Microalgae may be a powerful biopharmaceutical production platform that is still in its infancy of development. The research done in this project tested the feasibility of creating algal cell packs, a novel immobilised microalgae transient production platform for the expression of recombinant protein. First it had to be established whether the available plant expression vectors could be used for the transfer of genetic material into packed microalgae. The method showed successful transfer of the neomycin phosphotransferase II resistance gene (nptII). Further experiments analysed the plant expression vectors pTRAc and pRIC3.0 for expression of enhanced green fluorescent protein (EGFP) in Scenedesmus spp. by western blotting. Possible replication of the plant geminivirus-derived pRIC3.0 was then confirmed by comparing to replication in Nicotiana benthamiana by quantitative polymerase chain reaction (qPCR). Western blot results indicated EGFP expression in N. benthamiana but not in Scenedesmus. By using PCR the presence of EGFP DNA in Scenedesmus was detected but qPCR showed no increase of the pRIC3.0 replicon. Despite no detection via antibodies of EGFP in Scenedesmus, green fluorescence was observed. These initial results showed promise and points to a system that requires optimisation for increased transfection rates and protein expression. Following on from the initial work, the project set out to determine the feasibility of expressing a recombinant anti-Ebola viral inhibitor protein in three different plant based platforms namely N. benthamiana, a microalgal genus, Desmodesmus and a BY2 tobacco plant-cell culture. Protein expression was compared between the Desmodesmus algal cellpack, N. benthamiana plant expression system and BY-2 plant cell packs by western blotting. Four designs of the viral inhibitor fused to the maize ƴ-zein protein body inducing protein, ZERA, were expressed in trace quantities. Transient expression was more prominent in the algal cell packs than in N. benthamiana and BY-2 cells. The algal cell pack system may potentially be a powerful tool to test recombinant protein expression in a range of microalgal hosts via Agrobacterium-mediated genetic transfection. The future development of recombinant protein expression platforms could be enhanced by rapid testing of protein production in different species. Refinement needs to be done on the algal cell pack to increase transfection efficiency and expression in microalgae to produce commercially viable quantities of heterologous protein.

The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.

BackgroundAll cells carry a transmembrane proteoglycan calledsyndecan. Syndecans influence many functions like cell migration, cell adhesionand cell proliferation and it is involved in cellular signaling andtumourigenesis. The common features of differentiation in twomesenchymal tumor cell types, malignant mesothelioma cells and fibrosarcoma cells,are connected to the synthesis of syndecans. By studying the overexpression ofsyndecan-1 we hope to discover new features of the syndecan-1 molecule that wecan add to the puzzle of mesenchymal tumors. Methods and findingsMalignant mesothelioma cells and fibrosarcoma cellswere cultured and transfected with full-length- and truncated syndecan-1 constructs.To detect the expression of syndecan-1 on RNA level Rt-Q-PCR was conductedfollowed by immunocytochemical analysis to establish the syndecan-1 expressionon protein level. The result showed a 2-7 fold increase of syndecan-1 in thetransfectants comparing to the control. The proliferation of transfectants was analyzedby cell proliferation assay and cell cycle analysis. All transfectants showed alower proliferation rate comparing to the controls and a slight increase inG0/G1 phase. Because of the high structural similarities ofsyndecan family members, I studied how overexpression of syndecan-1 affected theother syndecans using Rt-Q-PCR. Syndecan-2 and -4 were downregulated in thetransfectants carrying syndecan-1 ectodomain, whereas the truncated versionshad the opposite effect. The expression of syndecan-bound heparan sulfate wasstudied by FACS and indicated an upregulation for heparan sulfate whenmeasuring internal- and membrane bound syndecans simultanesly. ConclusionsIn this study I haveshown that overexpression of full-length syndecan-1 and the different truncatedvariants, had similar profound effects on mesenchymal cell proliferation. Syndecan-1also influences the other members of the syndecan family suggesting a complexregulation.

The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.