When a multicellular organism suffers damages to tissues/organs it heals itself by either substituting the lost cellular matrix by scar formation or by regenerating the lost tissue. Regeneration likely occurs by a recapitulation of the developmental process that formed the organ. Many processes regulating organ development are based on epithelial-mesenchymal interactions and a strict control of organ specific stem/progenitor cells. Elucidation of the molecular basis of these processes is therefore vital in order to develop novel therapies in regenerative medicine. The LIM homebox gene Lhx2 is interesting in this context since Lhx2 has been shown to be important for the formation of several organs by regulating epithelial-mesenchymal interactions and progenitor cell function. Targeted inactivation of Lhx2 leads to a lethal anemia due to malformed liver and severe neural abnormalities such as hypoplasia of the forebrain and anophtalmia. Thus, elucidation of the mechanisms of the function of Lhx2 in different organ systems would give important insights into the molecular mechanisms regulating epithelial-mesenchymal interactions and stem/progenitor cell function. To elucidate the function of Lhx2 in the hematopoietic system Lhx2 was initially expressed in hematopoietic progenitor cells derived from ES cells differentiated in vitro using retroviral vectors. This approach led to the generation of hematopoietic stem cell (HSC)-like cell lines suggesting that Lhx2 could impact HSC function. However neither the specificity nor the efficiency of the Lhx2-induced phenotype could be determined using this approach. To be able to elucidate the function of Lhx2 in the hematopoietic system, an ES cell line with inducible Lhx2 expression was generated. Lhx2 expression induces self-renewal of a distinct hematopoietic progenitor cell from which HSC-like cell lines were established. Down-regulation of Lhx2 in these HSC-like cell lines leads to a rapid loss of stem cell character, providing a good model to study the molecular function of Lhx2 in hematopoietic stem/progenitor cells. A global gene expression analysis was performed comparing the Lhx2+ stem cell population to the Lhx2- differentiated progeny. This approach identified genes putatively linked to self-renewal/differentiation of HSCs. A considerable proportion of the genes showed an overlapping gene expression pattern with Lhx2 expression in tissue of non-hematopoietic origin suggesting that Lhx2 function in stem/progenitor cells partly overlap with Lhx2 function during organ development. In order to define other Lhx2-dependent progenitor cell populations and to generate a tool to analyze the function of Lhx2 in organ development a new transgenic mouse model was generated. By using a specific part of the Lhx2 promoter to drive expression of Cre recombinase in vivo (Lhx2-Cre mice) we have been able to define the first eye committed progenitor cells in the forebrain. By using the Lhx2-Cre mice it will be possible to distinguish the function of genes during eye development from their function in the patterning of the forebrain e.g. the eye field transcription factors. Conditional inactivation of Lhx2 in these eye specific progenitor cells causes an immediate developmental arrest. The transgene is also active in Lhx2-/- embryonic forebrain, but re-expression of Lhx2 in Lhx2-/- progenitor cells only promote formation of retinal pigment epithelium cells. Analysis of genes expressed by the Lhx2+ stem cell population allowed us to define novel genes putatively linked to Lhx2 function in eye development. Thus, we have defined the progenitor cells in the forebrain committed to eye development and the expansion and patterning of these progenitors are dependent on Lhx2. Although commitment to eye development is Lhx2-independent, Lhx2 might be important for the acquisition of the oligopotent fate of these progenitor cells.

The RAS-MAPK syndromes are a group of clinically and genetically related disorders, characterized by cardiac defects, facial dysmorphism, cutaneous abnormalities and neurocognitive impairment. The pathogenesis is dysregulation of the RAS-MAPK pathway, and several genes within the pathway are involved. The present thesis aimed at identifying genetic causes in three of the RAS-MAPK syndromes - Noonan syndrome (NS), cardio-facio-cutaneous syndrome (CFC) and Neurofibromatosis-Noonan syndrome (NFNS) - and at correlating genotype with phenotype. A mutation analysis of six genes associated with the RAS-MAPK syndromes in NS and CFC patients revealed mutations in 10/31 patients. The results suggested more complex genetic overlap and genetic heterogeneity among these syndromes than previously believed. Subsequently, gene dosage imbalances of seven RAS-MAPK-syndrome-related genes were investigated in mutation-negative patients. A multiplex ligation-dependent probe amplification strategy was developed that excluded copy number changes of these genes as a common mechanism in NS. Genetic causes of clinical variability in NS were investigated where an atypical and severe NS patient was described. In addition, multiple café-au-lait (CAL) spots affected the patient and four otherwise healthy family members. Molecular analysis of four candidate genes revealed a previously described de novo PTPN11 mutation and an inherited NF1 variant in the patient. Neither of them explained the CAL spots trait, which consequently represented a distinct entity. The results suggested that the atypical and severe phenotype in the patient could be a consequence of an additive effect. Finally, a family displaying NFNS was investigated clinically and molecularly revealing a novel mutation in the GAP-domain of NF1. Furthermore, the results suggested that other RAS-MAPK-syndrome-related genes are not involved in NFNS. A proposal of prioritizing the GAP-domain of NF1 in NFNS was presented. Conclusively, these studies contribute to further understanding of the RAS-MAPK syndromes and facilitate the diagnostic process and future prognosis prediction.

Among the great challenges in biology are the precise quantification of specific sets of proteins and analyses of their patterns of interaction on a much larger scale than is possible today. This thesis presents a novel protein detection technique - proximity ligation - and reports the development and application of a nucleic acid amplification technique, RCA. Proximity ligation converts information about the presence or co-localization of specific proteins to unique sets of nucleic acid sequences. For detection of target proteins or protein complexes the coincident binding by pairs or triplets of specific protein-binding reagents are required. Oligonucleotide-extensions attached to those binding reagents are joined by a DNA ligase and subsequently analyzed by standard molecular genetic techniques. The technique is shown to sensitively detect an assortment of proteins using different types of binders converted to proximity probes, including SELEX aptamers and mono- and polyclonal antibodies. I discuss factors important for using the technique to analyze many proteins simultaneously. Quantification of target molecules requires precise amplification and detection. I show how rolling circle amplification, RCA, can be used for precise quantification of circular templates using modified molecular beacons with real-time detection. The combination of proximity-probe templated circularization and RCA results in a sensitive method with high selectivity, capable of visualizing individual immobilized proteins. This technique is used for localized detection of a set of individual proteins and protein complexes at sub-cellular resolution.

All aspects of cell life are regulated by signal transduction mechanisms. This thesis describes the regulatory roles of a few key signal transduction molecules involved in three major biological responses. The studied pathways include platelet derived growth factor (PDGF)-BB induced transformation of murine fibroblasts, interferon (IFN)-γ stimulated monocyte activation and all-trans retinoic acid (ATRA) induced myeloid differentiation. We found that intact phosphoinositide 3OH-kinase (PI3K) activity is essential in the signaling pathway that leads to the morphological alterations and migration pattern characteristic of PDGF-BB transformed NIH/sis and NIH/COL1A1 fibroblasts. Furthermore, our data indicated that the small Rho-GTPase, Rac1 is the predominant mediator of these signals downstream of PI3K. The study of the IFN-γ induced activation of monocytic U-937 cells showed that upregulation of the high affinity receptor for IgG (FcγRI) is dependent on the coordination of several regulatory events: the PKR-mediated serine 727 phosphorylation of Stat1, the expression of the hematopoietic lineage specific transcription factor PU.I, and the activation of the NFκB pathway. ATRA-induced differentiation and cell cycle arrest are impaired in U-937 sublines expressing phosphorylation deficient Stat1 (Stat1Y701F and Stat1S727A). The findings in paper III indicated that the expression pattern of the myeloid specific transcription factors Stat2, ICSBP and c/EBPε was altered in the sublines and that intact Stat1 activation is critical for maintaining the balance of the transcriptional network during ATRA induced terminal differentiation. Finally, ATRA-induced differentiation and growth arrest were blocked by treatment with the IKKα/β inhibitor BMS345541 or by ectopic expression of the NFκB super repressor IκBα (S32A/S36A). The fact that IκB(AA) sublines differentiated normally in response to vitamin D3, showed that NFκB inhibition specifically affected ATRA induced responses. Notably we suggest that the activity of the NFκB pathway may interfere with the differentiation process via a direct effect on the RAR/RXR mediated transcription.

The incidence of cutaneous malignant melanoma has increased dramatically in Caucasians the last few decades, an increase that is partly explained by altered sun exposure habits. For the individual patient, with a localized disease, the tumor thickness of the excised lesion is the most important prognostic factor. However, there is a need to identify characteristics that can place patients in certain risk groups. In this study, the protein expression of multiple proteins in malignant melanoma tumors was studied, with the aim of identifying potential new candidate biomarkers. Representative samples from melanoma tissues were assembled in a tissue microarray format and protein expression was detected using immunohistochemistry. Multiple cohorts were used and for a subset of proteins the expression was also analyzed in melanocytes in normal skin and in benign nevi. The immunohistochemical staining was evaluated manually and for part of the proteins also with an automated algorithm. The protein expression of STX7 was described for the first time in tumors of the melanocytic lineage. Stronger expression of STX7 and SOX10 was seen in superficial spreading melanomas compared with nodular malignant melanomas. An inverse relationship between STX7 expression and T-stage was seen and between SOX10 expression and T-stage and Ki-67, respectively. In a population-based cohort the expression of MITF was analyzed and found to be associated with prognosis. Twenty-one potential biomarkers were analyzed using bioinformatics tools and a protein signature was identified which had a prognostic value independent of T-stage. The protein driving this signature was RBM3, a protein not previously described in malignant melanoma. Other markers included in the signature were MITF, SOX10 and Ki-67. In conclusion, the protein expression of numerous potential biomarkers was extensively studied and a new prognostic protein panel was identified which can be of value for risk stratification.

The human body is composed of trillions of cells closely working together to maintain a functional organism. Every cell is unique in molecular composition and can acquire genetic variations that might cause it to turn pathological. It is essential to develop improved tools to better understand the development of normal and disease tissue, ideally enabling single-cell expression studies in preserved context of complex tissue with single-nucleotide resolution. This thesis presents the development and application of a new in situ method for localized detection and genotyping of individual transcripts directly in cells and tissues. The described technique utilizes padlock probes and target-primed rolling circle amplification and is highly suitable for sensitive in situ analysis. First, a new strategy for directed cleavage of single stranded DNA was investigated, e.g. nucleic acid targets with extended 3´ ends, for successful initiation of rolling circle amplification. The presented cleavage strategy is simple and applicable for subsequent enzymatic reactions, e.g. ligation and polymerization. Specific cleavage of long target overhangs was demonstrated in synthetic oligonucleotides and in genomic DNA and the detection efficiency was substantially increased. For multiplex detection and genotyping of individual transcripts in single cells, a new in situ method was developed. The technique showed a satisfactorily detection efficiency and was later applied as a general mutation analysis tool for detection of KRAS point mutations in complex tumor tissue sections, e.g. formalin-fixed, paraffin-embedded tumor tissues and cytologic tumor imprints. Mutation status was assessed in patient samples by in situ padlock probe detection and results were confirmed by DNA-sequencing.  Finally, the method was adapted for simultaneous detection of individual mRNA molecules and endogenous protein modifications in single cells using padlock probes and in situ PLA. This assay will be useful for gene expression analysis and exploration of new drugs with vague effector sites. To our knowledge, no other technique exists today that offers in situ transcript detection with single-nucleotide resolution in heterogeneous tissues. The method will especially be suitable for discrimination of highly similar transcripts, e.g. splice variants, SNPs and point mutations, within gene expression studies and for cancer diagnostics.

Intra- and intercellular information is communicated by posttranslational modifications (PTMs) and protein-protein interactions, transducing information over cell membranes and to the nucleus. A cells capability to respond to stimuli by several highly complex and dynamic signaling networks provides the basis for rapid responses and is fundamental for the cellular collaborations required in a multicellular organism. Having received diverse stimuli, being positioned at various stages of the cell cycle or, for the case of cancer, containing altered genetic background, each cell in a population is slightly different from its neighbor. However, bulk analyses of interactions will only reveal an average, but not the true variation within a population. Thus studies of interacting endogenous biomolecules in situ are essential to acquire a comprehensive view of cellular functions and communication. In situ proximity ligation assay (in situ PLA) was developed to investigate individual endogenous protein-protein interactions in fixed cells and tissues and was later applied for detection for PTMs. Progression of signals in a pathway can branch out in different directions and induce expression of different target genes. Hence simultaneous measurement of protein activity and gene expression provides a tool to determine the balance and progression of these signaling events. To obtain this in situ PLA was combined with padlock probes, providing an assay that can interrogate both PTMs and mRNA expression at a single cell level. Thereby different nodes of the signaling pathway as well as drug effects on different types of molecules could be investigated simultaneously. In addition to regulation of gene expression, protein-DNA interactions present a mechanism to manage accessibility of the genomic DNA in an inheritable manner, providing the basis for lineage commitment, via e.g. histone PTMs. To enable analyses of protein-DNA interactions in situ we developed a method that utilizes the proximity dependence of PLA and the sequence selectivity of padlock probes. This thesis presents new methods providing researchers with a set of tools to address cellular functions and communication in complex microenvironments, to improve disease diagnostics and to contribute to hopefully finding cures.

Antibodies are the gold standard molecular recognition elements and a cornerstone of molecular biology. They are used in immunoassays to precisely measure a specific analyte, but certain targets are especially challenging. Difficult targets include small molecules and molecules that do not induce an immune response. Aptamers are short oligonucleotides that can form 3-dimensional structures and bind targets with high specificity. Aptamers are smaller and more flexible than antibodies and could therefore solve this problem. In contrast to antibodies, aptamers are synthetically produced, so they can have affinity for molecules that do not induce an immune response. This also makes them cheaper, faster and more ethical to produce. They are also easily modified and have the ability to renature and can therefore be reused.  Our conclusions are that aptamers can outperform antibodies, especially for small molecule targets, and that the synthetic production of aptamers gives them a further advantage over antibodies. Our report compares several different types of detection methods that use aptamers and we conclude that fluorescence-based methods are the most easy to use with basic lab equipment, can be made similar to the ELISA kits in addition to giving highly sensitive detection. We describe a variety of fluorescence-based detection strategies but the optimal method will depend on the specific aptamer and target. The report also includes an ethical analysis where antibodies and aptamers are compared. This report is commissioned by Mercodia AB, a company that develops, manufactures and distributes immunoassays for biomarkers within the field of metabolic disorders. They commissioned this report in order to give an overview of how aptamers interact with their target, and also to compare aptamer-based detection strategies with sensitivity prioritized over selectivity.  This was done by literature research.

Den ökande koldioxidhalten i atmosfären har lett till ett ökat intresse för användning av solljus, ett exempel är inom kemi. Att använda ljus i kemi kan leda till nya och intressanta kemiska reaktioner och kan tillämpas i användningsområden som exempelvis fotokatalys och fotovoltaik. Genom att kombinera de kraftfulla egenskaperna av ljus i kemi med den höga selektivitet som karaktäriserar proteiner kan intressanta och kraftfulla maskinerier erhållas, så kallade fotoaktiva proteiner. Att studera de fotoinducerade beteendena i fotoaktiva proteiner är däremot icke-trivialt eftersom många utmaningar uppstår från ett experimentellt och teoretiskt perspektiv. Denna uppsats fokuserar på det teoretiska perspektivet. Simulationer av beteenden hos fotoaktiva proteiner leder till utmaningar relaterade till den stora variationen av tids- och längdskalor involverade, där allt ifrån ultrasnabba och lokaliserade excitationsprocesser till storskaliga strukturförändringar som utspelar sig på större tidsskalor. Detta, tillsammans med det faktum att det inte finns en "svart låda" som vi kan använda för nya teoretiska studier motiverar ett försiktigt tillvägagångssätt för teoretiska studier av fotoaktiva proteiner. I detta projekt görs en teoretisk undersökning av två nyligen framställda fotosensibiliserande proteiner (PSP2 och PSP3). De skapades för att härma huvudprocesserna av fotosyntesen i plantor. Fotosensibiliserande proteinerna skiljer sig med en enda aminosyra (position 203), som är en aspartat i PSP2 och en tyrosin i PSP3. Även fast proteinerna är strukturellt lika så uppvisar de olika fotoinducerade beteenden. Målet med denna studie är att klargöra för de mekanistiska detaljerna bakom dessa olikheter. Som ett första steg i att teoretiskt studera PSP2 och PSP3 upprättar vi ett protokoll för teoretisk förberedelse av proteinerna. Detta protokoll används sedan för att teoretiskt undersöka fotofysiken i dessa två proteiner med hjälp av kvantmekaniska/ molekylär mekaniska simuleringar. Våra resultat ger en första ordningens kartläggning av de exciterade tillstånden i de två proteinerna. Vi erhåller en intressant laddningsöverföring tillstånd i PSP3 som involverar tyrosinen och proteinets kromofor, med en energi lägre än det tillstånd ansvarig för absorption av ljus. Denna laddningsöverföring saknas i PSP2 och kan därför vara anledningen till de olika fotoinducerade beteendena i proteinerna. Detta kräver dock fortsatta studier av de två systemen. / The increase of carbon dioxide levels in the atmosphere have resulted in an increasing interest in using solar light for different purposes, one being chemistry. The introduction of light in chemistry can drive new and exciting chemical reactions, finding applications in many fields such as photocatalysis and photovoltaics. Combining the aspects of light in chemistry together with the high selectivity that characterizes proteins make for interesting and powerful machineries, so-called photoactive proteins. However, studying the behaviors of photoactive proteins is a non-trivial task and many challenges arise from both an experimental and a theoretical point of view. This thesis takes a theoretical perspective. The challenges associated with simulating photoactive protein behavior originate from the wide range of time and length scales involved, ranging from ultrafast and localized excitation processes to large-scale structural changes occurring on longer timescales. This, together with the fact that there is no black box that we can use for novel theoretical studies of photoactive proteins motivates a careful approach for theoretical studies of photoactive proteins. In this project, we conduct a theoretical investigation of two photosensitizer proteins (PSP2 and PSP3), recently engineered to capture the essence of plant photosynthesis. The photosensitizer proteins differ by only one residue (position 203), which is an aspartate in PSP2 while a tyrosine in PSP3. Although structurally similar, the proteins demonstrate different photoinduced behaviors. This study aims to shed light on the mechanistic details underlying these differences. As a first step to study PSP2 and PSP3 computationally, we develop a computational protocol for protein preparation. The protocol is then used for a theoretical investigation of the photophysics of the two proteins using quantum mechanics/molecular mechanics simulations. Our results provide a first mapping of the electronic-state manifold of the two proteins. We find an intriguing charge-transfer state in PSP3, involving the tyrosine and the protein chromophore, located below bright state responsible for light absorption. This state is absent in PSP2 and could therefore be the reason for the different photoinduced behavior of the two proteins. However, this requires further studies of the two systems.

In drug development, molecular docking methods aim at characterizing the binding of a drug-like molecule to a protein. In a typical drug development process, a docking task is repeated millions of time, which makes optimization efforts essential. In particular, modern heterogeneous architectures, such as GPUs, allow for significant acceleration opportunities. AutoDock-GPU, a state-of-the-art GPU-accelerated molecular docking software, estimates the geometrical conformation of a docked ligand-protein complex by minimizing an energy-based scoring function. Our profiling results indicated that a reduction operation, which is performed several millions times in a single docking run, limits performance in AutoDock-GPU. Thus, we proposed a method to accelerate the block-level sum reduction of four-element vectors by using matrix operations. We implemented our method to make use of the high throughput capabilities offered by NVIDIA Tensor Cores to perform matrix operations. We evaluated our approach by designing a simple benchmark, and achieved a 4 to 7-fold runtime improvement compared to the original method. We then integrated our reduction operation into AutoDock-GPU and evaluated it on multiple chemical complexes on three GPUs. This evaluation allowed to assess the possibility to use half-precision reduction operations in parts of AutoDock-GPU code, without detrimental effects on the simulation result. In addition, our implementation achieved an average 27% improvement on the overall docking time during a real-world docking run. / Vid läkemedelsutveckling syftar molekylär dockningsmetoder till att karakterisera bindningen av en läkemedelsliknande molekyl till ett protein. I en typisk läkemedelsutvecklingsprocess upprepas en dockinguppgift miljontals gånger, vilket gör optimeringsinsatser nödvändiga. Framför allt moderna heterogena arkitekturer som GPU:er ger betydande accelerationsmöjligheter. AutoDock-GPU, en modern GPU-accelererad programvara för molekylär dockning, uppskattar den geometriska konformationen hos ett ligand-protein-komplex genom att minimera en energibaserad poängsättningsfunktion. Våra profileringsresultat visade att en reduktionsoperation, som utförs flera miljoner gånger i en enda dockningskörning, begränsar prestandan i AutoDock-GPU. Vi har därför föreslagit en metod för att accelerera summareduktionen på blocknivå av vektorer med fyra element med hjälp av matrisoperationer. Vi implementerade vår metod för att utnyttja den höga genomströmningskapacitet som erbjuds av NVIDIA Tensor Cores för att utföra matrisoperationer. Vi utvärderade vårt tillvägagångssätt genom att utforma ett enkelt testfall och uppnådde en 4- till 7-faldig förbättring av körtiden jämfört med den ursprungliga metoden. Vi integrerade sedan vår reduktionsoperation i AutoDock-GPU och utvärderade den på flera kemiska komplex på tre GPU:er. Denna utvärdering lät oss bedöma möjligheten att använda reduktionsoperationer med halvprecision i delar av AutoDock-GPU-koden, utan negativa effekter på simuleringsresultatet. Dessutom uppnådde vår version en genomsnittlig förbättring på 27% av den totala dockningstiden under en riktig dockningskörning.