The clathrin-independent endosomal system is required for cellular homeostasis and specialized modifications of the plasma membrane such as cell spreading and polarization. Clathrin-independent endocytosis (CIE) has been demonstrated in adherent cells including fibroblasts and epithelial cells. However, non-adherent cells also have highly dynamic clathrin-independent pathways, which have not been well described. Here, I have characterized Arf6-associated clathrin-independent endocytosis (CIE) in the human T cell line Jurkat and identified it's importance in immunological synapse formation. Our findings indicate that the CIE pathway is similar in Jurkat cells as compared to other cell types including rates of endocytosis and sorting after internalization. Two GTPases, Arf6 and Rab22, have been shown to regulate the clathrin-independent endosomal system and play a role in cell spreading. We found that wild type and constitutively active Arf6 co-localized with CIE cargo in resting T cells. Arf6 constitutively active mutant inhibited CIE cargo internalization but not internalization of CME cargo. Rab22 co-localized with CIE cargo at the endocytic-recycling compartment. Expression of the dominant negative Rab22 mutant also inhibited internalization of MHCI indicating it plays a direct role in CIE cargo internalization. T cells must modify their membranes to specifically interact with antigen presenting cells. To establish the role of CIE in this process, we then examined the role of Arf6 and Rab22 in T cell/antigen presenting cell conjugate formation. Both expression of dominant negative or constitutively active mutants of Arf6 reduced T cell conjugate formation while expression of only the Rab22 dominant negative mutant inhibited T cell/APC conjugate formation. Furthermore, T cells expressing the dominant negative mutant of Rab22 were not able to spread on antibody-coated coverslips that normally cause T cell activation. These results indicate that the clathrin independent endosomal system is required for membrane remodeling events necessary for T cell conjugate formation and T cell spreading during activation. I also conducted a proteomics screen to identify binding partners of CIE cargo proteins. I identified multiple proteins that could possibly play a role in CIE internalization and discovered a subset of proteins that specifically interact with A cargo proteins and not B cargo proteins. It is possible they could play a role in cargo retention at the plasma membrane or sorting after internalization. Three proteins of interest that interact with A cargo include NHERF-1 and ezrin, which participate in actin arrangements, and Dlg-1, a known scaffolding protein for synaptic vesicles. Ezrin and Dlg-1 co-localize with the CIE cargo protein CD98 in HeLa cells indicating that they could be interacting in cells.

<p>Targeted radiotherapy (TRT) of cancer is a promising approach that enables selective treatment of tumour cells, while sparing normal tissue. The humanized monoclonal antibody A33 (huA33) is a potential targeting agent for TRT of colorectal cancer, since its antigen is expressed in more than 95 % of all colorectal carcinomas. The aim of this thesis was to evaluate the therapeutic potential of the two huA33-based TRT-conjugates, ¹⁷⁷Lu-huA33, and ²¹¹At-huA33.</p><p>The conjugates ¹⁷⁷Lu-huA33, and ²¹¹At-huA33, bound specifically to colorectal cancer cells, both <i>in vitro</i> and <i>in vivo</i>. A dose dependent cytotoxic effect of ²¹¹At-huA33 was also demonstrated <i>in vitro</i>. From a therapeutic perspective, both conjugates had a favourable biodistribution in tumour-bearing nude mice, with high tumour uptake and a low uptake in normal organs (with the exception of an expected thyroid uptake of ²¹¹At). After injection of ²¹¹At-huA33, the blood absorbed a slightly higher dose than the tumour, but for ¹⁷⁷Lu-huA33, the tumour received a 12 times higher dose than blood. Two days after intravenous injection of ¹⁷⁷Lu-huA33 in tumour-bearing mice, the tumours could be clearly visualised by gamma camera imaging, with very low interference from normal tissue radioactivity. In an experimental therapy study, also performed in tumour-bearing mice, there was an excellent therapeutic effect of ¹⁷⁷Lu-huA33. About 50 % of the treated animals were tumour free 140 days after injection of ¹⁷⁷Lu-huA33, while none of the non-radioactive controls survived beyond 20 days after injection of treatment substances.</p><p>In conclusion, this thesis demonstrates that the therapeutic conjugates ¹⁷⁷Lu-huA33, and ²¹¹At-huA33, are promising targeting agents that might help improve therapy of colorectal cancer.</p>

The DEAD-Box RNA helicase family is a conserved group of enzymes that function in gene expression through ATP-dependent RNA unwinding and ribonucleoprotein (RNP) remodeling. DEAD-Box helicases function in multiple cellular processes, including pre-mRNA processing, translation, mRNA export, and mRNA decay. Although DEAD-Box proteins are critical for gene expression, much of their mechanistic activities are poorly understood. DEAD-Box proteins have increasingly been linked to tumorigenesis in humans, and better defining their activity at the mechanistic level will aid in understanding the underlying disease pathology. In this study, we used the model organism Saccharomyces cerevisiae to study the human DEAD-Box protein DDX3 orthologue, Ded1, and its role in translation initiation during cellular stress. Recently, we have found that Ded1 is an important mediator of the cellular stress response in a TOR-dependent manner. TOR regulates protein synthesis dependent on energy availability in the cell by regulating the assembly of the eukaryotic translation initiation complex. Human DDX3 has been found to interact with translation initiation complex subunit eIF4E and Ded1 has been found to interact with the translation initiation complex subunit eIF4G. In this study, we examined the purported interaction region between Ded1 and eIF4G on the C-terminus of Ded1 and found that ded1 Δ591-604 prevents eIF4G degradation under rapamycin treatment and confers resistance to rapamycin-induced growth inhibition. We also examined putative regulatory phosphorylation sites in the purported Ded1 eIF4G binding region. We propose that the Ded1/eIF4G interaction is critical for the repression of translation by Ded1 and that eIF4G degradation may be regulated by Ded1 under stress conditions.

<p><strong>Background: </strong>Lung cancer is the leading cause of cancer-related death and one of the most common cancer types worldwide. Epidermal growth factor receptor (EGFR) has been shown to be an important therapeutic target in non-small cell lung cancer. Kirsten rat sarcoma viral oncogene homologue (KRAS) is a downstream signalling molecule in the EGFR pathway. Lung cancer patients with <em>EGFR </em>mutations respond to tyrosine EGFR inhibitor therapy, in contrast, patients with <em>KRAS </em>mutations do not benefit of such treatment.</p><p><strong>Methods: </strong>This study investigates the frequency of <em>EGFR </em>and <em>KRAS </em>mutations in non-small cell lung cancer patients. Fifty-one lung cancer patients with primary non-small cell lung cancer diagnosed between 1995 and 2005 in the Uppsala-Örebro region were analysed by Sanger sequencing and Pyrosequencing to determine the mutation status of these genes.</p><p><strong>Results: </strong>Five <em>EGFR </em>mutations were found in four patients (8%), two deletions in exon 19, one point mutation in exon 20 and two point mutations in exon 21. <em>KRAS </em>mutations were found in 12 patients (24%), ten codon 12 mutations and two codon 61 mutations.</p><p><strong>Conclusions: </strong>This study confirms previous observations regarding the frequency of <em>EGFR </em>and <em>KRAS </em>mutations in non-small cell lung cancer. Mutations in <em>EGFR </em>and <em>KRAS </em>were mutually exclusive, indicating that both mutations present relevant tumorigenic genomic aberrations.</p>

<p>Proximity ligation is a recently established technique that can provide answers to questions about the concentration, localization, interactions, modifications and functions of proteins. The method enables sensitive protein measurements with a detection limit in the low femtomolar range in complex biological samples. In proximity ligation, the challenge of detecting specific proteins is converted to the analysis of specific DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins, and to form amplifiable tag sequences upon ligation when brought in proximity. Protocols for the conversion of monoclonal or polyclonal antibodies into proximity probes through the attachment of oligonucleotide sequences are described in the thesis. In addition, the thesis describes the adaptation of the proximity ligation technology for detection of microbial pathogens, analysis of interactions between proteins and nucleic acids, and of inhibition of receptor-ligand interactions. </p><p>Nucleic acid amplification allows specific detection of pathogens with single-copy sensitivity. There are many circumstances, however, when analysis of pathogen surface antigens or the antibody response can provide increased diagnostic value. Proximity ligation reactions were used to measure numbers of virus and bacteria by detection of viral or bacterial surface proteins. Detection sensitivities similar to those of nuclear acid-based detection reactions were achieved directly in infected samples for a parvovirus and for an intracellular bacterium. </p><p>Biological processes are orchestrated by interactions of proteins with molecules in their environment, and investigations of interactions between proteins and other biomolecules are thus of great importance. Protocols were established for very specific and sensitive homogeneous-phase analysis of interactions between proteins and specific nucleic acid sequences. Finally, the proximity ligation mechanism was used to monitor interactions between VEGF-A and two of its receptors, VEGFR-1 and VEGFR-2, and to characterize the effects of agents disrupting this interaction.</p>

<p>Improved methods are needed to interrogate the genome and the proteome. Methods with high selectivity, wide dynamic range, and excellent precision, capable of simultaneously analyzing many biomolecules are required to decipher cellular function. This thesis describes a molecular and microfluidic toolbox designed with those criteria in mind. It also presents a tool for graphical representation of nucleic acid sequences.</p><p>Proximity ligation is a novel protein detection method that requires dual and proximate binding of two oligonucleotide-tagged affinity reagents to a protein or protein complex in order to elicit a signal. The responses from such recognition reactions are the formation of specific nucleic acid reporter molecules that are subsequently amplified and quantitatively detected. </p><p>A scalable microfluidic platform suitable for fluorescence detection, cell culture, and actuation is also described. The platform uses rapid injection molding to produce microstructures in thermoplastic materials. By applying a thin layer of silica to the structures, a lid made of silicone rubber coated onto a thermoplastic support can be covalently bonded to generate enclosed channels.</p><p>A method is presented for precise biomolecule counting, termed “amplified single-molecule detection”. The method preserves the discrete nature of biomolecules, converting specific molecular recognition events to fluorescence-labeled micrometer-sized objects that are enumerated in microfluidic channels. </p><p>I also present a novel microarray-based detection method. To attain high selectivity and a wide dynamic range, the method is based on dual recognition with enzymatic discrimination and amplification. Upon target recognition in solution, DNA probes are subjected to thousand-fold amplification in solution, followed by selective detection on arrays and another hundred-fold amplification of reporter molecule created from the first amplification reaction. </p><p>Lastly, I describe a novel graphical representation of nucleic acid sequences using TrueType fonts that can be of value for visual inspection of DNA sequences and for teaching purposes</p>

<p>Padlock probes and proximity ligation are two powerful molecular tools for detection of nucleic acids and proteins, respectively. Both methods result in the formation of DNA reporter molecules upon recognition of specific target molecules. These reporter molecules can be designed to include tag sequences that can be analyzed by techniques for nucleic acid analysis. Herein, I present a dual-tag microarray (DTM) platform that is suitable for high-performance analyses of DNA reporter molecule libraries, generated by padlock and proximity probing reactions. The DTM platform was applied for analysis of mRNA transcripts using padlock probes, and of cytokines using proximity ligation. The platform drastically improved specificity of detection, and it allowed precise measurements of proteins and nucleic acids over wide dynamic ranges.</p><p>The thesis also presents two techniques for multi-probe analyses of biomolecules: the triple-specific proximity ligation assay (3PLA) for protein analyses, and the spliceotyping assay for mRNA analyses. 3PLA allows highly specific measurements of as little as hundreds of target protein molecules by interrogating three target epitopes simultaneously. In spliceotyping the exon composition of individual transcripts are represented as a series of tag sequences in DNA reporter molecules, via a series of target-dependent ligation reactions. Next, the splicing patterns along individual transcripts can be revealed by amplified single molecule detection and step-wise decoding.</p>

Proximity ligation is a recently established technique that can provide answers to questions about the concentration, localization, interactions, modifications and functions of proteins. The method enables sensitive protein measurements with a detection limit in the low femtomolar range in complex biological samples. In proximity ligation, the challenge of detecting specific proteins is converted to the analysis of specific DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins, and to form amplifiable tag sequences upon ligation when brought in proximity. Protocols for the conversion of monoclonal or polyclonal antibodies into proximity probes through the attachment of oligonucleotide sequences are described in the thesis. In addition, the thesis describes the adaptation of the proximity ligation technology for detection of microbial pathogens, analysis of interactions between proteins and nucleic acids, and of inhibition of receptor-ligand interactions. Nucleic acid amplification allows specific detection of pathogens with single-copy sensitivity. There are many circumstances, however, when analysis of pathogen surface antigens or the antibody response can provide increased diagnostic value. Proximity ligation reactions were used to measure numbers of virus and bacteria by detection of viral or bacterial surface proteins. Detection sensitivities similar to those of nuclear acid-based detection reactions were achieved directly in infected samples for a parvovirus and for an intracellular bacterium. Biological processes are orchestrated by interactions of proteins with molecules in their environment, and investigations of interactions between proteins and other biomolecules are thus of great importance. Protocols were established for very specific and sensitive homogeneous-phase analysis of interactions between proteins and specific nucleic acid sequences. Finally, the proximity ligation mechanism was used to monitor interactions between VEGF-A and two of its receptors, VEGFR-1 and VEGFR-2, and to characterize the effects of agents disrupting this interaction.

Padlock probes and proximity ligation are two powerful molecular tools for detection of nucleic acids and proteins, respectively. Both methods result in the formation of DNA reporter molecules upon recognition of specific target molecules. These reporter molecules can be designed to include tag sequences that can be analyzed by techniques for nucleic acid analysis. Herein, I present a dual-tag microarray (DTM) platform that is suitable for high-performance analyses of DNA reporter molecule libraries, generated by padlock and proximity probing reactions. The DTM platform was applied for analysis of mRNA transcripts using padlock probes, and of cytokines using proximity ligation. The platform drastically improved specificity of detection, and it allowed precise measurements of proteins and nucleic acids over wide dynamic ranges. The thesis also presents two techniques for multi-probe analyses of biomolecules: the triple-specific proximity ligation assay (3PLA) for protein analyses, and the spliceotyping assay for mRNA analyses. 3PLA allows highly specific measurements of as little as hundreds of target protein molecules by interrogating three target epitopes simultaneously. In spliceotyping the exon composition of individual transcripts are represented as a series of tag sequences in DNA reporter molecules, via a series of target-dependent ligation reactions. Next, the splicing patterns along individual transcripts can be revealed by amplified single molecule detection and step-wise decoding.

The cardiovascular system is the first functional organ to develop during embryogenesis. As the embryo reaches above a certain size, passive diffusion of gases and nutrients is no longer compatible with efficient growth. During embryogenesis, endothelial progenitor cells (angioblasts) are recruited from the primitive streak mesoderm and instructed to express vascular endothelial growth factor receptor-2 (VEGFR-2). This thesis examines the roles played by VEGFR-2 in the events through which a subpopulation of embryonic stem (ES) cells differentiate into endothelial cells and form the vasculature. We show that ES cells gene targeted for VEGFR-2 (flk1-/-) develop immature endothelial cells (ECs), precursors, when differentiated in vitro as embryoid bodies (EBs). The flk1-/- ECs are unresponsive to VEGF-stimulation and consistently fail to form vessels. However, when co-cultured with wild type ES cells in chimeric EBs, flk1-/- endothelial precursors are guided by wild type ECs to form transient, chimeric vascular structures. Use of lentivirus in an add-back approach allowed reconstitution of VEGFR-2 expression in flk1-/- ES cells, and rescue of vasculogenesis and sprouting angiogenesis. We propose that recruitment to the endothelial lineage is not dependent on VEGFR-2, although this receptor tyrosine kinase appears indispensible for EC integrity, survival and for differentation of endothelial precursors into mature ECs formating a stable vasculature. Neuropilin-1 (NRP1) and heparan sulfate proteoglycans (HSPGs) function as co-receptors for VEGFs. The co-receptors influence, qualitatively and quantitatively, the intracellular signal relayed by VEGFR-2 but it is unclear how. We examined the contribution of NRP1 to VEGFR-2 signaling in EB cultures, in zebrafish and in mice. Only NRP1-binding VEGFs were able to promote sprouting angiogenesis and formation of properly branched vascular tubes, supported by pericytes. Downstream of VEGFR-2/NRP1 activation, we identified recruitment of p38MAPK in signal transduction regulating sprouting angiogenesis.