Gastroesophageal cancer is the sixth leading cause of cancer-related death worldwide. The 2 most common histologies are squamous cell carcinoma and adenocarcinoma, which has seen an increase in incidence correlating with an increase in obesity in developed countries. Gastroesophageal adenocarcinoma has a preponderance to metastasize early, making it a highly lethal cancer with a low 5-year survival rate of similar to 15-25%. Therefore, for the majority of patients, treatment focuses on palliation and prolongation of survival. Combination chemotherapy regimens, mostly platinum-based, have only modestly prolonged survival in patients with stage IV disease. Recently, it was discovered that the activation of the HER2 receptor plays an important role in a minority of adenocarcinomas of the distal esophagus and stomach. This introduced the treatment option of trastuzumab (Herceptin), a monoclonal antibody directed at the HER2 receptor, which has demonstrated improvement in overall and progression-free survival as noted in the ToGA trial. Currently, the role of Herceptin therapy beyond first-line therapy and outside of combination regimens is not well established. In this case report we review 2 cases of patients with gastroesophageal cancer, with HER2 overexpression, who achieved a robust response to trastuzumab in combination with chemotherapy and were able to maintain a durable response with maintenance trastuzumab monotherapy. (c) 2017 The Author(s) Published by S. Karger AG, Basel.

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Membrane proteins are essential for life, and roughly one-quarter of all open reading frames in sequenced genomes code for membrane proteins. Unfortunately, our understanding of membrane proteins lags behind that of soluble proteins, and is best reflected by the fact that only 0.5% of the structures deposited in the protein data-bank (PDB) are of membrane proteins. This discrepancy has arisen because their hydrophobicity - which enables them to exist in a lipid environment - has made them resistant to most traditional approaches used for procuring knowledge from their soluble counter-parts. As such, novel methods are required to facilitate our knowledge acquisition of membrane proteins. In this thesis a generic approach for rapidly obtaining information on membrane proteins from the classic bacterial encyclopedia Escherichia coli is described. We have developed a Green Fluorescent Protein C-terminal tagging approach, with which we can acquire information as to the topology and ‘expressibility’ of membrane proteins in a high-throughput manner. This technology has been applied to the whole E. coli inner membrane proteome, and stands as an important advance for further membrane protein research.

Systematic genomic studies in the budding yeast S. cerevisiae have greatly increased our capacity to conduct functional profiling of the eukaryotic genome. I describe a new method that makes use of yeast “Barcoder” strains to uniquely tag strains in a yeast overexpression collection (FLEX) and to systematically examine the effects of gene overexpression on cell fitness. This novel system is compatible with the so-called Synthetic Genetic Array (SGA) method, which automates yeast genetics and allows introduction of marked query alleles of interest into arrayed collections of yeast mutants. I identified SDL interactions for two key regulatory kinases, Dun1 and Mck1, using my system. I also used my array and approach to identify SDL interactions for Dun1 that are only manifest in the presence of DNA damage. These studies demonstrate the utility of the pooled SGA-SDL method for systematic discovery of condition-specific genetic interactions in conserved biological pathways.

Systematic genomic studies in the budding yeast S. cerevisiae have greatly increased our capacity to conduct functional profiling of the eukaryotic genome. I describe a new method that makes use of yeast “Barcoder” strains to uniquely tag strains in a yeast overexpression collection (FLEX) and to systematically examine the effects of gene overexpression on cell fitness. This novel system is compatible with the so-called Synthetic Genetic Array (SGA) method, which automates yeast genetics and allows introduction of marked query alleles of interest into arrayed collections of yeast mutants. I identified SDL interactions for two key regulatory kinases, Dun1 and Mck1, using my system. I also used my array and approach to identify SDL interactions for Dun1 that are only manifest in the presence of DNA damage. These studies demonstrate the utility of the pooled SGA-SDL method for systematic discovery of condition-specific genetic interactions in conserved biological pathways.

The human epidermal growth factor receptor-2 (HER2) protein has been associated with breast cancer progression and the HER2 status can be used to determine the type of treatment for each breast cancer patient. The purpose of this study was to examine the HER2 protein and gene statuses in breast cancer tissue samples using two methods and analyze the concordance between them. Ten paraffin-embedded, formaldehyde-fixed breast cancer tissue samples from the Biobank at the Department of Pathology and Cytology at Sundsvall Hospital were analyzed in this study. All samples were from women born between 1931 and 1976. The methods used were immunohistochemistry (IHC) to visualise the HER2 protein and silver in situ hybridization (SISH) to detect gene amplification. The IHC staining method is an indirect detection of the HER2 protein using antibodies. The SISH method used in this study is a Dual ISH which detects both the HER2 gene and the centromere region of Chromosome 17 on the same tissue slide. A HER2 gene/Chromosome 17 ratio was calculated according to the manufacturer’s instructions. This ratio was used to determine HER2 gene status. Out of ten samples, seven were positive with IHC and three were negative. The results from the SISH staining exposed a gene amplification in three of the IHC positive samples, while seven samples did not contain any amplified HER2 genes. The conclusion was that the concordance between IHC and SISH for HER2 was 60 percent.

Nasopharyngeal carcinoma is a commonly occuring tumor in Southern China. However, the genetic basis underlying its tumorigenicity is still unclear. In eukaryotic cells, progression of the cell cycle is regulated by interactions of cyclins, cyclin dependent kinase (CDKs) and CDK inhibitors (CDKIs). These cell cycle-regulator proteins play important roles in growth of both normal and tumor cells. Many human tumors exhibit deregulation of one or more genes which involved in regulation of cell cycle progression. p27Kip1, a member of the Cip/Kip family, inhibits both cyclin D-CDK4, and cyclin E-CDK2 complexes and regulates progression of the cell cycle from G1 to S phase. Although p27Kip1 gene mutations are rare in human tumors, low expression of p27Kip1 are observed in several cancers, such as colon, breast and esophagus. In our previous study, p27Kip1 shown lower expression in two NPC cell lines compared with NNE and 293 (HEK). A doxycycline inducible construct, pBIG2r/p27Kip1, included a full length of human p27Kip1 cDNA was transfected into two NPC cell lines. Expression of several cell cycle-related genes were analyzed. By increasing p27Kip1 in NPC cell lines, we found that the G1 phase and the doubling time were lengthened. Protein expression of cyclin E and CDK2 were up-regulated. These data suggest that the overexpression of p27Kip1 might be cause NPC cells to arrest at G1 phase and might lead to apoptosis.

Serine/Arginine-rich proteins (SR-proteins) have been well characterized in metazoans and in the flowering plant Arabidopsis thaliana. But so far no attempts on characterizing SR-proteins in the moss Physcomitrella patens have been done. SR-proteins are a conserved family of splicing regulators essential for constitutive- and alternative splicing. SR-proteins are mediators of alternative splicing (AS) and may be alternatively spliced themselves as a form of gene regulation. Three novel SR-proteins of the SR-subfamily were identified in P. patens. The three genes show conserved intron-exon structure and protein domain distribution, not surprising since the gene family has evidently evolved through gene duplications. The SR-proteins PpSR40 and PpSR36 show differential tissue-specific expression, whereas PpSR39 does not. Tissue-specific expression of SR-proteins has also been seen in A. thaliana. SR-proteins determine splice-site usage in a concentration dependent manner. SR-protein overexpression experiments in A. thaliana and Oryza sativa have shown alteration of splicing patterns of endogenous SR-proteins. Overexpression of PpSR40 did not alter the splicing patterns of PpSR40, PpSR36 and PpSR39. This suggests that they might not be a substrate for PpSR40. These first results of SR-protein characterization in P. patens may provide insights on the SR-protein regulation mechanisms of the common land plant ancestor.

Malaria caused by the protozan parasite Plasmodium is one of the most serious infectious diseases in the developing world. It is estimated that malaria causes an annual mortality rate of ~627,000. New drugs are urgently required, as the incidence of resistance is spreading rapidly. Glycosylphosphatidylinositol (GPI) anchored proteins decorate the merozoite surface and several of which, including merozoite surface proteins - 1 and -2 have previously been shown to be essential for erythrocyte invasion and parasite survival. Plasmodium GPI-anchors contain a glycan core consisting of four mannose residues. Therefore, the enzymes involved in the synthesis of activated mannose, guanidine diphosphomannose pyrophosphorylase (GDP-Man PP) and dolichol phosphate mannose synthase (DPMS), are thought to be crucial for GPI-anchor biosynthesis and as such potential drug targets. Double homologous recombination has been exploited to test whether PfGDP-Man PP and PfDPMS are essential during the erythrocytic portion of the parasite's life cycle. Additionally, overexpression parasite lines for both enzymes have been generated and have shown that the regulation of the two enzymes are intricately linked. Focused metabolomics by multi-reaction monitoring of the overexpression lines suggests that the fucosylation pathway may have a novel function within the parasite, possibly as a dynamic store for activated fucose/mannose. In order to determine the cellular concentration of key metabolites within the parasite, the volumes of the intra-erythrocytic stages have been determined and show that the concentration of metabolites in the ring stage parasites is substantially higher than previously thought. Furthermore, the sub-cellular localisation of GDP-Man PP and DPMS has been determined by immunofluorescence assay. The recombinant expression of DPMS in E. coli allowed its active site residues to be probed as well as establishing a platform for inhibitors to be screened against the enzyme. Finally, inhibitors of the T. brucei DPMS enzyme have been screened against the P. falciparum parasites in culture.

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