1.1 125 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Feiruz Alamiri author Fredric Carlsson supervisor Degree Projects in Molecular Biology LURS00013 department Mycobacterium tuberculosis- invades to kill<br /> <br /> Tuberculosis is a leading cause of death globally. The causative agent, Mycobacterium tuberculosis, resides primarily in so-called macrophages, a type of immune cell that normally serves to kill invading bacteria. The mycobacterial ESX-1 type VII secretion system is critical for ability of the bacterium to survive and grow in these cells. Moreover, ESX-1 is known to induce secretion of proinflammatory cytokines, including IL-1ß, as well as cell death to infected cells. Because these functions may have important roles during infection, this project aimed to gain insight into mechanistic basis for ESX-1-induced IL-1 ß secretion and cell death, respectively. <br /> <br /> To study these processes we employed genetically modified macrophages and Mycobacterium marinum (Mm) – a close genetic relative of M. tuberculosis – that were analyzed in experimental infections in vitro. The extent of IL-1 ß secretion and cell death in infected macrophages was determined by measuring the amount of a cell death marker (the protein lactate dehydrogenase, LDH) and IL-1β in the culture medium. <br /> <br /> ESX-1 dependent IL-1β secretion <br /> Mm infection of macrophages lacking key components of the inflammasome system (responsible for secretion of IL-1 ß) revealed a significant ESX-1 dependent IL-1β secretion via the inflammasome proteins Caspase-1 and ASC. As has not been pointed out before, Caspase-11 was shown not to be involved in this ESX-1 regulated secretion. <br /> <br /> CYPD-dependent cell death <br /> CYPD dependent cell death by ESX-1 <br /> The Cyclophillin D (CYPD) protein is involved in a specific type of cell death called CYPD-dependent necrosis. This cell death is characterized by CYPD regulated pore formation in the outer mitochondrion layer (the cellular energy providing machinery) resulting in loss of cell balance, cellular swelling and burst. <br /> <br /> <br /> Treatment of macrophages with titrated amounts of CYPD inhibitor (Cyclosporin A, CsA) resulted in a dose-dependent inhibition of ESX-1-mediated cell death. These results indicate that ESX-1 induce cell death via CYPD-dependent processes, and imply a previously unrecognized functional interaction between ESX-1 and the host cell mitochondria. <br /> <br /> Advisor: Fredric Carlsson <br /> Degree project 45 credits in microbial immunology 2015 <br /> Department of immunology, Lund University http://lup.lub.lu.se/student-papers/record/7363808/file/7365765.pdf application/pdf 929512 yes 2015 eng Biology and Life Sciences 7363808 2015-06-17T09:12:55+02:00 2015-06-18T14:04:28+02:00 2015-06-17T10:12:00+02:00 1 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Samuel Cerps author Associate Professor Lena Uller supervisor Degree Projects in Molecular Biology LURS00013 department Introduction: Asthma is a chronic inflammatory lung disease, which are associated with airflow obstructions. Asthma exacerbations are commonly caused by respiratory viral infections and lack efficient treatment options. Rhinovirus-induced exacerbations have been characterized by occurrence of necrotic cells, which may cause pathogenic effects. We hypothesized that house dust mite (HDM)-induced allergic inflammation primes the airway to aggravated responses to viral infection involving activation of cell death pathways: apoptosis, necrosis and necroptosis.<br /> <br /> Methods: The bronchial epithelial cell line BEAS-B was used to study apoptosis in vitro. Wild type (C57BL/6), Myd88-/-, NfκB-/-, IRF3-/- and IFN-β-/- mice were administered saline or HDM intranasally for three weeks to induce experimental asthma in vivo. Double stranded (ds) RNA was given for additional 3 days to mimic viral infection and induce exacerbation. Lung tissue apoptosis was evaluated using Western blot for cleaved caspase-3 and PARP together with TUNEL-staining on lung sections. Lung expression of RIP3 was determined to explore occurrence of necroptosis (regulated necrosis).<br /> <br /> Results: TUNEL analysis revealed that HDM mainly induced cell death in recruited inflammatory cells which were further aggravated in IFN-β-/- mice but absent in NfκB-/- and Myd88-/- mice. Exacerbation evoked by PIC aggravated the HDM-induced lung inflammation and increased the apoptotic markers cleaved caspases-3 and PARP compared to control (p&lt;0.05). The necrosis marker RIP3 was induced by PIC alone in animals without prior HDM challenge but was not further increased at exacerbation. Furthermore RIP3 followed the same pattern as the pan necrotic marker lactate dehydrogenase levels in bronchoalveolar lavage fluid. We could confirm that PIC induced apoptosis in BEAS-2B cells in vitro.<br /> <br /> Conclusion: A mixture of both apoptotic and necrotic pathways are activated during exacerbation. This suggests that cell death pathways are involved in asthma exacerbations and may thus be molecular targets for therapeutic intervention. Periods of Asthma Attacks Lead to Increased Cell Death<br /> <br /> There is a coordinated balance between new and dying cells in the body controlled by different signals. The cells in the body can either die in a silent manner or burst in an inflammatory way that can be destructive for the organism. There are many diseases that are caused by problems in the cell death machinery involving both the silent and non-silent forms of death. Asthma is a chronic disease causing the lungs to react more easily to things that are not necessary harmful. This reaction leads to obstruction of the airways, which makes it harder to breath. Sometimes asthmatics have periods called exacerbations, during which they have more frequent asthma attack. The cold virus commonly causes these periods because asthmatics have deficient signals involved in fighting the virus. It is not known if the signals present in an asthmatic lung contribute to cell death or if cell death is actually one of the mechanisms that cause the disease, especially exacerbations<br /> <br /> A mouse model helps us understand the impact of cell death in the context of asthma<br /> In an experiment, both the silent and the non-silent form of cell death were studied by looking at specific markers of cell death in an asthma and exacerbation model. A viral mimic that was used instead of a real virus, induced both the silent and the non-silent form of cell death. During exacerbation there was an increase in the expression of the markers of the silent form of cell death compared to non-asthmatic mice that received the viral mimic. It was found that an important signal involved in fighting viral infections was also needed for survival of immune cells in asthmatic mice.<br /> <br /> These findings suggest that an asthma exacerbation caused by viral infection leads to an increased cell death compared to what we see in a healthy person just having a cold. If this increased death is manifested as death of the protective outer cell layer in the lungs together with impaired survival of cells fighting the virus, a consequence would be that viruses could reach further down in the lungs thereby causing an asthma attack. Therefore the cell death machinery could be an important drug target for treating asthma exacerbations.<br /> <br /> Advisors: Lena Uller and Hamid Akbarshahi<br /> Master´s Degree Project 60 credits in Molecular Biology, 2016<br /> Department of Biology, Lund University. http://lup.lub.lu.se/student-papers/record/8891276/file/8891284.pdf application/pdf 10647167 yes 2016 eng Biology and Life Sciences 8891276 2016-09-09T11:28:42+02:00 2016-09-09T11:37:07+02:00 2016-09-09T11:37:07+02:00 2 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Malou Arvidsson author Sonja Aits supervisor Degree Projects in Bioinformatics LURS00014 department Cell Death Detection in Images<br /> <br /> What if we could kill cancer cells? The fact is that we already can, at least to some extent. Many cancer treatments targets cancer cells and kill them by causing them to commit suicide. To be able to do this even better, and minimize adverse effects, information about the biological regulation behind cell death needs to be known, and far from everything has been discovered. Apart from cancer treatments, cell death plays a big role in many other diseases, e.g., Alzheimer’s disease and stroke. Learning more about cell death can help in the discovery of treatments and cures for all these diseases.<br /> <br /> Every living thing is built by cells, and the cells needs to die to make room for newer cells, remove damaged cells or prevent the spread of cells that are mutated and could become cancer cells. A cell consists of many small parts (organelles) and a core called nucleus in which the DNA is stored. A dying cell goes through changes in appearance such as increased or decreased cell size or changes in its shape. The nucleus of the cell may also increase or decrease in size and other organelles within the cell may also change during the cell death process. These changes can be visualized in a microscope by using specific colour stains for the cells.<br /> <br /> The human genome consists of around 20 000 genes. Each gene has one or several functions and some of these genes’ functions are involved in cell death. Mutations in these genes thus change the controlled cell death process. To identify the genes which are involved in controlling cell death, one can make use of methods which disable genes one at a time and then look for cell death changes in a microscope <br /> <br /> In this project, I have worked with a large microscopy image set, for which all human genes have been disabled, one at a time, before staining them with cell death-specific markers. The image set consists of millions of images, - too many to look at and analyse manually. To analyse all these images, automated processes are required. I have therefore used a machine learning method, to locate all cell objects in the images, count them and measure the size of them in a few seconds per image.<br /> The final product of the project was a program to predict, and evaluate many images in an automated process, which can be applied in batches to the whole image set in parallel, so that the analysis is sped up.<br /> <br /> The program was tested on 30 000 images, half of them were images of cells known to be in a cell death process. The analysis of these images showed that large differences could be detected between dying cells and healthy cells, meaning that the program is now ready to be used on the entire dataset to gain insights about which genes are controlling cell death processes. <br /> <br /> <br /> Master’s Degree Project in Bioinformatics 45 credits 2021<br /> Department of Biology, Lund University<br /> <br /> Advisor: Sonja Aits<br /> Department of Experimental Medical Science, Lund University http://lup.lub.lu.se/student-papers/record/9075474/file/9075475.pdf application/pdf 19719570 yes 2021 eng Biology and Life Sciences 9075474 2022-02-16T16:36:43+01:00 2022-02-16T16:41:27+01:00 2022-02-16T16:41:27+01:00 3 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Marlene Lochner author Lynn Wong supervisor Degree Projects in Molecular Biology LURS00013 department A LASTING ANTI-TUMOR RESPONSE<br /> <br /> Cancer and its treatment have been a hot topic in research for quite a while. Despite, it being well researched, researchers still have not come up with one magic bullet. Will they ever though, is the question? Or should we be satisfied with several working treatments for individual cancers and patients? If we were only this far, since there are still some cancer types that we have no cure for yet, although there are many new targets for cancer treatment coming out.<br /> <br /> In our study we have looked at compounds called SMAC mimetics (SM) which target proteins that inhibit apoptosis (IAPs) as shown in the figure. SM lead to the degradation of those IAPs and therefore, are able to induce cell death, namely apoptosis. There are several different modes of cell death. Apoptosis is believed to be silent and regulated, whereas necroptosis and pyroptosis are thought to induce an inflammatory response which is activating the immune system. Furthermore, as shown in the figure, the cytokine tumor necrosis factor (TNF) can signal through TNF receptor 1 (TNFR-1) stimulating cell death. Thus, we wondered how and if TNF or/and SMs can induce a type of cell death in cancer cells which is activating the immune system and eliciting a lasting immune response.<br /> <br /> Different mouse tumor cell lines were utilized in our experiments to test them for sensitivity towards death upon TNF or SM treatment. To further define the cell death, we determined proteins involved by an inhibitor screen and by protein expression. This way, we observed that proteins involved in apoptosis were expressed upon SM+TNF treatment in cancer cell lines which died. To investigate if the mentioned treatment could induce a lasting anticancer response, experiments to determine immunogenicity were conducted. Results showed that upon SM+TNF treatment in one of the tested cancer cell lines the treatment could possibly induce immune activation through release of damage-associated molecular patterns. However, upon culturing the cancer cells with immune cells we could not see an increase in immune cell activation.<br /> <br /> Immunogenic cell death and a lasting-cancer response are hard to be defined in an in vitro system as we used. However, in this isolated system it was easier to define modes of cell death and release of certain immunogenic stimulants. Nonetheless, to further find the characteristics of the cancer that decide if immunogenic cell death is induced or not upon SM+TNF treatment, other techniques might need to be applied. To transfer the gained knowledge to humans and cancer therapy a closed system such as animals, 3D organoids or organ-on-a-chip might be the mode of choice. As it seems from literature and our research, SM+TNF will also not present a magic bullet for inducing a lasting anti-tumor response.<br /> <br /> Master’s Degree Project in Molecular Biology, 60 credits, 2021<br /> Department of Biology, Lund University<br /> <br /> Advisor: Prof. Dr. Lynn Wong<br /> Institute of Experimental Immunology, University of Zurich http://lup.lub.lu.se/student-papers/record/9066645/file/9066646.pdf application/pdf 9302232 yes 2021 eng Biology and Life Sciences 9066645 2021-10-08T11:14:50+02:00 2021-10-13T11:19:29+02:00 2021-10-13T11:19:29+02:00 4 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Salma Kazemi Rashed author Sonja Aits supervisor Degree Projects in Bioinformatics LURS00014 department Lysosomes are cell organelles in charge of degradation. Lysosomal membrane permeabilization (LMP) causes the leakage of cathepsins and other digestive enzymes into the cytosol from lysosomal cavities. This usually causes the initiation of cell death. LMP can be triggered by many stimuli and this could potentially be used in new therapeutic approaches for removing cancer cells. However, excessive LMP also contributes to numerous diseases, e.g. neurodegeneration, stroke, and some infectious diseases. There are therefore great potential therapeutic benefits in LMP regulation. However, there is currently not enough knowledge in this field because of the long lack of good assays for measuring LMP. However, a novel assay has recently been developed that enabled detection of individual permeabilized lysosomes (the galectin LMP assay). My host group has now used the assay together with other cell death markers in a genome-wide high-content imaging screen to elucidate which proteins regulate LMP and cell death. The aim of this project is to analyse the screen image data set for extracting information regarding genes functioning in the cell death process. This was done by machine learning and compared to the traditional approach to analysing high-content imaging screens using CellProfiler. In the first step of the project, I explored the characteristics of the data set. Most of the images had black, non-informative background that made the detection of small, sub-cellular features, a big challenge even for the human vision. Besides, analyzing such a huge data set with that amount of redundancy in order to extract sparse information by machine learning algorithms, was practically challenging. Thus, I extracted a uniform, random subset of images and made cutouts centered on one cell to test different objectlevel analysis approaches. Unsupervised clustering with different feature reduction techniques was examined to find different types of cell death. However, no distinct clusters were observed. I therefore created an online game for citizen-science based image annotation to obtain a data set with labels for supervised learning algorithms. This is ready to be launched. When comparing new machine learning based approaches with conventional methods such as CellProfiler, we found that the new approaches were more time-consuming and challenging due to the technical issues such as the need for a large annotated training data set and super computational resources and a lack of established workflows. During the continuation of the project it will be determined if machine learning methods are nevertheless superior due to their high capability of exploring deeper and more accurate features of the data. Analysing a large microscopy dataset related to cell death using artificial intelligence<br /> <br /> Cell death is an essential process for quality control in our organism as it eliminates infected or damaged cells. Too much or too little cell death, however, is problematic and contributes to many diseases, e.g. stroke, degenerative diseases or cancer. In this project, we are trying to determine in how many ways cells can die and how the different types of cell death are regulated by analyzing several million cell images that my host group had previously taken in a large microscopy experiment. We were particularly interested in the status of the lysosomes, small compartments enclosed by a membrane, which are involved in different types of cell death. Lysosomes can lose their membrane integrity and release harmful substances into the main part of the cell causing its death, which is called lysosome membrane permeabilization (LMP). <br /> <br /> To evaluate the cell images I used different computational strategies and tools including machine learning, a type of artificial intelligence. The total size of the image set was so large (about 23 terabytes) that I could not do anything without the help of high-capacity Swedish storage and computing servers and also had to work with only a fraction of the images at this point. I first explored the characteristics of some representative images using mathematical techniques. Then, I resorted to a variety of machine learning techniques. I started with different unsupervised methods, which are methods that try to find patterns in the data. I tried to use these methods to group the cell images by similarity in order to understand which different types of dead or dying cells exist. However, no clear groups were seen with any of the techniques I used. The reason this approach was not successful could be that large parts of the images were black background. <br /> <br /> I therefore switched to supervised methods which rely on example images classified by a “supervisor”. To obtain enough labeled images, which is too time consuming for a few biology experts, I designed a citizen science based online game, called Cell Hunter, that I will launch shortly. In the game, ordinary people will label cell images according to several cell death-related characteristics defined by biology experts. The website for this game will be shared in public so that everyone who wants can join. In the future, I will use the labelled images from the game to train artificial intelligence tools called deep neural networks that can learn to detect the different cell death characteristics and classify the entire set of several million images.<br /> <br /> This project is a part of a larger project that aims to find gene networks that are effective in regulating cell death. This can provide new approaches to treat the many diseases that are associated with higher or lower than usual cell death rate. <br /> <br /> Master’s Degree Project in Bioinformatics 60 credits 2019<br /> Department of Biology, Lund University<br /> Advisor: Sonja Aits, Unit/Department: Cell Death and Lysosomes, Faculty of Medicine/Department of Experimental Medical Science, Lund University http://lup.lub.lu.se/student-papers/record/9006926/file/9006929.pdf application/pdf 25071117 yes 2020 eng Biology and Life Sciences 9006926 2020-03-20T14:24:58+01:00 2020-03-20T14:35:26+01:00 2020-03-20T14:35:26+01:00 5 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Luisana Duque author Catharina Svanborg supervisor Degree Projects in Biology LURS00012 department Current cancer treatments are associated with high mortality because anticancer drugs are highly toxic and have numerous side effects. Human α-lactalbumin Made Lethal to Tumour cells (HAMLET) is protein-lipid complex that kills tumor cells and spares healthy differentiated cells. HAMLET interacts with a broad range of cellular targets. Our group has shown that the cell death induced by HAMLET can be correlated with a caspase independent pathway, a transcriptional response characterized by the activation of ion fluxes and p38 MAPK signalling pathway. There are still many cellular events in response to HAMLET that need to be address. HAMLET is isolated from a casein fraction of human milk and Casein Kinase 1 (CK1) has been shown to phosphorylate several proteins contained in casein. Furthermore CK1 has been showed to phosphorylate the partially unfolded state of α-lactalbumin compared to its native state. For this reason we wanted to investigate if there was any functional interaction between HAMLET and CK1. Here we showed by a protoarray, confocal imaging, and co-IP assays that HAMLET interacts with CK1 isoforms. Treatment of cancer cells with HAMLET triggered a marked change in distribution of CK1 isoforms. CK1δ was found to accumulate in a network of filaments corresponding to the cytoskeleton, as confirmed by CK1δ colocalization with α-actinin1. This was explained by HAMLET inhibition of CK1δ dependent phosphorylation of Tau. CK1α and CK1ε accumulated in the perinuclear region of the cell. The perinuclear compartment was identified as the endoplasmic reticulum (ER) as confirmed by CK1α colocalization with the ER marker, calnexin. HAMLET triggered a marked ER stress response by activation of all the three main branches of the Unfolded Protein Response (UPR): activation of the inositol requiring protein 1 (IRE1), the protein kinase RNA-like endoplasmic reticulum kinase (PERK) and the activating transcription factor 6 (ATF6). Involvement of CK1 in the ER stress response triggered by HAMLET still needs to be clarified. Furthermore, inhibition of CK1 isoforms with the CK1 specific inhibitor IC261 caused partial inhibition in HAMLET induced CK1 distribution and caused synergistic cell death. Finally, ion fluxes which are essential for HAMLET-induced tumor cell death partially prevented CK1 change in distribution. Casein Kinase 1 and HAMLET functional interaction in Cancer Cells <br /> <br /> Casein Kinase 1 (CK1) are enzymes that regulate many signaling pathways in cells. A malfunction in CK1 activity has been proved to lead to cancer development. HAMLET is a potential drug that could be used for the treatment of cancer, because it selectively kills tumor cells and it spares the healthy ones. For this reason we wanted to investigate if there was any interaction between CK1 and HAMLET, and if this interaction existed, how does it contributes to the tumor cell death triggered by HAMLET. <br /> <br /> Cell death under different conditions was studied with ATP lite and Presto Blue assays. HAMLET and CK1 interaction was studied with a Protoarray, Co-immunoprecipitation assay, immunohistochemistry and confocal imaging. Immunohistochemistry and confocal imaging were also performed to observe CK1 distribution in response to HAMLET. Phosphorylation levels of CK1 targets after treatment of tumor cells with HAMLET were quantified with a phosphoarray. Finally to see if HAMLET triggered an Endoplasmic Reticulum (ER) stress response in tumor cells which can lead to cell death, techniques like transcriptomics, Western Blot, and RT-PCR were done to analyze different genes and proteins that are known to be involved in the ER stress response. <br /> <br /> Several CK1 isoforms were found to interact directly with HAMLET. HAMLET dramatically changed the localization of CK1 in cells. CK1δ was found to accumulate in the cytoskeleton. This was explained by HAMLET inhibition of CK1δ dependent phosphorylation of the microtubules associated protein Tau. On the other hand, CK1α and CK1ε accumulated in a cellular organelle, the endoplasmic reticulum (ER), which has a fundamental role in protein folding. HAMLET triggered a marked ER stress response through the activation of different proteins that have key roles in trying to recover the normal functions of the ER. Involvement of CK1 in the ER stress response triggered by HAMLET still needs to be clarified. Furthermore, inhibition of CK1 isoforms with the CK1 specific inhibitor IC261 caused a partial inhibition in HAMLET induced CK1 change in distribution and caused synergistic cell death. Finally, ion fluxes, which are essential for HAMLET-induced tumor cell death, partially prevented CK1 change in distribution. <br /> <br /> The results of this study are a good approach to understand many of the cellular events that are involved in HAMLET’s tumouricidal effect. In addition it suggests that drugs directed against CK1 family members could be used for the treatment of cancer. <br /> <br /> <br /> <br /> <br /> Advisor: Prof. Catharina Svanborg<br /> Master´s Degree Project in Biology, 45 credits <br /> Department of Biology, Lund University http://lup.lub.lu.se/student-papers/record/8052647/file/8052675.pdf application/pdf 1893244 yes 2015 eng Biology and Life Sciences 8052647 2015-10-07T10:57:23+02:00 2015-10-07T11:05:24+02:00 2015-10-07T11:05:24+02:00 6 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Mariam Miari author Sonja Aits supervisor Degree Projects in Bioinformatics LURS00014 department When technology meets cell biology<br /> <br /> Lysosomes are small bags present in our cells that are filled with destructive molecules, normally needed for removing cell garbage. In some cases, these bags rupture and their contents leak into the interior of the cell leading to cell death. This can contribute to many diseases, such as Alzheimer’s disease. However, rupturing lysosomes intentionally, can also be a strategy to target cancer cells! To understand lysosomes and cell death better and develop therapies that target them, we need to be able to test how thousands of therapeutic drugs and genetic changes affect them. Ruptured lysosomes, and many other structures important for cell death, like the cell nucleus, can be observed in a microscope but measuring them by hand is too time-consuming. In our project, we used the computer to analyze microscopic images to automatically identify bone cancer cells and structures inside the cells, such as nuclei and lysosomes. Besides, we assessed the possibility of training a computer program to distinguish images of healthy cells from the ones with ruptured lysosomes<br /> <br /> Before visualizing the cells under the microscope, cells had been dyed along with their subcellular structures with specific dyes. Once the desired structures became visible, images had been captured in a microscope and we analyzed these images with a software, called CellProfiler. The software helped us to identify the target structures, to count them, and to know their sizes and shapes as well as their brightness patterns. Since CellProfiler needs a lot of human intervention to find the best settings, we also compared it to another method that is based on artificial intelligence, which learns on its own from example images where the cell structures have been outlined manually. Lastly, we wanted the computer to distinguish between healthy cells and those with ruptured lysosomes, so we ‘trained’ another artificial intelligence model to do so, i.e. we provided it with a large number of images of each type so that it can learn to extract m meaningful patterns that distinguish them.<br /> <br /> We found that the computer performed very well in the tasks that humans used to do manually in the past. Both methods identified the cells and structures inside them quickly and efficiently. This result is very important, because it means that spending so much time and effort on studying cells images and on counting the cells and subcellular structures is not necessary anymore. We found that the artificial intelligence method, which is very self-dependent, generally outperformed CellProfiler, which still relies on the researcher in almost all the steps of analysis. Our second main result showed that the trained artificial intelligence model was able to distinguish images of healthy cells from the ones with ruptured lysosomes (i.e. probably dying cells) with very high accuracy<br /> <br /> Master’s Degree Project in Biology/Molecular Biology/Bioinformatics 45crs.<br /> Department of Biology, Lund University<br /> Advisor: Sonja Aits<br /> Cell death, lysosomes, and artificial intelligence group/ Biomedical Centre (BMC) http://lup.lub.lu.se/student-papers/record/9066762/file/9066763.pdf application/pdf 1914088 yes 2021 eng Biology and Life Sciences 9066762 2021-10-12T15:07:17+02:00 2021-10-12T15:15:52+02:00 2021-10-12T15:15:52+02:00 7 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Parisa Esmaeili author Thi Hien Tran supervisor Catharina Svanborg supervisor Degree Projects in Molecular Biology LURS00013 department Human alpha-lactalbumin made lethal to tumor cells (HAMLET) is a complex formed by human alpha-lactalbumin and oleic acid that displays tumoricidal activity but does not kill healthy, differentiated cells. As the sequence of alpha-lactalbumin is conserved among mammals, lactalbumins from other species are expected to form oleic acid complexes with similar activity. This study investigated the tumoricidal activity of Bovine alpha-lactalbumin made lethal to tumor cells (BAMLET). Bovine alpha-lactalbumin was shown to form a complex with oleic acid, which exhibited tumoricidal properties similar to HAMLET against several cancer cell lines. The cellular uptake of BAMLET was quantified, using confocal imaging and Western blots. A rapid internalization pattern was identified within 30 min. Effects on cell viability were also rapid as shown by ATP lite and Presto Blue assays. Long-term cell death was confirmed using the colony formation assay. The cancer cells response to BAMLET was affected by different media compositions, providing tools to further study the mechanisms of cell death and how to maintain the activity of BAMLET under different conditions. Mechanism of BAMLET in cancer cell death<br /> <br /> Human alpha-lactalbumin made lethal to tumor cells (HAMLET) is the first member of a family of protein-lipid complexes with broad tumoricidal effects against cancer cells of different origins. The complex of HAMLET consists of partially unfolded alpha lactalbumin from human breast milk and oleic acid. Bovine alpha lactalbumin has 85% homology with human alpha lactalbumin and has been shown to form oleic acid complexes with tumoricidal activity. This study examined the properties of BAMLET (Bovine alpha-lactalbumin made lethal to tumor cells).<br /> <br /> This study first compared the tumoricidal effects of BAMLET to HAMLET and the peptide-oleate complex alpha1-oleate. BAMLET killed cancer cells in a dose dependent manner with an efficacy similar to HAMLET, as shown by the ATPlite, PrestoBlue and Clonogenic assay. About 90% cell death was recorded at the highest concentration, after 1hour treatment. In contrast, there was no significant tumoricidal effect by bovine alpha-lactalbumin or oleic acid alone. <br /> <br /> To further examine the tumoricidal effect of BAMLET, on different cancer cell lines, lung carcinoma cells (A549) and two colorectal adenocarcinoma cell lines (DLD1 and HT29) were examined. The cells were treated with different concentrations of BAMLET and all cell lines were sensitive as shown by a reduction in ATP and PrestoBlue. The colony forming ability of cells decreased with increasing concentrations of BAMLET.<br /> <br /> Furthermore, Western blot and live cell imaging were used to analyze the uptake of BAMLET by the different cell lines. A time- and dose-dependent uptake of BAMLET was detected in A549, DLD1 and HT29 cells. By live cell imaging rapid aggregation of BAMLET around the cell membrane was detected, followed by cytoplasmic uptake with a diffuse cytoplasmic localization pattern and finally BAMLET was shown to translocate into the nuclei of tumor cells. <br /> <br /> The results confirm that BAMLET resembles HAMLET in terms of its tumoricidal activity. BAMLET will therefore be further explored as a potential prophylactic or therapeutic agent.<br /> <br /> Master’s Degree Project in Molecular Biology 30 credits 2019<br /> Department of Biology, Lund University<br /> <br /> Advisor: Hien Tran and Catharina Svanborg.<br /> Advisors Unit/Department: BMC/ Division of Microbiology, Immunology and Glycobiology - MIG http://lup.lub.lu.se/student-papers/record/9006842/file/9006843.pdf application/pdf 1712828 yes 2020 eng Biology and Life Sciences 9006842 2020-03-18T14:36:54+01:00 2020-03-18T14:46:33+01:00 2020-03-18T14:46:33+01:00 8 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Patrick Kavanagh author Luis Quintino supervisor Degree Projects in Molecular Biology LURS00013 department Parkinson Disease (PD) is a neurodegenerative disease affecting many neurons in the brain. Particularly nigrostriatal Dopamine (DA) neurons of the substantia nigra pars compacta (SNpc) appear to be the epicenter of the disease. Cell death of these neurons is believed to be the primary cause for motor defects and many of the symptoms seen in PD patients. Many experimental therapies look to prevent such cell death and subsequent disease. Leading this field is the Glial Derived Neurotropic Factor (GDNF) that has show much promise in animal models and clinical trials.<br /> <br /> Ret is the primary receptor for GDNF in the DA neurons and increasing it may provide protection in PD similar to that seen in studies where exogenous GDNF is delivered or expressed. This would be achieved by providing DA neurons with sufficient amounts of RET, allowing efficient activation of the required intracellular GDNF/Ret signalling pathways in target cells (Figure1).<br /> <br /> Here we use Lentiviral Vectors to deliver the RET gene to DA neurons. We then use a destabilizing domain (DD) to provide sufficient regulations of the Ret protein. DD is conjugated to the Ret protein and is ubiqutinated marking the entire construct for degradation. This can be prevented by providing trimethoprim which binds the DD preventing its degradation and allowing protein activity. <br /> <br /> Preventing 6OHDA lesion<br /> Here we use a 6-hydroxydopamine (6OHDA) in rats to model PD and determine the effects of DDRET expression on motor symptoms and cell death. We see a failure of the DD conjugation to provide sufficient regulation of the Ret protein. However, there is apparent ability of Ret to provide protection to the required cell types when assessed by tyrosine hydroxylase (TH) a common marker for DA neurons.<br /> <br /> Master’s Degree Project in Molecular Biology 60 credits 2016<br /> Department of Biology, Lund University<br /> <br /> Advisor: Luis Quintino<br /> Advisors Lund University- Department of Experimental Medical Science http://lup.lub.lu.se/student-papers/record/8895229/file/8895234.pdf application/pdf 868267 yes 2016 eng Biology and Life Sciences 8895229 2016-11-18T11:16:49+01:00 2016-11-18T11:40:25+01:00 2016-11-18T11:40:25+01:00 9 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Katie Laschanzky author Fredric Carlsson supervisor Degree Projects in Molecular Biology LURS00013 department Unravelling type I interferon signalling during Mycobacterial infection<br /> <br /> In 2018 alone, 1.2 million deaths worldwide were due to tuberculosis (TB), an infection, usually of the lung, which is caused by the bacterium Mycobacterium tuberculosis. While antibiotic treatments have been available for decades, antibiotic resistance, as well as the lack of an effective vaccine has prevented the eradication of TB. M. tuberculosis has evolved alongside humans since the start of civilization; the bacillus has adapted to life as pathogens by developing many methods of evading human the immune system. One such survival strategy employed by M. tuberculosis is using a type of immune cell- the macrophage- as a host to survive within the body. Macrophages are present in lung tissue and normally act as first responders to infection. They recognize microbial threats (as well as damaged and dead cells) and attempt to remove them through the process of phagocytosis. Once M. tuberculosis is recognized, it is taken up by the cell into a compartment called the phagosome. Normally the macrophage can eliminate bacteria though the harsh, acidic environment of the phagosome, however M. tuberculosis is unusual in that it can exit this compartment and live freely within the cell and avoid being digested.<br /> <br /> Phagosomal escape has been found to be essential for causing disease in humans and mice. This process is made possible by a group of genes in the region of difference 1 (RD1) area of the M. tuberculosis genome. These genes make up the ESX-1 type VII secretion system which, while still not fully understood, has been found to cause type I interferon (IFN) production. Type I IFN production is normally involved in a protective signaling pathway found during viral infection. However, when it is triggered during bacterial infections such as TB, it has found to be largely harmful. During this signaling response as set of over 300 genes is regulated in attempt to combat infection.<br /> <br /> In our lab M. marinum, a close relative of M. tuberculosis, is used as a model organism to attempt to understand the role of various genes involved in the host response to intracellular infection. The gene serpina3g, which encodes the protein Spi2A, is very highly expressed during M. marinum infection and is regulated by type I IFN signalling. Spi2A is a serpin (serine protease inhibitor) which inactivates cathepsin B, an enzyme which degrades host cell proteins. Under normal conditions, cathepsin B is isolated a cellular compartment called the lysosome, but when cells become stressed, this compartment may burst and cause damage to the cell. When a lysosome bursts (ie. due to damage from infection), the cell starts being rapidly broken down and a cell death pathway called necrosis may rapidly occur. Necrosis during M. tuberculosis infection has been found to result in the release of bacteria from the dying cell, intensifying disease.<br /> <br /> Our hypothesis is that an increase of cellular Spi2A production may play a role in limiting cathepsin B-driven cell death. If an increase in Spi2A limits the amount of damage caused by cathepsin B released during infection, it is possible that necrotic cell death could be limited. If this is the case, damaged cells would be able to trigger a controlled form of cell death, called apoptosis instead. Understanding the cell death pathways involved during mycobacterial infection is an essential step in investigating the mycobacterial disease progression on a molecular level.<br /> <br /> Master’s Degree Project in Molecular Biology 60 credits 2020<br /> Department of Biology, Lund University<br /> Advisor: Fredric Carlsson<br /> Molecular Cell Biology; Microbiology group http://lup.lub.lu.se/student-papers/record/9015135/file/9015144.pdf application/pdf 834118 yes 2020 eng Biology and Life Sciences 9015135 2020-06-09T14:16:04+02:00 2020-06-09T14:21:49+02:00 2020-06-09T14:21:49+02:00 10 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Eirinaios Gkika author Professor Sara Ek supervisor Degree Projects in Molecular Biology LURS00013 department types of B-cell lymphomas. However, the role of MYC in mantle cell lymphoma (MCL), a clinically diverse and incurable disease, needs to be further elucidated. The aim of this study is to evaluate MYC mRNA deregulation and explore the mechanism behind PLK-1 induced cell death, as a target for MYC-driven MCL. To achieve this, RNAscope® is used to detect and quantify MYC mRNA expression in a population-based cohort (BLISS) diagnosed with MCL and correlate it to patient outcome, MYC protein expression and other clinicopathological data. Additionally, PLK-1 is investigated as a candidate target to treat MYC-driven MCL patients by testing two anti-cancer compounds (Rigosertib, Volasertib). MYC expression was quantified by RNAscope® in 87 diagnostic MCL samples. The results revealed not only inter-patient but also intra-patient heterogeneity. A total of 25 patients were shown to overexpress MYC. These patients had significantly (p &lt; 0.0001) worse clinical outcome than patients that did not overexpress MYC, with median survival time of 1.19 years and 5.29 years, respectively. MYC mRNA levels were strongly associated with MYC protein levels (R=0.74 p &lt; 0.001). Additionally, MYC overexpression was significantly correlated to higher risk of death (HR=3.81, 95% CI 1.95-5.52) and also with PLK-1 expression levels (R=0.56, p &lt; 0.001). PLK-1 influences the MYC-dependent kinase network, hence was tested as a potential target to reduce MYC expression in MCL-derived cell lines, using two anti-PLK-1 compounds (Rigosertib and Volasertib). Volasertib was more potent and induced effective cell death and proliferation inhibition with lower IC50 values in 4/5 cell lines compared to rigosertib. Volasertib, but not rigosertib, was shown to reduce both PLK-1 and MYC levels in 3/5 cell lines. In summary, it is shown that MYC overexpression is linked to worse patient outcome and can be used as a high-risk marker to stratify patients based on MYC status. This strengthens the use of PLK-1 as an indirect target for MYC-driven high-risk mantle cell lymphoma. A druggable target as a weapon against an aggressive type of blood cancer<br /> <br /> Cancer is the second most common cause of death worldwide. Mantle cell lymphoma is a type of blood cancer that affects the defenders of an organism, the immune system. The immune system is important for an organism to fight viruses and bacteria. The disease is caused by multiple alterations in a specific type of cells of the immune system, the so-called B-cells. These alterations result in aggressive production and dysfunctionality of the B-cells. The aim of this study is to investigate the role of a gene named MYC, that when overproduced, is observed to contribute to worse survival and death of patients diagnosed with different types of cancer. But why is this important to study? The short answer is that the role of MYC mRNA is less investigated in mantle cell lymphoma and this type of cancer does not have any treatment approaches that target patients with MYC deregulations. Therefore, it is important to also investigate possible targets that minimize the negative effects of MYC overproduction. This leads to the second aim which is to test anti-cancer drugs that can indirectly reduce the amount of the overproduced MYC protein levels. These drugs block a protein named PLK-1 which has been observed to regulate MYC production levels.<br /> <br /> So, how does MYC affect mantle cell lymphoma? First it is important to detect and quantify MYC on cancer tissue collected from mantle cell lymphoma patients. This is done by using a method called RNAscope®, which allows to label and detect MYC. The tissue was then visualized and analysed with a software to quantify MYC. The results showed that high MYC levels are correlated to poor patient outcome. MYC also correlates to PLK-1 and other important dysfunctional components that contribute to a more aggressive disease. These results show that MYC is important to be further investigated to find possible treatment targets.<br /> <br /> What about the second aim? Is it possible to verify that PLK-1 blockage reduces elevated MYC levels? This was shown here by treating in vitro mantle cell lymphoma models using drugs that inhibit PLK-1. Afterward, we evaluated the effect of the drugs by measuring cell death induction and the protein levels of MYC and PLK-1. We showed that one of the drugs, volasertib, was able to reduce both PLK-1 and MYC levels. We were able to show a correlation between PLK-1 and MYC in patient material and because PLK-1 inhibition seems to reduce MYC levels in cell lines, we suggest that PLK-1 could be potentially used as a target to treat MCL patients with high MYC levels.<br /> <br /> Master’s Degree Project in Molecular Biology 60 credits 2022<br /> Department of Biology, Lund University<br /> <br /> Advisor: Sara Ek, Joana de Matos Rodrigues<br /> Advisors Department: Dept. of Immunotechnology, Lund University http://lup.lub.lu.se/student-papers/record/9085976/file/9085983.pdf application/pdf 872510 yes 2022 eng Biology and Life Sciences 9085976 2022-06-09T10:53:36+02:00 2022-06-09T11:01:47+02:00 2022-06-09T11:01:47+02:00 11 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Kyriaki Smyrilli author Adriana Mañas Nuñez supervisor Daniel Bexell supervisor Degree Projects in Molecular Biology LURS00013 department Inducing iron-dependent death to treat high-risk childhood cancer<br /> <br /> Neuroblastoma (NB) is the most common pediatric extracranial solid tumor and one of the deadliest, accounting for 15% of pediatric cancer deaths. In Europe there are 10.5 cases per million, with the median age at diagnosis being 17 months. In some cases, tumors regress spontaneously but in others they relapse and become resistant to treatment. Regardless of the intense treatment, survival rate for high-risk NB is still less than 50%. Hence, there is a high need for improved treatments which will not lead to resistance. This study aimed to test the potential of three drugs which induce an iron-dependent cell death, known as ferroptosis, to treat high-risk NB. These drugs were also combined with the currently used chemotherapeutic protocol to test for potential enhanced sensitivity against aggressive resistant NB. <br /> <br /> Background, aims and significance of this study <br /> NB cells have a higher metabolic need for iron. This enhanced metabolism promotes aggressive growth but can also produce damaging byproducts called reactive oxygen species (ROS). To deal with ROS, cancer cells also have enhanced compensatory antioxidant systems to prevent damage and cell death. We can take advantage of this dependency from a therapeutic standpoint, as the inhibition of these antioxidant systems would potentially sensitize NB cells to ferroptosis, an iron-dependent type of cell death. In this study we have tested three drugs that inhibit different stages of these systems and we have used patient-derived models called organoids, which are 3D cultured spheres of cancer cells. These models derived from high-risk NB patients and are known to be highly resistant to normal chemotherapy, making them perfect to test novel therapeutics. <br /> <br /> Main results <br /> The ferroptosis-inducing drugs were more efficient in inhibiting the cell growth of the resistant models, compared to the current standard-of-care. Two of the drugs seemed more promising and were each combined with the current standard-of-care to test for possible enhanced sensitivity. One combination led to a decreased sensitivity in both models, known as an antagonistic effect. The other combination moderately increased the inhibition of cell growth caused by each of the drugs individually, known as an additive effect. RNA analysis of the later combination showed an upregulation of mechanisms which lead to increased intracellular iron levels and a downregulation of a drug efflux pump. This drug efflux pump has been associated with NB chemoresistance. Hence, this data suggests that this drug combination promotes ferroptosis and increases the sensitivity of resistant NB tumours by decreasing drug efflux.<br /> <br /> Conclusions <br /> This study revealed the potential of ferroptosis-inducing drugs to treat high-risk resistant NB. Combination of the current standard-of-care with one of the ferroptosis-inducing drugs tested, led to enhanced sensitivity, providing an alternative approach to treat this complex disease. <br /> <br /> Master’s Degree Project in Molecular Biology 45 credits 2022<br /> Department of Biology, Lund University<br /> <br /> <br /> Advisors: Adriana Mañas Nuñez and Daniel Bexell<br /> Advisors Unit/Department: Division of Translational Cancer Research, Medicon Village, Building 404 http://lup.lub.lu.se/student-papers/record/9103305/file/9103306.pdf application/pdf 1593704 yes 2022 eng Biology and Life Sciences 9103305 2022-11-17T12:02:18+01:00 2022-11-17T12:05:53+01:00 2022-11-17T12:05:53+01:00 12 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Simon Mathis Kønig author Elena Papaleo supervisor Matteo Lambrughi supervisor Thilde Bagger Terkelsen supervisor Degree Projects in Bioinformatics LURS00014 department Apoptosis is a vital physiological defence mechanism against tumorigenesis and critical for embryogenesis, tissue homeostasis, and discharging damaged or infected cells. Protein members of the B-cell lymphoma-2 (Bcl-2) family regulates the cellular commitment to cell survival or programmed cell death by intrinsic (mitochondrial) apoptosis. In response to intracellular stresses, this apoptotic balance is governed by interactions on the outer mitochondrial membrane, by three distinct subgroups; the activator/sensitizer BH3 (Bcl-2 homology 3)-only proteins, the pro-survival inhibitor proteins, and the pro-apoptotic executioner proteins. Inhibition of apoptosis, e.g., deregulation or functional impairment, by pro-survival proteins can lead to imbalance in tissue homeostasis and overexpression of pro-survival proteins can be oncogenic. The critical role in homeostasis and tumorigenesis, coupled with progress in structural elucidation of the protein-protein interaction in the Bcl-2 family has lead to pro-survival Bcl-2 proteins being considered promising therapeutic targets in anticancer treatments. A better comprehension of the transcriptomic signature and molecular mechanism underlying pro-survival Bcl-2 proteins in different cancer types, could help to clarify their role in cancer development and will likely guide advancement in drug discovery, targeting these proteins. Here, we shed light on the molecular mechanisms underlying the pro-survival Bcl-2 proteins by proposing a `multiscale&#39; approach to cancer bioinformatics. We will start from transcriptomic signatures of the pro-survival proteins and their interaction partners, in breast cancer patients. We will then zoom in, at the molecular and structural level, on the most interesting interactions, using what we call `the computational microscope&#39; for cancer biology. Through this approach we aim at uncovering the gene expression landscape and assess the structural and functional effects of mutations on the binding affinity and structural stability. Moreover, we apply a high-throughput in silico mutagenesis approach to identify functionally important residues in the pro-survival members and their interactors. Understanding the role of pro-survival Bcl-2 proteins in cancer<br /> <br /> Programmed cell death or apoptosis is the process in which a cell commits &quot;suicide&quot;. It is a vital physiological process and essential to human development. When a cell is damaged, apoptosis allows that cell to be disposed of, in an orderly way that protects surrounding cells from harmful cell content. When the process of apoptosis is disrupted, cells that should be eliminated may survive and these may exhibit excessive cellular proliferation, resulting in the development of cancer. The cellular decision making-process, between cell survival and cell death is controlled by protein members of the B-cell lymphoma-2 (Bcl-2) family.<br /> <br /> Regulation of apoptosis is orchestrated by interactions between three distinct subgroups of the Bcl-2 family: (i) the activator/sensitizer BH3 (Bcl-2 homology 3)-only proteins, (ii) the pro-survival, cell death inhibitor proteins, and (iii) the pro-apoptotic cell death executioner proteins. The BH3-only proteins are responsible for sensing cellular damage, whereupon they either activate the pro-apoptotic executioner members or sensitizes the pro-survival inhibitor proteins. Both of which leads to the elimination of the damaged cell. Pro-survival Bcl-2 proteins exploit their function by binding to pro-apoptotic proteins, preventing elimination of damaged cells. <br /> <br /> The interaction between the three subgroups of proteins is mediated through a small amino acid sequence termed the BH3-motif (a motif is a widespread sequence pattern that has a biological function). The inhibition of apoptosis by pro-survival members can enable cancer cells to escape cell death and pro-survival members of the Bcl-2 family have been found to be overexpressed in a range of cancers. That is, the relative abundance of pro-survival proteins has been found to be higher, in some cancers, in respect to normal tissue. <br /> Because of their pivotal role as inhibitors of apoptosis, pro-survival Bcl-2 members are considered to be promising therapeutic targets in anti-cancer treatments. That is, the intriguing prospective in designing drugs that can induce cancer cell death by preventing these members from inhibiting apoptosis. Though progress has been made in the development of drugs (BH3-mimetics) mimicking the BH3-only sensitizers, common for these drugs are that they have not been successful in discriminating between different pro-survival members.<br /> Additional knowledge of the abundance of different pro-survival members in different cancer types, coupled with a better understanding of their interaction and functionality on a molecular level, will provide a solid foundation for the development of BH3-mimetics with increased sensitivity and specificity. <br /> <br /> We have proposed a computational workflow to shed light on the role of the pro-survival Bcl-2 members, by bridging two of the major branches of cancer bioinformatics: (i) analysis of high-throughput sequencing data, and (ii) molecular modeling.<br /> We started from transcriptomic profiles of the pro-survival proteins in cancer patients, with the aim of clarifying their abundance in cancer tissue, compared to normal tissue. <br /> Next, we zoomed in at the molecular and structural level of the pro-survival members and their interactors, with what we call ‘the computational microscope’ for cancer biology, allowing us to predict amino acids important for protein-protein interactions.<br /> <br /> To demonstrate this approach, we conducted a study using breast cancer and found a pro-survival protein member to be overexpressed in breast cancer compared to normal tissue. Moreover, we identified several interactors containing small linear sequences resemblaning the BH3-motif found in BH3-only proteins. <br /> <br /> Our study highlights the prospects of an integrative bioinformatic approach for an increased understanding of the pro-survival Bcl-2 proteins and their interactions.<br /> <br /> <br /> Master’s Degree Project in Bioinformatics 45 credits 2018<br /> Department of Biology, Lund University<br /> <br /> Advisors: Thilde Bagger Terkelsen, Matteo Lambrughi and Elena Papaleo<br /> Danish Cancer Society Research Center, Computational Biology Laboratory, Copenhagen, Denmark http://lup.lub.lu.se/student-papers/record/8953112/file/8953117.pdf application/pdf 8610554 yes 2018 eng Biology and Life Sciences 8953112 2018-06-26T15:03:49+02:00 2018-06-26T15:23:45+02:00 2018-06-26T15:23:45+02:00 13 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Jong Chan Lim author Sonja Aits supervisor Degree Projects in Bioinformatics LURS00014 department Cell death is an important field of biology that is linked to many other topics in medicine and biology. One of the methods to investigate cell death is through high content screening (HCS) which makes use of robotic microscopy. By this technology, cell death associated organelle membrane permeabilisation and the localisation of certain dyes can be visualised and elucidate a substantial amount of information. In HCS, an extremely large amount of images is created and much like how factories must filter out poor quality products, poor quality images must also be filtered out. Having tools for image processing and quality control available leads to easier and more accurate results in downstream processing. Here, specialised solutions were developed to detect different aberrations such as out of focus images, corrupted images, and images with dead pixels. A method for finding out of focus images was developed using the laplace operator to measure the frequency of edges in an image. In addition, machine learning was implemented using Tensorflow to train models to detect aberrant images. The best model showed an accuracy of 96% when prediction was performed on new images in the corrupted image category. An effective model could not be trained for the dead pixel category. However, this aberration type is scarce. Finally, all of the tools developed were compiled into an organised Python package for streamlined usability. Filtering Good and Poor Quality Images of Cells to Research Lysosomes<br /> <br /> With today’s technologies, we are surrounded by an immeasurable number of images that need to be analysed by computers. These images play a big part in the life sciences. Different types of images (like cells under a microscope or brain scans taken using advanced machines) can help in learning more about cells or various diseases.<br /> <br /> The Aits Lab focuses on how cell death and a part of the cell called the lysosome can cause different diseases, so that eventually we can learn how to combat these diseases. One of the approaches that is used is analysing images of cells and lysosomes. Machine learning is an approach used to train a computer program to make predictions by having it learn from data. At present it is a very popular way to find patterns in science. An example of how machine learning can be used is to have a computer program that can tell you if a picture has a dog or a cat. In our study, we want to know if the image is a good quality image or a poor quality image. An example of the images we work with is shown below, with different parts of the cell represented by different colours.<br /> <br /> Consider a person learning how to make fried rice. In order to do this, the person reads 100 fried rice recipes. However, imagine that in 5 of the recipes, there was a typo where it said “mice” instead of “rice.” This might confuse the person but since there are only five instances, they would assume it’s a typo. However, if 60 of the recipes said “mice”, then they might end up making fried mice instead of fried rice. In the same way, if we’re teaching machines to do something and the data is of poor quality, the machine won’t learn the way we want it to. <br /> <br /> Testing Methods for Three Different Error Types<br /> Different approaches were evaluated for three different error types: blurry images, corrupted images, and images with dead pixels. An accurate method was developed for the blurry images involving measuring how blurry an image was and then setting a threshold which would exclude images that were too blurry. Although it can still be improved, it was able to predict blurry images almost perfectly with the right settings. For corrupted images, a machine learning model was trained to predict corrupted images and had over 95% accuracy. We experimented with different methods for detecting the dead pixel error, but none gave good results.<br /> <br /> These methods can be further developed and then used to find and remove poor quality images from our data set. This way, when we’re working with images later on, we’ll know we’re working with good quality images. <br /> <br /> Master’s Degree Project in Bioinformatics BINP52 credits 60<br /> Department of Biology, Lund University<br /> <br /> Advisor: Dr. Sonja Aits<br /> Advisors Department of Experimental Medicine http://lup.lub.lu.se/student-papers/record/9031410/file/9031412.pdf application/pdf 3199091 yes 2020 eng Biology and Life Sciences 9031410 2020-10-27T15:38:52+01:00 2020-10-27T16:06:23+01:00 2020-10-27T16:06:23+01:00 14 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Karthik Ganganna author Anders P Håkansson supervisor Degree Projects in Molecular Biology LURS00013 department Antibiotic resistant bacteria are emerging worldwide and have endangered the effectiveness of antibiotics. This problematic development has accelerated infections with antibiotic resistant bacteria and to counter this, the World Health Organization (WHO) recently published a report listing twelve antibiotic resistant pathogens that required the most attention. One of those is Streptococcus pneumoniae (the pneumococcus), a gram-positive, respiratory tract pathogen. Pneumococci cause many types of infections, some of which are serious and life-threatening, affecting the lungs, blood, brain, sinuses or middle ears. Increasing emergence of antibiotic resistant pneumococci poses a threat to treatment of these infections. Human milk contains multiple bioactive proteins that confer antimicrobial activity apart from its nutritional and growth stimulatory activity in infants. HAMLET is a protein-lipid complex of human α-lactalbumin and oleic acid that has a bactericidal activity against certain bacteria. To understand HAMLET’s bactericidal activity, we used S. pneumoniae in our studies. HAMLET caused dissipation of the pneumococcal membrane polarity and membrane integrity and killed the bacteria. Depolarization was accompanied by calcium and sodium transport and HAMLET’s activity was effectively inhibited with transport inhibitors and potentiated with ionophores. Inhibition of glycolysis with 2-deoxyglucose, sensitized the bacteria to HAMLET and suggested a role for ATP in maintaining membrane polarity. We also observed that kinase activity was required for HAMLET-induced depolarization and death, and that HAMLET modulated kinase activity in pneumococci. The relative importance of each of these events during HAMLET-induced bacterial death could lead to identifying novel targets for future antimicrobial targets. New paths into HAMLET pneumococci treatment<br /> <br /> Antibiotic resistant bacteria are a major threat to human health. In the US alone, antibiotic resistant organisms have caused approximately 2.9 million infections resulting in 35,000 deaths. In Europe, deaths from antibiotic resistant organisms increased from 2007 to 2015 by almost 250%. Additionally, misuse and overuse of antibiotics are accelerating the spread of resistant organisms compared to past decades and has increased the burden on health care systems around the world.<br /> <br /> In response to the present situation, the World Health Organization (WHO) in collaboration with pharmaceutical companies, recently compiled and presented a list of twelve highly problematic pathogens. These pathogens were prioritized according to their antibiotic resistance level, and their prevalence in society. Since then, governments around the world, including Sweden, have invested into solving the problem of antibiotic resistance. One of the twelve pathogens listed by the WHO is Streptococcus pneumoniae, also known as pneumococcus. S. pneumoniae is one of the many pathogens located in the respiratory tract, usually as bacterial communities called biofilms, where a majority of the spread of resistance genes occur. Pneumococcal infection is often triggered through interactions with respiratory viruses that lead to secondary bacterial infections. The Spanish flu is one example of this interaction, where millions died from a secondary pneumococcal infection due to a novel influenza A virus. Currently, pneumococci cause nearly 500 million illnesses resulting in 1.6 million deaths each year, and an increasing portion of these infection are caused by antibiotic resistant organisms. As antibiotics are becoming less effective, novel strategies to treat and prevent infections with antibiotic resistant strains are needed. <br /> <br /> Infants, through breast feeding, are protected from bacterial and viral infections, in both the gastrointestinal and respiratory tracts. In human milk, a protein-fatty acid molecule was identified, and named ‘Human Alpha-lactalbumin Made LEthal to Tumor cells’, or in short - HAMLET. HAMLET is antibacterial, but also has the ability to weaken bacterial resistance to antibiotics through re-sensitization of bacteria. This makes it an interesting candidate for medical treatment. However, the mechanisms of HAMLET-induced bacterial death and antibiotic-sensitization are not completely clear. <br /> <br /> In our studies, we investigated HAMLETs effect on the pneumococcal membrane. As previously shown, HAMLET caused membrane depolarization and rupturing, we also verified that calcium and sodium were involved in the process. We also tested HAMLET’s activity on glucose starved pneumococci, as glucose is its main energy source, efficiency of HAMLET on starved pneumococci was higher. We also verified that a ‘kinase’ protein, known to be involved in bacterial death, was involved in HAMLET’s activity using a kinase inhibitor. Surprisingly, it was found that calcium and sodium were not required in kinase activation with HAMLET. This has implications on future research, as it presents new paths of inquiry into improved treatments of pneumococcal infections.<br /> <br /> Master’s Degree Project in Molecular Biology - 30 credits, 2021<br /> Department of Biology, Lund University<br /> <br /> Advisor: Anders P Håkansson, Department of Translational Medicine, Malmö http://lup.lub.lu.se/student-papers/record/9059284/file/9059288.pdf application/pdf 816981 yes 2021 eng Biology and Life Sciences 9059284 2021-06-28T11:16:32+02:00 2021-06-28T11:26:13+02:00 2021-06-28T11:26:13+02:00 15 info:srw/schema/1/mods-v3.3xml studentPublicationsM2 Linn Denison author Associate Professor Gabriela Godaly supervisor Degree Projects in Molecular Biology LURS00013 department Effect of zinc-transporter blockers to induce intoxication in Mycobacteria<br /> <br /> The World Health Organisation estimated of 10 million new cases of Tuberculosis in 2019 and an annual death rate of 1.4 million. This makes the disease the leading cause of death brought about by one infectious agent, namely Mycobacteria tuberculosis (Mtb). Because of the emergence of multi resistant bacterial strains, substandard vaccines, long treatment periods and difficulties in diagnostic latent infections, the need to develop new, alternative drugs is urgent. The bacteria are spread between hosts via air born aerosol and mostly affect the pulmonary tracts where they are captured by cells of the immune system. In an attempt to rid the host of bacteria, these immune cells overload them with zinc ions to induce intoxication. The bacteria, in turn, respond with zinc transportation out of their cells through pumps located in their membrane. Mtb is dependent on these pumps for survival in these elevated levels of zinc, hence, inhibiting activity of such pumps could lead to an induction of metal poisoning and clearance of Mtb. <br /> <br /> During this project, the efficiency of seven different inhibitors for such zinc pumps was evaluated. Through monitoring the growth and survival of the Mtb relative Mycobacteria bovis Bacillus Calmette-Guérin (BCG) in the presence of different concentrations of these inhibitors, their respective minimum inhibitory concentration (MIC) was determined. The MIC is defined as the lowest concentration of inhibitor where no BCG growth can be observed. The method for analysis involves a luminous, detectable signal from live BCG which allows for detection of growth. Furthermore, a method based on a detectable colour change was used, where such change implied bacterial survival. <br /> <br /> The test yielded rather ineffectual results with high variance of growth inhibition between different replication. The BCG was clearly affected negatively by the presence of the inhibitors and growth decreased during treatment with them all, although to what degree differed markedly during treatment with the same inhibitor. However, complete inhibition was not observed and therefore, the MIC could not be determined. Suggestively, repeated tests may give more reliable results. <br /> <br /> To investigate the inhibitors respective suitability as antibiotic agents their toxicity on human cells were examined. By using white blood cells, altered to express a detectable signal in response to inflammation, any inflammation caused by the inhibitors present at different concentrations could be quantified. Additionally, the amount of live blood cells after the same inhibitor treatment was examined. The results from these tests show minimal induction of inflammation during exposure to all inhibitors, cellular death on the other hand, was seen to a greater extent for most inhibitors except compound 7, indicating toxicity for all but one inhibitor. However, the toxicity test needs to be repeated for statistical relevance. <br /> <br /> Supervisor: Gabriela Godaly<br /> Co-supervisor: Komal Umashankar Rao<br /> Bachelor thesis, MOBK01, 15 ETC, 2021<br /> Department of Molecular Biology, Lund University http://lup.lub.lu.se/student-papers/record/9060090/file/9060091.pdf application/pdf 1946050 yes 2021 eng Biology and Life Sciences 9060090 2021-06-30T16:09:44+02:00 2021-06-30T16:15:42+02:00 2021-06-30T16:15:42+02:00 16 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Vendela Rissler author Elena Papaleo supervisor Degree Projects in Bioinformatics LURS00014 department Cancer genetics<br /> <br /> Our body consists of billions of cells. As new cells continuously are produced, some other are dying. This homeostatic balance is crucial for maintaining healthy tissues in any living organism. Different cells have different purposes, red blood cells for example transport oxygen around our body, while neural cells can send signals to and from our brain to ensure movement, and skin cells keep our body intact. The function different cells have depend on their “production manual”, called DNA. From DNA, a different set of genes for different types of cells are available. Those genes can be transcribed into RNA-chains, which will work as templates in a production of mainly proteins. Many RNA-chains in a cell can therefore correlate to a high need of that protein at that time. Thus, by analysing changes in number of RNA-chains, researchers can learn a lot about tissue specific cellular behaviour. <br /> <br /> As DNA plays an important role in cell function, damage to it can have severe consequences. Dysfunctional proteins or changes in number of RNA-chains are some outcomes. This is what happens when a healthy cell become a cancer cell. Due to cancerous mutations, those cells will start to divide at a very high rate, and at the same time inactivate their own death. These cells also lose their specific purpose, and are instead inducing harm to surrounding tissues. Spreading of cancer cells will further damage other tissues as well, and by doing so the cancer have evolved to become malignant. A lot of cancer treatments today are targeting different cancer features such as cell division. The problem with this is that some healthy cells in our body share those features, making them drug targets as well. Hence, a lot of side effects come with cancer treatment due to healthy cells continuously being killed off. More efficient cancer drugs are desired, but to develop such further knowledge about differences between cancer and healthy cells is needed. <br /> <br /> By analysing cancer specific RNA quantity and DNA mutations, a more detailed picture on differences between cancer and healthy cells can be captured. In this project, a group of proteins regulating cell death have been genetically analysed with a main focus on their mutations and differences in number of RNA-chains. As cancer cells are known for their resistance of death, those proteins are believed to have a central role in cancer development. A mapping of their alterations in 18 cancer types, subtypes and tumour stages has been performed to further conclude this. That, alongside with analyses on how they can regulate or compensate for each other, have provided a greater picture on how and why they might be suitable for future cancer treatments. <br /> <br /> Master’s Degree Project in Bioinformatics 60 credits 2019<br /> Department of Biology, Lund University<br /> Advisor: Elena Papaleo<br /> Danish Cancer Society Research Center, Computational Biology Laboratory, Copenhagen, Denmark http://lup.lub.lu.se/student-papers/record/8989788/file/8989789.pdf application/pdf 3392919 yes 2019 eng Biology and Life Sciences 8989788 2019-07-03T14:11:38+02:00 2019-07-03T14:17:03+02:00 2019-07-03T14:17:03+02:00 17 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Keshav Kher author Zaal Kokaia supervisor Sara Palma Tortosa supervisor Degree Projects in Molecular Biology LURS00013 department Stroke is a highly debilitating disease of the central nervous system occurring as a result of cerebral ischemia – deprivation of a cerebral tissue from blood that supplies it with oxygen and glucose, causing the cells in affected tissue to die, bringing about a large number of downstream effects and responses. Localized infarction in the brain also triggers tissue death and related responses in remote, directly unaffected, yet connected parts of brain. In case of a cortical stroke, a secondary degeneration in some areas of thalamus has been described, also known as “secondary thalamic degeneration”. This phenomenon also encompasses an elaborate immune response activated against this degeneration. The whole process of cell death and entailing immune response is highly dynamic and progresses in stages that are marked by specific cellular and molecular characteristics. We aimed to unravel this progress from stroke onset to acute stage followed by a chronic stage. To this end, we mimicked a clinical stroke in a rodent model. The primary focus of this study is to characterize the dynamic changes in the secondary degenerative lesions in thalamus at the cellular level, focusing specifically on the chronic phase of stroke. Immunohistochemistry followed by tissue imaging was employed as the main technique to immunostain and visualize the cells present in the region of interest. An artificial intelligence-based software was then applied to quantify these changes at cellular level with reference to different parameters like loss in cell number, area, density etc. High magnification images were then taken in selected sections to observe the morphological changes at cellular level in greater detail. The obtained results show significant changes in terms of neuronal loss as well as demyelination of neuronal axons in the ventro-posterior nucleus of the thalamus as a consequence of cortical stroke. A cascade of inflammatory response was also observed, mediated by astrocytes and microglia. This study uncovers some of these basic changes and an extensive delineation of the processes at molecular and possibly genetic level is warranted. Dynamics of Secondary Degeneration and Inflammation post a Cortical Ischemic Stroke<br /> <br /> The rapid progress and innovations in medical field have markedly enhanced the average survivable age of human population. This has caused an increase in incidence of age-related diseases, the risks of which grow with advancing age. One such condition is stroke which is known to be the second-most common cause of death worldwide and a major cause of disability. Ischemic stroke, which involves about 85% of stroke cases is a result of occlusion by a clot or a lipid plaque, of a blood vessel that supplies blood to the brain.<br /> Brain comprises different types of cells apart from its fundamental units called neurons. These other types of cells include oligodendrocytes, astrocytes and microglia (as well as infiltrating macrophages). A blockage in the brain, thus deprives it of oxygen and nutrients leading to damage and ultimately death of some cell types. The brain as a tissue is an elaborately connected one, where an injury at a primary site brings about neuropathologic changes in nearby and distant unaffected parts of brain. Such neuropathologic changes also include some other cell types responding to it by mounting an inflammatory response. This is called secondary damage which also includes degradation of myelin and one site of this damage is the thalamus. Thus, a stroke in the superficial layers of brain for example, also damages the inner, deeper parts like thalamus leading to development of sensory disorders, cognitive impairment and poor neurofunctional outcomes. The timeline or the course of these events however, is not well understood resulting in lack of therapies. Thus, the goal of this project is to develop a temporal analysis of the events occurring in thalamus after an ischemic stroke, allowing us to obtain valuable information to identify the ideal therapeutic time-window in the treatment of stroke focused on avoiding symptoms associated with secondary degeneration. <br /> <br /> Approach and findings…<br /> Clinical stroke was mimicked in a rodent model by artificially obstructing a specific blood vessel in the brain. Immunohistochemistry was then used as the main technique to stain the brain tissue in order to note the changes quantitatively as well as qualitatively that happen in an acute stage at 3 months compared to the changes that can be observed chronically at 6 months. Quantitative analysis was done using an AI-driven software called Visiopharm®.<br /> Our main findings include a significant loss of neurons in a particular part of thalamus also leading to its shrinkage, possibly beginning at a time point before 3 months, but progressing to completion by 6 months. The loss of neurons is an effect of neuronal death which eventually also causes degradation of myelin that surrounds the neuronal axons. The debris produced by this damage triggers an activation and transforms astrocytes and microglia into a reactive cell type that results in an intricate inflammatory response. Both the cell types change expression of their proteins allowing them to change morphology and bringing about processes like gliosis, phagocytosis and formation of glial scar that consequentially clears the debris caused by dead cells, while also limiting the damage in brain and preventing it from spreading to healthy tissue. This inflammatory response however, as expected, is observed to be subside by 6 month-time-point compared to 3 months. These results give a valuable insight into stroke progression which can be used to design ideal therapies to target specific cell types and/or processes based on the time-point a patient is brought in for treatment.<br /> <br /> Master’s Degree Project in Molecular Biology, 60 credits 2023-2024, Department of Biology, Lund University<br /> <br /> Advisors: Zaal Kokaia, Sara Palma-Tortosa, Laboratory of Stem Cells &amp; Restorative Neurology, Lund Stem Cell Centre, Department of Clinical Sciences, Faculty of Medicine, Lund University Biomedical Centre http://lup.lub.lu.se/student-papers/record/9176134/file/9176135.pdf application/pdf 3410166 yes 2024 eng Biology and Life Sciences 9176134 2024-10-04T15:57:04+02:00 2024-10-04T16:02:31+02:00 2024-10-04T16:02:31+02:00 18 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Raghda Ibrahim author Martin Dalin supervisor Degree Projects in Molecular Biology LURS00013 department Potential use of circulating tumor DNA as a biomarker in children’s cancer<br /> <br /> Pediatric cancer is the second most common cause of death in children in the developed countries, even though a highly improvement in diagnostic tools and treatment strategies occurred in the last decade. Cancer patients have to endure long treatments with many side effects, with possible multiple surgeries, and radiation. Treatment complications result in different physical and psychological consequences that affect the childhood of cancer survivors and their family relationship or lead to death. Thus, one of the greatest challenges in pediatric cancer management is to find strategies to help doctors to find a better balance between sufficient treatment and least side effects to avoid over or under treatment. So our research can possibly help children who need more treatment to receive more and maybe not die as well as those who are recovering well and don’t need as much treatment. <br /> <br /> In the human body, cells are continuously dying. During this process, small pieces of DNA can leak into the blood circulation. This DNA is called cell-free DNA (cfDNA). When cfDNA comes from a cancerous cell it receives the name of circulating tumor DNA (ctDNA). CtDNA can be detected and monitored by analyzing plasma and may be an indicator of the presence of cancer in the body. We aimed to investigate the potential use of ctDNA as a biomarker for treatment response and disease recurrence in childhood cancer by analyzing the correlation between ctDNA levels and clinical outcome during treatment and follow up. For this reason, we set up a pilot study where we analyzed ctDNA in the plasma of 9 children with different types of cancer. To achieve that, we used a novel technique called “Simple, Multiplexed, PCR-based barcoding of DNA for Sensitive mutation detection using Sequencing’’ (SiMSen–Seq). After the completion of this pilot study, we will have a better understanding of the potential of ctDNA as a biomarker in treatment response and disease recurrence. <br /> <br /> <br /> Master’s Degree project 60 credits in Molecular Genetics and Biotechnology 2020 <br /> Department of Biology, Lund University <br /> Subject of degree project: Translational Cancer Research <br /> Supervisor: Martin Dalin http://lup.lub.lu.se/student-papers/record/9028602/file/9028604.pdf application/pdf 4695744 yes 2020 eng Biology and Life Sciences 9028602 2020-09-08T14:36:04+02:00 2020-09-08T14:45:33+02:00 2020-09-08T14:45:33+02:00 19 info:srw/schema/1/mods-v3.3xml studentPublicationsH2 Mònica Sánchez Guixé author Stefan Johansson supervisor Degree Projects in Molecular Biology LURS00013 department Abstract<br /> <br /> Autosomal recessive cerebellar ataxias (ARCA) are a group of rare monogenic diseases with a polygenic ethology, characterized mainly by the neurodegeneration of the cerebellum that causes the ataxia phenotype. Ataxias as well as many other neurodegenerative diseases are suspected to be caused by alterations in the protein homeostasis that evoke protein accumulation in cells leading to toxicity and cell death.<br /> <br /> Using whole exome sequencing in one family, with three siblings affected with ARCA, we identified one homozygous mutation in STUB1 (STIP-1 homology and U-box containing protein) as novel cause of their disorder. Hereinafter, a second family with one affected patient was included in the study: a compound heterozygous with two novel mutations in STUB1. STUB1 encodes CHIP (C-terminus of Hsc70 Interacting Protein), a co-chaperone and an E3-Ubiquitin Ligase that links protein folding and proteasome mediated degradation, binding to heat shock proteins and ubiquitinating their misfolded client proteins to send them to degradation avoiding their aggregation.<br /> <br /> In the present study, we functionally characterized the p.Asn65Ser (CHIP-N65S) mutation identified in patient 1 (family 1) and the p.Glu28Lys (CHIP-E28K) mutation identified in patient 2 (family 2), both which are located in the binding domain important for chaperone interactions. The second mutation seen in patient 2 is predicted to lead to a premature stop –codon (CHIP-K144*). We compared their properties with a mutation that was published during our work as the cause of the Gordon-Holmes syndrome - a syndrome characterized by ARCA and hypogonadotropic hypogonadism. We expressed all four CHIP variants as recombinant proteins and performed an in vitro ubiquitination assay to analyse their ability to ubiquitinate Hsc70. The results show that mutated CHIP variants have an impaired capacity to ubiquitinate proteins. Immunoblotting using CHIP specific antibody on extracts from patient primary fibroblast cultures showed reduced protein steady states for all mutated variants. The CHIP-N65S mutant protein from patient 1 was further analysed in a co-immunoprecipitation assay to analyse the binding with Hsc70, as well as an analysis of the conformational state given that it migrates at a different rate in comparison with the wild type protein as seen in SDS-PAGE.<br /> <br /> This impaired function of CHIP is suspected to cause aberrant protein aggregation that causes toxicity and cell death, where the neurodegeneration of the cerebellar areas would turn into the ataxia phenotype in the patients. Popular science summary:<br /> <br /> Recessive mutations in STUB1 cause cerebellar ataxia<br /> <br /> Autosomal recessive cerebellar ataxias (ARCA) constitute a group of rare disorders characterized by ataxia due to cerebellum neurodegeneration. Here we characterize three novel mutations in the STUB1 (STIP1 homology and U-box containing protein) gene as the cause of ARCA in two families. STUB1 encodes CHIP (C-terminus of Hsc70 interacting protein), a protein involved in the protein turnover system of the cell. Aggregation of misfolded proteins seems to cause toxicity and cell death that leads to neurodegeneration of the cerebellum, causing gait impairment.<br /> <br /> Many neurodegenerative diseases have protein aggregation as a common feature that leads to<br /> toxicity and cell death. CHIP functions as a link between protein folding and proteasomal mediated degradation. It binds to heat shock proteins (chaperones), such as Hsc70 (Heat shock cognate 70), that are important for stabilising proteins. When CHIP binds Hsc70, it normally adds small molecules called ubiquitin (ubiqutination) to the client proteins of Hsc70.This process facilitates the degradation of misfolded proteins via proteasome (Figure 1). Thus, we hypothesise that impaired CHIP could lead to toxic protein aggregation.<br /> <br /> In this study, we present two ARCA families with recessive mutations in the STUB1 gene: one family with a homozygous mutation (CHIP-N65S) and a second family with two different mutations (CHIP-144* and CHIP-E28K, compound heterozygous). We expressed the predicted proteins and analysed their ubiquitination activity to characterize the function of CHIP in the patients. Furthermore, in the first family we performed studies to reveal whether the mutation caused a weaker binding with Hsc70.<br /> <br /> Fibroblasts from patients in the two families revealed lower CHIP steady states in comparison with normal fibroblasts. In vitro ubiquitination assay showed impaired function in CHIPN65S from family one (probably due to weaker binding with heat shock proteins) and a complete loss of function of CHIP-144* from family two (encoding a truncated protein), while CHIP-E28K from family two presented similar activity as the wild type protein. These results show that the phenotype of the patients is likely caused by impaired CHIP function: a combination of the inability to ubiquitinate and the lower steady states of the protein in vivo. A consequence could be that some misfolded proteins are less efficiently degraded and begin to form protein aggregates. If these protein aggregates are toxic to the cell they could cause neurodegeneration in the cerebellum, explaining the observed ataxia in these patients.<br /> <br /> Advisor/s: Stefan Johansson, Per Knappskog<br /> Master´s Degree Project 60 credits in Molecular Genetics as an Erasmus program exchange at the University of Bergen,<br /> Norway 2013/2014<br /> Department of Biology, Lund University http://lup.lub.lu.se/student-papers/record/4451006/file/4451033.pdf application/pdf 1030311 yes 2014 eng Biology and Life Sciences 4451006 2014-05-27T13:57:09+02:00 2014-05-27T14:44:17+02:00 2014-05-27T14:44:17+02:00 20 1.1department exact v1000601 and deathanddepartmentexactv1000601srw.ServerChoicescrdeath