Leukocyte cell-derived chemotaxin 2 (LECT2) is a zinc-binding multi-functional protein comprising three disulfide bonds, that is involved in multiple disorders of worldwide concern. Recently LECT2 was found to be involved in amyloidosis (ALECT2) and is believed to be the third most common form of systemic amyloidosis. The disease progression of ALECT2 is relatively slow, and the aggregation assembly is foremostly associated with the kidneys and the liver, but also other organs in the later onset of the disease. This study involved developing a protocol for producing His6-TEV-LECT2 including expression in E.coli BL21(DE3), refolding, and purification. The protocol resulted in a sufficient yield for initial measurements for characterization and biophysical analysis with the following methods: mass spectrometry (MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), and fluorimetry. The produced protein was characterized as LECT2 predominantly in its oxidized form. A brief biophysical analysis was made where LECT2 started to unfold already at physiological temperature with a midpoint at 50°C. Additionally, under chemical denaturation LECT2 unfolded with a midpoint of 3 M urea in a cooperative transition without any intermediates. Further on, wavelengths for monitoring the unfolding and the aggregation simultaneously were identified. The unfolding process occurred under 20 sec in 6 M urea and correlates with a double-exponential model. The LECT2 aggregates resemble protofibril-like structures and aggregates species from monomer up to hexamer were found, suggesting simple monomeric addition towards a growing fibril as the aggregation mechanism. The content of aggregates in the sample was notably decreased upon disulfide bond reduction highlighting the importance of further investigating the role of the disulfide bonds in the destabilization and aggregate formation of LECT2.

No description available.

Cell lines are widely used as in vitro model systems and substitute for animal experiments. The frequently used Caco-2 cell line is considered to reflect characteristics of differentiated intestinal epithelium. However, the need to culture the cells with fetal calf serum (FCS) induces a high variability, risk of contamination and is ethically disputed. We tested the culture of Caco-2 cells with human platelet lysate (PL) instead of FCS. We compared cell viability and differentiation by measuring ATP levels, gene and protein expression of specific markers in total cell extracts, brush border membrane vesicles (BBM) and lipid rafts (LR). Cell viability was slightly enhanced in cells grown with PL compared to FCS. The cells differentiated to an intestinal phenotype like the cells cultured in FCS, as indicated by the similar gene expression levels of hexose and protein transport proteins and the structural protein VILLIN. BBM showed a comparable distribution of the intestinal hydrolases, indicating a maintained cell membrane polarity. The distribution of the marker protein FLOTILLIN-2 in LR was also similar. We conclude that PL is an exquisite and suitable replacement for FCS in the culture of Caco-2 cells that can eliminate many disadvantages incurred due to the use of FCS.

No description available.

No description available.

Lysosomal storage diseases are genetically inherited diseases caused by the dysfunction of lysosomal enzymes. In a normal cell, lysosomal enzymes cleave specific macromolecules as they are transported to the lysosome. However, in diseased cells, these lysosomal enzymes are either absent or malfunctioning, causing macromolecular substrates to accumulate, becoming toxic to the cell. Over fifty lysosomal storage diseases have been identified, collectively occurring in one out of 7,700 live births. We investigated the lysosomal enzyme β-galactosidase (β-gal). In order to study the biochemistry and enzymology of this protein a robust expression system was needed. The GLB1 gene has been inserted into Pichia pastoris creating high protein expressing cell lines. The result of this work will yield a high expression system for β-gal, which can then be subjected to structural and biochemical studies.

Yes / Studies have been conducted to highlight the anti-inflammatory and immunosuppressive properties of synthetic cannabinoids as well as their potential for cartilage repair. Various wound healing techniques can be used to investigate the mechanisms of chondrocyte repair in monolayers or three dimensional tissues constructs. In this work the effect of WIN55, 212-2 (WIN-2) on nitric oxide (NO) and matrix metalloproteinase-2 (MMP-2) expressed by wounded chondrocyte monolayers was investigated. Moreover, expression of collagen type-I and type-II, fibronectin and S100 proteins were detected using immunofluorescence and quantitatively verified using ELISA based techniques following treatment with 1 μM and 2 μM of WIN-2. Treating chondrocytes with 1 μM of WIN-2 significantly increased expression of collagen type-II, fibronectin and S100, and significantly reduced collagen type-I expressions as compared to the control groups. On the other hand, both concentrations of WIN-2 significantly reduced the expression of the inflammation markers NO and MMP-2 in a dose dependent manner. These findings highlight the potential use of the synthetic cannabinoids for improving cartilage healing properties as well as acting as an anti-inflammatory agent which could be used to enhance tissue engineering protocols aimed at cartilage repair.

Porcine Epidemic Diarrhea Virus (PEDV) was first detected in Europe in the 1970s, but did not emerge in the United States until 2013. When it arrived, it ran rampant due to the lack of previous exposure, causing the death of 7-8 million neonatal piglets and $900 million to $1.8 billion in losses to the U.S. pork industry in 2013 and 2014. This virus causes diarrhea and vomiting which leads to dehydration and in extreme cases, death. Neonatal piglets rely heavily on passive lactogenic immunity from their mother's milk, thus making them especially vulnerable to this disease. Within 2-3 days of infection during the initial outbreak, there was a 90-95% mortality rate among these weaning piglets. Additionally, this virus is highly contagious, with high rates of fecal shedding during infection. To control the outbreak, the USDA had approved two emergency-relief vaccines, but both have proved to be ineffective at preventing disease or reducing fecal shedding. These vaccines are still available today. As such, it is necessary to develop a vaccine that will be effective at preventing illness and viral shedding. PEDV is a single-stranded RNA virus made of four major subunits: a structural spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. The one most studied and of particular interest is the S protein as it facilitates the virus' attachment and entry into the host cell. The S protein is made of two domains, the S1 domain which allows for protein interactions between the virus and the host cell, and the S2 domain which allows for membrane fusion. Because of the S1's role in protein interaction, it is often the target of potential vaccines. Within the S1 domain, it's C-terminal domain encodes for the receptor binding domain (RBD), which is why the S1 CTD is the target of this study. In this study we focused on the expression, purification, and immunogenicity testing of the CTD protein using T7 Express E. coli as the expression host. We used PCR, gel electrophoresis, Sanger Sequencing, western blots, and mass spectrometry to ensure that the protein was being expressed properly. The future goal is to use this protein as the antigen in a future nanoparticle-based PEDV vaccine. / Master of Science / In 2013, Porcine Epidemic Diarrhea Virus (PEDV) emerged in the United States, causing an estimated $900 million to $1.8 billion in damages to the pork industry and the death of 7 to 8 million newborn piglets in just one year. This virus causes diarrhea and vomiting which causes dehydration and death, and newborn piglets are particularly vulnerable. During the initial outbreak, two emergency-relief vaccines were approved but have not been proven effective against the disease. Thus, it is of great importance to develop a vaccine that is both effective and safe. Therefore, our task was to express, purify, and test the immunogenicity of a segment of the PEDV spike protein to be used as the antigen of a future nanoparticle-based vaccine.

Synthesizing proteins for use in therapeutics is restrained by, in part, contaminants in in vivo expression systems and limited production capacity of in vitro systems. Cell free expression (CFE) systems have emerged as a potential alternative for protein expression because of the inherently lower contents of contaminants, and their flexible modular design that allows the addition of factors that aid in synthesis of complex products. Here, we investigate and establish an in-house Escherichia coli-based cell free protein synthesis (CFPS) system, explore different CFPS commercial kits, develop assays to test performance of these systems and identify potential rules that dictate expression levels. Using CFE, we were able to test different vectors and conditions of system, as well as scale-up protein synthesis reactions. In conclusion, this work shows that CFPS is a functional and easy-to-use platform and can potentially meet the requirements for the synthesis of therapeutics.

Our understanding of the complexity of the brain is limited by the data we can collect and analyze. Because of experimental limitations and a desire for greater detail, most investigations focus on just one aspect of the brain. For example, brain function can be studied at many levels of abstraction including, but not limited to, gene expression, protein interactions, anatomical regions, neuronal connectivity, synaptic plasticity, and the electrical activity of neurons. By focusing on each of these levels, neuroscience has built up a detailed picture of how the brain works, but each level is understood mostly in isolation from the others. It is likely that interaction between all these levels is just as important. Therefore, a key hypothesis is that functional units spanning multiple levels of biological organization exist in the brain. This project attempted to combine neuronal circuitry analysis with functional proteomics and anatomical regions of the brain to explore this hypothesis, and took an evolutionary view of the results obtained. During the process we had to solve a number of technical challenges as the tools to undertake this type of research did not exist. Two informatics challenges for this research were to develop ways to analyze neurobiological data, such as brain protein expression patterns, to extract useful information, and how to share and present this data in a way that is fast and easy for anyone to access. This project contributes towards a more wholistic understanding of the fruit fly brain in three ways. Firstly, a screen was conducted to record the expression of proteins in the brain of the fruit fly, Drosophila melanogaster. Protein expression patterns in the fruit fly brain were recorded from 535 protein trap lines using confocal microscopy. A total of 884 3D images were annotated and made available on an easy to use website database, BrainTrap, available at fruitfly.inf.ed.ac.uk/braintrap. The website allows 3D images of the protein expression to be viewed interactively in the web browser, and an ontology-based search tool allows users to search for protein expression patterns in specific areas of interest. Different expression patterns mapped to a common template can be viewed simultaneously in multiple colours. This data bridges the gap between anatomical and biomolecular levels of understanding. Secondly, protein trap expression patterns were used to investigate the properties of the fruit fly brain. Thousands of protein-protein interactions have been recorded by methods such as yeast two-hybrid, however many of these protein pairs do not express in the same regions of the fruit fly brain. Using 535 protein expression patterns it was possible to rule out 149 protein-protein interactions. Also, protein expression patterns registered against a common template brain were used to produce new anatomical breakdowns of the fruit fly brain. Clustering techniques were able to naturally segment brain regions based only on the protein expression data. This is just one example of how, by combining proteomics with anatomy, we were able to learn more about both levels of understanding. Results are analysed further in combination with networks such as genetic homology networks, and connectivity networks. We show how the wealth of biological and neuroscience data now available in public databases can be combined with the Brain- Trap data to reveal similarities between areas of the fruit fly and mammalian brain. The BrainTrap data also informs us on the process of evolution and we show that genes found in fruit fly, yeast and mouse are more likely to be generally expressed throughout the brain, whereas genes found only in fruit fly and mouse, but not yeast, are more likely to have a specific expression pattern in the fruit fly brain. Thus, by combining data from multiple sources we can gain further insight into the complexity of the brain. Neural connectivity data is also analyzed and a new technique for enhanced motifs is developed for the combined analysis of connectivity data with other information such as neuron type data and potentially protein expression data. Thirdly, I investigated techniques for imaging the protein trap lines at higher resolution using electron microscopy (EM) and developed new informatics techniques for the automated analysis of neural connectivity data collected from serial section transmission electron microscopy (ssTEM). Measurement of the connectivity between neurons requires high resolution imaging techniques, such as electron microscopy, and images produced by this method are currently annotated manually to produce very detailed maps of cell morphology and connectivity. This is an extremely time consuming process and the volume of tissue and number of neurons that can be reconstructed is severely limited by the annotation step. I developed a set of computer vision algorithms to improve the alignment between consecutive images, and to perform partial annotation automatically by detecting membrane, synapses and mitochondria present in the images. Accuracy of the automatic annotation was evaluated on a small dataset and 96% of membrane could be identified at the cost of 13% false positives. This research demonstrates that informatics technology can help us to automatically analyze biological images and bring together genetic, anatomical, and connectivity data in a meaningful way. This combination of multiple data sources reveals more detail about each individual level of understanding, and gives us a more wholistic view of the fruit fly brain.