In this study the aim was to optimize the production of the Affibody fusion-protein Z03358- ABD094-(S4G)3-IL2 with regard to the amount of soluble protein produced. However, problems with reproducibility with this protein and the chosen expression system were encountered. Therefore, expression of the His-tagged Affibody His6-(Z05477)2 was evaluated using the same expression system as well as expression in another well characterized expression system. Both target proteins are of therapeutic interest. One of the proteins is an IL2 fusion protein (Z03358-ABD094-(S4G)3-IL2) that bind the platelet-derived growth factor receptor β (PDGFR-β). PDGF signaling is of interest in cancer treatment where, among other things, the effects of PDGF on tumor angiogenesis is researched. The His6-(Z05477)2 protein has a classified target but is developed as a therapeutic in the area of inflammation and autoimmune disease. Both model proteins are known to be difficult to purify due to low solubility. The two E. coli expression systems investigated and compared were BL21(DE3) and Lemo21(DE3). The fusion protein Z03358-ABD094-(S4G)3-IL2 was produced in BL21(DE3) in inclusion bodies with a yield of 4.95 g/l. An optimized process for the expression of His6-(Z05477)2 using BL21(DE3) was developed with a yield of 6.6 g/l soluble protein after expression at 30°C for 6 h.

<p>Grade 2 gliomas are malignant brain tumours affecting otherwise healthy adults. Although the long-term prognosis is poor, many patients are well and may have a high quality of life for several years. There is, however, a large variability in the natural course of the disease which makes it essential to identify patients who might benefit from early surgery or radio-therapy. The aim of the present thesis was to define new and clinically useful prognostic markers that may assist in the initial treatment decision and in patient follow-up.</p><p>A retrospective study of 189 patients with gliomas WHO grade 2 showed no advantage in survival of early tumour resection or radiotherapy, and confirmed that histological subtype and patient age are the most important predictors of survival (I). In 89 patients, the pre-treatment uptake of 11C-methionine (MET) measured with positron emission tomography (PET) was identified as a prognostic marker for survival (II). At the time of tumour progression, irradiated tumours demonstrated signs of a residual radiotherapeutic effect that correlated with the pre-treatment uptake of MET (III). Pre-treatment uptake of MET may, therefore, be important both in predicting the natural course of the disease and the response after treatment. Immunohistochemical staining of 40 tumour samples showed an inverse association between the number of tumour cells expressing platelet-derived growth factor alpha receptor (PDGFRa) and survival (IV). Also, a reduction was observed in the number of receptor-positive cells after malignant transformation, supporting the prognostic value of PDGFRa.</p><p>Lumbar puncture was performed in eight patients with newly diagnosed low-grade gliomas to identify three important growth factors in tumour development. Neither PDGF nor vascular endothelial growth factor (VEGF) were detected in the cerebrospinal fluid (CSF), and fibroblast growth factor 2 (FGF-2) was measurable at extremely low concentrations in two of the patients (V). A proteome screening of the CSF, using two-dimensional gel electrophoresis and mass spectrometry, detected alpha 2-HS glycoprotein at significantly higher concentrations than in a control group (VI). This glycoprotein emerges as a novel substance in glioma research and may be of great interest because of its suggested involvement in the embryonic development of the neocortex.</p>

<p>Midgut carcinoid tumours are rare with an incidence of 0.5-2.1/100 000. The primary tumour is usually small and grows slowly but has almost always set metastases at diagnosis. When radically operated, most patients will eventually recur in their disease. </p><p>We evaluated different methods in detecting recurrent disease in 61 malignant midgut carcinoid tumours that had been radically operated. Thirty-eight patients have been diagnosed with a recurrence. In 32/38 of these patients P-Chromogranin A was the first method to indicate a recurrence. We therefore recommend using P-CgA in the work up in these patients.</p><p>We investigated characteristics, survival and independent factors that could be of bad prognostic value. We found that in our 284 malignant midgut carcinoid tumours, 208/284 (73%) had distant metastases and 30/284 (11%) had carcinoid heart disease. Median survival was 115.5 months and five-year survival was 77%. In a multivariate analysis liver metastases and carcinoid heart disease were poor prognostic factors.</p><p>We performed a phase II study with octreotide pamoate investigating the clinical effect in 12 malignant midgut carcinoid tumours in a progressive phase. We found that 9/12 (75%) were stabilised for a median duration of 12 months. We think that this is a good effect considering the advanced stage.</p><p>We investigated the frequency of four different tyrosine kinase receptors, platelet derived growth factor receptor (PDGR) α and β, epidermal growth factor receptor (EGFR) and c-kit, in 36 malignant midgut carcinoid tumours with immunohistochemistry. We found that 13/34 (38%) tumour samples expressed PDGFRα, 29/33 (88%) PDGFRβ, 24/33 (73%) EGFR, whereas none expressed c-kit. This implicates that midgut carcinoid tumours might be susceptible to treatment with tyrosine kinase receptor inhibitors. </p>

Midgut carcinoid tumours are rare with an incidence of 0.5-2.1/100 000. The primary tumour is usually small and grows slowly but has almost always set metastases at diagnosis. When radically operated, most patients will eventually recur in their disease. We evaluated different methods in detecting recurrent disease in 61 malignant midgut carcinoid tumours that had been radically operated. Thirty-eight patients have been diagnosed with a recurrence. In 32/38 of these patients P-Chromogranin A was the first method to indicate a recurrence. We therefore recommend using P-CgA in the work up in these patients. We investigated characteristics, survival and independent factors that could be of bad prognostic value. We found that in our 284 malignant midgut carcinoid tumours, 208/284 (73%) had distant metastases and 30/284 (11%) had carcinoid heart disease. Median survival was 115.5 months and five-year survival was 77%. In a multivariate analysis liver metastases and carcinoid heart disease were poor prognostic factors. We performed a phase II study with octreotide pamoate investigating the clinical effect in 12 malignant midgut carcinoid tumours in a progressive phase. We found that 9/12 (75%) were stabilised for a median duration of 12 months. We think that this is a good effect considering the advanced stage. We investigated the frequency of four different tyrosine kinase receptors, platelet derived growth factor receptor (PDGR) α and β, epidermal growth factor receptor (EGFR) and c-kit, in 36 malignant midgut carcinoid tumours with immunohistochemistry. We found that 13/34 (38%) tumour samples expressed PDGFRα, 29/33 (88%) PDGFRβ, 24/33 (73%) EGFR, whereas none expressed c-kit. This implicates that midgut carcinoid tumours might be susceptible to treatment with tyrosine kinase receptor inhibitors.

Grade 2 gliomas are malignant brain tumours affecting otherwise healthy adults. Although the long-term prognosis is poor, many patients are well and may have a high quality of life for several years. There is, however, a large variability in the natural course of the disease which makes it essential to identify patients who might benefit from early surgery or radio-therapy. The aim of the present thesis was to define new and clinically useful prognostic markers that may assist in the initial treatment decision and in patient follow-up. A retrospective study of 189 patients with gliomas WHO grade 2 showed no advantage in survival of early tumour resection or radiotherapy, and confirmed that histological subtype and patient age are the most important predictors of survival (I). In 89 patients, the pre-treatment uptake of 11C-methionine (MET) measured with positron emission tomography (PET) was identified as a prognostic marker for survival (II). At the time of tumour progression, irradiated tumours demonstrated signs of a residual radiotherapeutic effect that correlated with the pre-treatment uptake of MET (III). Pre-treatment uptake of MET may, therefore, be important both in predicting the natural course of the disease and the response after treatment. Immunohistochemical staining of 40 tumour samples showed an inverse association between the number of tumour cells expressing platelet-derived growth factor alpha receptor (PDGFRa) and survival (IV). Also, a reduction was observed in the number of receptor-positive cells after malignant transformation, supporting the prognostic value of PDGFRa. Lumbar puncture was performed in eight patients with newly diagnosed low-grade gliomas to identify three important growth factors in tumour development. Neither PDGF nor vascular endothelial growth factor (VEGF) were detected in the cerebrospinal fluid (CSF), and fibroblast growth factor 2 (FGF-2) was measurable at extremely low concentrations in two of the patients (V). A proteome screening of the CSF, using two-dimensional gel electrophoresis and mass spectrometry, detected alpha 2-HS glycoprotein at significantly higher concentrations than in a control group (VI). This glycoprotein emerges as a novel substance in glioma research and may be of great interest because of its suggested involvement in the embryonic development of the neocortex.

Activated receptor tyrosine kinases recruit many signalling proteins to initiate downstream cell proliferation and survival pathways, including phosphatidylinositol 3-kinase (PI3K), a heterodimer consisting of a p85 regulatory protein and a p110 catalytic protein. Our laboratory has previously shown the p85α protein also has in vitro GTPase activating protein (GAP) activity towards Rab5 and Rab4, small GTPases that regulate vesicle trafficking events for activated receptors. Expression of a p85α protein containing an arginine to alanine substitution at position 274 (p85R274A) that affects its GAP activity, caused sustained levels of activated platelet-derived growth factor receptors (PDGFRs), enhanced downstream signalling, and resulted in cellular transformation. Together with other data, this suggested that in p85R274A-expressing cells, PDGFRs are more rapidly trafficked through the endocytic pathway, which reduces opportunities for sorting events necessary for receptor degradation. Our laboratory has observed previously that p85 was capable of binding to both Rab5-GDP, as well as Rab5-GTP, which is an atypical characteristic of GAP proteins, whereas p110β had previously been reported to bind Rab5-GTP selectively. Based on these observations, this thesis project was designed to test the hypothesis that both proteins contributed GAP activity towards Rab5, with p85 providing a catalytic arginine residue (R274) and p110β providing switch stabilization functions specific to the GTP-bound state. To accomplish the thesis objective, cells expressing individual p85 defects (lacking GAP activity, R274A; or lacking p110-binding ability through deletion of residues 478-513, Δ110) were compared to cells expressing a double mutant missing both functions. Stable clonal NIH 3T3 cell lines were generated and selected in G418 and clones expressing similar levels of FLAG-tagged p85 wild type or mutants compared to the control cell lines (NIH 3T3, FLAG-vector control, p85 wild type, and p85R274A) were chosen for analysis. A time-course of PDGF stimulation showed that cells expressing p85R274A or p85Δ110+R274A have sustained phosphorylation levels of the PDGFR, reduced rates of PDGFR degradation and sustained MAPK/Erk signalling. Contrary to the cellular transformation previously reported for p85R274A-expressing cells, expression of p85Δ110+R274A did not lead to cellular transformation. These divergent results suggest that p85-associated p110 serves two functions. As the catalytic subunit of PI3K, one function is the localized generation of PI3,4,5P3 lipids at the plasma membrane for Akt activation, and possibly during receptor endocytosis where it could impact MAPK/Erk activation/deactivation kinetics and cell transformation. These results support a second function for p110 in the regulation of PDGFR activation/deactivation kinetics and PDGFR half-life, both strongly influenced by alterations in PDGFR trafficking. This suggests that p110β may regulate PDGFR trafficking by providing Rab5-GTP switch stabilization that complements the catalytic arginine residue (R274) within p85, and that p85α and p110β work together as a Rab5 GAP. The role of PDGFR in the localization of the RabGAP function of p85 to specific subcellular compartments was also examined. It was hypothesized that PDGFR may help localize the RabGAP function of p85 to vesicles containing Rab5 or Rab4 through the binding of p85 to phosphorylated tyrosine residues on activated PDGFR. Stable cell lines expressing individual p85 defects (lacking GAP activity, R274A; or lacking PDGFR-binding ability through site-directed mutation of residues 358 and 649 from arginine to alanine, ΔR; or a double mutant missing both functions) demonstrated that p85R274A or p85ΔR+R274A expression leads to sustained PDGFR activation and signalling, and to delayed PDGFR degradation in response to PDGF stimulation. The sustained signalling observed resulted in cellular transformation in cells expressing p85R274A or p85ΔR+R274A. The data suggests that PDGFR does not play a role in the localization of the RabGAP activity of p85. The findings of this study elucidates important non-canonical functions of the PI3K heterodimer and contributes to our understanding of how specific mutations in both p85 and p110β within regions implicated in the regulation of RabGAP activity can alter signalling events and lead to enhancement of tumour-associated phenotypes.

Nous avons découvert que TRF2 est aussi sur-exprimée au niveau des cellules endothéliales de nombreux types de cancers humains alors qu’elle n’est pas détectable dans les vaisseaux des tissus sains adjacents. Des cellules endothéliales extraites de tumeurs ex-vivo manifestent une expression supérieure de TRF2, une migration et une prolifération accrues et une aptitude à former des tubules élevée, comparées aux endotheliums isolées de tissus sains. La sur-expression de cette protéine in vitro dans des cellules endothéliales primaires et ex-vivo entraine l’augmentation de la prolifération, de la migration et de la capacité de ces dernières à former des tubules. La diminution de l’expression de TRF2 conduit à l’effet inverse. Par ailleurs, la modulation de l’expression de TRF2 n’affecte pas la proportion de cellules apoptotiques. De même, les variations des niveaux d’expression de TRF2 n’induisent aucune réponse aux dommages à l’ADN et les modifications des facultés angiogéniques sont indépendantes d’ATM. Les effets angiogéniques de TRF2 semblent donc distincts des fonctions télomériques. Etant donné que le facteur de transcription WT1 (Wilms’ tumour suppressor 1) est fortement exprimé dans les vaisseaux de tumeurs humaines et régule les propriétés angiogéniques des cellules endothéliales, nous nous sommes penché sur la régulation potentielle de TRF2 par WT1. WT1 se lie en effet sur le promoteur de TRF2 pour activer sa transcription. Enfin, nous avons démontré que l’activité angiogénique de TRF2 réside en partie dans sa capacité à se fixer sur le promoteur du gène codant pour le récepteur angiogénique à activité tyrosine kinase PDGFRβ et à activer sa transcription. / We discovered that TRF2 is expressed in endothelial cells of many human cancer types but not in the vessels of healthy adjacent tissues. Endothelial cells derived from tumours ex vivo exhibited a significantly increased TRF2 expression, and a higher migration, proliferation and tube formation potential as endothelium obtained from healthy tissues. In vitro TRF2 over-expression in primary or ex vivo endothelial cells resulted in an increased proliferation, migration and tube formation, while silencing of TRF2 led to the opposite results. No changes in apoptosis could be observed. Interestingly, modulation of TRF2 in endothelium does not induce DNA damage responses and the observed changes in the angiogenic behaviour are ATM –independent. The angiogenic effects of TRF2 seem therefore to be uncoupled from its telomeric function. Since the transcription factor WT1 (Wilms’ tumour suppressor 1) is highly expressed in human tumour vessels and mediates angiogenic properties of endothelial cells, we investigated whether TRF2 expression could be regulated by WT1. Indeed, WT1 binds the TRF2 promoter and activates its transcription. Finally, we demonstrated that TRF2 promotes angiogenesis by binding to the promoter of the gene encoding for the angiogenic tyrosine kinase receptor PDGFRβ and activating its transcription.

A neuraminidase 1 (Neu1) é a enzima que regula o catabolismo de sialoglicoconjugados nos lisossomos. A deficiência da Neu1 é a base da sialidose, doença grave associada a um amplo espectro de manifestações, incluindo hipotonia e fraqueza muscular. Camundongos com deficiência de Neu1 desenvolvem degeneração muscular caracterizada principalmente por atrofia, invasão das fibras musculares por fibroblastos e expansão da matriz extracelular. A Neu1 controla a proliferação de fibroblastos de pacientes por meio da desialilação dos receptores de PDGF e IGF. Além disso, há enzimas lisossomais que são moduladas pela Neu1, tais como as catepsinas, que são capazes de degradar componentes musculares e estariam excessivamente ou erroneamente ativas (sialiladas) em decorrência da deficiência de Neu1. O objetivo deste trabalho foi identificar se o fenótipo da musculatura esquelética de camundongos Neu1-/- poderia estar associado à atividade do IGF-1R, PDGFR e/ou à sialilação de catepsina B, através da análise histológica e proteica de músculos esqueléticos e fibroblastos de camundongos Neu1+/+ e Neu1-/- tratados com inibidores de IGF1-R e PDGFR. O estudo da expressão proteica de catepsina B foi realizado nos músculos tratados com os inibidores de IGF-1R e PDGFR, e nas frações citosólica e lisossomal de fibroblastos tratados com neuraminidase exógena. Em comparação com camundongos Neu1+/+, os músculos de animais Neu1-/- apresentam menor área de fibra, peso corporal, expressão de pAkt e maior expressão de catepsina B; e os fibroblastos Neu1-/- exibem maior proliferação e expressão de pAkt. A inibição do IGF-1R em camundongos Neu1-/- aumentou a área das fibras musculares, expressão de pAKt e diminuiu a expressão de catepsina B; em relação aos fibroblastos Neu1-/-, entretanto aumentou a proliferação celular com diminuição de pAkt. A inibição do PDGFR em músculos de camundongos Neu1-/- levou ao aumento da expressão de pAkt, da área das fibras, com diminuição de pERK e catepsina L, quando comparados com os controles Neu1-/-; a mesma inibição in vitro conduziu à diminuição da expressão de pAkt, pERK e proliferação. A catepsina B encontra-se bastante ativa na fração lisossomal e o tratamento com neuraminidase foi eficaz na correção de seu peso molecular e compartimentalização lisossomal. De forma geral, o fenótipo muscular de camundongos Neu1-/- parece estar relacionado com a atividade de IGF-1R e PDGFR, e a catepsina B hipersialilada é potencialmente deletéria para o músculo esquelético / Neuraminidase 1 (Neu1) is an enzyme that regulates the catabolism of sialoglycoconjugates in lysosomes. Neu1 deficiency is the basis of sialidosis, a severe disease associated with a broad spectrum of manifestations, including hypotonia and muscle weakness. Neu1 deficient mice develop muscular degeneration characterized by atrophy, invasion of muscle fibers by fibroblasts, and expansion of the extracellular matrix. Neu1 controls the proliferation of fibroblasts from patients through the desialylation of PDGF and IGF receptors. In addition, lysosomal enzymes are modulated by Neu1, such as cathepsins, which degrade muscle components and are excessively or erroneously active (sialylated) as a result of Neu1 deficiency. The aim of this study was to identify whether skeletal muscle phenotype of Neu1-/- mice may be associated with IGF-1R, PDGFR and/or sialylation of cathepsin B, through protein and histological analysis of skeletal muscles and fibroblast from Neu1+/+ and Neu1-/- mice treated with IGF-1R and PDGFR inhibitors. The study of cathepsin B protein expression was performed in skeletal muscles treated with IGF-1R and PDGFR inhibitors, and in the cytosolic and lysosomal fractions of fibroblasts treated with exogenous neuraminidase. Compared with Neu1+/+ animals, Neu1-/- muscles showed smaller muscle fiber area, body weight, pAkt expression and higher cathepsin B expression; and Neu1-/- fibroblasts exhibited increased proliferation and expression of pAkt. The inhibition of IGF-1R Neu1-/- mice increased the area of muscle fibers, expression of pAkt and decreased expression of cathepsin B; but, considering Neu1-/- fibroblasts, there was increased cell proliferation with reduction of pAkt. The inhibition of PDGFR in muscles of Neu1-/- mice led to increased expression of pAkt, muscle fiber area, with decreased expression of pERK and cathepsin L, when compared with the Neu1-/- controls; the same inhibition in vitro led to reduced expression of pAkt, pERK and cell proliferation. Cathepsin B presented high activity in the lysosomal fraction and the treatment with neuraminidase was effective in the correction of its molecular weight and lysosomal compartmentalization. In general, the muscular phenotype of Neu1-/- mice is possibly related to IGF-1R and PDGFR activity, and oversialylated cathepsin B is potentially deleterious for the skeletal muscle

A neuraminidase 1 (Neu1) é a enzima que regula o catabolismo de sialoglicoconjugados nos lisossomos. A deficiência da Neu1 é a base da sialidose, doença grave associada a um amplo espectro de manifestações, incluindo hipotonia e fraqueza muscular. Camundongos com deficiência de Neu1 desenvolvem degeneração muscular caracterizada principalmente por atrofia, invasão das fibras musculares por fibroblastos e expansão da matriz extracelular. A Neu1 controla a proliferação de fibroblastos de pacientes por meio da desialilação dos receptores de PDGF e IGF. Além disso, há enzimas lisossomais que são moduladas pela Neu1, tais como as catepsinas, que são capazes de degradar componentes musculares e estariam excessivamente ou erroneamente ativas (sialiladas) em decorrência da deficiência de Neu1. O objetivo deste trabalho foi identificar se o fenótipo da musculatura esquelética de camundongos Neu1-/- poderia estar associado à atividade do IGF-1R, PDGFR e/ou à sialilação de catepsina B, através da análise histológica e proteica de músculos esqueléticos e fibroblastos de camundongos Neu1+/+ e Neu1-/- tratados com inibidores de IGF1-R e PDGFR. O estudo da expressão proteica de catepsina B foi realizado nos músculos tratados com os inibidores de IGF-1R e PDGFR, e nas frações citosólica e lisossomal de fibroblastos tratados com neuraminidase exógena. Em comparação com camundongos Neu1+/+, os músculos de animais Neu1-/- apresentam menor área de fibra, peso corporal, expressão de pAkt e maior expressão de catepsina B; e os fibroblastos Neu1-/- exibem maior proliferação e expressão de pAkt. A inibição do IGF-1R em camundongos Neu1-/- aumentou a área das fibras musculares, expressão de pAKt e diminuiu a expressão de catepsina B; em relação aos fibroblastos Neu1-/-, entretanto aumentou a proliferação celular com diminuição de pAkt. A inibição do PDGFR em músculos de camundongos Neu1-/- levou ao aumento da expressão de pAkt, da área das fibras, com diminuição de pERK e catepsina L, quando comparados com os controles Neu1-/-; a mesma inibição in vitro conduziu à diminuição da expressão de pAkt, pERK e proliferação. A catepsina B encontra-se bastante ativa na fração lisossomal e o tratamento com neuraminidase foi eficaz na correção de seu peso molecular e compartimentalização lisossomal. De forma geral, o fenótipo muscular de camundongos Neu1-/- parece estar relacionado com a atividade de IGF-1R e PDGFR, e a catepsina B hipersialilada é potencialmente deletéria para o músculo esquelético / Neuraminidase 1 (Neu1) is an enzyme that regulates the catabolism of sialoglycoconjugates in lysosomes. Neu1 deficiency is the basis of sialidosis, a severe disease associated with a broad spectrum of manifestations, including hypotonia and muscle weakness. Neu1 deficient mice develop muscular degeneration characterized by atrophy, invasion of muscle fibers by fibroblasts, and expansion of the extracellular matrix. Neu1 controls the proliferation of fibroblasts from patients through the desialylation of PDGF and IGF receptors. In addition, lysosomal enzymes are modulated by Neu1, such as cathepsins, which degrade muscle components and are excessively or erroneously active (sialylated) as a result of Neu1 deficiency. The aim of this study was to identify whether skeletal muscle phenotype of Neu1-/- mice may be associated with IGF-1R, PDGFR and/or sialylation of cathepsin B, through protein and histological analysis of skeletal muscles and fibroblast from Neu1+/+ and Neu1-/- mice treated with IGF-1R and PDGFR inhibitors. The study of cathepsin B protein expression was performed in skeletal muscles treated with IGF-1R and PDGFR inhibitors, and in the cytosolic and lysosomal fractions of fibroblasts treated with exogenous neuraminidase. Compared with Neu1+/+ animals, Neu1-/- muscles showed smaller muscle fiber area, body weight, pAkt expression and higher cathepsin B expression; and Neu1-/- fibroblasts exhibited increased proliferation and expression of pAkt. The inhibition of IGF-1R Neu1-/- mice increased the area of muscle fibers, expression of pAkt and decreased expression of cathepsin B; but, considering Neu1-/- fibroblasts, there was increased cell proliferation with reduction of pAkt. The inhibition of PDGFR in muscles of Neu1-/- mice led to increased expression of pAkt, muscle fiber area, with decreased expression of pERK and cathepsin L, when compared with the Neu1-/- controls; the same inhibition in vitro led to reduced expression of pAkt, pERK and cell proliferation. Cathepsin B presented high activity in the lysosomal fraction and the treatment with neuraminidase was effective in the correction of its molecular weight and lysosomal compartmentalization. In general, the muscular phenotype of Neu1-/- mice is possibly related to IGF-1R and PDGFR activity, and oversialylated cathepsin B is potentially deleterious for the skeletal muscle

In 2001 the human proteome organization (HUPO) was created with the ambition to identify and characterize all proteins encoded in the human genome according to several criteria; their expression levels in different tissues and under different conditions; the sub-cellular localization; post-translational modifications; interactions, and if possible also the relationship between their structure and function.When the knowledge of different proteins and their potential interactions increases, so does the need for methods able to unravel the nature of molecular processes in cells and organized tissues, and ultimately for clinical use in samples obtained from patients. The in situ proximity ligation assay (in situ PLA) was developed to provide localized detection of proteins, post-translational modifications and protein-protein interactions in fixed cells and tissues. Dual recognition of the target or interacting targets is a prerequisite for the creation of a circular reporter DNA molecule, which subsequently is locally amplified for visualization of individual protein molecules in single cells. These features offer the high sensitivity and selectivity required for detection of even rare target molecules. Herein in situ PLA was first established and then employed as a tool for detection of both interactions and post-translational modifications in cultured cells and tissue samples. In situ PLA was also adapted to high content screening (HCS) for therapeutic effects, where it was applied for cell-based drug screening of inhibitors influencing post-translational modifications. This was performed using primary cells, paving the way for evaluation of drug effects on cells from patient as a diagnostic tool in personalized medicine. In conclusion, this thesis describes the development and applications of in situ PLA as a tool to study proteins, post-translational modifications and protein-protein interactions in genetically unmodified cells and tissues, and for clinical interactomics.