Weight loss (WL) interventions postpone age-related diseases and improve health span in a wide variety of species including humans, monkeys, rodents, flies and yeast. These interventions such as dietary/caloric restriction (DR/CR) or bariatric surgery in formerly obese humans lead to a reprogramming of adipose tissue physiology with a better adipokine profile, insulin sensitivity and beneficial effects on adipose stem cells (ASCs) as protection against DNA damage, improvement of survival capacity, extension of replicative lifespan and holding adipogenesis at a low rate (Mitterberger et al,. 2014, Exp. Gerontol.). The research project is WL target genes in ASCs from subcutaneous White Adipose Tissue.

To find WL target genes, a microarray analysis was performed by the lab to establish the global gene expression pattern in ASCs of calorically restricted formerly obese (WLD), normal weight (NWD) and obese donors (OD). From this screening, it appears that the gene Sushi Domain Containing 2 (SUSD2) is upregulated after WL in ASCs of WLD compared to NWD and OD. As a consequence, the goal of the project is to study the function of SUSD2 in ASCs. The hypothesis is that the downregulation of SUSD2 in ASCs induce hyper-activation of mitogenic pathway which leads to premature senescence of ASCs. To address this hypothesis, genomic, transcriptomic and proteomic technologies, modern techniques of molecular and cell biology will be employed.

As an ESR of the MSCA program ARDRE, her current research is focused on the study of skin aging and the role of dysfunctional mitochondrial metabolism and mitophagy in the triggering of senescence, a 3D culture model subjected to different stressors (UV, air pollutants) will be established. Her work is also focused on the investigation of FAHD1, a newly identified human mitochondrial enzyme, studying its role in various metabolic shifts, and its possible consequences on the functioning of the electronic transport chain and cell proliferation.

Lucas enrolled in the ARDRE program under the supervision of Prof. Thedieck to combine theoretical and experimental approaches in exploring the interplay of mTOR signaling with energy metabolism and neuronal function in the context of ageing.

The team is working on the hydrozoan freshwater polyp Hydra, which is part of Cnidarians as a sister-group of Bilateria. Hydra is a powerful model in biology for axial patterning, regeneration studies and stem cell biology, and allows us to understand ancestral mechanisms diverged from early metazoans. This polyp reproduces asexually and has a 

strong regeneration ability thanks to a hydrozoan-specific multipotent stem cell population, called the interstitial stem cells. Dynamic of this stem cell population is very constant in terms of cell proliferation, movement and proportions of cell type during asexual reproduction. Stem cell decision-making in this simple model raise an interest in the scientific community to understand the underlying genetic regulation system. Two core regulators of stem cells are the Wnt/ β-Catenin signalling pathway and the Myc oncogene, which are both highly conserved through evolution. The PhD project will aim to decipher these regulation mechanisms at cellular and molecular level in Hydra. In a first part, we want to establish Myc mutants by genetic interference, with Knock-down (siRNA) and Knock-out (CRISPR/Cas9) methods, or establish long-term and stable transgenic lines, in order to interfere with Myc. In a second part, we will use these successful Myc-inhibited Hydra and take advantage of transgenic β-Catenin over-expressing Hydra to decipher stem-cell regulation. The cell cycle and dynamics of interstitial stem cells will be analysed. Then, we will analyse at the molecular level by using transcriptomic analysis at single cell resolution. Finally, we want to extend this work depending on the result to decipher the molecular network and stem cells dynamics which control the decision making. We expect to collaborate with other ARDRE PhD candidates about related topics like repressive mechanisms of TCF/ β-Catenin or mitochondria dysfunction in ageing Hydra.

Ageing of organisms can be defined as continuous deterioration resulting in higher risk of mortality and loss of endurance over time. Deterioration and viability loss can be induced by “artificial ageing” methods in seeds of higher plants, enabling studying the underlying molecular processes associated with life and death – whether a seed will live, germinate and grow into a new plant, or if it will die. Hydra, a member of the Cnidaria (hydrozoa), is a small freshwater polyp (Fig. 1) with seeming “immortality” due to its remarkable ability to divide. This high potential of asexual reproduction together with its capability to regenerate makes it another exciting model for studying regeneration, cell death and senescence. However, our understanding of the common denominators inducing ageing – or regeneration – is compromised by the lack of molecular tools to diagnose “ageing” of tissues and organisms.

 

Figure 1: Thermal image of imbibed seeds of different viability using the method described in Kranner et al.  2010, PNAS; figure 2: Sexually induced male Hydra oligactis

In this project we will assess ageing-associated changes in the cellular redox environment and elucidate molecular mechanisms of ageing and regeneration in plant seeds and Hydra sp. Non-invasive techniques will be used to separate highly viable, aged and dead seeds (Fig. 2)  to predict seed viability prior to germination, in conjunction with state-of-the-art GC-MS-based metabolite profiling.

The activation of Wnt signalling is of central role in the pluripotency context. The Wnt pathway is present in almost all animals from sponges to human. It is well conserved evolutionary and plays important roles in the activation of target genes in a variety of cellular processes, such as cell proliferation, axis formation, embryonic patterning, organ development and stem cell renewal. Novel repressive functions of its nuclear effectors influencing gene expression and cell fate choice were discovered by the host lab in Ciona intestinalis, where Tcf and its cofactor β-catenin can directly repress the gata.a binding activity at GATA sites. More recently, a mechanistic model was suggested where Tcf7/β-catenin repress the gata.a transcriptional activity by preventing gata.a from binding to its site through the formation of a ternary complex, but several questions remain open to discussion. We hypothesize that Tcf alone can bind GATA sites independently of βcatenin and that a GATA site signature likely determines diverse degrees of repression translating into a fine tuning for tissue specific expression levels at the various GATA target genes in different regions of the embryo. Such signature may be determined by overlapping incomplete Tcf sites resulting in the masking of the GATA sites by Tcf to weaken the gata.a binding strength. The aims of this project are to refine the repressive signature of Tcf/βcatenin on different GATA sites by correlating the GATA signature with known activities of target genes. To test the repressive mechanism in other contexts in Ciona and to define a possible conservation in stemness in other species, we will isolate and analyse cis-regulatory regions of ß-catenin/Tcf repressed target genes in the Ciona context and probe for a conserved biochemical interaction between the Wnt nuclear effectors and to target DNA.

Pancreatic acinar cells make up for the largest number of cells in the pancreas. There are various studies showing the outstanding growth capacity and interesting cell fate plasticity of acinar cells. A previous research using zebrafish models for acinar cells ablation could discover a new population of cells in the exocrine pancreas expressing ptf1a as an early pancreatic development marker yet negative for acinar cell maturation markers. These progenitor-like cells seemed to be involved in acinar cell regeneration after near complete ablation. Our goal in this project is to further describe the identity and function of this cell population using single-cell, molecular and in-vivo imaging approaches and to look for the other possible early progenitors involved in exocrine pancreas regeneration.

In this project, we will study the regeneration processes of the adult human brain by deploying a novel approach of direct conversion of human fibroblasts into induced NSCs (iNSCs), which allows the (partial) maintenance of the ageing profile of the cells. The reprogramming trajectories will be studied over time at single cell level, assessing the transcriptomic and epigenetic profile of the cells. This experimental design will lead to valuable knowledge into stem cell reprogramming in the human brain and allow the establishment of a rejuvenation/ reprogramming cell atlas. A second aim of the project will be to decipher the molecular mechanisms that are involved in age-dependent changes in regeneration and the potential pathological mechanisms leading to neurodegenerative disorders. For that, the ageing profiles of iNSCs will be compared to profiles of induced pluripotent stem cells (iPSCs), iPSC- derived NSCs and their parental fibroblasts. For this purpose, single-cell age-related DNA methylation signatures will be analyzed and compared between the groups. In order to answer these complex scientific questions, state-of-the-art technologies are going to be employed. The innovative method of direct conversion of adult differentiated cells into iNSCs and induced Neurons (iNs) will be used and subsequently a serial combinatorial cell-indexing approach (CellTagging) will be performed, followed by single cell transcriptomics (high-throughput droplet microfluidics, scRNA-seq), high resolution lineage analysis, DNA methylation ageing analysis as well as neuronal functionality assays.

Natural products are small molecules produced by living organisms and they have been used as a source of drugs for a long time. Interestingly, many available drugs that are used as antibiotics, analgesics, antimalarials and even as anti-cancer agents are natural products or their derivatives. The scientific and technological advances during the years, have dramatically increased the number of drugs of natural origin that are now at our disposal. The total synthesis of these products has not only allowed the obtaining of compounds difficult to isolate but it also lays the foundations to fully synthetic natural product derivatives, which depict valuable compounds for the investigation of structure-activity relationships also known as SARs. 

Particularly, marine organisms such as corals and sponges are a rich source of small molecules that present biological activity.  The relatively low natural abundance of most of the bioactive compounds and the fact that many marine organisms are largely unculturable, make the development of drugs from marine natural products extremely difficult. Furthermore, extensive harvesting of wild marine invertebrates would have a huge impact on the marine environment. Therefore, the total synthesis of these molecules is crucial for further extensive biological investigations and/or medicinal applications. This fact determines the social, economic and ecological aspects of our work.

In this context, one example of soft corals that have been a rich source of bioactive natural products are the members of the Xenia genus (Figure 2), that includes 28 species that live in the shallow water of tropical reefs in the Indo-Pacific.

Figure 2: Soft corals of the Xenia genus, Photograph by Dawson, distributed under a CC-BY 2.0 license 

These corals are the producers of the so-called Xenia diterpenoids, which have been shown to exhibit a broad spectrum of biological activities ranging from anti-cancer to anti-microbial activities. Xenia diterpenoids represent a unique and challenging target for total synthesis. The primary challenge lies in the successful incorporation of the E- or Z-cyclononene fragment. From a synthetic point of view, the main task of xenicanes’ synthesis is to obtain the 6,9-bicyclic core structure. Previous successful total synthesis approaches towards this scaffold have been accomplished, relying on macrocyclization, ring expansion, ring contraction or cross coupling strategies.

Based on previous experience in the total synthesis of natural products containing a medium sized ring, this project aims to develop a divergent approach toward different congeners of the Xenia diterpenoids. The strategy relies on an intramolecular alkylation to forge the pivotal 9-membered ring, starting from a common advanced intermediate. In addition, the route should allow to access fully synthetic analogs, which are not accessible by semi-synthesis.

During the study, we try to link the functional changes of calcium channels with their amino acid sequences via simulations of these channels at molecular level.
 

Voltage-dependent calcium channels (Cav) are multidomain proteins selectively permeable to calcium ions. In the human body ten different types of Cav with different biophysical properties and tissue distribution controls a variety of calcium-dependent physiological processes, including muscle contraction, hormone or neurotransmitter release, gene expression as well as learning and memory. Cav dysfunction also contributed to human diseases such as severe neurodevelopmental syndrome with and without congenital endocrine symptoms. Therefore, selective and potent channel inhibitors appear as an attractive pharmacological approach to treat these disorders. So far only non-selective inhibitors of these channel types are available. Aim of my thesis is to characterize the pharmacological properties of such channels using an interdisciplinary approach combining biophysical and pharmacological experiments with molecular modeling techniques to discover selective channel inhibitors in order to provide a better understanding to the channels’ physiological behavior and discover potential drugs for clinical application.

Lichens are a symbiosis of a fungus (usually an Ascomycete), termed “mycobiont”, and one or more green microalgae and/or cyanobacteria, called “photobiont”. Secondary lichen products have been broadly employed in folk medicine, and screening campaigns revealed the occurrence of lichen metabolites with antibiotic, antimycotic, antibacterial, antiviral, antitumor, analgesic, antipyretic, antidiabetic, anticoagulant, antithrombotic, antioxidant, cardio-protective and anti-inflammatory properties. Regardless of the well-known bioactive properties of lichen compounds, most lichens are not commonly consumed by humans, due to the risk of intoxication. There are nevertheless some lichen species across the world, which are used as a food source, e.g. Cetraria islandica, Umbilicaria esculenta or Bryoria fremontii. One of the main drawbacks lichens present as a source for new bioactive products is their biomass production. Due to their slow growth rate and difficulties in cultivation, the production of substantial amounts of the compounds of interest is still challenging. It is noteworthy that the secondary metabolite profile of batches produced in culture might differ from those produced in nature, because lichenized fungi produce their typical “lichen products” almost exclusively in the presence of their compatible photobionts and under specific stressing environmental conditions. Biotechnological applications to produce new pharmaceuticals would require the development of novel culture techniques for edible lichens, by which the production of the desired secondary metabolites can be induced. Alternatively, compounds of interest might be synthesized. During this thesis, it is planned to compare secondary metabolite profiles of lichen extracts from species collected in nature and grown in culture. In a first attempt C. islandica, which is used traditionally as herbal cough remedy, but also for the treatment of dysentery and chronic digestive disturbances, will be investigated. Secondary metabolites of interest will be isolated, their structures assigned, and their pharmacological profile studied. Particular attention will be given to their effects on inflammation inhibition, humane tyrosinase inhibition, enhancement of wound healing (e.g. MMP-9 inhibition), and their activity on tumor cells. In a second task, the metabolic faith of secondary lichen metabolites after oral intake will be investigated, taking advantage of in vitro assays that simulate digestion. Besides C. islandica, further edible lichens growing in different areas of Europe, including the Austrian Alps, will be studied.