cell migration and attachment. I realized that only cells lacking both MacroD2 and TARG1 had tremendous defects in cell migration and attachment. In order to identify how cell migration and attachment were deregulated in cells lacking MacroD2 and TARG1, I tested epidermal growth factor receptor (EGFR) signaling as a possible deregulated pathway. I found that cells lacking both enzymes did not increase cell migration in response to EGF treatment and that EGFR was accumulated in perinuclear foci after EGF treatment. In summary, I could show that cells lacking MacroD2 and TARG1 had defects in cell migration and attachment, as well as deregulated EGFR signaling. The fact that MacroD2 and TARG1 can compensate for each other in cell migration, attachment, and EGFR signaling suggests that they perform at least partially redundant functions in unstressed cells

Modulation of the Wnt pathway at single-cell level uncovers diverging functional domains in the ciliary marginal zone of medaka

The continuous life-long growth of the fish retina is fuelled by neural stem cells located within the ciliary marginal zone (CMZ). These stem cells are characterised by their multipotency and ability to self-renew. Through asymmetric divisions, the neural stem cells give rise to progenitor cells with restricted proliferation potential that ultimately commit to terminally differentiated neurons of the mature retina. In this process, pharmacological manipulation suggests that Wnt signalling acts on both cell proliferation and differentiation, but a refined analysis is missing. Therefore, I present here a detailed analysis of Wnt signalling localisation and function within different cell types and their lineages in the post-embryonic CMZ of medaka. In this thesis, I show that Wnt ligands are expressed by cells of the retinal pigment epithelium, which is located directly adjacent to the CMZ. Wnt/β-catenin signalling activity is restricted to stem cells, whereas β-catenin independent Wnt/LRP6 signalling extends to dividing progenitor cells. To address the role of Wnt signalling in proliferation and differentiation of specific stem and progenitor cells, I created transgenic lines that allow inducible clonal labelling combined with upregulation of Wnt signalling in individual cells. My lineage tracing experiments suggest that Wnt upregulation has diverging effects on stem and progenitor cells. First, stem cells lose stemness characteristics presumably through induction of apoptosis or symmetric division. Second, progenitor cells reacquire the capacity to self-renew, but their pre-existing fate restrictions are irreversible. Finally, committed progenitors shift their fate and/or change their division mode and proliferation characteristics upon Wnt upregulation. Taken together, my results indicate that Wnt signalling functionally divides the CMZ into stem cells, non-committed and committed progenitors, which has far reaching implications for Wnt functions in other stem cell niches

Improvement of the Digestibility of Sulfated Hyaluronans by Bovine Testicular Hyaluronidase: a UV Spectroscopic and Mass Spectrometric Study

Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are important, natural polysaccharides which occur in biological (connective) tissues and have various biotechnological and medical applications. Additionally, there is increasing evidence that chemically (over)sulfated GAGs possess promising properties and are useful as implant coatings. Unfortunately, a detailed characterization of these GAGs is challenging: although mass spectrometry (MS) is one of the most powerful tools to elucidate the structures of (poly)saccharides, MS is not applicable to high mass polysaccharides, but characteristic oligosaccharides are needed. These oligosaccharides are normally generated by enzymatic digestion. However, chemically modified (particularly sulfated) GAGs are extremely refractive to enzymatic digestion. This study focuses on the investigation of the digestibility of GAGs with different degrees of sulfation by bovine testicular hyaluronidase (BTH). It will be shown by using an adapted spectrophotometric assay that all investigated GAGs can be basically digested if the reaction conditions are carefully adjusted. However, the oligosaccharide yield correlates reciprocally with the number of sulfate residues per polymer repeating unit. Finally, matrix-laser desorption and ionization (MALDI) MS will be used to study the released oligosaccharides and their sulfation patterns

Corrigendum: Abundance and Potential Biological Removal of Common Dolphins Subject to Fishery-Impacts in South Australian Waters

Conservation management of wildlife species should be underpinned by knowledge of their distribution and abundance, as well as impacts of human activities on their populations and habitats. Common dolphins (Delphinus delphis) are subject to incidental capture in a range of Australia’s commercial fisheries including gill netting, purse seining and mid-water trawling. The impact these fishery interactions have on common dolphin populations is uncertain, as estimates of abundance are lacking, particularly for the segments of the populations at risk of bycatch and in greater need of protection. Here we used double-observer platform aerial surveys and mark-recapture distance sampling methods to estimate the abundance of common dolphins in 2011 over an area of 42,438 km2 in central South Australia, where incidental mortality of common dolphins due to fisheries bycatch is the highest. We also used the potential biological removal (PBR) method to estimate sustainable levels of human-caused mortality for this segment of the population. The estimated abundance of common dolphins was 21,733 (CV = 0.25; 95% CI = 13,809–34,203) in austral summer/autumn and 26,504 in winter/spring (CV = 0.19; 95% CI = 19,488–36,046). Annual PBR estimates, assuming a conservative maximum population growth rate of Rmax = 0.02 and a recovery factor of Fr = 0.5 for species of unknown conservation status, ranged from 95 (summer/autumn) to 120 dolphins (winter/spring), and from 189 (summer/autumn) to 239 dolphins (winter/spring) with an Rmax = 0.04. Our results indicate that common dolphins are an abundant dolphin species in waters over the central South Australian continental shelf (up to 100 m deep). Based on the 2011 abundance estimates of this species, the highest estimated bycatch of common dolphins (423 mortalities in 2004/05) in the southern Australian region exceeded the precautionary PBR estimates for this population segment. Recent bycatch levels appear to be below PBR estimates, but low observer coverage and underreporting of dolphin mortalities by fishers means that estimates of dolphin bycatch rates are not robust. The effects of cumulative human impacts on common dolphins are not well understood, and thus we recommend a precautionary management approach to manage common dolphin bycatch based on local abundance estimates

The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria

Mitochondrial gene expression in Saccharomyces cerevisiae is responsible for the production of highly hydrophobic subunits of the oxidative phosphorylation system. Membrane insertion occurs cotranslationally on membrane-bound mitochondrial ribosomes. Here, by employing a systematic mass spectrometry-based approach, we discovered the previously uncharacterized membrane protein Mrx15 that interacts via a soluble C-terminal domain with the large ribosomal subunit. Mrx15 contacts mitochondrial translation products during their synthesis and plays, together with the ribosome receptor Mba1, an overlapping role in cotranslational protein insertion. Taken together, our data reveal how these ribosome receptors organize membrane protein biogenesis in mitochondria