<p>Protein kinases are essential in cell signaling pathways and are well-validated targets for cancer therapeutics and detection of activity levels. Yet, there remains a critical need for kinase inhibitors with high specificity and potency. The development of DNA-encoded library (DEL) technology dramatically facilitates the discovery of ligands to therapeutically relevant proteins. The preparation of combinatorial libraries followed by stringent selections can be exploited to rapidly generate hit molecules that bind to a large variety of targets.</p> <p>A combinatorial library of peptidomimetics is prepared and subjected to a selection for enriching molecules that can serve as substrates for tyrosine kinase Src. Non-natural substrate molecules are recognized by the anti-phosphotyrosine antibody during the selection. Using biophysical characterization assays including ADP-Glo and NMR, the resulting hits are investigated as novel peptide-substrate competitive inhibitors, as well as specific chemical probes that would benefit kinase activity detection. An ester derivative of the lead compound SrcDEL10 demonstrates cellular activity with inhibition of Src-dependent signaling in cell culture. Subsequently, our effort extends to parallel selections with a highly diverse-scaffold DEL on three cancer-related tyrosine kinases. Several hit molecules are validated with differential phosphotransfer activities among Src, Lyn, and Syk. Studies on the structure activity relationship of hit molecules produce selective kinase substrates with the lowest molecular weights reported to date. Potential bisubstrate inhibitors, showing above 8-fold Src selectivity over Lyn, are designed based on structures of selective substrates.</p> <p>Meanwhile, high sensitivity of DNA sequence analysis enables the development of specific and multiplexed activity assays. Using the substrate selection strategy, we develop a DNA-based kinome activity profiling assay using DNA conjugates of tyrosine kinase peptide substrates. Selective enrichment of phosphorylated probes enables activity detection by either quantitative PCR (qPCR) or parallel DNA sequencing. Results with detecting recombinant kinases demonstrated a low (~50 pM kinase) limit of detection. A library of 96 DNA-substrate conjugates enabled multiplexed tyrosine kinase assays in cell lysates in a manner analogous to peptide microarrays. This DNA-based assay potentially empowers the detection of tumor biomarkers with high specificity, lower detection limit, multiplexing capability, and high cost-effectiveness.</p> <p>Together, this research uses DNA-based technologies to assist developing new therapeutics and diagnostics, drug target validation, unveiling drug mechanisms of action, and understanding the role of protein phosphorylation in disease progression.</p>

Spatiotemporal metabolic modeling of microbial metabolism is a step closer to achieving higher dimensionalities in numerical studies (in silico) of biofilm maturation. Dynamic Flux Balance Analysis (DFBA) is an advanced modeling technique because this method incorporates Genome Scale Metabolic Modeling (GSMM) to compute the biomass growth rate and metabolite fluxes. Biofilm thickness is pertinent because this variable of biofilm maturation can be measured in a laboratory (in vitro). Pseudomonas aeruginosa (P. aeruginosa) is the model bacterium used in this computational model based on previous research conducted by Dr. Michael Henson, available GSMMs, and the societal significance of patients suffering from P. aeruginosa airway infections. Spatiotemporal Flux Balance Analysis (SFBA) will be the computational method used in this thesis to simulate biofilm growth. Another level of accuracy will be introduced to SFBA which is a dynamic finite difference grid that will vary relative to the biofilm’s velocity of expansion/contraction. This novel idea is governed by a differential equation that defines the biofilm’s velocity and updates the spatial dependency of the finite difference grid which has never been done while utilizing GSMM. Environmental conditions (bulk concentrations of metabolites) are altered to investigate how varying nutrients (glucose, oxygen, lactate, nitrate) affected biofilm maturation.

The human genome is characterized by an extent of functions that act further than its genetic role. Indeed, the genome can also affect cellular processes by nongenetic means through its physical and structural properties, specifically by exerting mechanical forces that shape nuclear morphology and architecture. The balancing between two chromatin compartments with antagonist functions, namely Transcriptional and Polycomb condensates, is required for preserving nuclear mechanical properties and its perturbation is causative of the pathogenic condition Kabuki syndrome (KS) (Fasciani et al., 2020). KS is a rare monogenic disease caused by the haploinsufficiency in the KMT2D gene encoding for MLL4, a H3K4-specific methyltransferase important for the regulation of gene expression. By interrogating the effect of KMT2D haploinsufficiency in Mesenchymal Stem Cells (MSCs) we discovered that MLL4 loss of function (LoF) impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture and the cell mechanoresponsiveness during differentiation (Fasciani et al., 2020). These results suggest that altered nuclear mechanics rely on chromatin architecture and could potentially lead to changes in cell responses to external mechanical stimuli. In the present work, we investigated the role of Transcriptional and Polycomb condensates in tuning nuclear responses to different external mechano-physical conditions. To affect nuclear mechanics, we employed the use of several mechanical devices (e. g. substrate stiffness, microchannels with constrictions, and cell confinement). We found that Polycomb and Transcriptional condensates are modulated by changes in substrate rigidity in healthy conditions and that MLL4 LoF impairs the MSCs nuclear condensates-driven mechanical response. Furthermore, we observed that MLL4 LoF impacts nuclear adaptation to confined spaces by incrementing susceptibility to nuclear envelope rupture. We also showed that the increased nuclear fragility in MLL4 LoF is accompanied by an alteration of cell migratory capacity and survival rate. Altogether these findings suggest that MLL4 LoF impairs cell responses to external mechanical stimuli, shedding light on the pathological connection between the altered cell mechanoresponsiveness during differentiation and KS phenotype in terms of skeletal and cartilage anomalies.

Biomolecular condensates are dynamic intracellular structural units or distinct reaction spaces that can form by condensation of their constituents from the cytoplasm or the nucleoplasm. It is generally not clear yet, how dynamic, continuum-like condensate properties relevant for large-scale intracellular organisation emerge from the interplay of proteins and nucleic acids on the level of few individual molecules. With this work, we expand the portfolio of methods to investigate the role of protein-nucleic acid interactions in biomolecular condensates by introducing optical tweezers-based mechanical micromanipulation of single DNA molecules combined with confocal fluorescence microscopy to the field. We used this approach to characterise how the two landmark proteins1 Fused in Sarcoma and Heterochromatin Protein 1 form condensates with single DNA molecules. Fused in Sarcoma (FUS) is a key protein for various aspects of the nucleic acid metabolism and evidence is accumulating that biomolecular condensation is crucial for both, its physiological functions and its role in pathological aggregate formation. In this thesis, we directly visualised the formation of FUS condensates with single molecules of ssDNA and dsDNA. We showed that the formation of these microcondensates is based on nucleic acid scaffolding. We explored their mechanical properties and found that the mechanical tension that (FUS dsDNA) condensates can withstand or exert is in the range below 2 pN. We further demonstrated that already on this fundamental scale and with limited amounts of constituent molecules, dynamic properties like shape relaxations, reminiscent of viscoelastic materials, can emerge. Heterochromatin Protein 1 (HP1) is a prototypic chromatin organising factor that is in particular involved in the formation of dynamically compacted heterochromatin domains. HP1 forms biomolecular condensates and compacts DNA strands in vitro. In this work, we measured the influence of HP1 on the mechanical properties of individual DNA molecules and demonstrated the response of HP1-DNA condensates to different environmental conditions. We contributed a methodological framework to characterise viscoelastic-like systems on the single molecule level. Taken together, our optical tweezers-based approach revealed structural and mechanical properties of prototypic protein-DNA condensates and hence helped to elucidate mechanisms underlying their formation in unprecedented spatiotemporal and mechanical detail. We anticipate that this method can become a valuable tool to investigate how large-scale intracellular organisation based on protein-nucleic acid condensation emerges from interactions between individual building blocks.

No description available.

One of the essential requirements of biomolecular modeling is an accurate description of water as a solvent. The challenge is to make this description computationally facile -- reasonably fast, simple, robust and easy to incorporate into existing software packages, yet accurate. The most rigorous procedure to model the effect of aqueous solvent is to explicitly model every water molecule in the system. For many practical applications, this approach is computationally too intense, as the number of required water atoms is on an average at least one order of magnitude larger than the number of atoms of the molecule of interest. Implicit solvent models, in which solvent molecules are replaced by a continuous dielectric, have become a popular alternative to explicit solvent methods. However, implicit solvation models often lack various microscopic details which are crucial for accuracy. One such missing effect that is currently missing from popular implicit models is the so called effect of charge hydration asymmetry (CHA). The missing effect of charge hydration asymmetry -- the asymmetric response of water upon the sign of solute charge -- manifests a characteristic, strong dependence of solvation free energies on the sign of solute charge. Here, we incorporate this missing effect into the continuum solvation framework via the conceptually simplest Born equation and also in the generalized Born model. We identify the key electric multipole moments of model water molecules critical for the various degrees of CHA effect observed in studies based on molecular dynamics simulations using different rigid water models. We then use this gained insight to incorporate CHA first into the Born model, and then into the generalized Born model. The proposed framework significantly improves accuracy of the hydration free energy estimates tested on a comprehensive set of varied molecular solutes -- monovalent and divalent ions, small drug-like molecules, charged and uncharged amino acid dipeptides, and small proteins. We finally develop a methodology to resolve the issue with unacceptably large uncertainty that stems from a variety of fundamental and technical difficulties in experimental quantification of CHA from charged solutes. Using the proposed corrections in the continuum framework, we untangle the charge-asymmetric response of water from its symmetric response, and further circumvent the difficulties by extracting accurate estimate propensity of water to cause CHA from accurate experimental hydration free energies of neutral polar molecules. We show that the asymmetry in water's response is strong, about 50% of the symmetric response. / Ph. D.

The research presented in this thesis includes the development of designs, materials, and fabrication processes and the results of characterization experiments for a meso-scale hydrogel-based lipid bilayer array system. Two design concepts are investigated as methods for forming Droplet Interface Bilayer (DIB) arrays. Both concepts use a base of patterned silver with Ag/AgCl electrodes patterned onto a flat polymer substrate. In one technique, photopolymerizable hydrogel is cured through a mask to form an array of individual hydrogels on top of the patterned electrodes. The other technique introduces a second type of polymer substrate that physically supports an array of hydrogels using a set of microchannels. This second substrate is fitted onto the first to contact the hydrogels to the electrodes. The hydrogels are used to support and shape droplets of water containing phospholipids, which self-assemble at the surface of the droplet when submerged in oil. Two opposing substrates can then be pushed together, and a bilayer will form at the point where each pair of monolayers come into contact. The photopatterning technique is used to produce small arrays of hydrogels on top of a simple electrode pattern. Systems utilizing the microchannel substrate are used to create mesoscale hydrogel arrays of up to 3x3 that maintained a low resistance (~50-150 kΩ) connection to the substrate. Up to three bilayers are formed simultaneously and verified through visual observation and by recording the current response behavior. Arrays of varying sizes and dimensions and with different electrode patterns can be produced quickly and inexpensively using basic laboratory techniques. The designs and fabrication processes for both types of arrays are created with an eye toward future development of similar systems at the microscale. / Master of Science

Yes / Ancient human mobility at the individual level is conventionally studied by the diverse application of suitable techniques (e.g. aDNA, radiogenic strontium isotopes, as well as oxygen and lead isotopes) to either hard and/or soft tissues. However, the limited preservation of coexisting hard and soft human tissues hampers the possibilities of investigating high-resolution diachronic mobility periods in the life of a single individual. Here, we present the results of a multidisciplinary study of an exceptionally well preserved circa 3.400-year old Danish Bronze Age female find, known as the Egtved Girl. We applied biomolecular, biochemical and geochemical analyses to reconstruct her mobility and diet. We demonstrate that she originated from a place outside present day Denmark (the island of Bornholm excluded), and that she travelled back and forth over large distances during the final months of her life, while consuming a terrestrial diet with intervals of reduced protein intake. We also provide evidence that all her garments were made of non-locally produced wool. Our study advocates the huge potential of combining biomolecular and biogeochemical provenance tracer analyses to hard and soft tissues of a single ancient individual for the reconstruction of high-resolution human mobility. / The Danish National Research Foundation; The Carlsberg Foundation, L'Oreal Denmark-UNESCO; The ERC agreement no. 269442

No / The first Raman spectrometers to be used for in situ analysis of planetary material will be launched as part of powerful, rover-based analytical laboratories within the next 6 years. There are a number of significant challenges associated with building spectrometers for space applications, including limited volume, power and mass budgets, the need to operate in harsh environments and the need to operate independently and intelligently for long periods of time (due to communication limitations). Here, we give an overview of the technical capabilities of the Raman instruments planned for future planetary missions and give a review of the preparatory work being pursued to ensure that such instruments are operated successfully and optimally. This includes analysis of extremophile samples containing pigments associated with biological processes, synthetic materials which incorporate biological material within a mineral matrix, planetary analogues containing low levels of reduced carbon and samples coated with desert varnish that incorporate both geo-markers and biomarkers. We discuss the scientific importance of each sample type and the challenges using portable/flight-prototype instrumentation. We also report on technical development work undertaken to enable the next generation of Raman instruments to reach higher levels of sensitivity and operational efficiency.

No / Scytonemin-imine, a novel derivative of scytonemin, has been isolated and identified very recently and proposed to serve as a photoprotective biomarker for certain bacteria growing under intense photon flux density. This study predicts theoretically the Raman spectrum of scytonemin-imine by density functional theory calculations and provides comparison of major bands to those of scytonemin, the parent compound for which both the experimentally characterized and theoretically predicted spectra exist in the literature. It is proposed to be an addendum to the collection of our previous work on scytonamin and its derivatives to facilitate recognition of the diagnostic Raman spectral signatures for scytonemin-imine.