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Bovine herpesvirus-1 (BoHV-1) is associated with the syndrome bovine respiratory disease, which is the major cause of morbidity and mortality within feedlots in Australia and around the world. Currently there are no vaccines that completely prevent BoHV-1 infections and viral shedding. The most efficacious vaccines used are live attenuated which have the potential to revert to wild type and cause disease. DNA vaccines are ideal vaccine candidates as they not only induce humoral and cellular immunity, they are also inexpensive and easy to produce. However, DNA vaccines although efficacious in small animal models have not yielded similar success in large animals. The inconsistent translation of DNA vaccines to large animal models, including cattle, has been associated with poor delivery of the vaccine to the nuclei of cells which is required for antigen transcription. Recently, the human herpesvirus-1 protein VP22 (hVP22) was demonstrated to exhibit the uncommon capacity to spread intercellularly from the cell of expression to the nuclei of neighbouring cells in a golgi and energy independent process. This process was very efficient with hVP22 being identified in all cells of a monolayer after transfection. hVP22 was quickly used to promote the efficiency of DNA vaccines by fusing the hVP22 gene with antigen genes in the vaccine resulting in the increased delivery of the antigenic protein to neighbouring cells. The fusion protein was subsequently degraded and presented as peptides on the cell surface in association with major histocompatibility complex (MHC) class II molecules that lead to an increase in fusion protein specific antibody production. This pathway, although successful augmenting the humoral response, did not increase the amount of antigen presented on MHC class I molecules which is essential for protection against intracellular pathogens. This thesis describes the development of a methodology whereby VP22, fused to a DNA binding protein, was hypothesised to increase the number of cells the DNA vaccine was delivered to and then to facilitate the transport of the DNA vaccine to their nuclei. A homologue of hVP22 has been identified in BoHV-1 and the capacity of the BoHV-1 protein to spread intercellularly and localise in the nuclei of cells was determined in this thesis using a novel and definitive model. Although retaining similar translocation capabilities to hVP22 the BoHV-1 VP22 homologue could not be expressed in bacteria and was subsequently not able to be used to demonstrate the proposed vaccine concept. hVP22 instead was fused to the DNA binding protein, Gal4, for bacterial expression. The purified fusion protein was demonstrated to bind not only oligonucleotides encoding the Gal4 binding sequence but also to a model DNA vaccine encoding Gal4 binding sequences in vitro. However, application of the hVP22 fusion protein:vaccine complex alone or condensed with poly-L-lysine to mammalian cells did not promote the delivery of the DNA vaccine to the nuclei of cells. As part of the DNA vaccine development for BoHV-1 the first nucleotide sequence of the Unique Short region of the Australian BoHV-1 strain V155 (8925 nucleotides) was determined. The sequence information generated permitted insights into epitopes contained within BoHV-1 antigens, particularly glycoprotein D which has been identified as the most appropriate glycoprotein for the purpose of vaccination. Furthermore, comparison of the Unique Short sequence variations between different subtypes of BoHV-1 provided molecular data that may be associated with the observed variation in virulence. Further optimisation of the methodology described in this study is required to facilitate the delivery of the DNA vaccine into cells by the VP22 fusion protein. The future development of strategies that utilise polypeptides to augment delivery of DNA vaccines into cells and then to facilitate the transport of the vaccine to the nuclei of cells, resulting in increased antigen expression, may ultimately lead to the successful application of this vaccine technology in animal models.

Protein transduction domains (PTDs), such as TAT from human immunodeficiency virus (HIV) or VP22 from herpes-simplex-virus-1, have been shown to deliver a myriad of molecules, including synthetic small molecules, peptides and proteins in vivo and in vitro. The protein transduction processes mediated by TAT or VP22 are highly efficient and occur in many types of cells with low toxicity. The anti-tumor proteins to be investigated are abrin A chain (ABR-A) and Apoptin. ABR-A is the toxophoric subunit of plant toxin abrin from the seeds of Abrus precatoriusa. ABR-A is a potent inhibitor of translation, but not toxic to cells due to its lack of the cell-binding B chain. Apoptin is a protein derived from chicken anemia virus and has been proved to be selectively cytotoxic to various tumor cells but not to normal cells. The tumor-specific activity of Apoptin is correlated with its nuclear localization in tumor. In this study, we employed VP22 PTDs to promote the entry of natural toxins, such as ABR-A or Apoptin, into tumor cells, thereby to enhance their anti-tumor effects. We generated and characterized green fluorescent protein (GFP)-, hemagglutini (HA)-, and VP22-fused expression constructs for ABR-A and Apoptin, to evaluate the gene delivery effect of ABR-A/Apoptin genes in non-transformed NIH3T3 cells and tumor cells, including Hela and A375 melanoma cells. Gene delivery of ABR-A led to growth inhibition by 50~70% in transformed and non-transformed cells. In contrast, Apoptin gene delivery exhibited cytotoxicity only in tumor cells. The cytotoxicity of ABR-A and Apoptin gene delivery was enhanced when fused with VP-22. Furthermore, the depletion of APAP1 reduced the cytotoxic effect of Apoptin gene delivery. In the future, the anti-tumor effect of these novel PTD-toxin vectors will be explored in cell culture as well as animal model. We hope these studies will open a new avenue for cancer therapy.

O Diabetes Mellitus tipo I (DM1) é causado pela destruição auto-imune das células β pancreáticas, encontradas na porção endócrina do pâncreas, constituída pelas ilhotas pancreáticas. As células β são responsáveis pela produção e liberação de insulina, um hormônio que promove a internalização da glicose pelas células. Junto com outros hormônios, a insulina é um dos principais reguladores do nível de glicose sanguinea (glicemia). Uma das terapias utilizadas para o tratamento do DM1 é o transplante de ilhotas pancreáticas. Entretanto, um dos maiores problemas em relação a esta terapia é a falta de massa celular adequada para ser infundida no paciente. Uma tentativa para solucionar este problema, é o desenvolvimento de fontes alternativas de células produtoras de insulina, como as células-tronco, que possuem a capacidade de se diferenciarem em diversos tipos de células, inclusive nas produtoras de insulina. Pax4 é um dos fatores de transcrição responsáveis pela diferenciação de células β , sendo essencial para o apropriado desenvolvimento e maturação destas, constitui um bom candidato para induzir a diferenciação de células-tronco em células produtoras de insulina in vitro. Para introduzir o Pax4 nas células-tronco, sem provocar alterações no genoma das células diferenciadas, em virtude dos potenciais efeitos indesejáveis de vetores que se integram ao genoma celular, recorreu-se às proteínas contendo domínio de transdução (PTDs), que são capazes de carregar a proteína Pax4, através da membrana, diretamente para o interior das células. As PTDs são pequenas sequências peptídicas que permitem a translocação de proteínas através de membranas celulares e sua internalização em células-alvo. Uma das PTDs mais comumente estudadas é a VP22, produto do gene UL49 do Herpes Simplex vírus tipo I. Portanto, a proteína de fusão VP22.Pax4 permitiria que o Pax4 fosse inserido em células-tronco, possibilitando que este fator de transcrição ative a transcrição de certos genes que aumentariam a eficiência de diferenciação das células-tronco em células produtoras de insulina. Para tal, amplificamos e clonamos o cDNA do Pax4 a partir do RNA das células RINm5f de insulinoma murino, construímos o vetor pVP22.Pax4, o qual foi transfectado em células CHO, que passaram a produzir a proteína de fusão VP22.Pax4. Após o tratamento de células-tronco com a proteína de fusão VP22.eGFP e análise por microscopia confocal, comprovamos que a VP22 é capaz de tranduzir a proteína de fusão também neste tipo celular. Portanto, incorporamos a um dos passos do protocolo de diferenciação de células-tronco em células produtoras de insulina, utilizado em nosso laboratório, a co-cultura com células CHO produtoras de VP22.Pax4. Observamos que a introdução do Pax4 leva a formação de um número maior de agregados celulares (clusters) produtores de insulina. Concluímos, então, que a utilização da VP22 como ferramenta para internalização de proteínas em células-tronco é viável e que a adição do Pax4 pode trazer melhorias para protocolos que busquem a produção de células produtoras de insulina. / Diabetes Mellitus type 1 (DM1) is caused by an auto-imunne destruction of the pancreatic β cells, found in the endocrine portion of the pancreas, known as pancreatic islets. These β cells are responsible production and release of insulin, a hormone which promotes glucose internalization by cells. Along with other hormones, insulin is a major regulator of blood glucose levels (glycemia). One of the therapeutical strategies used to treat DM1 is pancreatic islet transplantation. One of the major problem related to this therapy is the lack of adequate cell mass to be infused into the pacients. An attempt to solve this problem is the development of an alternative source of insulin-producing cells by differentiation of stem cells, which display this differentiating potential. Pax4 is one of the transcription factors responsibles for β cell differentiation, being essential for its proper development and maturation, therefore being a good candidate to induce stem cell differentiation into insulin producing cells in vitro. A promising alternative to avoid the alterations of the differentiated cells genome due to its undesirable effects of integrating vectors, but yet allowing the Pax4 to act in diferentiation within the cells are the proteins with a transduction domain (PTDs), which would have the ability to lead the Pax4 protein directly into the cells. The Pax4 could thus act in the nucleus and generate specific transcriptional responses. The PTDs are small peptide sequences which allow translocation of proteins across cell membranes and their internalization into target cells. One of the most commonly studied PTDs is the VP22, a product of the UL49 gene from Herpes Simplex vírus type I. Therefore, the VP22.Pax4 fusion protein would transduce Pax4 into the stem cells, thus allowing the transcription activation of certain genes by Pax4, leading to improvement in the process of stem cells differentiation into insulin-producing cells. To this end, we cloned the Pax4 cDNA from RINm5f murine insulinoma cells, constructed the pVP22.Pax4 vector and transfected this construct into CHO cells, which then produced the VP22.Pax4 fusion protein. Upon verifying that VP22 was also able to transduce proteins into stem cells, by confocal microscopy analysis, after the treatment of these cells with the fusion protein VP22.eGFP, we incorporated the fusion protein VP22.Pax4 to one of the steps of the protocol used for stem cells diferentiation into insulin producing cells in our lab, by co-culturing with CHO cells producing VP22.Pax4. We observed that the addition of Pax4 led to the formation of a higher number of insulin producing cell clusters, therefore we conclude that VP22 may be used as a tool to internalize proteins into stem cells, and that the addition of Pax4 may improves protocols seeking the production of insulin-producing cell

O Diabetes Mellitus tipo I (DM1) é causado pela destruição auto-imune das células β pancreáticas, encontradas na porção endócrina do pâncreas, constituída pelas ilhotas pancreáticas. As células β são responsáveis pela produção e liberação de insulina, um hormônio que promove a internalização da glicose pelas células. Junto com outros hormônios, a insulina é um dos principais reguladores do nível de glicose sanguinea (glicemia). Uma das terapias utilizadas para o tratamento do DM1 é o transplante de ilhotas pancreáticas. Entretanto, um dos maiores problemas em relação a esta terapia é a falta de massa celular adequada para ser infundida no paciente. Uma tentativa para solucionar este problema, é o desenvolvimento de fontes alternativas de células produtoras de insulina, como as células-tronco, que possuem a capacidade de se diferenciarem em diversos tipos de células, inclusive nas produtoras de insulina. Pax4 é um dos fatores de transcrição responsáveis pela diferenciação de células β , sendo essencial para o apropriado desenvolvimento e maturação destas, constitui um bom candidato para induzir a diferenciação de células-tronco em células produtoras de insulina in vitro. Para introduzir o Pax4 nas células-tronco, sem provocar alterações no genoma das células diferenciadas, em virtude dos potenciais efeitos indesejáveis de vetores que se integram ao genoma celular, recorreu-se às proteínas contendo domínio de transdução (PTDs), que são capazes de carregar a proteína Pax4, através da membrana, diretamente para o interior das células. As PTDs são pequenas sequências peptídicas que permitem a translocação de proteínas através de membranas celulares e sua internalização em células-alvo. Uma das PTDs mais comumente estudadas é a VP22, produto do gene UL49 do Herpes Simplex vírus tipo I. Portanto, a proteína de fusão VP22.Pax4 permitiria que o Pax4 fosse inserido em células-tronco, possibilitando que este fator de transcrição ative a transcrição de certos genes que aumentariam a eficiência de diferenciação das células-tronco em células produtoras de insulina. Para tal, amplificamos e clonamos o cDNA do Pax4 a partir do RNA das células RINm5f de insulinoma murino, construímos o vetor pVP22.Pax4, o qual foi transfectado em células CHO, que passaram a produzir a proteína de fusão VP22.Pax4. Após o tratamento de células-tronco com a proteína de fusão VP22.eGFP e análise por microscopia confocal, comprovamos que a VP22 é capaz de tranduzir a proteína de fusão também neste tipo celular. Portanto, incorporamos a um dos passos do protocolo de diferenciação de células-tronco em células produtoras de insulina, utilizado em nosso laboratório, a co-cultura com células CHO produtoras de VP22.Pax4. Observamos que a introdução do Pax4 leva a formação de um número maior de agregados celulares (clusters) produtores de insulina. Concluímos, então, que a utilização da VP22 como ferramenta para internalização de proteínas em células-tronco é viável e que a adição do Pax4 pode trazer melhorias para protocolos que busquem a produção de células produtoras de insulina. / Diabetes Mellitus type 1 (DM1) is caused by an auto-imunne destruction of the pancreatic β cells, found in the endocrine portion of the pancreas, known as pancreatic islets. These β cells are responsible production and release of insulin, a hormone which promotes glucose internalization by cells. Along with other hormones, insulin is a major regulator of blood glucose levels (glycemia). One of the therapeutical strategies used to treat DM1 is pancreatic islet transplantation. One of the major problem related to this therapy is the lack of adequate cell mass to be infused into the pacients. An attempt to solve this problem is the development of an alternative source of insulin-producing cells by differentiation of stem cells, which display this differentiating potential. Pax4 is one of the transcription factors responsibles for β cell differentiation, being essential for its proper development and maturation, therefore being a good candidate to induce stem cell differentiation into insulin producing cells in vitro. A promising alternative to avoid the alterations of the differentiated cells genome due to its undesirable effects of integrating vectors, but yet allowing the Pax4 to act in diferentiation within the cells are the proteins with a transduction domain (PTDs), which would have the ability to lead the Pax4 protein directly into the cells. The Pax4 could thus act in the nucleus and generate specific transcriptional responses. The PTDs are small peptide sequences which allow translocation of proteins across cell membranes and their internalization into target cells. One of the most commonly studied PTDs is the VP22, a product of the UL49 gene from Herpes Simplex vírus type I. Therefore, the VP22.Pax4 fusion protein would transduce Pax4 into the stem cells, thus allowing the transcription activation of certain genes by Pax4, leading to improvement in the process of stem cells differentiation into insulin-producing cells. To this end, we cloned the Pax4 cDNA from RINm5f murine insulinoma cells, constructed the pVP22.Pax4 vector and transfected this construct into CHO cells, which then produced the VP22.Pax4 fusion protein. Upon verifying that VP22 was also able to transduce proteins into stem cells, by confocal microscopy analysis, after the treatment of these cells with the fusion protein VP22.eGFP, we incorporated the fusion protein VP22.Pax4 to one of the steps of the protocol used for stem cells diferentiation into insulin producing cells in our lab, by co-culturing with CHO cells producing VP22.Pax4. We observed that the addition of Pax4 led to the formation of a higher number of insulin producing cell clusters, therefore we conclude that VP22 may be used as a tool to internalize proteins into stem cells, and that the addition of Pax4 may improves protocols seeking the production of insulin-producing cell