Innate Immunity in Insects, Function and Regulation of Hemolin from Hyalophora cecropia

Insects are useful models for the study of innate immune reactions and development. The distinction between recognition mechanisms preceding the breakdown of apoptotic cells during metamorphosis, and the breakdown of cells in response to infections, is unclear. Hemolin, a Lepidopteran member of the immunoglobulin superfamily, is a candidate molecule in self/nonself recognition. This thesis investigates hemolin function and hemolin gene regulation at a molecular level. We investigated the binding and cell adhesion properties of hemolin from H. cecropia and demonstrated that the proteins could homodimerize in presence of calcium. Moreover, a higher molecular weight membrane form of hemolin was present on hemocytes. These results, taken together with an earlier finding that soluble hemolin inhibits hemocyte adhesion, indicated that the secreted hemolin could modulate hemocyte aggregation in a competitive manner in the blood. In addition, hemolin was expressed in different tissues and at different developmental stages. Since hemolin is expressed both during development and during the immune response, its different regulatory factors must act in concert. We found that the third intron contains an enhancer, through which Dif, C/EBP and HMGI synergistically activate a reporter construct in vitro. We concluded that the enhancer is used during infection, since the κB-site is crucial for an immune response. Interestingly, we also found that the active form of the steroid hormone, ecdysone, induces the hemolin gene transcription in vivo, and in addition, acts synergistically during bacterial infection. Preliminary in vivo results indicate a secondary effect of ecdysone and the importance of hormone receptor elements in the upstream promoter region of hemolin. To explore the use of Drosophila as a genetic tool for understanding hemolin function and regulation, we sought to isolate the functional homologue in this species. A fly cDNA library in yeast was screened using H. cecropia hemolin as bait. The screen was not successful. However, it did lead to the discovery of a Drosophila protein with true binding specificity for hemolin. Subsequent characterization revealed a new, highly conserved gene, which we named yippee. Yippee is distantly related to zinc finger proteins and represents a novel family of proteins present in numerous eukaryotes, including fungi, plants and humans. Notably, when the Drosophila genome sequence was revealed, no hemolin orthologue could be detected. Finally, an extensive Drosophila genome chip analysis was initiated. The goal was to investigate the Drosophila immune response, and, in contrast to earlier studies of artificially injected flies, to examine a set of natural microbes, orally and externally applied. In parallel experiments viruses, bacteria, fungi and parasites were compared to unchallenged controls. We obtained a unique set of genes that were up-regulated in the response to the parasite Octosporea muscadomesticae and to the fungus Beauveria bassiana. We expect both down-regulated and up-regulated genes to serve as a source for the discovery of new effector molecules, in particular those that are active against parasites and fungi.

Multivariate spectroscopic methods for the analysis of solutions

In this thesis some multivariate spectroscopic methods for the analysis of solutions are proposed. Spectroscopy and multivariate data analysis form a powerful combination for obtaining both quantitative and qualitative information and it is shown how spectroscopic techniques in combination with chemometric data evaluation can be used to obtain rapid, simple and efficient analytical methods. These spectroscopic methods consisting of spectroscopic analysis, a high level of automation and chemometric data evaluation can lead to analytical methods with a high analytical capacity, and for these methods, the term high-capacity analysis (HCA) is suggested. It is further shown how chemometric evaluation of the multivariate data in chromatographic analyses decreases the need for baseline separation. The thesis is based on six papers and the chemometric tools used are experimental design, principal component analysis (PCA), soft independent modelling of class analogy (SIMCA), partial least squares regression (PLS) and parallel factor analysis (PARAFAC). The analytical techniques utilised are scanning ultraviolet-visible (UV-Vis) spectroscopy, diode array detection (DAD) used in non-column chromatographic diode array UV spectroscopy, high-performance liquid chromatography with diode array detection (HPLC-DAD) and fluorescence spectroscopy. The methods proposed are exemplified in the analysis of pharmaceutical solutions and serum proteins. In Paper I a method is proposed for the determination of the content and identity of the active compound in pharmaceutical solutions by means of UV-Vis spectroscopy, orthogonal signal correction and multivariate calibration with PLS and SIMCA classification. Paper II proposes a new method for the rapid determination of pharmaceutical solutions by the use of non-column chromatographic diode array UV spectroscopy, i.e. a conventional HPLC-DAD system without any chromatographic column connected. In Paper III an investigation is made of the ability of a control sample, of known content and identity to diagnose and correct errors in multivariate predictions something that together with use of multivariate residuals can make it possible to use the same calibration model over time. In Paper IV a method is proposed for simultaneous determination of serum proteins with fluorescence spectroscopy and multivariate calibration. Paper V proposes a method for the determination of chromatographic peak purity by means of PCA of HPLC-DAD data. In Paper VI PARAFAC is applied for the decomposition of DAD data of some partially separated peaks into the pure chromatographic, spectral and concentration profiles.