Mammalian Scribble is a target for proteins encoded by human papilloma virus, retro- and flaviviruses. Tick-borne encephalitis virus (TBEV) is a flavivirus that have evolved distinct strategies to escape antiviral responses. Information of how flaviviruses intrude on cell integrity comes from understanding of the roles that host-factors play when they interfere with viruses. The first part of this thesis describes a novel interaction between the TBEVNS5 protein and Scribble. The importance of the interaction was demonstrated by RNAi-mediated depletion of Scribble, which prevented suppression of JAK-STAT signaling by NS5. Together, these results define Scribble as a novel target for NS5. TBEV is known to cause central nervous system disease TBE in humans that can lead to cognitive dysfunction. A unifying theme in CNS related diseases are defects in neuronal extensions. We therefore addressed the effects of TBEV expression in PC12 cell differentiation, which is characterized by extensive neurite growth. Our data show that TBEVNS5 suppresses neurite outgrowth through the Rho GTPase Rac1. These findings provide evidence that Rac1 is an indirect target of NS5 in neurite inhibition. Scribble was recently implicated in spine morphogenesis. Thus, we tested the role of Scribble in neurite elongation. Depletion of Scribble in PC12 cells, reduced neurite density but increased length of those remaining. Moreover, Scribble bound components in the Ras/ERK cascade in a growth factor dependent manner. Together, these results demonstrate that Scribble controls neurite elongation by scaffolding MAPK components. Moreover, as loss of dendritic spines, actin-rich protrusions on neurons, is a feature in cognitive dysfunction we speculate that cognitive dysfunction in TBE might involve disturbed Scribble expression by NS5. We also investigated the binding between NS1 of Influenza A virus and Scribble. The PDZ domains of Scribble are usually selective for specific C-terminal motifs in proteins. Because NS1 has a canonical PDZ motif we tested if binding to Scribble depends on this motif. We found that Scribble binds NS1; the association is dependent on the NS1 C-terminus that is recognized by PDZ3-4 of Scribble. Together, these results suggest that Scribble is a target for the H5N1 NS1 protein / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 3: Manuscript. Paper 4: Manuscript.

Our work on RGMb demonstrates a clear and new role in the developing chick visual system. RGMb is expressed in distinct areas of the developing visual system: retinal ganglion cells (RGCs) of the retina, which are the only cells in the visual system that extend axons to the brain, as well as newly differentiated neuronal cells within the optic tectum (OT), the primary target of RGC axons. Knockdown of RGMb in RGCs at embryonic day 2 (E2) resulted in aberrant axon projection at E17, indicating that RGMb is required for axon development. Furthermore, knockdown of RGMb in the optic tectum at E5 resulted in disrupted cellular migration at E9, demonstrating that RGMb is involved in correct cell migration. Lastly, we demonstrated that RGMb binds to the Fibronectin III (3,4) domain of Neogenin, which provides a basis for determining the mechanism through which RGMb exerts its biological effects.

Our work on RGMb demonstrates a clear and new role in the developing chick visual system. RGMb is expressed in distinct areas of the developing visual system: retinal ganglion cells (RGCs) of the retina, which are the only cells in the visual system that extend axons to the brain, as well as newly differentiated neuronal cells within the optic tectum (OT), the primary target of RGC axons. Knockdown of RGMb in RGCs at embryonic day 2 (E2) resulted in aberrant axon projection at E17, indicating that RGMb is required for axon development. Furthermore, knockdown of RGMb in the optic tectum at E5 resulted in disrupted cellular migration at E9, demonstrating that RGMb is involved in correct cell migration. Lastly, we demonstrated that RGMb binds to the Fibronectin III (3,4) domain of Neogenin, which provides a basis for determining the mechanism through which RGMb exerts its biological effects.

Sprouting endothelial cells (ECs) use soluble and insoluble cues to guide migration and expand the existing vascular network to meet changing trophic needs of the tissue during angiogenesis. A noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique was used to optically image endothelial sprouting morphogenesis in three dimensional (3D) collagen matrices with simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Sprout advancement and lumen expansion companying with ECM alteration were the synergistic results of membrane-associated matrix metalloproteinase and cell traction evidenced by proteinase inhibition and Rho-associated kinase (p160ROCK) inhibition experiments. These physical EC-ECM interactions suggest that ECM mechanical properties may influence angiogenic responses. In a 3D angiogenesis model, we measure angiogenic responses as a function of collagen matrix stiffness by inducing collagen cross-linking with microbial transglutaminase (mTG). Collagen matrices stiffen with both mTG treatment and incubation time as evidenced with biaxial mechanical test results and collagen TPF intensity increases with mTG treatment and that the ratio of TPF/SHG correlates with biaxial tested mechanical stiffness. SHG and optical coherence microscopy (OCM) are further used to show that other physical properties of the matrix do not change with mTG treatment, thus providing the same density but different stiffness with which to measure angiogenic responses. Stiffer matrices promote angiogenesis with more invading sprouts that invade deeper. No differences in lumen size were observed between control and mTG stiffened 3D cultures, but there was evidence of greater matrix remodeling in stiffer gels using NLOM. Results of this study show angiogenic responses are enhanced with increasing stiffness and suggest that these properties may be used in tissue engineering and regenerative medicine applications to engineer angiogenesis.

Aggregating proteoglycans (PG) bearing chondroitin sulfate (CS) side chains are well-known inhibitors of neural plasticity and associate with hyaluronan and tenascin-R to form a complex of extracellular matrix (ECM) in the central nervous system (CNS). Little is known about whether proteolytic cleavage of the core protein affects neural plasticity. Several members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of metalloproteinases are glutamyl-endopeptidases that cleave aggregating PGs. Our initial studies determined that neural cultures secrete a brevican-containing matrix, and that these neural cultures also produced ADAMTS4, a protease that cleaves brevican. Furthermore, this brevican-containing matrix in astrocytes could be modulated by treatment with transforming growth factor beta (TGFbeta) through the inhibition of the activity of the ADAMTSs.Once it was established that neural cultures produce a brevican-rich matrix, we s ought to utilize this matrix to determine whether cleavage of aggregating PGs, especially brevican, by the ADAMTSs influences neurite outgrowth in cultured neurons. Transfection of rat neurons with ADAMTS4 cDNA induced longer neurites, and interestingly, this effect proved to be independent of the proteolytic action of the ADAMTSs. Addition of recombinant ADAMTS4 or ADAMTS5 protein to immature neuronal cultures similarly enhanced neurite extension, an action dependent on the activation of extracellular signal-related kinase (ERK)1/2 (MAP kinase 42/44), resulting in the first evidence that ADAMTSs may induce intracellular signaling events. Studies of dendritic spine morphology and levels of synaptic proteins in response to ADAMTS4 treatment were also undertaken. Neuronal cultures treated with ADAMTS4 showed increased length of dendritic spines and increased percent of immature spines detected. A concurrent decrease in post-synaptic protein staining was detected on the neurites of yo ung neurons overexpressing ADAMTS4 or expressing proteolytically-inactive mutant ADAMTS4 protein. Thus, ADAMTS4 may promote plasticity in neurons in vitro by preventing the formation, maturation, and/or stabilization of synapses. Overall, these experiments provide evidence that implicate the ADAMTSs as mediators of neural plasticity, and while primarily known only as proteases, these studies demonstrate that the ADAMTSs exert actions distinct from these proteolytic properties that require the induction of intracellular signaling events.

The growth of a neuronal dendritic tree depends on the neuron’s internal state and the environment within which it is situated. Different types of neuron develop dendritic trees with specific characteristics, such as the average number of terminal branches and the average length of terminal and intermediate segments. A key aspect of the growth process is the construction of the microtubule cytoskeleton within the dendritic tree. Neurite elongation requires assembly of microtubules from free tubulin at the growth cone. The stability of microtubule bundles is an important factor in determining how likely it is for a growth cone to split to form new daughter branches. Microtubule assembly rates and bundle stability are controlled by microtubule-associated proteins, principally MAP2 in dendrites. Extending previous work (Hely et al, J. Theor. Biol. 210:375-384, 2001) I have developed a mathematical model of neurite outgrowth in which elongation and branching rates are determined by the phosphorylation state of MAP2 at the tips of each terminal branch. Tubulin and MAP2 are produced in the cell body and transported along the neurite by a combination of diffusion and active transport. Microtubule (dis)assembly at neurite tips is a function of tubulin concentration. The rate of assembly depends on the amount of unphosphorylated MAP2 bound to the microtubules and linking them together. Phosphorylation of MAP2 destroys its linking capability and destabilises the microtubule bundles. Each terminal has a probability of branching that depends on the phosphorylation of MAP2 which, in turn, is a function of calcium concentration. Results from this model show that changes in the (de)phosphorylation rates of MAP2 affect the topology of the final dendritic tree. Higher phosphorylation promotes branching and results in trees with many short terminal branches and relatively long intermediate segments. Reducing phosphorylation promotes elongation and inhibits branching.

Current therapy for chronic kidney disease (CKD) is limited; however, recent studies have shown that a subpopulation of cells derived from the bone marrow, known as early outgrowth cells (EOCs), are able to attenuate kidney injury. Here we examined the efficacy of a modular tissue engineering system whereby the EOCs might be easily removed in the event of malignant change. While modular therapy mimicked the effects seen with standard EOC therapy, the modules degraded allowing the encapsulated EOCs to enter systemic circulation. Given the presumed egress of EOCs, we explored an alternative strategy for kidney protection. Here we investigated the long-term effectiveness of administering the conditioned medium (EOC-CM) that contains the factors the EOCs secrete, rather than the cells themselves. In these studies, repeated administration of EOC-CM attenuated the structural and functional manifestations of kidney injury suggesting that this approach may provide an effective and feasible, cell-free approach for CKD.

Current therapy for chronic kidney disease (CKD) is limited; however, recent studies have shown that a subpopulation of cells derived from the bone marrow, known as early outgrowth cells (EOCs), are able to attenuate kidney injury. Here we examined the efficacy of a modular tissue engineering system whereby the EOCs might be easily removed in the event of malignant change. While modular therapy mimicked the effects seen with standard EOC therapy, the modules degraded allowing the encapsulated EOCs to enter systemic circulation. Given the presumed egress of EOCs, we explored an alternative strategy for kidney protection. Here we investigated the long-term effectiveness of administering the conditioned medium (EOC-CM) that contains the factors the EOCs secrete, rather than the cells themselves. In these studies, repeated administration of EOC-CM attenuated the structural and functional manifestations of kidney injury suggesting that this approach may provide an effective and feasible, cell-free approach for CKD.

Recent advances in cellular reprogramming have enabled the generation of embryoniclike cells from virtually any cell of the body. These inducible pluripotent stem cells (iPSCs) are capable of indefinite self-renewal while maintaining the ability to differentiate into all cell types. Nowhere will this technology have a greater impact than in the ability to generate disease and patient-specific cell lines. Here we explore the capacity of human iPSCs reprogrammed from peripheral blood endothelial progenitor cells lines to differentiate into atrial myocytes for the study of patient specific atrial physiology. Methods and Results: Late outgrowth endothelial progenitor cells (EPCs) cultured from clinical blood samples provided a robust cell source for genetic reprogramming. Transcriptome analysis hinted that EPCs would be comparatively more amenable to pluripotent reprogramming than the traditional dermal fibroblast. After 6 passages, EPCs were transduced with a doxycycline inducible lentivirus system encoding human transcription factors OCT4, SOX2, KLF4 and Nanog to permit differentiation after removal of doxycycline. The high endogenous expression of key pluripotency transcripts enhanced the ease of iPSC generation as demonstrated by the rapid emergence of typical iPSC colonies. Following removal of doxycycline, genetically reprogrammed EPC-iPSC colonies displayed phenotypic characteristics identical to human embryonic stem cells and expressed high levels of the pluripotent markers SSEA-4, TRA1-60 and TRA1-81. After exposure to conditions known to favor atrial identity, EPC- iPSC differentiating into sheets of beating cardiomyocytes that expressed high levels of several atrial-specific expressed genes (CACNA1H, KCNA5, and MYL4). Conclusions: EPCs provide a stable platform for genetic reprogramming into a pluripotent state using a doxycycline conditional expression system that avoids reexpression of oncogenic/pluripotent factors. Human EPC-derived iPSC can be differentiated into functional cardiomyocytes that express characteristic markers of atrial identity.

Neovibsanin A and B are natural products which induced neurite outgrowth in PC12 cells. They belong in the neovibsane class under the rare vibsane natural product family, whose structures are characterized by polycyclic, polyoxygenated cores. Based on a proposed biosynthesis, the synthetic strategy towards neovibsanin A and B involved synthesizing a key enone intermediate. Initial investigation using this intermediate lead to the total synthesis of 2-O-methylneovibsanin H. Crucial to this concise synthesis was an acid-catalyzed, one-pot, four-step cascade reaction. Modifying the reaction condition leads to a different five-step cascade pathway, resulting in the total synthesis of 4,5-bis-epi-neovibsanin A and B. The synthetic trials and tribulations encountered on the road to these final compounds are explored. It is envisaged that other related neovibsane natural products may arise based on this synthetic sequence. 4,5-Bis-epi-neovibsanin A and B, as well as several other structural analogues collected during the synthesis, were biologically assayed using NGF-stimulated PC12 cells. All compounds induced a significant proportion of neurons to extend neurite processes compared to control cultures. The structure-activity relationship studies indicated that the tricyclic core, as well as the 3,3-dimethylacroyl enol ester side chain, may be responsible for promoting a biological response.