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.

El Ad es el vector más utilizado en ensayos clínicos con humanos. Para evitar la respuesta inmune celular inducida por los Ad de 1ª y 2ª generación, se han generado los vectores de 3ª generación, también llamados gutless o helper dependientes. Para producir estos vectores se necesitan tres elementos fundamentales: un Ad gutless con un gen terapéutico o marcador de interés; un Ad helper que aporte las proteínas virales necesarias in trans y; una línea celular permisiva para la producción de Ad. Los Ad gutless, al no contener ninguna región viral codificante, no generan respuesta inmune celular y tienen una capacidad de hasta 36 Kpb. Se ha demostrado que la expresión de los genes que incorporan puede durar toda la vida del organismo. Sin embargo, si bien presentan grandes ventajas, su uso en ensayos clínicos con humanos todavía no ha sido viable debido a dos grandes inconvenientes: la contaminación por Ad helper y su producción a gran escala. Para solventar el problema de la contaminación por Ad helper, en este trabajo se propone un nuevo sistema de generación de Ad gutless basado en la recombinasa ?C31-attB/attP. Los Ad helper generados llevan flanqueada su señal ? por las secuencias attB/attP. ?C31 es una recombinasa unidireccional con lo que una vez realizada su función, al escindir la señal de empaquetamiento, evita la reacción inversa. Esta característica supone una ventaja frente a otras recombinasas como Cre ó FLPe. Sorprendentemente, al incorporar la secuencia attB entre el extremo ITR del Ad y su señal de empaquetamiento, los Ad helper generados alargan su ciclo viral hasta las 56-60 horas, sin embargo, ello no afecta la replicación eficiente del genoma viral y la producción de proteínas virales. Asimismo, se ha demostrado que tanto el proceso de empaquetamiento como el de la maduración de la partícula viral están afectados. Se ha observado que la clonación de una segunda señal de empaquetamiento en el extremo 3' normaliza los niveles de producción de los Ad controles, confirmando así que el genoma no queda retenido en ninguna región nuclear. Ensayos de EMSA han mostrado que diferentes proteínas celulares se unen a la secuencia attB y probablemente la unión de una de ellas impida el correcto empaquetamiento del genoma adenoviral. Por todo ello, el empaquetamiento diferencial por tiempo de los Ad helper-attB/attP generados ha sido aprovechado para la producción de Ad gutless acotando su producción a las 36 horas (tiempo en el que un Ad control completa su ciclo viral). Sin embargo, en las producciones de Ad gutless, los niveles de contaminación por Ad helper fueron elevados y éstos aumentaban significativamente en los sucesivos pasos de amplificación. El análisis del extremo 5' del Ad helper confirmó que éste recombinaba con el Ad gutless por la señal de empaquetamiento perdiendo las secuencias de recombinación y así su capacidad de empaquetarse más lentamente. Sin embargo, la inversión de la señal de empaquetamiento supuso la demostración de que este efecto es fácilmente evitable lo que convierte al Ad helper Ad5/FC31.Cre.?R en una buena herramienta para la producción de Ad gutless. / Adenovirus is the most used vector in human clinical trials. In order to overcome cellular inmune response evoked by first and second generation adenovirus, third generation, also called gutless or helper-dependent adenovirus have been generated. Gutless adenovirus production needs three basic elements: a gutless adenovirus with a therapeutic or marker gene; a helper adenovirus which provide all viral proteins in trans and; a permisive cell line to produce adenovirus. Gutless adenovirus, without any codificant viral region, don't evoke cellular inmune response and can incorporate DNA inserts up to 36 Kb. It has been reported that the expresion of incorporated genes can last the whole life of the organism. Nevertheless, its use in human clinial trials is not suitable due two important inconvenients: helper adenovirus contamination and up-scale processes. To solve helper adenovirus contamination problem, this present work propose a new adenovirus gutless generation system based on ?C31-attB/attP recombinase. Helper adenovirus generated have flanked its packaging signal (?) by attB/attP sequences. ?C31 is an unidirectional recombinase which avoid reverse reaction. This characteristic is an important advantage in front of other recombinases such as Cre or FLPe. Surprisingly, attB sequence incorporated between Ad-ITR and ? lengthens adenovirus cycle up to 56-60 hours, However, this effect don't affect efficient genome replication or protein shyntesis. Moreover, it has been shown that packaging and maturation processes are affected. It has been observed that the cloning of a second ? in the 3'-ITR normalize production levels in comparison to control adenvovirus, proving adenovirus genome is not trapped in any nuclear region. EMSA assays have shown different cellular proteins interact with attB sequence and likely the interaction of one of this cellular proteins impairs the correct packaging of adenovirus genome. For this reason, differential packaging in time of attB/attP-helper adenovirus generated have been used to produce gutless adenovirus limiting production times at 36 hours (time when control adenovirus finish its viral cycle) . However, in gutless adenovirus productions, helper adenovirus contamination levels were high and they increase significantly in the successive amplification steps. The 5' extreme analysis showed helper and gutless adenovirus recombine by their ? loosing recombination sequences and, in this way, helper adenovirus slow packaging capacity. Nevertheless, the inversion of ? showed this effect can be easily avoided which make Ad5/FC31.Cre.?R helper adenovirus a good tool for gutless adenovirus production.

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.

<p>The adenoviruses are nonenveloped double stranded DNA viruses, which cause enteric dysentary and respiratory infection. Adenovirus has become a focus of the water treatment community because of its apparent resistance to ultraviolet disinfection; it is the basis for stringent new EPA regulations regarding all viruses in both surface and ground waters. Most of the work done so far, however, has involved the use of monochromatic (254 nm) low pressure (LP) UV sources and subsequent assay of viral infectivity in cell culture models. LP UV lamps primarily damage DNA, while polychromatic UV sources may damage other parts of the virus as well. Recent research has shown that these newer, polychromatic UV sources--such as medium pressure (MP) UV--are more effective than monochromatic LP UV for disinfection of adenovirus. The objectives of this work were to study adenoviral response to UV using both LP and MP UV as well as using both standard cell culture infectivity assays and more direct methods of assessment based on molecular biology. These include quantitative long PCR for assessment of DNA damage and SDS-PAGE for assessment of protein damage; transmission electron microscopy was used to examine the structure of UV treated viral particles. This work was only the second significant study to show the response of adenoviruses to medium pressure UV and the first to thoroughly examine the response of adenoviruses to both LP and MP UV using cell culture-independent methods. Results confirm that adenovirus is sensitive to MP UV when assayed in cell culture; they show that LP and MP UV are equally effective at inducing damage to the adenoviral genome and that MP UV is more effective than LP UV at damaging the viral proteins. This work helps deepen our understanding of UV disinfection of adenovirus.</p> / Dissertation

The Wnt signaling pathway is highly conserved and participates in many important cellular functions including differentiation, embryonic development and tissue generations. £]-catenin, the central mediator of the Wnt signaling, interacts with the TCF/LEF family of transcription factors in the nucleus and initiates downstream gene transcription. In addition, £]-catenin is known as a proto-oncogene implicated in numerous cancers including colorectal, cervical, endometrial and skin cancer. Chibby (Cby) is evolutionarily conserved in many species and acts as a repressor of Wnt/£]-catenin signaling. In our previous study, we have established that Cby over-expression attenuated £]-catenin translocation to nucleus and its transcriptional activity. Thus, it was hypothesized that Cby may possess potential tumor suppressing capabilities. In the present study, we first explored endogenous Cby expression status in human cervical cancer cells: HeLa and SiHa cell lines. It was observed that Cby mRNA and protein levels were significantly down-regulated in both cancer lines compared with primary cervical cells. We then conducted functional assays of tumorigenicity on both cells using adenoviral-encoded Cby and its NLS (nuclear localization signaling) deleted variant (Cby∆NLS). It was found that gene delivery of Cby or Cby∆NLS inhibited the proliferation, invasiveness, and colony forming in HeLa and SiHa cells. Immunofluorescent analysis revealed that Cby or Cby∆NLS gene transfer reduced the expression of Ki-67, a cell proliferative marker. Furthermore, Cby or Cby∆NLS restoration induced apoptosis and perturbed cell cycle progression in both cervical cancer cells. Finally, Cby over-expression decreases the expression of £]-catenin/TCF4 regulated genes such as c-myc and PCNA, which might contributed to the anti-neoplastic mechanism for Cby in cervical cancer cell lines. Our results strongly suggest that Cby may serve as a tumor suppressor gene during cervical carcinogenesis, and may facilitate in creation of new therapeutic methods.

The use of gene-based techniques to produce antinociceptive molecules has been actively investigated for treatment of neuropathic pain and trauma of central nervous system. Among the endogenous opioids, b-endorphin (b-EP) is the most potent one, which is derived from pro-opiomelanocortin (POMC). In addition to b-endorphin, POMC is also the precursor of many neuropeptides such as adrenocorticotropin hormone (ACTH), melanocyte-stimulating hormone (a-MSH), ¡Ketc. Appropriate administration of POMC gene is essential for the success of its clinical application. Thus, gene transfer approach seems to be suitable for continuous supply of b-endorphin to alleviate intractable pain. Recombinant adenovirus was used as gene delivery system for POMC because of its high titer, wide host range, and transduction efficiency. In the present study, we have generated and characterized the recombinant adenovirus encoding POMC (Ad-POMC) by PCR and western blot analysis, and detect the presence of opioid peptides including ACTH, a-MSH and b-EP by RIA and chemilluminiscent assay. GH3 cells infected with Ad-POMC showed significantly higher levels of ACTH, b-endorphin, and a¡VMSH comparing with cells of control groups. By using Ad-GFP, the optimal MOI for adenovirus vector to infect neuronal GH3 cells, glial C6 cells, hepatoma Hep3B cells, smooth muscle G8 cells, fibroblast CCD-965K cells, and endothelial EA.hy926 cells was determined at 50, 500, 50, 500, 500, and 200, respectively. The results of determining the efficiency of POMC processing in different types of cells after in vitro cell cultures gene delivery indicated that peripheral cells, though at a lower extent, are capable of cleaving POMC and releasing opioid peptides after POMC gene delivery like neuronal cells of central nervous system. In formalin test, the intrathecal POMC gene delivery significantly decreased the magnitude of the formalin-evoked flinching response phase 1 (P < 0.05) and phase 2 (P < 0.001) when compared with rats receiving saline or Ad-GFP. In conclusion, the intrathecal POMC gene delivery can produce effectively attenuation on the inflammatory pain response. So far, there have been various gene delivery studies confirming the potential role of POMC in antinociception. In the future, more experiments will be needed to characterize the effects of POMC expression on cellular lipid metabolism. This will enable us to evaluate the therapeutic potential of POMC on treatment of obesity.

Obesity is a disorder of energy imbalance and the most prevalent nutritional diseases in developed countries. Besides, obesity is also strongly associated with health problems such as type 2 diabetes (NIDDM), hypertension, hyperlipidaemia, cardiovascular diseases and cancers. However, the defects in lipid metabolism underlying obesity-related disorders are extremely complicated. Thus, extensive studies on the mechanism of endogenous fatty acids synthesis would be one of the keys to elucidate molecular pathogenesis of obesity. In liver or adipose, fatty acid synthase (FAS) utilizes acetyl-CoA, malonyl-CoA and NADPH to synthesize long-chain fatty acids (C16 or C18), which can be converted to triglycerides and stored as fat. During lactation, thioesterase II (TE II) expresses in mammary glands and interacts with FAS to produce medium-chain fatty acid (primarily C10) in milk, which provides immune protection and energy for the newborn. TE II causes premature termination of fatty acid synthesis catalyzed by FAS and releases medium-chain fatty acids. Unlike long-chain fatty acids, medium-chain fatty acids can enter mitochondria directly for beta-oxidation to generate ATP, thus provide energy more efficiently. Since TE II gene expression is under strict regulation, we utilized adenovirus gene transfer techniques to deliver and express TE II in hepatocytes. It was postulated that expression of TE II in hepatocytes might result in the increase of ATP and reduction of long-chain fatty acids, subsequently decrease the fat production. Recombinant adenovirus was used as gene delivery system for TE II because of its high titer, wide host range, and transduction efficiency. In the present study, we have generated and characterized the recombinant Ad-TE II by PCR, western blot analysis, and enzymatic assay, respectively. By using Ad-GFP, we have determined the optimal multiplicity of infection (MOI) for adenovirus to infect HepG2 cells is about 100-200. Adenovirus-mediated TE II expression in hepatocytes was demonstrated by western blot as well as TE II enzymatic assay. We have demonstrated that the adenovirus-mediated TE II expression was slightly cytotoxic to hepatocytes. Besides, an increase of free fatty acids, asparate transaminase, lactate dehydrogenase levels, as well as ATP synthesis was also noted in the TE II-expressed hepatocytes. The enhanced the release of asparate transaminase (AST/GOT) and lactate dehydrogenase (LDH) after TE II expression in the hepatocytes further supported its cytotoxcity to hepatocytes. In the future, we will carry out experiments to further characterize the effects of TE II expression on cellular lipid metabolism through adenovirus gene delivery. We hope that the present studies will not only provide further insights into mammalian lipid metabolism, but also enable us to evaluate the therapeutic potential of TE II on the treatment of obesity and its related disorders.

Adenoviruses, particularly types 4 and 7, are associated with febrile respiratory illness (FRI) outbreaks in US military basic trainees. Vaccines against these two serotypes controlled FRI in basic trainees until production ceased in the mid-1990s. After contracting a new manufacturer, adenovirus vaccination of military basic trainees resumed in 2011. The purpose of this dissertation was to assess the cost-effectiveness of using the new adenovirus type 4 and type 7 vaccines for the prevention of FRI in US military basic trainees from the perspective of each military branch. Two decision tree models comparing adenovirus vaccination to no adenovirus vaccination were used for this dissertation. The first model is similar to previous models used to assess the cost-effectiveness of the adenovirus vaccine in the military, where the outcome is number of FRI hospitalizations prevented. The second model created for this dissertation used information gathered from published literature and conversations with experts on the adenovirus vaccine. The outcome for the second model was number of training days lost (TDL) averted. Results from part I indicated that adenovirus vaccination of basic trainees was cost-effective as measured by FRI hospitalizations prevented in all US military service branches but the Coast Guard. The model showed that reintroducing the adenovirus vaccine to basic trainees saved the Army $5.8 million, the Navy, $1 million, the Marine Corps, $238,000, and the Air Force, $5.2 million, annually. In addition, adenovirus vaccination prevented 1,221, 543, 317, 677 cases of FRI hospitalization annually in the Army, Navy, Marine Corps, and Air Force respectively. In part II of this study, adenovirus vaccination of basic trainees was the dominant strategy as measured by TDL averted in all US military service branches but the Marine Corps and the Coast Guard. Results indicate that it would cost approximately $37.63 and $563.78 per TDL averted for the Marine Corps and Coast Guard respectively. Both models used for this dissertation provide evidence supporting the cost-effectiveness of using the adenovirus vaccine in US basic trainees in all services but the Coast Guard. / text