| 1. |
- Cengic, Ivana
(författare)
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Synthetic biology approaches for improving production of fatty acid-derived compounds in cyanobacteria
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The environmental consequences associated with the use of fossil-sourced fuels and chemicals have brought with it a realization that future development must move in a more sustainable direction. Currently available biofuels or renewably produced chemical, such as bioethanol or biodiesel, are produced from microbial fermentation of sugar-rich crops or by chemical conversion of natural oils or fats. However, these strategies are not sustainable in the long run as fuel and chemical production competes with food supply and arable land usage. Instead of relying on photosynthetic feedstocks that require further conversion, one can engineer photosynthetic cyanobacteria to produce a product of interest directly from CO2 and sunlight. The first part of this thesis aimed to develop new synthetic biology tools for the model cyanobacteria Synechocystis sp. PCC 6803. The second part of the thesis focused on evaluating the regulation of fatty acid synthesis in cyanobacteria, and the production of fatty acid-derived chemicals in Synechocystis.In paper I, fusion of small affinity proteins (Affibodies) to the major type IV pili protein was shown to mediate successful surface display of the affibody. This surface display strategy was further shown to allow inter-species binding between Synechocystis and Escherichia coli or Staphylococcus carnosus displaying complementary polymerizing affibodies.In paper II, a CRISPR-interference tool was successfully implemented in Synechocystis for inducible gene repression. Further, its multiplexing ability was proven by simultaneous repression of up to four aldehyde reductase/dehydrogenase genes. In paper III, this established CRISPRi tool was used to target and repress native pathways competing with heterologous fatty alcohol production in Synechocystis. Repressing the gene encoding the PlsX phosphate acyltransferase allowed re-direction of carbon-flux from membrane lipids to fatty alcohol production, with a final best strain producing 10.4 mg g-1 DCW octadecanol and hexadecanol.In paper IV, the transcriptional response towards perturbations within the fatty acid synthesis pathway was evaluated for the two model cyanobacteria Synechocystis and Synechococcus elongatus PCC 7942. Preliminary results indicate that blocking fatty acid synthesis initiation/elongation causes a transcriptional response of the involved pathway genes only in S. elongatus PCC 7942, indicating differential transcriptional responses in these two strains.In paper V, metagenomically sourced aldehyde deformylating oxygenase (Ado) variants were evaluated for their alka(e)ne synthesizing ability. Several of these novel Ado enzymes outperformed the generally well-performing Ado from S. elongatus when relating alka(e)ne production to the soluble enzyme amount.
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| 2. |
- Anfelt, Josefine
(författare)
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Metabolic engineering strategies to increase n-butanol production from cyanobacteria
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The development of sustainable replacements for fossil fuels has been spurred by concerns over global warming effects. Biofuels are typically produced through fermentation of edible crops, or forest or agricultural residues requiring cost-intensive pretreatment. An alternative is to use photosynthetic cyanobacteria to directly convert CO2 and sunlight into fuel. In this thesis, the cyanobacterium Synechocystis sp. PCC 6803 was genetically engineered to produce the biofuel n-butanol. Several metabolic engineering strategies were explored with the aim to increase butanol titers and tolerance.In papers I-II, different driving forces for n-butanol production were evaluated. Expression of a phosphoketolase increased acetyl-CoA levels and subsequently butanol titers. Attempts to increase the NADH pool further improved titers to 100 mg/L in four days.In paper III, enzymes were co-localized onto a scaffold to aid intermediate channeling. The scaffold was tested on a farnesene and polyhydroxybutyrate (PHB) pathway in yeast and in E. coli, respectively, and could be extended to cyanobacteria. Enzyme co-localization increased farnesene titers by 120%. Additionally, fusion of scaffold-recognizing proteins to the enzymes improved farnesene and PHB production by 20% and 300%, respectively, even in the absence of scaffold.In paper IV, the gene repression technology CRISPRi was implemented in Synechocystis to enable parallel repression of multiple genes. CRISPRi allowed 50-95% repression of four genes simultaneously. The method will be valuable for repression of competing pathways to butanol synthesis.Butanol becomes toxic at high concentrations, impeding growth and thus limiting titers. In papers V-VI, butanol tolerance was increased by overexpressing a heat shock protein or a stress-related sigma factor.Taken together, this thesis demonstrates several strategies to improve butanol production from cyanobacteria. The strategies could ultimately be combined to increase titers further.
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| 3. |
- Danielsson, Frida, 1984-
(författare)
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Integration of RNA and protein expression profiles to study human cells
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cellular life is highly complex. In order to expand our understanding of the workings of human cells, in particular in the context of health and disease, detailed knowledge about the underlying molecular systems is needed. The unifying theme of this thesis concerns the use of data derived from sequencing of RNA, both within the field of transcriptomics itself and as a guide for further studies at the level of protein expression. In paper I, we showed that publicly available RNA-seq datasets are consistent across different studies, requiring only light processing for the data to cluster according to biological, rather than technical characteristics. This suggests that RNA-seq has developed into a reliable and highly reproducible technology, and that the increasing amount of publicly available RNA-seq data constitutes a valuable resource for meta-analyses. In paper II, we explored the ability to extrapolate protein concentrations by the use of RNA expression levels. We showed that mRNA and corresponding steady-state protein concentrations correlate well by introducing a gene-specific RNA-to-protein conversion factor that is stable across various cell types and tissues. The results from this study indicate the utility of RNA-seq also within the field of proteomics.The second part of the thesis starts with a paper in which we used transcriptomics to guide subsequent protein studies of the molecular mechanisms underlying malignant transformation. In paper III, we applied a transcriptomics approach to a cell model for defined steps of malignant transformation, and identified several genes with interesting expression patterns whose corresponding proteins were further analyzed with subcellular spatial resolution. Several of these proteins were further studied in clinical tumor samples, confirming that this cell model provides a relevant system for studying cancer mechanisms. In paper IV, we continued to explore the transcriptional landscape in the same cell model under moderate hypoxic conditions.To conclude, this thesis demonstrates the usefulness of RNA-seq data, from a transcriptomics perspective and beyond; to guide in analyses of protein expression, with the ultimate goal to unravel the complexity of the human cell, from a holistic point of view.
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| 4. |
- Darmanis, Spyros, 1983-
(författare)
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Solid-phase Proximity Ligation Assays : High-performance and multiplex protein analyses
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein biomarkers circulating in blood hold the promise of improved diagnosis, prognosis and follow-up of treatment of disease via minimally invasive procedures. For the discovery and validation of such biomarkers, methods are needed that can facilitate parallel, highly specific and in-depth analysis of the blood proteome. The work presented in this thesis intends to develop and apply such assays, building on the concept of the proximity ligation assay (PLA). In paper I, I present an easy and non-expensive alternative for the conjugation of oligonucleotides to antibodies via biotin-streptavidin-biotin interaction. This approach can be used when large sets of antibodies and/or oligos need to be validated for their performance as probes in PLA reactions. In paper II, a solid-phase variant of PLA (SP-PLA) for the detection and quantification of proteins in blood is presented. SP-PLA exhibited an improved limit of detection compared to commercial ELISA assays by two orders of magnitude. In addition SP-PLA exhibited a broader dynamic range by at least one order of magnitude and required only 5 μl of sample, rendering the method very well suited for analyses of precious bio-banked material. Last but not least, SP-PLA was used to validate the diagnostic potential of GDF-15 as a biomarker for cardiovascular disease in a set of cardiovascular disease patients and healthy controls. Paper III discusses the development of a multiplex SP-PLA (MultiPLAy) for the simultaneous detection of 36 proteins in just 5 μl of sample. MultiPLAy exhibited an improved LOD when compared to state-of-the-art bead-based sandwich assays. Most importantly, we observed only a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that much higher levels of multiplexing will be possible. The assay was used to identify putative biomarkers in sample cohorts of colorectal cancer (CRC) and cardiovascular disease (CVD). Subsequent multivariate analysis revealed previously known diagnostic biomarkers. Furthermore, we successfully applied next-generation sequencing as a readout for the protein assays, allowing for the first time digital recording of protein profiles in blood. In paper IV, we investigated the suitability of prostasomes as blood biomarkers in patients with prostate cancer using a newly developed PLA assay (4PLA) that utilizes five binders for the detection of complex target molecules. The assay successfully detected significantly elevated levels of prostasomes in blood samples from prostate cancer patients prior to radical prostatectomy, compared to controls and men with benign biopsy results.
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| 5. |
- Edfors, Fredrik, 1988-
(författare)
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Targeted proteomics methods for protein quantification of human cells, tissues and blood
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The common concept in this thesis was to adapt and develop quantitative mass spectrometric assays focusing on reagents originating from the Human Protein Atlas project to quantify proteins in human cell lines, tissues and blood. The work is based around stable isotope labeled protein fragment standards that each represent a small part of a human protein-coding gene. This thesis shows how they can be used in various formats to describe the protein landscape and be used to standardize mass spectrometry experiments. The first part of the thesis describes the use of antibodies in combination with heavy stable isotope labeled antigens to establish a semi-automated protocol for protein quantification of complex samples with fast analysis time (Paper~I). Paper II introduces a semi-automated cloning protocol that can be used to selectively clone variants of recombinant proteins, and highlights the automation process that is necessary for large-scale proteomics endeavors. This paper also describes the technology that was used to clone all protein standards that are used in all of the included papers. The second part of the thesis includes papers that focus on the generation and application of antibody-free targeted mass spectrometry methods. Here, absolute protein copy numbers were determined across human cell lines and tissues (Paper III) and the protein data was correlated against transcriptomics data. Proteins were quantified to validate antibodies in a novel method that evaluates antibodies based on differential protein expression across multiple cell lines (Paper IV). Finally, a large-scale study was performed to generate targeted proteomics assays (Paper V) based on protein fragments. Here, assay coordinates were mapped for more than 10,000 human protein-coding genes and a subset of peptides was thereafter used to determine absolute protein levels of 49 proteins in human serum. In conclusion, this thesis describes the development of methods for protein quantification by targeted mass spectrometry and the use of recombinant protein fragment standards as the common denominator.
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| 6. |
- Fasterius, Erik, 1987-
(författare)
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Exploring genetic heterogeneity in cancer using high-throughput DNA and RNA sequencing
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- High-throughput sequencing (HTS) technology has revolutionised the biomedical sciences, where it is used to analyse the genetic makeup and gene expression patterns of both primary patient tissue samples and models cultivated in vitro. This makes it especially useful for research on cancer, a disease that is characterised by its deadliness and genetic heterogeneity. This inherent genetic variation is an important aspect that warrants exploration, and the depth and breadth that HTS possesses makes it well-suited to investigate this facet of cancer.The types of analyses that may be accomplished with HTS technologies are many, but they may be divided into two groups: those that analyse the DNA of the sample in question, and those that work on the RNA. While DNA-based methods give information regarding the genetic landscape of the sample, RNA-based analyses yield data regarding gene expression patterns; both of these methods have already been used to investigate the heterogeneity present in cancer. While RNA-based methods are traditionally used exclusively for expression analyses, the data they yield may also be utilised to investigate the genetic variation present in the samples. This type of RNA-based analysis is seldom performed, however, and valuable information is thus ignored.The aim of this thesis is the development and application of DNA- and RNA- based HTS methods for analysing genetic heterogeneity within the context of cancer. The present investigation demonstrates that not only may RNA-based sequencing be used to successfully differentiate different in vitro cancer models through their genetic makeup, but that this may also be done for primary patient data. A pipeline for these types of analyses is established and evaluated, showing it to be both robust to several technical parameters as well as possess a broad scope of analytical possibilities. Genetic variation within cancer models in public databases are evaluated and demonstrated to affect gene expression in several cases. Both inter- and intra-patient genetic heterogeneity is shown using the established pipeline, in addition to demonstrating that cancerous cells are more heterogeneous than their normal neighbours. Finally, two bioinformatic open source software packages are presented.The results presented herein demonstrate that genetic analyses using RNA-based methods represent excellent complements to already existing DNA-based techniques, and further increase the already large scope of how HTS technologies may be utilised.
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| 7. |
- Hjelm, Barbara, 1983-
(författare)
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Epitope mapping of antibodies towards human protein targets
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis, based on five research papers, presents results from development and evaluation ofmethods for identifying the interaction site of antibodies on their antigens and the functional investigation of these in different assays. As antibodies have proven to be invaluable tools in diagnostics, therapy and basic research, the demand of characterizing these binding molecules has increased. Techniques for epitope mapping in a streamlined manner are therefore needed, particularly in high throughput projects as the Human Protein Atlas that aims to systematically generate two antibodies with separate epitopes towards all human proteins. In paper I we describe an approach to map the epitopes of polyclonal and monoclonal antibodies for the first time using staphylococcal display. This method was combined with peptide scanning and alanine scanning using suspension bead arrays, to create a streamlined approach of highresolution characterization of epitopes recognized by antibodies as demonstrated in paper II. Single epitopes were identified for the monoclonal antibodies and several (one to five) separate epitopes scattered throughout the antigen sequence were determined for each polyclonal antibody. Further, antibodies of different species origin showed overlapping binding epitopes. In paper III we studied the epitope patterns of polyclonal antibodies generated with the same antigen in different animals. Although common epitope regions could be identified the exact epitope pattern was not repeated, as some epitopes did not reoccur in the repeated immunizations. In paper IV, a potential biomarker for colon cancer, RBM3, was investigated using validated antibodies by epitope mapping and siRNA analysis. Finally, in paper V, a method for generating epitope-specific antibodies based on affinity purification of a polyclonal antibody is described. The generated antibodies were used in several immunoassays and showed a great difference in functionality. Paired antibodies with separate epitopes were successfully generated and could be used in a sandwich assay or to validate each other in immunohistochemistry. Taken together, in these studies we have demonstrated valuable concepts for the characterization of antibody epitopes.
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| 8. |
- Hober, Andreas, 1992-
(författare)
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Development of novel affinity enrichment strategies for clinical applications using selected reaction monitoring
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are key components of any living organism and an essential part of life itself. They can provide cells with structure and perform life-sustaining intracellular reactions. As organisms grow more complex, this task expands even further. The proteins’ areas of responsibility suddenly also include communication and coordination between cells and throughout entire organisms, such as the human body. Everything that can be touched and felt on a living organism is composed of millions and millions of proteins tightly packed together. They are even the molecules responsible for propagating the signals that make up the sense of feeling. Understanding the role of proteins in the complex system of life is essential for understanding what makes up a healthy human and what causes disease. This knowledge makes up the foundation of modern medicine, and to further this knowledge, allowing for new treatments and preventative interventions, the study of proteins is crucial. The large-scale study of proteins, proteomics, is an extensive field of research where a vast toolbox of technologies has been implemented. The foundation for this toolbox is made up of mass spectrometry- and affinity-based technologies.In this thesis, both mass spectrometry-based proteomics and affinity-based proteomics will be explored. The first part, Paper I and Paper II, describe the use of selected reaction monitoring for measuring proteins of clinical relevance in human blood plasma. The second part, Paper III and Paper IV, highlight the importance of validating reagents used for affinity-based proteomics and how this can be achieved in a high throughput manner. Lastly, Paper V showcases how a combined strategy, relying on both affinity-based proteomics and mass spectrometry-based proteomics, can capitalize on the best properties of each technology and how this combined strategy can even be utilized for diagnostic purposes.
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| 9. |
- Karlsson, Max
(författare)
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Mapping and annotating the mammalian body-wide protein-coding gene expression
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A central aim of fundamental research is to create conditions necessary for fueling further research and innovation. Our understanding of basic biology is central for future developments of tools for diagnosing, monitoring, and treating disease. This doctoral thesis focuses on mapping the mammalian protein-coding gene expression in healthy cells and tissues, and annotation of genes based on their expression patterns, specificity, location, and function. This has in large part been achieved by using large scale transcriptomic and proteomic profiling to describe the gene expression landscape that defines the identities of the great diversity of cells present in mammals. Characterization of gene expression across different tissues and cell types provide fundamental tools to enable the exploration, summary, and ultimately, the annotation of the mammalian proteome, which is still incomplete.The studies comprising this thesis have contributed to the Human Protein Atlas, an online open-access portal for proteomic and transcriptomic data, with the aim to profile each human protein-coding gene to create a spatial map of the molecular organization of the human body, providing basic tools for the scientific community. Paper I comprises an effort to catalogue all proteins that are actively secreted from cells; defining the human secretome. Paper II entails the deep characterization and annotation of the protein-coding transcriptome of 18 peripheral immune cell types. Paper III describes the, to date, most comprehensive tissue-based transcriptomic profiling of protein-coding genes in 98 tissues of the increasingly important model animal pig. Paper IV extends previous tissue-based maps of the human protein-coding genome by integration of 13 single cell transcriptome datasets. Paper V explores the human protein-coding genome in a clustering-based annotation of co-expressed genes across single cells and tissues to provide a framework for finding previously unknown functional relationships between genes by the principle of “guilt-by-association”.In summary, the work described here entails a small contribution to the grand effort of spatially mapping proteins across tissues and cell types, for building a framework of biological knowledge that can lead to increased understanding of the constituents that make us humans.
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| 10. |
- Seijsing, Johan, 1986-
(författare)
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Rational and Combinatorial Engineering of Affinity Proteins Towards Therapeutical Applications
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein engineering has had an immense impact on the development of biological drugs, including replacement therapies with engineered versions of insulin or factor VIII to treat diabetes or bleeding disorders, and monoclonal antibodies to treat cancer and various other malignancies. Now, with the next generation of treatment modalities coming up, including monoclonal reagents based on alternative scaffolds, gene and cell therapies, the importance of protein engineering to tailor-make these treatments is likely to increase further.The neonatal Fc receptor (FcRn) is widely expressed in the body. One of the receptor's interesting functions is to rescue immunoglobulin G (IgG) and serum albumin (SA) from degradation by cells in contact with blood. When serum proteins are endocytosed by a cell, they are transported via the endosome to the lysosome for degradation. However, IgG and SA associate with the FcRn already at the slightly acidic pH of the endosome followed by transport back to the cell surface. There the complex encounters the neutral pH of the blood, at which the binding affinity to FcRn is lost, and IgG and SA are released back into circulation. In the main part of this thesis, efforts are described aiming to take use of, or block, the FcRn recycling mechanism to control the serum circulation half-life of proteins.In the first study (Study I), a robust expression strategy for human FcRn was designed and evaluated. The extracellular domain was produced in the human SKOV3 cell-line after facile lentiviral delivery of the expression cassettes. This lead to continuous expression of secreted protein that could be purified to homogeneity by a single affinity chromatographic step, using the intrinsic pH-dependent interaction between FcRn and IgG, where the latter was immobilized in a column. The amount of purified protein was 1.4 mg per liter medium. The protein was characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay. The results suggested a fully functional and stable protein of high purity. In addition, the gene encoding full-length FcRn as a fusion to eGFP was delivered to HeLa cells utilizing the same lentiviral system. Subsequent analysis by flow cytometry and confocal fluorescence microscopy indicated a wide distribution of eGFP/FcRn expression among the cells. Binding of IgG and HSA was found to correlate well with the amount of eGFP/FcRn expressed by the cells.In a following study (Study II), the goal was to generate affinity proteins interacting with human FcRn in a pH-dependent manner similar to that of FcRn's natural ligands. The affinity proteins used are denoted affibody molecules, a class of small alternative scaffold proteins with a three-helical structure. Affibody molecules were selected from a combinatorial library displayed on phage where binding took place at pH 5.5 and elution was performed at pH 2.2 or 8.0. Selected variants were characterized by developed in vitro and cell based assays, and some were found to have the desired pH-dependent binding to FcRn. In vivo studies in mice showed that the serum half-life of a model protein, genetically fused to the FcRn binding affibody molecules, was extended up to almost three-fold compared to a control protein (from 33 to 91 hours).In a subsequent study (Study III), the use of a FcRn binding affibody molecule to reduce, rather than prolong, the serum half-life of proteins was explored. Here, the rationale was to investigate if injection of a FcRn binding affibody could hinder endogenous IgG from being rescued by FcRn, which could lead to depletion of IgGs by lysosomal degradation. In autoimmune diseases, such depletion of IgG would include also pathogenic IgG and could thus mitigate the symptoms of the disease. Using cell based assays, it was found that one affibody molecule, selected in Study II, could readily block IgG from binding both human and murine FcRn. A following in vivo study in mice showed that systemic injection of the molecule reduced the amount of endogenous IgG by 39%.In a fourth study (Study IV), the goal was to use a different class of affinity proteins to regulate the level of an enzyme in the brain. More specifically, artificial zinc finger-based transcription factors regulating the level of GAD67, which is the rate-limiting enzyme in synthesis of gamma-aminobutyric acid (GABA), were designed. Imbalances in GABA-signaling is involved in different diseases, including Parkinson's disease and epilepsy, and regulation of GAD67 at particular sites in the brain might be a route to ameliorate symptoms associated with such diseases. ELISA-based investigation showed that one of the designed zinc fingers, denoted G3, bound selectively to its intended target DNA sequence. A construct encoding G3 fused to a general transcriptional activator (VP64) was delivered to PC12-cells, using a lentivirus-based gene delivery system, resulting in a significant up-regulation of endogenous GAD67 expression. The same construct was subsequently delivered to the striatum of rats, with an induced disease model of Parkinson's disease. Western blot of striatal samples showed a significant up-regulation of GAD67 expression in lesioned striatum compared to intact striatum, and a tendency towards up-regulation compared to lesioned striatum.Taken together, the protein engineering efforts described in this thesis concerning affinity proteins binding other proteins or DNA, has the potential to find use in drug development and may benefit patients in the future.
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