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For its immune system to function properly, the human body relies on a multitude of signaling processes executed by many different types cells. The most common one of these cell types is the neutrophil. Neutrophil cells patrol the blood and once encountering pathogens, they react to kill it making use of their armory contained in different types of granules. The neutrophil serine proteases are an important part of this armory and they can, for instance, be released in the phagosome containing a phagocytosed pathogen to be killed. Proteases, like the neutrophil serine proteases, are enzymes that hydrolyze peptide bonds and as such cut their substrate peptides. They can have a variety of functions but need to be tightly controlled as aberrant activity of a protease in question may lead to disease. For example, aberrant control of the neutrophil serine proteases can play a part in diseases such as chronic obstructive pulmonary disease, cancer and Alzheimer dementia. This regulation, however, means that protease presence does not necessarily equal protease activity. Therefore, activity-based probes have been developed as a special tool enabling distinguishing of active from inactive protease. To achieve this activity-direction, these small molecule probes contain an electrophilic group that reacts with the active site residue of an active protease. The probes eventually stay connected to the protease and usually also contain a tag, either a dye or a group allowing for the attachment of a dye, to make the protease-probe complex visible. Furthermore, quenched probes exist which turn fluorescent only after having reacted with a protease. In this work we design, develop and apply new activity-based probes directed against the neutrophil serine proteases. In chapter 2, we develop amino phosphinates as irreversible inhibitors for serine proteases. These are similar to amino phosphonates which find wide application as serine protease inhibitors. We establish a facile synthetic path towards the phosphinates and incorporate different carbon substituents at the phosphorus and evaluate their influence on activity, thereby assessing their reactivity and fit into the protease active site. We show that phosphinates incorporating a phenyl substituent are better inhibitors than analogous phosphonates and perform docking experiments giving evidence on a preferred configuration at the phosphorus. We then go on to use these novel phosphinate inhibitors as reactive group for serine protease activity-based probes in chapter 3. The phosphinates are fused to a linker and a tag and then used on neutrophil serine proteases neutrophil elastase, proteinase 3 and cathepsin G as well as the digestive protease chymotrypsin. We are able to separate different diastereomers of some of the probes and show that these possess distinct labeling capacities providing indirect proof for the docking results of parent inhibitors. Finally, we use some of these probes for imaging of neutrophil serine proteases in primary human neutrophils. Chapter 4 is directly related to the one before and follows a similar path. Here we apply the same phosphinate warhead but use it to develop quenched activity-based probes. These contain a quencher furnishing a dark molecule. The quencher gets expelled from the molecule upon reaction with protease restoring fluorescence. We make quenched probes for neutrophil elastase and cathepsin G, show that they can be used for purified protease labeling and labeling of protease in neutrophil lysates. Furthermore, we show unquenching of one of the probes by neutrophil elastase. This chapter establishes the phosphinate scaffold as a new and only the second group that allows synthesis of quenched activity-based probes for serine proteases. Lastly, in chapter 5 we develop quenched activity-based probes for the three most abundant neutrophil serine proteases, this time using the already established mixed phosphonate group as the reactive group. We make a set of probes, demonstrate labeling of purified protease and in neutrophil lysates as well as in the supernatant of neutrophils stimulated for degranulation. We then go on to use the probe designed to target neutrophil elastase in a live-cell imaging experiment of neutrophils undergoing NETosis. As we can see background fluorescence in this experiment, we go on to improve the probe, fitting a different quencher and dye and end with establishing its labeling capacity. Finally, we show using this new probe in a similar live-cell experiment of neutrophils undergoing NETosis. However, this experiment needs to be improved and is thus placed in chapter 6, the general discussion, as an outlook to future experiments we plan to perform.

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In this project the student will theoretically analyze how cell division is spatially and temporally coordinated. Using computational modeling, the student will first uncover how multiple functional motifs (such as bistable switches) can optimally work together to create different desired cell responses in time. Second, the student will develop models to show how the cell cycle is dynamically coordinated in space. Special attention will be paid to studying how the spatial regulation of cell division is influenced by changes in its surroundings, such as differences in size and shape of the cells, as well as in its internal structure (i.e. cytoskeleton, nucleus, and centrosomes). The modeling efforts are expected to interact synergistically with experiments that are carried out by others in the lab.

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The ubiquitously expressed Cx43 isoform is one of the major building blocks of gap junction channels or hemichannels in the heart, brain and bone. Gap junction channels connect the cytoplasm of two neighboring cells, thereby exchanging signaling molecules and metabolites, whereas hemichannels mediate paracrine signaling by releasing ATP and other signaling molecules into the extracellular environment. The opening of Cx43 hemichannels must be tightly controlled to avoid loss of metabolic, energetic and ionic gradients, resulting in cell demise.The regulation of Cx43-based gap junctions has been extensively studied, revealing that their activity is controlled by intramolecular interactions between the C-terminal tail (CT) of Cx43 and the second part of the intracellular loop (L2). A “ball-and-chain” model was proposed in which binding of the CT tail to the L2 region results in closing of the Cx43-based gap junctional channels. Until recently, the mechanisms controlling Cx43-hemichannel activity remained unresolved. However, our lab found that similar loop/tail interactions exist in Cx43 hemichannels as in gap junctions but with an opposite functional outcome, whereby loop/tail interactions are essential for Cx43-hemichannel opening. Loss of loop/tail interactions, either by genetically deleting the CT of Cx43 or by physiological conditions (like increases in [Ca2+]i of 1 µM and higher) that elicit actomyosin contractility, results in loss of Cx43-hemichannel activity. This can be overcome by the last 9 amino acids of the CT (CT9), which targets the L2 region, and more specifically the Gap19 region within this sequence, in Cx43.First, we identified the residues present in the CT9 region responsible for L2 interaction and for mediating Cx43-hemichannel activity. We found that the interaction of the CT9 region with the L2 region critically depended on the presence of Asp378 and Asp379 residues and of Pro375 and Pro377 residues. Also, altering these residues in CT9 into Ala residues rendered TAT-CT9 (a cell-permeable version of CT9) ineffective in restoring the hemichannel activity of Cx43M239-based hemichannels (CT-deleted variant) and Cx43-hemichannel activity in conditions of actomyosin contractility. These findings correlate with the highly positively charged Gap19 region within the L2 domain, the presumed target of CT9.Second, despite the fact that only the last 9 amino acids were sufficient to restore the activity of Cx43-hemichannels lacking the complete CT, deletion of this region in the CT of Cx43 only mildly impacted the interaction with Gap19/L2. We therefore hypothesized that another region within the CT contributes to such interaction. Here, we identified the SH3-binding domain as a second region within the CT, contributing to the loop/tail interaction. TAT-SH3 peptide was able to restore the activity of CT-truncated Cx43-hemichannels or of Cx43-hemichannels in conditions of increased actomyosin contractility. Furthermore, the ability of TAT-SH3 to restore CT-truncated Cx43-hemichannels depended on the presence of Gap19, since deletion of the latter rendered the channels resistant to functional modulation by TAT-SH3. Deletion of either the SH3-binding domain or the CT9 domain in Cx43 reduced hemichannel activity, while deletion of both domains completely abolished Cx43-hemichannel activity.Third, we know that the CT of Cx43 is not only important for channel regulation, but is also important for Cx43 trafficking. Here, we studied the contribution of autophagy, a lysosomal degradation pathway, for Cx43-hemichannel turnover by exposing cells to nutrient starvation, a condition known to trigger autophagic flux. Our data show that nutrient starvation results in a rapid decline of Cx43-protein levels, both as gap junctions and hemichannels, and of Cx43-hemichannel function, which could be partially rescued by Bafilomycin A1, an autophagy inhibitor acting at the level of the lysosomes.In conclusion, our study (i) identified key residues present in the CT9 region responsible for functional modulation of Cx43-hemichannels; (ii) elucidated the SH3-binding domain as a novel determinant underlying loop/tail interactions critical for Cx43-hemichannel function; and (iii) proposed autophagy as a turnover pathway of Cx43-hemichannels in particular in conditions of nutrient starvation.

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This project aspires 1. to demonstrate cancer cells dual addiction to anti-apoptotic Bcl-2 by exploiting the properties of BH3 mimetics and BH4-targeting tools 2. to screen and/or develop novel, in vivo applicable petidomimetics and TAT-IDPs peptide derivatives targeting Bcl-2 via its BH4-domain via a Celltox-TM Green Cytotoxicity Assay 3. to establish the effect of TAT-IDPs and derivatives on the survival and function of platelets from healthy volunteers and cancer patients.

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Dynamic events of protein and lipid phosphorylation and dephosphorylation are involved virtually in all cellular signaling networks and transduction pathways. Kinases are the enzymes that catalyze phosphorylation reactions, while phosphatases are responsible for phosphate hydrolysis from the substrates. Defective or inappropriate activity of phosphatases contributes to the development of many human diseases, including cancer. PRL-3 is a plasma membrane-associated dual specificity phosphatase that exhibits a highly restricted and tightly regulated expression pattern. Additionally, PRL-3 is an emerging prognostic marker for cancer progression and a promising therapeutic target. Although in the last decade numerous clinical studies have extensively validated the causative role of PRL-3 in cancer metastasis, to date its substrate(s) and the direct downstream transduction pathways affected by PRL-3 overexpression are still elusive.The first part of this work concerns the in vitro characterization of PRL-3 phosphatase activity towards phosphopeptides and phosphoinositides. We show that PRL-3 does not present any activity against the library of phosphopeptides tested, while it robustly dephosphorylates the phosphoinositide PtdIns(4,5)P2. Moreover, our experimental results and molecular docking studies suggest that PRL-3 is a phosphatidylinositol 5-phosphatase. Finally, structure-activity relationship studies correlate the PRL-3 phosphatase activity toward PI(4,5)P2 with its ability to promote cell migration.The second part of this work concerns the characterization of PRL-3 oncogenic activity with respect to epithelial cell polarity. Using an organotypic 3D-culture system, in which epithelial cells form highly organized spherical cysts, we show that overexpression of PRL-3 significantly affects epithelial morphogenesis leading to the development of cysts with ectopic lumens. Moreover, we prove that a remnant organelle from the cytokinesis process, the post-mitotic midbody, is the first polarization signal during cyst development. Finally, we show that PRL-3 over- expression generates major defects in epithelial cell polarization by interfering with the fate of post-mitotic midbodies.In conclusion, we show that PRL-3 could be a new phosphoinositide phosphatase and that its overexpression affects epithelial cell polarization by altering post-mitotic midbody fate, suggesting a novel mechanism for epithelial tumorigenesis.