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Dublin City University, Ireland

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Stage-onderwerp 1 2012-2013: Exploring the immunological responses to adjuvants and combined vaccine formulations in a human immune model based on primary human peripheral blood mononuclear cells
(Viral Immunology lab)
 
Stage-onderwerp 2 2012-2013: Drug dissolution modelling (School of computing, SCI-SYM Research Group)
 
Stage-onderwerp 3 2012-2013: Isolation and cloning of vacular stem cells (School of biotechnology Cell Biology)
Abstract Bachelor Project (FBT) Lab Sandra O’Neill 2017-2018: Immunomodulating properties of Fasciola hepatica-derived exosomes

Fasciola hepatica is known to induce fascioliasis, an important zoonotic disease with annual global costs ranging over 3 billion USD. Besides the high prevalence in humans, its victims are foremost ruminants such as cattle and sheep.

While fascioliasis is manageable through the use of chemical agents, resistance is spiking. Thankfully, new potential surrounding exosomes, as key mediators of cellular communication, have recently surfaced as possible vaccine candidates. While tegument and excretory-secretory products of Fasciola hepatica have extensively been studied, a lot remains unknown about exosomes and its effect on the host.

In the conducted research, the immunomodulating effects of F. hepatica-derived exosomes were studied on bone marrow-derived macrophages (BMMФ). The focus was put on classical (M1) and alternative activation (M2) pathways, induced or inhibited by exosomes. Based on the secretion of cytokines (ELISA), the representation of immunological markers (flow-cytometry), presence of intracellular protein transcripts (PCR + gel electrophoresis), the interaction with Toll-like receptors (western blot) and cytotoxicity (Resazurin-assay), a clearer picture of the immunomodulatory effect could be painted.

A pro-inflammatory effect was detected as early as fifteen minutes after BMMФ stimulation, along with increased TNF-α secretion and iNOS expression, while M2-markers remained low and unaltered. This study is the first to report the immunomodulatory effect of F. hepatica-derived exosomes on macrophages, resulting in M1-polarization.

Abstract Bachelor Project (FBT) Lab Paul Cahill 2017-2018The effect of gamma-secretase inhibitor on stem cells

As cardio vascular disease is the most common cause of death in the European Union last year, the aim of this project is to produce nanoparticles loaded with compound E which are able to block gamma-secretase and thereby blocking the Notch Signalling pathway in stem cells.

It is know that compound E is able to block the Notch Signalling pathway in breast cancer cells.  Many research has been performed of how nanoparticles can be made and how to characterize them using dynamic sight  scattering (DLS).

To achieve the aim several minor aims need to be set. First of all, nanoparticles loaded with compound E have to be made. The nanoparticles are characterized by using DLS (size determination) and HPLC (the amount of drug incorporated). After being sure that the drug is incorporated, the compound E is tested to be sure that the drug is being released and is active. This done by comparing the Hey1 levels (gen influenced by Notch signaling pathway between mouse vascular smooth muscle  cells (mVSMC) treated with blank and mVSMC treated with loaded nanoparticles (baseline experiment). 

The results out of the DLS show use that the nanoparticles are not identical in size. But from HPLC results there can be determined that a high percentage (>90%) of the drug is incorporated in the nanoparticle. From the results out of the baseline experiment can be shown that compound E is indeed blocking the Notch signalling pathway. 

The nanoparticles were not what was expected so further experimentation is needed to optimize the production of nanoparticles. The effects of compound E should be more research. Since only the effect of one concentration (55 µM) is research and only one time point has been seen. More research of the effect of time, dose and the present of a magnetic field to fully understand the release of the drug incorporated in nanoparticles.

Abstract traineeship (advanced bachelor of bioinformatics) 2017-2018: Integration of Gene Expression and Genome-Wide Association data

Background

Alzheimer’s disease (AD) is a neurodegenerative disease that causes dementia. It affects the brain by forming Tau-tangles and amyloid-b plaques. There are three major groups of risk factors to the disease: vascular, psychosocial and genetic. The genetic side of late onset Alzheimer’s disease is not yet well understood.
To understand what genes are involved, genome wide association studies (GWAS) have been done in the past. These studies only provide information about the pure genetics, but do not tell anything tissue specific. As a solution for that limitation, Haky Im Lab’s PrediXcan and MetaXcan come in. These methods implement tissue specific gene expression data in GWAS data.

Setup

PrediXcan is a method that takes raw GWAS data, a gene expression model of a specific tissue and a phenotype file. The GWAS data should be provided as dosage files, preferably per chromosome. PrediXcan provides a script to convert the raw GWAS data to dosage files.
MetaXcan takes GWAS summary statistics and a gene expression model of a specific tissue. The GWAS summary statistics file should contain the phenotype data (Figure 1).

The aim of the project was to run a MetaXcan analysis on International Genomics of Alzheimer's Project (IGAP) data. To confirm that PrediXcan and MetaXcan provide similar results, a comparison of the methods was done on Genetic and Environmental Risk in Alzheimer's Disease (GERAD) data. The raw GWAS data is available for GERAD. The comparison was convincing. The advantage of MetaXcan is that GWAS summary statistics are publicly more available than the raw data.

Results

10% of the genes that came up in the MetaXcan analysis on IGAP data were known as Alzheimer’s disease risk factor genes. 76% were genes in the same regions as known genes. The other 14% were genes outside of known regions. These genes could possibly be risk factors as well. To confirm these genes as being involved in AD, a differential expression analysis was done on data of the sequence read archive of NCBI. A dataset of 117 samples of AD and healthy brain samples of the fusiform gyrus was used for that(GSE95587). The results were not convincing.  

Future

The genes that have come up with the MetaXcan analysis will have to be confirmed with other data. On one side another gene expression reference will have to be used. There are gene expression datasets of the brain available on the database of Genotypes and Phenotypes (dbGAP) of the National Centre for Biotechnology Information (NCBI). An application will have to be filed for that. Haky Im Lab’s PredictDB has a pipeline available to train the prediction model, which has been adapted and tested to fit the server structure of DCU. On the other side more GWAS summary statistics data should be gathered.

Additional projects

In addition to the human AD project, there was a project about fascioliasis. A differential gene expression analysis was done on mice infected with Fasciola hepatica compared to control mice. Different pipelines were compared to test the reproducibility of the results that were already obtained by other people in the lab. The resulting genes were roughly the same, but the significance varied according to the statistical test that was used. The overall image of the gene set enrichment analysis showed that cell proliferation and digestion related pathways are downregulated in infected mice and immune system related pathways are upregulated.

Another small project was the comparison of Qiime and Qiime2. Both versions of microbiome analysis software. Qiime is being discontinued so the switch to Qiime2 has to be made.

Abstract traineeship (advanced bachelor of bioinformatics) 2016-2017: Analysis and fine-mapping of Alzheimer's Disease susceptibility loci

Alzheimer’s disease is the most common form of dementia. During a genome-wide association study by the International Genomics of Alzheimer’s Project (IGAP), more than 20 loci were identified that influence risk of Alzheimer’s disease. This project will refine the known genetic association of variants at multiple loci.

This will be achieved by performing imputation of un-genotyped variants at the identified loci in the case/control dataset GERAD (a subset of IGAP). Using the imputed data, association testing will be performed for each locus. This will determine if an association exists between a variant and Alzheimer’s, and the strength of that association. If a novel lead variant is identified at a locus, conditional analysis will be performed to determine the independence of the association signal from the IGAP lead variant at that locus.

The whole process will be automated to analyse each locus quickly and efficiently. To do this, a pipeline script is written in bash that uses the command line tools Plink, gtool, impute2, snptest and awk.

The next step is functional annotation for each of the candidate variants, to determine the potential molecular mechanism through which the candidate variant acts. For functional annotation the webtools 3DSNP and LDProxy are used.

As a side project we tried to determine candidate pleiotropic variants impacting AD and other traits through meta-analysis. This includes other GWAS data from other traits and trying to identify variants which show significance in both diseases.

 

Abstract bachelorproef 1 2015-2016: The effect of the endotoxin LPS on mesenchymal stem cell function and fate

Septic shock is an important disease that can lead to mortality. It is a well-known disease caused by bacteria. Hereby, starts an inflammatory reaction of the body against the bacteria. Lipopolysaccharide is a surface marker on gram negative bacteria that makes cells go under stress. C3H-mesenchymal stem cells mimic stem cells in the adventitial layer of the vessel wall. The effect of LPS on these stem cells in normal conditions are studied. Also, the ability of the stem cell to differentiate to smooth muscle cells and there inflammatory response is under investigation.

There are four proteins under investigation. Tumor necrosis factor-alpha is a cytokine that is activated during an inflammatory response and stimulates this response. Inducible nitric oxygen synthase releases nitric oxygen in the blood vessel during inflammation. It causes the blood vessel to vasodilate. Alpha-actin is a protein that is mostly found in the process of cell contraction. This protein is very important because of the fall in blood pressure during septic shock. If cells create more actin, there can be a bigger cell contraction in the blood vessels so the fall of blood pressure reverses. Calponin-1 stimulates the α-actin to start the muscle contraction.

Alpha-actin and calponin-1 are studied proteins to look at the ability of the stem cell to differentiate to smooth muscle cell and the inflammatory response in normal and differentiating conditions. Calponin-1 is measured by immunocytochemistry. Alpha-actin is measured by immunocytochemistry and western blotting. Inducible nitric oxygen synthase and tumor necrosis factor-alpha are measured to investigate the inflammatory response of the stem cell. Tumor necrosis factor-alpha is measured by enzyme-linked immunosorbent assay. Inducible nitric oxygen synthase is measured by immunocytochemistry and western blotting.

Cells in differentiation media don’t grow much because they are focused on differentiating instead of growing. The calponin-1 is more present in maintenance media than in differentiating media but it increases in differencing conditions during inflammatory reaction while in maintenance media it decreases. This show that more cells want to differentiate to smooth muscle cells to reverse the septic shock. The alpha-actin increases in maintenance and differentiation media to stimulate muscle contraction to reverse the septic shocks blood fall. Inducible nitric oxygen synthase directly increases in maintenance media and cause a big blood fall and triggers the septic shock. Inducible nitric oxygen synthase decreases during differentiating circumstances. The results showed an increase of tumor necrosis factor-alpha at 1 µg/ml LPS and decreases after that.

C3H-mesenchymal stem cells can be differentiating to smooth muscle cells because calponin-1 and alpha-actin increases during differentiating and inflammation circumstances. The cells do produce inducible nitric oxygen synthase and TNF-alpha to trigger an inflammation reaction. So the cells in the adventitial layer are affect by the inflammation and produce proteins to block this reaction.

 
Abstract bachelorproef 2 2015-2016Optimization of the production and purification of a recombinant protein

This thesis is about the optimization of the production and purification of a recombinant protein. The used recombinant protein for this work is Green fluorescent protein, produced by the Escherichia coli (E.coli) host.

The protocol lab BE383 forms the base for the experiments. To optimise that protocol several parameters will be changed and investigated. First the parameters for protein production are examined. An E.coli colony is incubated overnight and then inoculated in LB broth until the proper OD is reached. Afterwards, parameters can be changed and examined. Different E.coli hosts have first been tested to determine which one yields the best protein production. Once the right clone is chosen, colonies were grown for several hours to determine the optimal incubation time. To this end, samples were taken over a timespan of 72 hours. In the next step, different levels of IPTG were added to the broth with E.coli and samples were taken at different points in time. Next comes finding the right temperature to grow, since colonies grow at variable temperatures. Bacterial growth was monitored by taking the absorbance from the samples. The samples were set on a SDS-PAGE gel which was afterwards stained with instant blue.

The second part is the optimisation of the purification of the protein. A better cell lysis implies a better purification, which is why it was optimised. The original protocol was run with the improvements discovered in past experiments, one with the normal lysozyme to break the cells and the other one with the Bug buster. Samples were taken and then run on an IMAC column. The elusions were run on a SDS-PAGE gel and stained with instant blue. A protein assay, the Biuret method, is performed to calculate the total amount of the produced protein.

The new clone grows better and has a higher protein production than the old one. The results from the time-course show the highest amount of protein after 5 hours of incubation and cells still  grow as the absorbance is high. The highest protein production and absorbance can be seen at 37°C, which means that the cell grows best at that temperature. 100 µM IPTG induces protein production best since other concentrations induce less production or even block the cell growth. In the last result  the Bug buster implies the best cell lysis.

The results show that the following points can be optimised: use the new clone, with an incubation time up to 5 hours and Bug buster for the cell lysis. The IPTG level and temperature do not need to be optimised.

 
Samenvatting eindwerk 1 2014-2015: Expression and role of a Cyprinid herpesvirus-3 microRNA
MicroRNAs are small non-coding, single-stranded RNAs molecules found in plants and animals and they are important in the viral life cycle. Recently, microRNAs were identified in Cyprinid Herpesvirus-3. This virus infects carps and koi and leads to drastic consequences in the aquaculture. A miRNA called MR5057-miR-3P was found to be the most highly expressed miRNA discovered in CyHV-3. MR5057-miR-3p shows the ability to down-regulate the viral dUTPase gene by binding on 3’UTR or mRNA, this results in a degradation of dUTPase mRNA. dUTPase is an enzyme that may play an important role in regulating the life cycle of the virus.
This project is intended to mutate the binding site of the miRNA so that MR5057-miR-3p is no longer capable of binding. When this is successfully done the expression of the viral dUTPase in the presence of MR5057-miR-3p will be analysed to verify dUTPase down regulation by this miRNA.
The mutation of the binding site of the miRNA consist a few steps. The first step is a PCR based on site directed mutagenesis. This technique is useful for introducing a specific mutation into the plasmid at a specific site. This PCR is followed by ligase and DpnI treatment. The possible mutant plasmid is then transformed into E.coli cells followed by another PCR and a restriction digest to confirm the mutation. The next step is plasmid purification to purify the plasmid. A last PCR and restriction digest is done to be sure that the product of the plasmid purification contains the mutated plasmid.
The result of the first PCR to confirm if the plasmid was mutated was positive, but after the plasmid purification the result is negative. Some other test were done to be sure the plasmid purification worked well.
Because the last result was not what was expected, some other test has to be done before going to the next step. The RsaI enzyme and buffer should be tested if everything still works. When the plasmid is mutated and incorporated in the E.coli cells, co-transfection of non-mutated and mutated plasmid with chemically synthesised MR5057-miR-3p are prepared from transfected cells and electrophoreses by SDS-PAGE. The next step will be to see the level of recombinant dUTPase produced from the plasmids, the gel is blotted onto nitrocellulose and probed with an anti-flag antibody. This antibody can bind to the recombinant dUTPase.
If this whole project succeed, the hypothesis of the miRNA down-regulate the expression of CyHV-3 dUTPase can be confirmed.
 
Samenvatting eindwerk 2 2014-2015: Isolation and characterization of a cloned resident vascular stem cell population
Within the scientific research of vascular biology and pathology, the vascular smooth muscle cells, the multipotent vascular stem cells (MVSC) and the adventitial potential stem cells (APC) have an important role. For years, scientists thought that the vascular remodeling within the blood vessel wall in cardiovascular diseases was caused by dedifferentiated vascular smooth muscle cells. The discovery of two different types of stem cells within the vascular wall challenges this theory. The most recently discovered one is the multipotent vascular stem cell, which is expressed by multipotent vascular stem cell markers Sox10, Sox17 and S100b. They are cloneable, have telomerase activity and have the ability to differentiate into neural cells and mesenchymal stem cell-like cells.
By lineage tracing with a myosin heavy chain 11 marker it is proven that the proliferative smooth muscle cells do not arise from the dedifferentiation of mature smooth muscle cells. The multipotent vascular stem cells become proliferative in response to vascular injuries and differentiate into smooth muscle cells and chondrogenic cells, contributing to the vascular remodeling and cardiovascular diseases.
Atherosclerosis is a cardiovascular disease in which plaques accumulate and build up inside the arteries. The plaque is made up of fat, cholesterol, calcium and other substances that can be found in the blood. Over time the plaque hardens and narrows the arteries which limits the blood flow to the organs and the rest of the body. This can cause heart attacks and strokes. (“What Is Atherosclerosis? - NHLBI, NIH,” n.d.)
To perform research on dedifferentiation theory a 100% pure stem cell line is required. Therefore three methods are developed: the serial dilution, clonal ring isolation and semi solid medium.
The first method is based on cultivating cells at a very low density and diluting them until a theoretical chance of finding a single cell is reached. MVSCs require signaling factors from neighboring cells to grow which makes this technique not useable to created a cloned cell line.
The second method is again based on the principle of plating cells at a very low density but here a micro-environment is created by placing a cylinder over the single cell and letting it grow to a colony. Due to the same problem as with the serial dilution this technique cannot be used.
By culturing MVSCs in suspension a cloned cell cluster was isolated and thus be able to form a cloned stem cell line. Via the semi solid medium technique macroscopic colonies were formed and able to pick which are derived from a single cell.
The goal of this research is completed by culturing suspension MVSCs and growing them in semi solid medium which are then grown in cell culture flasks and screened if it is a  100% pure population. The cell line was positive for proteins; Sox10, Nestin, Sox17, S100b and beta-actin while being negative for SMMHC11.
The results are as expected and it can be concluded that a 100% pure cloned stem cell line is formed.
 
Samenvatting eindwerk 2013-2014: SNARE proteins in immune cells: A possible role for fats?
Fusing of the docked bilayers of eukaryotic cell membranes is mediated by SNARE proteins. These proteins are found in the membrane of the cells. There are two types of SNARE proteins, R-SNAREs and Q-SNAREs. R-SNAREs are mostly found on the vesicle membrane while Q-SNAREs are found on the target membrane. Q-SNAREs are subclassified as Qa-, Qb-, Qc- and Qbc-SNAREs. At time of writing, the mammalian SNARE family has 38 members. They are classified as Q-SNAREs, R-SNAREs and some are unclassified. When there is a dysfunction of the SNARE mechanism, this might contribute to diseases such as diabetes, pulmonary arterial hypertension and Parkinson’s disease.
In immunological cells, SNAREs are best documented in macrophages.  They regulate trafficking for several aspects of innate immune responses such as phagocytosis, tumor necrosis factor secretion and cross presentation of antigens. A continuous stream of carrier vesicles, with attendant SNAREs, are responsible for the transport of cytokines between intracellular compartments. Finally, the cytokines are transported to the cell surface where they are released.
In this project, dendritic cells were studied. Dendritic cells are professional antigen-presenting cells. They have the ability to induce a primary immune response in resting naïve T cells. Dendritic cells play a crucial role in initiating and amplifying the innate and adaptive immune response. Vti1a, VAMP3, VAMP8, STX3, STX6, STX8 are examples of SNAREs that are found on dendritic cells. In this project expression of STX3 is examined.
The omega 6 fatty acid arachidonic acid and the omega 3 acid docosahexaenoic acid facilitate SNARE interactions. These polyunsaturated fatty acids are released by phospholipase A. It is an enzyme that splits membrane phospholipids, resulting in release of several lipid metabolites. Stud-ies have shown that arachidonic acid and docosahexaenoic have a positive influence on the ex-pression of STX3. In this project it was examined what the effect of docosahexaenoic acid is on expression of STX3 in dendritic cells.
In the in vitro study presented here, a dendritic cell line (JAWS II DCs) was used. The cells were cultured for seven days in the presence of GMCSF. On day four docosahexaenoic acid was added. After seven days, the cells were stimulated with lipopolysaccharide. At different intervals, ranging from 1h to 24h, STX3 mRNA and protein levels were determined. Moreover, cytokine release in the cell culture medium was measured. STX3 protein expression was increased in cells treated with DHA while this is not noticeable for mRNA expression. The cytokine levels of IL-6 and TNFa did not show a significant difference when DHA was added. IL-10, IL-13, IL-12p70 and IL-27p28 production was increased when cells were treated with DHA. However this was not noticeable with all the time intervals. The conclusion of this project is that in dendritic cells the fatty acid docosahexaenoic acid has a positive effect on the expression of the Qa-SNARE STX3 on protein level.
 
Samenvatting eindwerk 1 2012-2013: Exploring the immunological responses to adjuvants and combined vaccine formulations in a human immune model on primary human peripheral blood mononuclear cells
The use of vaccines has become very common in everyday life so it is important to study the effects that vaccines have on the immune system. Recently an influenza vaccine developed against swine flu (influenza H1N1) (Pandemrix) was withdrawn from the market due to adverse responses to an adjuvant included in the vaccine formulation. In addition, as more and more vaccines are brought to the market it is proposed that vaccines will be combined to ensure uptake in the stakeholder populations. Therefore it is necessary to investigate what the human immune responses are to new formulation vaccines/adjuvants or how combining vaccines may interfere with each other in the human population. 
In this project a human model was developed to monitor innate and adaptive immune responses of an adjuvant associated with adverse outcomes and to monitor the outcome of combined vaccine formulations. The study was carried out in the Viral Immunology lab of Dublin City University and consisted of two objectives. One was to study the immunological effects of the adjuvant AS03 that was used in the H1N1 2009 pandemic vaccine Pandemrix. The Pandemrix vaccine has been associated with the sleeping disorder, Narcolepsy. The second objective was to examine the effectiveness of combining a pneumococcal vaccine and a seasonal influenza vaccine since research already conducted in Viral Immunology lab at DCU had shown results that the bacterial infection and the life virus strongly interfere with one another. 
To monitor the innate immune responses, CD14+ monocytes were isolated from human blood donations and incubated with different concentrations of the vaccines or with the different ingredients of the AS03 adjuvant used in the vaccine. When the innate stimulation was completed a co-culture with allogeneic mixed lymphocytes was carried out to study the adaptability of the T-cells against the vaccines. The cytokine production and cell maturation were measured by performing ELISA and flow cytometry.
The ELISA results of the adjuvanted Influenza vaccine showed increased innate IL-23 which may explain the link to the onset of Narcolepsy associated with the adjuvant. For the combined vaccine approach the ELISA showed that there was an interference in the innate immune responses particularly when the cells stimulated with the combined vaccines were compared to the immune responses from the cells stimulated only with the Pneumococcal vaccine. There were no interferences noted when examining the adaptive immune responses. The flow cytometry results showed that the combination formulation induces increased dendritic cell maturation of the CD14+ monocytes compared to the individual vaccines. The overall conclusion of the first project was that the adjuvant does produce adverse effects combined with the Influenza vaccine and the conclusion of the second project was that the combination of  Influenza  and Pneumococcal vaccines does interfere with individual efficacy and this needs to be taking into account when the combination vaccine  is developed.
 
Samenvatting eindwerk 2 2012-2013: Drug Dissolution Visualisation
The design of drug delivery systems is a rapidly evolving field whereby the formulation is redesigned according to therapeutic requirements. Targeted delivery, for instance, has become a necessity in certain cases. This has caused for a corresponding increase in the complexity of the drug delivery systems which, in turn, necessitates a lot of in vitro testing to investigate the effects of this systems’ composition, design and manufacturing process. These tests require a lot of time, effort and financial investment to reassure agencies such as the Food and Drug Administration that the formulation will behave as specified in human (in vivo). This process is known as in vitro in vivo correlation (IVIVC) and is where modelling comes in to try and address these problems in silico.
One of the research topics at the Sci-Sym (centre for Scientific Computing & Complex System Modelling) Research Center, at DCU, is aimed at developing and improving mathematical and computational models for drug delivery systems. Ultimately the goal is to accurately simulate the drug release over time and thereby gaining an understanding of the microscopic features of new complex drug designs, many of which are not amenable to in vitro testing. 
In the light of this project and given the diverse background of individuals involved in such collaborative activities in the area, there exists a drastic need for a powerful yet simplistic tool for the visualisation of these computational models. This visualisation has the potential to give a good indication of the power of actual models in representing the capabilities of the model.
 
Samenvatting eindwerk 3 2012-2013: The isolation and cloning of vascular stem cells
Vascular smooth muscle cells have an important role within vascular biology and pathology. Increased knowledge about these cells is therefore an important foundation for many vascular studies. For decades, scientists thought that smooth muscle cells proliferate through the de-differentiation theory. The discovery of stem cells within the vascular wall has challenged this theory. The new hypothesis suggests that up to four vascular stem cells (Sca adventitia progenitor cells, multipotent vascular stem cells, mesenchymal stem cells and endothelial to mesenchymal stem cells) are responsible for the repopulation of smooth muscle cells. Beside theoretical changes, many questions arise about the function, molecular differentiation pathway and their contribution to vascular pathology. In order to improve our knowledge on the role of vascular stem cells, the research at the school of biotechnology in Dublin City University, under supervision of Professor Paul Cahill, proposed to isolate and clone the two vascular stem cells, Sca-1+ and multipotent vascular stem cell.
The first vascular progenitor cell is termed Sca-1+ cell and is situated in the adventitia of the vascular wall. Previous research suggested that Sca-1+ cells have the ability to differentiate into smooth muscle and endothelial cells and contribute to the vascular disease, atherosclerosis. The first step in order to improve the knowledge about Sca-1+ cells is to generate these cells. Since these cells are not commercially available, Sca-1+ cells are developed in vitro by the stimulation of mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells with collagen IV. ES cells are undifferentiated inner cells isolated from blastocyst of an embryo while iPS cell are reprogrammed somatic cells with a stem cell-like phenotype. These cells can be developed from a specific (alive) donor, which significantly increases the therapeutic possibilities.
There is a slight difference in the generation method of Sca-1+ cells between ES and iPS cells. ES cells are purified before being exposed to collagen IV. The cultivation time on collagen IV varies between two and six days in order to determine the optimal exposure time. After the ES cells are cultivated on collagen IV, the present of Sca-1+ cells is determined by immunocytological analysis. The result of the time course confirmed the presence of Sca-1+ cells. The amount of Sca-1+ cells is the highest between day three and five. Using this information, the iPS cells are cultured on collagen IV during four days and Western blotting will confirm the presence of Sca-1+ cells.
The second vascular stem cell is termed multipotent vascular stem cell (MVSC). These progenitor cells are located in the tunica media of various blood vessels. MVSCs are characterized by present of three protein markers: Sox10, Sox17 and S100β with the absent of the myosin and calponin. Recent studies discovered the present of cells with a similar character in two embryonic smooth muscle cell lines: A10 and A7r5. The first step in order to investigate these MVSC-like cells is to clone the A10 and A7r5 population. Therefore two methods are developed: the serial dilution and cloning disk method.
The first method is based on the cultivation of a single cell per well, obtained by serial dilution in a 48 well plate. After 24h one well of the A10 cell line contain a single cells. Six days later, more wells of the A10 cell line contains a single cell clusters. This may be due to the convex bottom of the 48 well plate, resulting in hazy view of some cells. Finally three well with a cell population high likely derived from a single cells are cultured until the population is expanded to a number high enough to investigate the cells for the similarities with MVSCs. The A7r5 cell line contains a few wells with a single cell after one day but after five days the multiple cells have died instead of proliferating. These results indicate that the A7r5 cells need more signals from other cells in order to survive and are not clonable in comparison with A10.
In order to solve this problem, the other method uses a cloning disk to detach and transfer a cell clusters (± 15 cells), obtained from a single cell, to a new well in a 24 well plate. The cloning disk does detach the cells from the plate but they do not come loose in the 24 well plate. Further research about the functioning of the cloning disk has revealed that only a part of the attached cells come loose. Thus the cloning disks are only useful if a large amount of cells are detached. A possible solution to this problem is to let the cell cluster grow to ± 100 cells instead of 15 cells. Although this method has not succeeded in cloning cells, further optimization will make the cloning of A7r5 and A10 cell line an achievable goal.
The cloned A10 cells (since the cloning of A7r5 did not succeed) are then screened for the smooth muscle cells markers: smooth muscle-myosin heavy chain (SM-MHC) and calponin by immunocytological analysis. Both protein markers are characteristic for smooth muscle cells while they are absent in MVSCs. If one of the cloned A10 cells does not express calponin or SM-MHC, we termed these cells, MVSC-like cells. The present of the markers Sox17, Sox10 and S100β in the cloned cells will be investigated in future research.
 
Samenvatting eindwerk 1 2011-2012: Production of gold nanoparticles by Shewanella loihica PV-4 protein extract
The interest for the use of nanoparticles in biomedical applications such as bioimaging, biosensing, photothermal therapy and drug delivery has raised a lot during the last decades. This is mainly due the unique optical and electronic properties of the gold nanoparticles. The synthesis of the gold particles occurs by chemical and physical methods; most of these methods are realized under extreme conditions or make use of potentially environmental harmful compounds. For this reason there is a need for new methods that are less energy consuming and environmentally dangerous.
During this research project the synthesis of gold particles by Shewanella loihica PV-4 grown in a defined medium was explored by using different extracts. The formation of the gold nanoparticles was detected by optical spectrometry. The best results were achieved by using a heat killed extract and by dissolving the S. Loihica cells into distillated water. During the second part of the project the effect of electron donor lactic acid and hydrogen on the nanoparticle formation ratio was examined. Both have a visible effect on the formation of the gold nanoparticles. Adding hydrogen to the reaction has a positive effect on the speed of nanoparticles formation. Adjusting the concentration of lactic acid in the growth medium contributed to an increased nanoparticles formation speed and it slightly adjusted the size and shape of the nanoparticles. A drawback on the use of the lactic acid was however that lactic acid has the ability to reduce the gold by itself without formation of nanoparticles; therefore lactic acid concentrations above 100mM couldn’t be used.
 
Samenvatting eindwerk 2 2011-2012: Cloning and Expression of the rhizobactin 1021 Biosynthesis Regulon of Sinorhizobium meliloti 1021
Plants need nitrogen for their growth. The presence of nitrogen in soil is however, scarce. In response to this, certain plants have formed a close symbiotic relationship with Rhizobia bacteria. This symbiosis results in the formation of nitrogen fixing root nodules. Sinorhizobium meliloti is one of these bacteria and the main focus of this paper.
There is a high demand for iron in the symbiotic interaction between plant and bacteria. S. meliloti  extracts this iron from the soil with siderophores.
The genome of S. meliloti consists of chromosomal DNA along with two independent megaplasmids known as pSymA and pSymB. The pSymA S. meliloti 1021 plasmid contains the rhbABCDEF genes that form the operon that codes for the siderophore Rhizobactin 1021. While the genes, responsible for the Rhizobactin 1021 synthesis were first identified in 1989, the biosynthesis and transport  have only recently been determined in depth  by Dr. O'Connell M. and colleagues. However, there is still a query relating to the addition of an enoyl-lipid moiety to rhizobactin 1021 in that the protein responsible for this addition has not been definitively identified.
However, RhbG shows significant similarity to IucB and other acyl-CoA-dependent acyl transferases. It therefore seems likely that it codes for the  enoyl–lipid. The function of this lipid is unknown, but it has been speculated that it anchors the siderophore to the cell.
If the function of the rhbG gene can be determined, it may provide insight into the biosynthesis and ultimately the transport system of Rhizobactin 1021 and similar siderophores.

Address

Glasnevin 9
Dublin
Ireland
Glasnevin 9
Dublin
Ireland

Contacts

Traineeship supervisor
Enrico Marsili
Traineeship supervisor
Johnson Patricia
Traineeship supervisor
Crane Martin
Traineeship supervisor
Cahill Paul
paul.cahill@dcu.ie
Traineeship supervisor
Laura Collins
Traineeship supervisor
Brendan O’Connor
Denise Harold
denise.harold@dcu.ie
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