One of the major limitations in drug development and gene therapy for brain diseases is the natural defensive structure called the blood brain barrier (BBB), which prevents therapeutic polypeptide drugs and viral vectors from entering the brain. Intranasal delivery of therapeutic gene products into the brain offers a non-invasive alternative towards a feasible gene and protein therapy for neurological diseases. From recent studies involving axonal transport, it is tempting to speculate that therapeutic macromolecules including neurotrophic factors and viral vectors can be delivered into the brain by peripheral neurons, such as olfactory receptor neurons (ORNs), which span the BBB. It is thought that the nasal pathway into the brain involves two general mechanisms; intracellular (intraneuronal) or extracellular routes of transport. However the pathways involved have not yet been fully characterized. In this study I firstly investigated the temporal and spatial localisation pattern of both biotinylated and I125 labelled ciliary neurotrophic factor (CNTF) following nasal delivery into Sprague-Dawley rats. Results showed that intranasal delivered CNTF was transported to several brain regions by both intracellular axonal pathway through ORNs and the extracellular trigeminal pathway. Excess unlabelled CNTF competed for receptor binding in the olfactory mucosa confirming receptor mediated intracellular transport to the olfactory bulb via ORNs. Denervation of the olfactory mucosa prior to CNTF delivery failed to prevent CNTF transport to trigeminal and hypothalamic brain regions. Intranasal delivered CNTF was biologically active, resulting in activation of the STAT3 signalling pathway in the thalamus and hypothalamus. To examine the functional activity of intranasal delivered CNTF, I conducted a weight loss trial using an obese Zucker rat (OZR) model to test whether CNTF treatment caused body weight loss. Intranasal administration of CNTF resulted in reduced body weight in the CNTF treated OZR group compared to the BSA control group during the 12 day trial and for 3 days after. Intranasal delivery of CNTF may be a valuable method for the treatment of obesity. In the second study, I investigated the temporal and spatial expression of Enhanced Green Fluorescent Protein (EGFP) transferred by a single nasal delivery of either a recombinant adenovirus vector (Ad5CMV-EGFP) or an adeno-associated virus vector (AAV2-EGFP) into Sprague-Dawley rats. Adenovirus mediated EGFP expression was localized in ORNs throughout the olfactory epithelium after 24 hours. EGFP in the ORNs appeared to be anterogradely transported along their axons to the olfactory bulb and transferred in glomeruli to second-order neurons. EGFP was transferred to several brain regions including the cortex, hippocampus, and brainstem after 7 days. EGFP expression co-localized with Olfactory Marker Protein and was confirmed with EGFP immunofluorescence labelling and western blotting. AAV expressed EGFP localized in similar olfactory and brain regions 6 weeks after delivery. mRNA levels suggested that the AAV-EGFP construct was only incorporated into olfactory mucosa cells and the viral vector was not present in olfactory bulb and brain regions. In conclusion, this simple and non-invasive polypeptide and gene delivery method provides ubiquitous macromolecule distribution throughout the rodent brain and may be useful for the treatment of neurological disorders.

<p>To investigate mechanisms involved in virus-host interactions, global changes in host gene expression were examined during infection with adenovirus type 2 (Ad2) using cDNA microarray technology. </p><p>In paper I and II, transcriptional changes in HeLa cells were investigated during the early and late phase of infection, respectively. A limited number of genes, mainly implicated in cell growth and antiviral defence, were found to be differentially expressed in the early phase, whereas modulation of host cell gene expression during the late phase was augmented and mainly focused on growth inhibition and cell architecture. </p><p>The experimental set-up was then redesigned to follow transcriptional regulatory events in growth synchronised, human primary lung fibroblasts. The immediate response of the host cell within two hours of infection was investigated in paper III, revealing a transient induction of a small number of cellular alert genes. This was followed by an expanded time course presented in paper IV, which included gene expression profiling at eight consecutive time points throughout the infectious cycle. The results indicated that specific sets of cellular genes were targeted at different stages of the infection, and four distinct periods were identified. </p><p>In summary, the studies presented in this thesis demonstrate that adenovirus interferes with many cellular processes during the progression of infection to optimize the cellular environment for viral replication. These include cell cycle control, cell growth and growth inhibition, as well as DNA, RNA and protein metabolism. However, a transient induction of cellular genes involved in immune response and growth inhibition was observed before the onset of viral gene expression. During the very late stages of infection, the expression of a large number of genes involved in maintaining the cell structure was down-regulated, presumably to facilitate the spread of progeny virus.</p>

Activated Heat Shock Transcription Factor 1 (HSF1) has received attention in recent literature as a therapeutic effector in diseases of protein misfolding, as an immune modulating adjuvant in tumor regression, and as a trigger for gene therapy transcription. In its normal function, activated HSF1 enhances heat shock protein (Hsp) expression when additional molecular chaperoning is required (i.e., in situations of proteotoxic stress, including thermal stress) in a process known as the heat shock (HS) response. Thus, HSF1 acts as an environmental sensor, and a harness based on the biology of this capability enables transcription of genes for engineered purposes. The hypothesis of this thesis is that a harness of the heat shock response, when paired with a therapeutic mechanism, will refine novel therapies. Extensions to the concept of deliberately activating HSF1's normal functions for therapeutic purposes are examined through in vitro trials and in vivo preliminary studies that feature the use of HSF1 as a regulator of therapy. Successful in vitro work translated to pioneering preclinical studies, launched at the University of Florida's Center for Environmental and Human Toxicology. Collaboration supported the development of an innovative project to treat solid tumors using a recombinant virus system. The system was designed to facilitate intratumoral delivery of a previously characterized molecular switch, which was newly engineered to control cytotoxic gene transcription that produced dramatic consequences in cells of human origin. Central to the targeting of the in vivo therapy, is a transient, initial trigger: a thermal dose, delivered to solid tumors, which localizes HSF1 activation (a constitutively active mouse HSF1 construct was also produced to aid clarification of physiological consequences associated with deliberately upregulating HSF1 activity in vivo). Gene transcription was expected to ensue to both cause and sustain tumor regression through other regulatory elements of the molecular switch. Results demonstrated practical potential to achieve a therapeutic outcome of solid tumor regression and define contemporary challenges that continuing research directions (e.g.: production of additional viral vectors, an improved animal model, and a refined heat system) now confront in order to target and safely regulate even more potent, novel therapeutic agents.

To investigate mechanisms involved in virus-host interactions, global changes in host gene expression were examined during infection with adenovirus type 2 (Ad2) using cDNA microarray technology. In paper I and II, transcriptional changes in HeLa cells were investigated during the early and late phase of infection, respectively. A limited number of genes, mainly implicated in cell growth and antiviral defence, were found to be differentially expressed in the early phase, whereas modulation of host cell gene expression during the late phase was augmented and mainly focused on growth inhibition and cell architecture. The experimental set-up was then redesigned to follow transcriptional regulatory events in growth synchronised, human primary lung fibroblasts. The immediate response of the host cell within two hours of infection was investigated in paper III, revealing a transient induction of a small number of cellular alert genes. This was followed by an expanded time course presented in paper IV, which included gene expression profiling at eight consecutive time points throughout the infectious cycle. The results indicated that specific sets of cellular genes were targeted at different stages of the infection, and four distinct periods were identified. In summary, the studies presented in this thesis demonstrate that adenovirus interferes with many cellular processes during the progression of infection to optimize the cellular environment for viral replication. These include cell cycle control, cell growth and growth inhibition, as well as DNA, RNA and protein metabolism. However, a transient induction of cellular genes involved in immune response and growth inhibition was observed before the onset of viral gene expression. During the very late stages of infection, the expression of a large number of genes involved in maintaining the cell structure was down-regulated, presumably to facilitate the spread of progeny virus.

Photocatalytic oxidation is an alternative water treatment method under consideration for disinfecting water. Chlorine disinfection can form harmful byproducts, and some viruses (e.g. adenoviruses) are resistant to other alternative disinfection methods. Photocatalytic oxidation using nano-sized photocatalytic particles (e.g. TiO2, fullerene) holds promise; however, it is limited by its low efficiency and long required treatment times. This research focuses on improving virus inactivation by photocatalytic oxidation by modifying catalysts for improved activity, by analyzing virus inactivation kinetics, and by elucidating the inactivation mechanisms of adenovirus serotype 2 (AdV2) and bacteriophage MS2. Modifying TiO2 with silver (nAg/TiO2) or silica (SiO2-TiO2) improves the inactivation kinetics of bacteriophage MS2 by a factor of 3-10. nAg/ TiO2 increases hydroxyl radical (HO•) production while SiO2 increases the adsorption of MS2 to TiO2. These results suggest that modifying the photocatalyst surface to increase contaminant adsorption is an important improvement strategy along with increasing HO• production. The inactivation kinetics of AdV2 by P25 TiO2 is much slower than the MS2 inactivation kinetics and displays a strong shoulder, which is not present in the MS2 kinetics. nAg/TiO2 initially improves the inactivation rate of AdV2. SiO2-TiO2 reduces the AdV2 inactivation kinetics since adsorption is not significantly enhanced, as it is with MS2. Amino-C60 is highly effective for AdV2 inactivation under visible light irradiation, making it a good material for use in solar disinfection systems. The efficacy of amino-fullerene also demonstrates that singlet oxygen is effective for AdV2 inactivation. When exposed to irradiated TiO2, AdV2 hexon proteins are heavily damaged resulting in the release of DNA. DNA damage is also present but may occur after capsids break. With MS2, the host interaction protein is rapidly damaged, but not the coat protein. The kinetics of MS2 inactivation are rapid since it may quickly lose its ability to attach to host cells, while AdV2 kinetics are slower since the entire capsid must undergo heavy oxidation before inactivation occurs. Adenovirus inactivation likely occurs through breaching the capsid followed by radical attack of DNA and core proteins.

Photocatalytic oxidation is an alternative water treatment method under consideration for disinfecting water. Chlorine disinfection can form harmful byproducts, and some viruses (e.g. adenoviruses) are resistant to other alternative disinfection methods. Photocatalytic oxidation using nano-sized photocatalytic particles (e.g. TiO2, fullerene) holds promise; however, it is limited by its low efficiency and long required treatment times. This research focuses on improving virus inactivation by photocatalytic oxidation by modifying catalysts for improved activity, by analyzing virus inactivation kinetics, and by elucidating the inactivation mechanisms of adenovirus serotype 2 (AdV2) and bacteriophage MS2. Modifying TiO2 with silver (nAg/TiO2) or silica (SiO2-TiO2) improves the inactivation kinetics of bacteriophage MS2 by a factor of 3-10. nAg/ TiO2 increases hydroxyl radical (HO•) production while SiO2 increases the adsorption of MS2 to TiO2. These results suggest that modifying the photocatalyst surface to increase contaminant adsorption is an important improvement strategy along with increasing HO• production. The inactivation kinetics of AdV2 by P25 TiO2 is much slower than the MS2 inactivation kinetics and displays a strong shoulder, which is not present in the MS2 kinetics. nAg/TiO2 initially improves the inactivation rate of AdV2. SiO2-TiO2 reduces the AdV2 inactivation kinetics since adsorption is not significantly enhanced, as it is with MS2. Amino-C60 is highly effective for AdV2 inactivation under visible light irradiation, making it a good material for use in solar disinfection systems. The efficacy of amino-fullerene also demonstrates that singlet oxygen is effective for AdV2 inactivation. When exposed to irradiated TiO2, AdV2 hexon proteins are heavily damaged resulting in the release of DNA. DNA damage is also present but may occur after capsids break. With MS2, the host interaction protein is rapidly damaged, but not the coat protein. The kinetics of MS2 inactivation are rapid since it may quickly lose its ability to attach to host cells, while AdV2 kinetics are slower since the entire capsid must undergo heavy oxidation before inactivation occurs. Adenovirus inactivation likely occurs through breaching the capsid followed by radical attack of DNA and core proteins.

The L1 region of the porcine adenovirus (PAdV)-3 genome encodes a protein of 622 amino acids named IIIa. Although it binds a neighboring group of nine (GON) hexons at the capsid level and cement the icosahedral shell that contains the viral DNA, little is known regarding its function with respect to viral life cycle. Moreover, the known location of IIIa protein in the capsid may help to express targeting ligands for altering the tropism of PAdV-3. The objective of this study was to characterize the IIIa protein of porcine adenovirus Type 3 (PAdV-3). <p> In order to characterize the IIIa protein, polyclonal antisera were raised in rabbits against different regions of IIIa. Anti-IIIa sera detected a specific protein of 70 kDa in PAdV-3 infected cells using Western blot assay. Immunofluorescence studies indicated that IIIa is predominantly localized in the nucleus of PAdV-3 infected cells. Analysis of PAdV-3 IIIa using antibodies specific for N- and C- terminal domains of the protein suggested that although the N-terminus and C-terminal domains of IIIa are immunogenic, they are not exposed on the surface of PAdV-3 virions. These results were further confirmed by our inability to isolate a chimeric PAdV-3 virion containing a heterologous protein fused to the N-terminus or C-terminus of IIIa. <p>Functional analysis suggested that IIIa may transactivate the major late promoter and down regulate the early region (E) 1A promoter. In order to locate the domains of IIIa responsible for different functions, in-frame deleted/truncated forms of IIIa were constructed. Analysis of the deleted/truncated forms of IIIa suggested that a) the sequences located between amino acids 273-410 and between amino acids 410-622b) affect the nuclear localization and transactivation function respectively.<p>Since protein- protein interactions are important for the biological functions of the protein, we determined the interaction of PAdV-3 IIIa with other viral proteins. IIIa was found to interact with DNA binding protein (DBP), E3 13.7 kDa protein, hexon, fiber, and pIX. These results suggest that PAdV3 IIIa may do more in the viral life cycle than merely act as cement between the hexons to maintain capsid stability and may actually be involved in regulating early to late gene transcription at appropriate stages during viral infection.

Receptor tyrosine kinases (RTKs) are integral membrane proteins that regulate many functions including cell proliferation, cell survival, and cell death. They have been shown to be responsible for the uncontrolled growth of several cancers. RTKs phosphorylate downstream targets such as phosphatidylinositol 3 kinase (PI3K), a lipid kinase that is made up of two major subunitsp85 and p110. Receptor-mediated endocytosis delivers RTKs from the plasma membrane to late endosomes and lysosomes for degradation. This process is controlled by ESCRT proteins and Rab7. PI3K associates with PDGFR during endocytosis, and PI3K binding sites are necessary for the lysosomal trafficking of PDGFR. The smaller isoforms of the ankyrin 3 (Ank3) proteins bind p85. Ank3 overexpression was shown to increase PDGFR degradation, perhaps by controlling the targeting of PDGFR to late endosomes and lysosomes. Ank3 overexpression also reduced the RTK levels and cell proliferation rates of NIH 3T3 cells. We sought to investigate if cancer cells with RTK overexpression might be deficient in Ank3, and if overexpression of ankyrin 105 (Ank105), one of the smaller isoforms of Ank3, would reduce RTK levels and the tumorigenic properties of cancer cells. Two brain cancer cell lines showed reduced Ank105 levels associated with high RTK levels, while high levels of Ank105 associated with low RTK levels were found in normal brain cells. This suggested a loss of Ank105 in the cancer cells, which may have played a role in the cancer development process. We observed reduced RTK levels and anchorage-independent growth in cancer cells overexpressing HA-Ank105, however, most cells overexpressing a blank vector also showed the same results. An independent effect of the overexpression process was thought to play a role in influencing cell behavior. In the lung cancer cell line HCC827, however, there was significant reduction of anchorage-independent growth that was specific for HA-Ank105. There also appeared to be a significant reduction in the cell proliferation rate of T98G brain cancer cells following transfection with HA-Ank105. Furthermore, those cells overexpressing HA-Ank105 tended to die early in tissue culture, with those that survived losing their HA-Ank105 expression. Overall our results suggest a possible role for Ank105 in downregulating RTK levels and growth properties of cancer cells.

Bovine adenovirus (BAdV)-3 is a non-enveloped, icosahedral virus with a double-stranded DNA genome, and is being developed as a vector for vaccination of animals and humans. Expression of viral genes is divided into early, intermediate, and late phases. The late genes of BAdV-3 are grouped into seven families (L1 to L7) based on usage of common polyadenylation site(s). The L1 region of BAdV-3 encodes the 52K protein, a non-structural protein conserved among members of the family Adenoviridae. In human adenovirus (HAdV)-5, the 52K protein is involved in packaging of the viral DNA into the capsid. The N-terminal half of the protein has been proposed to mediate serotype specificity of DNA packaging. The objective of this study was to characterize the 52K protein of BAdV-3. <p> DNA sequence analysis revealed that the BAdV-3 52K open reading frame encodes a protein of 370 amino acids rather than 331 amino acids as previously reported. Western blotting with anti-52K serum detected the expression of a 40kDa protein at 24 to 72 hrs post-infection. BAdV-3 52K localized predominantly to the nucleus in BAdV-3 infected cells and in transfected cells in the absence of other viral proteins. Analysis of mutant 52K proteins revealed that residues 102-110 were necessary but not sufficient for nuclear import. This suggests that residues upstream or downstream of the identified 52K nuclear localization signal (NLS) are required, or that the function of the NLS is dependent on its conformation within 52K. <p> The nuclear import of 52K is significantly, but not completely, dependent on soluble factors, ATP, and temperature. A peptide competing for binding to importin beta and a peptide encoding the NLS of Ycbp80 were also able to inhibit nuclear import of 52K. However, a dominant negative mutant of Ran was unable to block 52K nuclear import. These results suggest that 52K uses a classical importin alpha/importin beta pathway for nuclear import. In support of this, a specific interaction between 52K and importin alpha-3 was detected. In addition, 52K was able to accumulate in the nucleus in the absence of soluble factors and ATP when the nuclear membrane was permeabilized with detergent. This suggests that, in addition to nuclear import by the importin alpha/importin beta pathway, 52K is able to accumulate in the nucleus by binding to nuclear components. <p> A yeast two-hybrid system identified interactions between BAdV-3 52K and pV, pVI, pVII, and IVa2. However, only the interaction with pVII could be confirmed by GST pulldown. 52K and pVII also interact during BAdV-3 infection. An interaction between 52K and pVII has previously been shown in HAdV-5 infected cells. <p> Mass spectrometry analysis of proteins co-precipitating with BAdV-3 52K identified a cellular protein, NFkB-binding protein (NFBP), which interacted with 52K. The interaction between NFBP and 52K was confirmed <i>in vitro</i> and <i>in vivo</i>. NFBP has been shown to be essential for ribosomal RNA (rRNA) processing. While NFBP is normally localized in the nucleolus, co-expression with 52K results in the redistribution of NFBP from the nucleolus to other parts of the nucleus. While this suggested that redistribution of NFBP by 52K could inhibit rRNA processing during BAdV-3 infection, we were unable to detect a difference in rRNA processing in cells expressing truncated or full-length 52K in the absence of other viral proteins. Since NFBP is a multi-functional protein, future experiments should focus on other possible biological functions of the interaction of NFBP with BAdV-3 52K.

The success of adenoviral mediated airway gene therapy is hindered by host immune responses against adenoviral vectors. Helper-dependent adenoviral vectors (HD-Ad) are devoid of viral coding sequences and have an improved safety profile compared to earlier generation adenoviral vectors. However, intranasal delivery of HD-Ad vectors potentiates a pulmonary adaptive immune response, described in chapter 2, which is a barrier to gene therapy. One of the ways to reduce the immunogenicity of HD-Ad vectors is to increase the efficiency of HD-Ad mediated gene transfer to the airways, which would lessen the immunogen availability, limiting immune response against HD-Ad vectors. In chapter 3, a viral formulation strategy using Nacystelyn and DEAE-Dextran to substantially increase the efficacy of adenoviral mediated gene transfer to the airways is described. To further reduce the immune response to HD-Ad vectors, I have developed two novel strategies to induce vector-specific tolerance. The first strategy, described in chapter 4, involves the use of dendritic cells (DCs) differentiated in presence of IL-10, which are refractory to HD-Ad induced maturation and instead prime generation of regulatory T cells which suppress HD-Ad induced T cell proliferation. Delivery of these DCs pulsed with HD-Ad vectors to mice results in induction of immunological tolerance along with sustained gene expression following multiple rounds of HD-Ad readministrations. The second strategy, described in chapter 5, involves delivery of apoptotic DCs followed by delivery of antigen towards which tolerance needs to be generated. Apoptotic DCs are readily taken up by viable DCs, which suppresses DC maturation and induces TGF-β1 secretion, driving generation of regulatory T cells towards the delivered antigen. This strategy has shown remarkable success in achieving tolerance towards ovalbumin. Therefore, these strategies can be used to induce immunological tolerance towards gene therapy vectors which will likely allow for sustained and long term therapeutic transgene expression.