It is evident that the study of proteins is crucial for a deeper understanding of how drug treatments affect the body. However, differential protein expression analysis, which can be described as the method of finding which proteins are affected by a treatment, faces some major challenges. First of all, because proteomics data typically comprise several thousand different proteins for just a small number of biological tissues, there are both problems concerning multiple comparisons and low statistical power. Secondly, proteomics data are prone to suffer high rates of missing values, which could bias the results. One approach to handle these issues, which is gaining popularity, is to apply Bayesian hierarchical modeling in order to pool information from the complete dataset of all proteins when making inferences for each protein individually. Yet, in practice, there seems to be essentially only one Bayesian hierarchical model that currently is being employed, which uses a conjugate prior for the error variances but has no prior for the coefficients or the missing values. Given this, the aim of the thesis is to investigate how the model can be improved by adding priors for the coefficients and the missing values. The results show that by adding a hierarchical prior for the coefficients prediction accuracy may be increased. Furthermore, the results show that by adding a prior for the missing values differently expressed proteins can be detected that otherwise would have been overlooked.

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development, and nervous system function. Previous studies have suggested that the regulatory domain (RD) of CaN binds Calmodulin (CaM) towards the N-terminal end of CaN. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the regulatory domain, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto inhibitory domain (AID) from the active site of CaN, which activates phosphatase activity. In the absence of calcium-loaded CaM, the RD is at least partially disordered, and binding of CaM induces folding in the RD. Previous studies have shown that an ?-helical structure forms in the N-terminal half of the RD, but organization may occur in the C-terminal region as well. Here, we are presenting a model for the structural transition of the full length RD as it binds to CaM. Using nuclear magnetic resonance (NMR) spectroscopy, we have successfully assigned >85% of the 15N, 13C?, 13C? and HN chemical shifts of the unbound, regulatory domain of CaN. Secondary chemical shifts support a model where the RD is highly disordered. Our study of the CaM and CaN interaction supports the formation of a distal helix in the region between the AID and calmodulin-binding region. Heat capacity changes upon binding predict that 43 residues fold when CaM binds to CaN, consistent with the formation of this distal helix. Paramagnetic relaxation enhancement (PRE) studies of this interaction suggest a potential binding mode where the distal helix binds to CaM near residues I10-A11. Mutagenesis in the distal helix disrupts PREs, further supporting this hypothesis. Together, these data suggest that the interactions between CaM and the distal helix of CaN can be important in regulation of phosphatase activity.

Cell-free protein synthesis (CFPS) using crude lysates has developed into a robust platform technology over the last 60 years to express numerous types of recombinant proteins. The open-nature, elimination of reliance on cell viability, and focus of all energy towards production of the protein of interest represent substantial advantages of CFPS over in vivo protein expression methods. CFPS has provided new opportunities across a series of research fields that include metabolic engineering, therapeutic and vaccine development, education, biosensors, and many more. In recent years, optimizations of CFPS have even allowed the platform to reach the industrial level of protein production. Although there have been many advancements toward CFPS development, the democratization of the platform to a wide variety of educational, research, and industrial institutions has lacked due to an absence of resources for new users as well as a limited number of developments toward redesigning the tedious and time-consuming protocols to generate robust cell extract. To address these challenges to CFPS implementation, a comprehensive review spanning numerous cell lines with their respective applications, methodologies, and reaction formats were provided in addition to detailed protocols outlining the process of going from E. coli cells to a completed CFPS reaction. Together, these resources provide the scientific community with easily accessible resources for CFPS implementation. Moreover, the aforementioned protocols were redesigned from a four-day process into one that may be completed in under 24-hour’s time with very little researcher oversight. The resulting workflow maintained the robustness of prior methods but generated 400% more extract compared to traditional methods via a set-it-and-forget-it approach. To date, the works presented herein have garnered tremendous viewership from the CFPS research community with a substantial following among all three of the articles. Moving forward, I anticipate that these works will continue to bring new users into the CFPS field through the ease of access to these resources and through the advance of the simplistic and reproducible new workflow for preparation of robust E. coli cell extract.

This thesis presents a method for integrating heterogeneous gene/protein datasets at the functional level based on Gene Ontology term similarity. Often biologists want to integrate heterogeneous data sets obtain from different biological samples. A major challenge in this process is how to link the heterogeneous datasets. Currently, the most common approach is to link them through common reference database identifiers which tend to result in small number of matching identifiers. This is due to lack of standard accession schemes. Due to this problem, biologists may not recognize the underlying biological phenomena revealed by a combination of the data but by each data set individually. We discuss an approach for integrating heterogeneous datasets by computing the similarity among them based on the similarity of their GO annotations. Then we group the genes and/or proteins with similar annotations by applying a hierarchical clustering algorithm. The results demonstrate a more comprehensive understanding of the biological processes involved.

No description available.

Yeast Rad1-Rad10 is a structure specific nuclease that processes branched double-strand break (DSB) repair intermediates; the persistence of which can impede normal DNA metabolism. The single strand annealing (SSA) mechanism of DSB repair acts when homologous repeats flank both sides of the DSB. End resection from the 5′ ends of the break exposes complementary sequences at the flanking repeats, which are annealed to form 3′ non-homologous flap structures. Saw1 recruits Rad1-Rad10 recruits to these 3′ non-homologous flaps, where Rad1-Rad10 incises the DNA and removes the flap. Saw1 has affinity towards branched DNA structures and forms a stable complex with Rad1-Rad10. The mechanism of both structure specific recruitment and nucleolytic activity of the Saw1-Rad1-Rad10 complex is currently unknown. To study this nuclease complex, we need to produce large quantities of pure, stable, and active recombinant protein. Using dynamic light scattering (DLS) and differential scanning fluorimetry (DSF)-based high throughput thermal stability assays, we have developed a method for large-scale production of recombinant Saw1. This optimized method has increased the stability and yield of protein, thereby allowing for future biochemical investigation of Saw1. Similarly, we have optimized the large-scale production of the higher molecular-weight complex (Saw1-Rad1-Rad10) and improved the homogeneity of the recombinant complex. We have also biochemically characterized the minimal branched DNA substrates for both Saw1 and Saw1-Rad1-Rad10. This work allows for biochemical investigation into the molecular mechanism of eukaryotic 3′ non-homologous flap removal during SSA. / Thesis / Master of Science (MSc)

Background Multiple myeloma is an incurable cancer disease that emerges from the bone marrow. Bone morphogenetic proteins (BMPs) are ligands that activates intracellular signaling pathways causing activation of transcription factors. Previous studies show that BMP treatment of myeloma cells induce apoptosis, a mechanism dependent on downregulation of c-MYC. BMPs uses different receptors on myeloma cells, but it is still unclear how the intracellular signaling pathway leading to apoptosis works. A recent whole genome CRISPR/Cas9 knockout screening suggested FOXO1 as a gene involved in the mechanism of apoptosis during BMP treatment. We therefore aimed to investigate further on how FOXO1 has an impact on BMP induced apoptosis. Methods Our hypothesis was that knockout of FOXO1 would protect the cells from apoptosis. To begin to address this issue we tested INA-6 FOXO1 knock-out cell clones that was generated before the start of the project and treated them with BMP-9 to look for effects on cell viability and protein expression. We measured cell viability using CellTiter-Glo® 2.0 Cell viability assay and expression of c-MYC and FOXO1 protein using Western blot. Results and conclusions Treatment with BMP-9 for 72 hours showed a decrease in viability of the cells, up to 98%. Protein expression of c-MYC was inhibited by BMP-9 treatment while a constant expression of FOXO1 was seen in all cells clones regardless of BMP treatment. Expression of FOXO1 in the FOXO1 knock-out cells indicates that the knock-out has not worked. More experiments are needed to clarify the role of FOXO1 in BMP-induced apoptosis.

The serpin antiplasmin (APL) is the primary inhibitor of plasmin, a proteinase that digests fibrin, the main component of blood clots. Most serpins are serine protease inhibitors, which undergo dramatic conformational change in forming a tight covalent complex with the target protease. Plasmin has been shown to be angiogenic through its protease activity, but it is also angiostatic, being the source of angiostatin, which inhibits angiogenesis. The main objective of our study is to obtain antiplasmin in large amounts, for crystallization and structure determination of APL and of its complex with plasmin, and for solution studies of the complex. Bacterially expressed APL will not be glycosylated, an advantage in crystallization trials.Bacterial expression of rAPL has been problematic. We have found that it can be greatly enhanced through the use of host E.coli cells that carry extra copies of genes for tRNAs coding for rarely used codons in E.coli that occur in high frequency in eukaryotic genes. Several vectors were screened for rAPL expression (pET19b, pET20b and pET28b). rAPL is expressed in high yield from a pET28b construct in host BL-21 RIPL codon plus cells. rAPL thus expressed accumulates as inclusion bodies, but can be solubilized using N-lauroyl sarcosine at pH11. Refolding and purification of rAPL is achieved by using a sizing column followed by a Nickel His-tag affinity column with an imidazole gradient. rAPL fractions thus obtained are stable at 4°C in the presence of EDTA. However, no inhibitor activity of this rAPL towards trypsin was observed, nor did it form inhibition complex with trypsin. The presence of trace protease and/or failure to fold correctly may be preventing recovery of inhibitory activity. A screen of various refolding buffers failed to yield soluble, stable APL.

Blood fluke Schistosoma mansoni is one of the most important human parasites. Proteolytic system of schistosoma is crucial for parasite - host interactions. Therefore some of the proteases became potential therapeutic targets. This work is focused on not yet characterized serine protease SmSP2. SmSP2 is newly discovered protease of S. mansoni, whose biological role is unknown. This protease is highly expressed in developmental stages parasitizing humans. SmSP2 was recombinantly expressed in prokaryotic and eukaryotic expression system (E. coli a P. pastoris) and purified using chromatographic methods. Recombinant SmSP2 was used for polyclonal antibody production. Conditions for refolding were optimized. Basic biochemical properties of the protease were detected and substrate amino acid preferences for P1 - P4 sites for single aminoacids were identified using synthetic fluorogenic peptides for positional scanning substrate combinatorial library (PS-SCL). (In Czech)

Chloride channel family has been shown to play a significant role in physiological homeostasis processes. The function mechanism of these proteins has not yet been clearly understood. Their deficiency or mutation causes serious human illnesses. Our understanding of the chloride channels' transporting mechanisms can lead to better treatment of these illnesses. As mammalian chloride channels are difficult to prepare in laboratory, the experiments are usually done on homologous chloride channels from prokaryotic organisms. The structures of prokaryotic chloride channels have been solved and moreover they are produced with high yields. Most experiments currently use protein crystallography and provide a static picture of the system. This thesis is focused on the study of structural changes of an E. coli chloride channel using hydrogen/deuterium exchange. This method enables us to monitor dynamic conformation changes dependent on pH and exchanged ions. The measurements were done for the protonated (pH 4.5) and deprotonated state (pH 7.5) and/or in the presence of various anions: Cl− , SCN− , I− , F− , TAR. (tartaric anion). The obtained results justified the theories explaining the function of chloride channel as Cl− /H+ antiporter and provided new findings. Subject words biochemistry, protein...