Abstract The purpose of the study was to evaluate model systems of cancer development and compare some of their critical features with cancer development in vivo. Ovarian and endometrial cancers in man were used as correlates. Tumor development in experimental animals, exposed to carcinogens and UV irradiation, showed the entire spectrum of tumor development as compared to spontaneous carcinomas: hyperplasia, dysplasia, benign papillomas and malignant squamous cell carcinomas. For short-term analysis of differentiated homogenous cell populations, the transplant model proved most useful. For long term analysis of effects of extraneous agents, the skin carcinogenesis model is probably the most rewarding. Analysis of proliferation markers in human tumor samples as studied by immunohistochemistry, showed that an increased expression of PCNA and Ki-67 was associated with poor prognosis in ovarian neoplasms. Analysis of cell proliferation in model tumors showed that the transplant model has a better sensitivity when compared to the animal skin model and the subcutaneous injection model, in that effect of changes in cell-host interaction on the location and extent of the proliferating cell population can be studied therein. The expression of some growth factors, their receptors, oncogenes and suppressor genes were studied in ovarian and endometrial carcinomas and in skin cancer model system in mouse exposed to carcinogens and UV irradiation. Variability in expression and methodological problems precluded detailed analysis of these markers in different models. The expression of TGFβ1, TGFβ2 and TGFβ3 was determined in normal human keratinocytes, and in 7 immortalized and ras-transfected benign and malignant keratinocyte cell lines, maintained as transplants and as subcutaneous tumors in nude mice. By differential immunohistochemical localization of TGFβ isoforms, we demonstrated that each isoform may serve specific roles in tumor development and progression. The complex nature of TGFβ expression prevented detailed analysis of isozymes in different models, the results in this study, however, indicated a similar pattern in the models analyzed. Morphological methods were used to determine relationship between epithelial growth and formation and deposition of collagens in the extracellular matrix in experimental models and human tumors. The composition of the mesenchyme differed in tumors originating from different cell lines reflecting functional interaction between epithelial cells and the mesenchyme in neoplastic development. Tumor-stroma interaction was distinct in human, comparable alterations were observed in experimental models, more so in transplants, less in subcutaneous tumors, affecting tumor growth and differentiation in the different models.

Natural diseases caused by keratin mutations are rare and have only been reported in humans. We have recently identified a heritable skin disorder in Norfolk terriers caused by a mutation in KRT10. Affected dogs have a tendency to form shallow erosions or blisters following mild trauma, which is first noted after the birthing process. As the dogs age, they display generalized hyperpigmentation and scaling that is most severe in the axillary and inguinal regions. The main histologic and ultrastructural features include: marked hyperkeratosis, epidermal hyperplasia, prominent vacuolation of the upper suprabasal layers, eosinophilic intracytoplasmic aggregates (keratin bundles), numerous and frequently enlarged keratohyaline granules, and epidermal hyperplasia. Analysis of an extended pedigree through seven generations confirmed an autosomal recessive mode of inheritance. The keratin 10 mutation was defined as a G-T point mutation in intron 5 that affected splicing at the boundary of exon 4 and intron 5. The primary outcome of the mutation was a 35 bp deletion in exon 4 caused by use of a cryptic splice site. Real-time PCR quantitation of KRT10 confirmed that this mutation led to premature mRNA decay and an average 35-fold decrease in KRT10 message. Organotypic cell culture techniques were used to establish in vitro models for normal and affected Norfolk terriers. After 21 days of culture, normal epidermis was cornified with a compact and multifocally parakeratotic stratum corneum. Affected epidermis largely reproduced the expected morphologic alterations. Immunoblotting and immunohistochemistry for keratin 10 protein and real-time PCR quantitation of KRT10 message showed significantly less keratin expression in vitro than in vivo suggesting that the differentiation program in vitro underwent significant alterations. A diagnostic PCR assay was established for detection of the carrier state. Global analysis of gene expression between normal, carrier and affected dogs was performed with DermArray cDNA microarrays. Affected and carrier dogs showed differential regulation of 320 and 298 genes, respectively, between normal dogs. In affected dogs, 217 were upregulated and 103 were downregulated. In carrier dogs, 222 were upregulated and 76 were downregulated. 72 genes (65 upregulated, 7 downregulated) were altered in both affected and heterozygous dogs.

Induced pluripotent stem cells (iPSCs) can be differentiated into multiple cell types in the body while maintaining proliferative capabilities. The generation of human iPSC-derived hepatocytes (iPSC-Heps) has resulted in a new source for hepatic cells. The current available options for human hepatocytes are primary human hepatocytes (PHHs) and cell lines. PHHs isolated from healthy human donors are difficult to obtain, while cell lines exhibit reduced hepatotoxic sensitivity. iPSC-Heps are being investigated as an alternative option as they are derived from a continuous, stable source and are able to maintain their original donor genotype, which opens the door for patient-specific studies. iPSC-Heps show promise for utilization in tissue engineering, hepatotoxicity studies as well as screening for patient-specific therapeutics. Various reports have concluded that iPSC-Heps exhibit reduced hepatocyte function in comparison to PHHs. Prior reports on iPSC-Heps have focused on improving their adult phenotype functions through variations in differentiation protocols or by altering their in vitro culturing environment. This thesis focuses on incorporating hepatic non-parenchymal cells to more closely mimic the tissue and cell architecture found in the liver tissue. We designed and assembled a 3D iPSC-Hep model that integrates liver sinusoidal endothelial cells, with the goal of achieving functional maturity. Hepatotoxicants were administered to our models and various hepatic markers were measured to analyze the toxic response. This work demonstrates the need for the inclusion of hepatic non-parenchymal cells in iPSC-derived liver tissues, specifically for hepatotoxicity applications. / Master of Science / Induced pluripotent stem cells (iPSCs) can be differentiated into multiple cell types in the body while maintaining proliferative capabilities. The generation of human iPSC-derived hepatocytes (iPSC-Heps) has resulted in a new source for hepatic cells. The current available options for human hepatocytes are primary human hepatocytes (PHHs) and cell lines. PHHs originating from healthy human donors are difficult to obtain, while cell lines may exhibit reduced hepatotoxic sensitivity to chemicals. iPSC-Heps are being investigated as an alternative option since they are derived from a continuous source and are able to maintain their original donor genetic make-up, allowing for patient-specific studies. iPSC-Heps can be used in tissue engineering, hepatotoxicity studies as well as screening for patient-specific therapeutics. Various reports have concluded that iPSC-Heps exhibit reduced function in comparison to PHHs. Prior reports on iPSC-Heps have focused on improving their function through variations in differentiation procedures or by changing their culture environment. This thesis focuses on incorporating other hepatic cells to more closely mimic the tissue and cell architecture found in the liver tissue. We designed and assembled a 3D iPSC-Hep model that integrates liver sinusoidal endothelial cells, with the goal of improving hepatocyte function. Chemicals were administered to our models and various hepatic markers were measured to analyze the toxic response. This work demonstrates the need for the inclusion of additional hepatic cell types in iPSC-derived liver tissues, specifically for hepatotoxicity applications.

In nature, cells are not randomly clustered to form tissues. The tissue is a more complicated system with functions that go beyond what any single cell type could accomplish. While studying single-cell mechanics and dynamics is relevant from an investigative point of view, this approach loses, or fail to gather information about the tissue. The tissue investigated in this study is the neurosensory retina which seeing as extension of the brain is a very convenient model for the central nervous system due to its accessibility. The retina is constantly subjected to different mechanical stresses from development to adulthood. Although the majority of the phenomena where mechanical stresses are involved are well-studied, the mechanics behind them is not well understood. However, knowledge about the ability of the retina to adjust to mechanical stresses is essential, for example, for improving retinal surgery. Establishing a method to mechanically probe the retina is a challenge due to the extremely delicate nature of this multilayered neural tissue and to the short-time survival ex vivo. The organotypic tissue culture is a powerful tool because it allows to maintain with high accuracy the complex multicellular anatomy and the microenvironment of the original tissue. One of the limitations of the organotypic culture techniques has been until recently due to the ability to use only post-natal/juvenile tissues for long-term culture. The importance of using adult tissue is incontestable when the investigation focuses on age-related pathologies such as vitreous shrinkage or macula degeneration. In this work, TiO2 nanotube arrays are presented as the innovative substrate for long-term organotypic culture of adult neural tissue. The retinal whole-mount of adult guinea pig and the brain slices of adult mouse were cultures for 14 days without showing any sign of edema or swelling. Furthermore, in order to study the behavior of the retinal tissue under shear stress new set-ups were designed. For the first time, the behavior of the retinal layers were observed showing that the retina does not act as an homogeneous material in response to an applied stress. The methods developed here can be used for future quantitative studies, to provide an exact knowledge of retinal biomechanics which will help retinal surgeons to optimize their methods.

Os tumores da cérvice-uterina, que representam uma das principais doenças ginecológicas em mulheres na idade reprodutiva em todo o mundo, estão etiologicamente associados com a infecção pelo papilomavírus humano (HPV). A progressão de uma lesão intraepitelial escamosa de baixo-grau (LSIL) a um carcinoma invasivo de cérvix uterina está acompanhada da degradação da matriz extracelular (MEC) devido à ação progressiva das metaloproteinases de matriz (MMP-2, MMP-9 e MMP-14) no processo de invasão e metástase. Entretanto, o balanço entre as MMPs e seus reguladores como RECK e TIMPs é necessário para controlar esta invasão. O objetivo deste projeto consiste em avaliar a atividade e a expressão das metaloproteinases 2, 9, e 14, e caracterizar a expressão do gene supressor de metástase RECK e do inibidor tecidual de metaloproteinases (TIMP-2), em modelo de queratinócitos humanos infectados com retrovírus recombinantes que expressam os oncogenes E6 e/ou E7 de HPV 16, em culturas cultivadas em monocamada e organotípicas. Para isso, utilizamos ensaios de real-time PCR, zimografia, western blot, imunocitoquímica, ensaio de ELISA e imunohistoquímica. Em culturas em monocamada observamos que as células que expressam as oncoproteínas E6E7 de HPV16 apresentaram menores níveis protéicos de RECK e TIMP-2 em relação ao controle pXLSN. Quando analisamos as culturas organotípicas, também observamos esta diminuição dos níveis de RNAm e protéicos de RECK em rafts que expressam E6E7, acompanhado pelo aumento da atividade de MMP-9, em relação ao controle. Também observamos que o tratamento das culturas com a citocina TNF aumenta a expressão gênica, protéica e atividade de MMP-9 em todas as linhagens analisadas. Além disso, os oncogenes E6 e/ou E7 não afetam a expressão e/ou atividade de MMP-2, MT1-MMP. Nossos dados demonstraram que a expressão das oncoproteínas E6E7 de HPV16 estão relacionadas com o desequilíbrio entre MMPS e seus inibidores, sugerindo que em uma fase pré-invasiva do carcinoma cervical, não somente as MMPs, mas, principalmente seus inibidores são críticos para início da progressão tumoral. / Cervical cancer is etiologically associated with to high-risk human papillomavirus (HPV) infection. It has been observed that matrix metalloproteinases (MMPs) -2, -9, and MT1-MMP are required for basement membrane degradation during cervical carcinoma progression. Moreover, a counterbalancing among MMPs and their regulators, such as TIMPs and RECK, is necessary to modulate invasion. In order to study the effect of HPV oncogenes on MMPs expression, primary human keratinocytes (PHKs) were infected with recombinant retroviruses expressing wild-type HPV16 E6 and/or E7 oncogenes and were used to seed monolayers and organotypic cultures. Quantitative real-time PCR (Q-PCR), western blot, zimography, immunocitochemistry, ELISA assay and immunohistochemistry were used to determine the expression level and activity of MMP-2, MMP-9, MT1-MMP and their inhibitors RECK and TIMP-2. We observed that cultures expressing E6E7 presented lower RECK and TIMP-2 protein levels than control keratinocytes. In addition, rafts cultures presented the same lower RECK levels additionally presenting higher MMP-9 activity than control. Furthermore, we observed that expression of E6 and/or E7 proteins do not affect MMP-2 and MT1-MMP protein levels and/or activity. We also observed that TNF treatment enhance the MMP-9 gene and protein expression and activity in all studied cell lines. Taken together, our results demonstrate that HPV16E6E7 expression is related with the unbalance between MMPs and their inhibitors, suggesting that in the initial steps of HPV-related cervical disease, not only MMPs but also RECK and TIMP-2 are critical for tumor progression.

A feature of prion disease and other protein misfolding neurodegenerative disease is the formation of amyloid plaques. Amyloid is commonly found in the brain of individuals who have died from prion disease and Alzheimer’s disease. The formation and purpose of amyloid in such diseases is poorly understood and it is not currently known whether the material is neurotoxic, neuroprotective or an artefact. Several methods are used to investigate the formation of amyloid both in vitro and in vivo. A cell free protein conversion assay has been optimised to gain insight into the protein misfolding pathway and prion infection has been introduced to a newly characterised whole brain organotypic slice culture model. Fibrillar, but not oligomeric, recombinant PrP species induce a seeding effect on amyloid formation in the protein conversion assay. Brain homogenate containing amyloid from a β-amyloid aggregation mouse model is demonstrated to have a similar effect to recombinant fibril seeds with a PrP substrate indicating a cross-seeding effect. A whole brain organotypic slice culture (BOSC) model has been developed and slices maintained in culture for up to 8 months. During this time slices remain viable with low levels of stress and thin down from 400μm to 30-50μm with morphological consequences. A prominent glial scar forms on the surface of the slice as a result of astrocyte activation and proliferation. The neuronal population decreases while the microglia have a consistent presence throughout time in culture. Replication of misfolded prion protein has been successfully demonstrated within whole BOSC following prion infection after 2 months in culture. The BOSC model represents an accessible short term in vitro model of the brain which can offer insights into protein misfolding in a complex multicellular context. Amyloid formation has been investigated in vivo using a β-amyloid misfolding mouse model following seeding with a range of recombinant protein and brain homogenate seeds. No seeding effect was observed in animals which had received intracerebral inoculations compared to control animals within the time frame of the experiment. A lack of overall amyloid within all animals at the final time point investigated suggests later time points are required for observation of seeding. The functional role of amyloid in protein misfolding neurodegenerative diseases remains unclear. From the cell free protein conversion assay oligomers do not form on the direct pathway towards amyloid in prion misfolding. BOSC provide an accessible and useful short term in vitro model which retains multiple characteristics of the brain. BOSC support replication of misfolded protein and amyloid formation therefore this model can now be utilised to investigate plaque growth and the effect of amyloid formation on surrounding cells. Results from these assays provide important information to guide future in vivo studies and aid the search for therapeutic intervention in these devastating diseases.

A infecção por HPV de alto risco é considerada um dos principais fatores de risco para o desenvolvimento do carcinoma do colo uterino, um das neoplasias mais freqüentes em mulheres de todo o mundo. As oncoproteínas E6 e E7 de HPV-16 são capazes de induzir a degradação dos genes supressores de tumor p53 e pRb, respectivamente. Mais do que isso, a expressão dessas oncoproteínas está relacionada a alterações na via de PI3K/AKT/mTOR. A proteína quinase mTOR apresenta importante papel no controle da tradução de proteínas e é considerada o principal mediador entre crescimento celular e proliferação. A ativação de mTOR é correlacionada à fosforilação das proteínas eIF4G1 e 4EBP1, aumentando assim a taxa de síntese de proteínas. A Rapamicina é um inibidor específico de mTOR e seus análogos apresentam potente atividade antiproliferativa em um grande número de células tumorais e tumores gerados em animais. Uma vez que as proteínas E6 e E7 são capazes de interagir com diversas proteínas da via que controla a atividade de mTOR optamos por investigar o efeito da rapamicina na proliferação de culturas organotípicas de queratinócitos expressando esses genes. Também avaliamos o efeito dos genes E6 e E7 na atividade de mTOR após o tratamento com essa droga. Para geração de culturas organotípicas de queratinócitos infectamos essas células com vetores retrovirais recombinantes contendo os genes E6 e E7 de HPV-16 em conjunto ou separadamente. Nós também avaliamos o papel da degradação de p53 e pRb na resposta à rapamicina através da utilização de mutantes de E6 e E7 incapazes de induzir a degradação dessas proteínas celulares. Após a infecção dos queratinócitos, os mesmos foram semeados em uma matriz de colágeno. Após 6 dias as culturas foram tratadas com 100ng/ml de rapamicina e permaneceram 60h em contato com a droga. Para análise por imunohistoquímica os tecidos foram fixados em formalina tamponada e emblocados em parafina. A reação de imunohistoquímica foi realizada utilizando os anticorpos contra BrdU, p-4EBP1 (ser 65), p-eIF4G1 (ser 1188) e pAKT (ser 473). Os resultados obtidos ilustram que a rapamicina apresenta efeito antiproliferativo em culturas de queratinócitos contendo o vetor vazio. Por outro lado, culturas contendo o gene E7 são resistentes ao efeito antiproliferativo dessa droga. Essa resistência parece estar relacionada à capacidade de E7 induzir a degradação da proteína pRb, uma vez que em queratinócitos expressando o mutante de E7, incapaz de induzir a degradação dessa proteína, não foi observada resistência. Além disso, a fosforilação de eIF4G e 4EBP1 indica que a expressão de E7 impede que a rapamicina seja capaz de inibir a atividade de mTOR. Esses resultados mostram, pela primeira vez, que o efeito antiproliferativo da rapamicina pode ser superado pela expressão de uma proteína viral, no caso a proteína E7 de HPV-16. / High-risk HPV infection has a major etiologic role in development and progression of cervical cancer, one of the most frequent forms of cancer among women worldwide. HPV-16 E6 and E7 oncoproteins are able to induce degradation of p53 and pRb tumor suppressor proteins respectively. Moreover, the expression of these oncoproteins is related to alterations in the PI3K/AKT/mTOR pathway. The cellular kinase mammalian target of Rapamycin (mTOR) is an important regulator of the cellular protein synthesis machinery and has emerged as a principal mediator of cell growth and proliferation. mTOR activation has been shown to stimulates eIF4G1 and 4EBP1 phosphorylation, thus increasing the rate of protein synthesis. Rapamycin is a specific inhibitor of mTOR signaling pathway and its analogues have demonstrated impressive activity against a broad range of human cancer derived cell lines in culture and in human tumor xenograft models. Since E6 and E7 target several proteins controlling the mTOR pathway we aimed to investigate the effect of Rapamycin in the proliferation of organotypic raft cultures expressing these genes. We also evaluated the effect of E6 and E7 genes in mTOR activity after rapamycin treatment. To generate organotypic culture of keratinocytes we infect these cells with recombinant retroviruses containing HPV-16 E6 and E7 together or separately. We also analyzed the role of p53 and pRb degradation in rapamycin responsiveness by using E6 and E7 mutants lacking the hability to inactivate these cellular proteins. After infection, keratinocytes were seeded on to a collagen matrix. After 6 days, these cultures were treated with 100ng/ml of Rapamycin for 60 hours. BrdU was added in the last 12 hours to evaluate proliferation. For immunohistochemistry analysis tissues were fixed in buffered formalin and embedded in paraffin. Immunohistochemistry reactions against BrdU, p-4EBP1 (ser 65), p-eIF4G1 (ser 1188) and p-AKT (ser 473) were performed The results show that proliferation of organotypic cultures of keratinocytes transduced with empty vector is inhibited by Rapamycin. On the other hand, cultures generated with keratinocytes transduced with E7 gene were completely resistance to the antiproliferative effect of Rapamycin. Moreover, we found that this antiproliferative effect was dependent of Rb degradation since the cells transduced with E7 mutant unable do induce Rb degradation were sensitive. In addition, eIF4G and 4EBP1 phosphorylation indicates that E7 expression impairs mTOR inhibition by rapamycin. AKT phosphorilation indicates that rapamycin resistance could be dependent of Rb inactivation induced by E7 expression. These results show for the first time that the Rapamycin antiproliferative effect is bypassed by the expression of a viral oncogene, in this case the HPV-16 E7. Moreover, E7 expression impairs rapamycin to inactivate mTOR.

In this study we aimed to investigate the effects of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) and rotenone neurotoxins on dopaminergic (DAergic) neuronal survival using ventral mesencephalic (VM) organotypic cell culture derived from postnatal rat pups (P4-5) and immunocytochemistry for tyrosine hydroxylase (TH) as a marker of DAergic cells. In addition, we examined the neuroprotective effects of glial cell line-derived neurotrophic factor (GDNF) on TH-ir cells exposed to MPTP and rotenone as a possible treatment for PD. The TH-ir cells in co-cultures with striatum (ST) as a target grew better then when VM was cultured alone and that TH-ir cells in co-cultures could be maintained without using conditioned and trophic media. We treated 7 day and 14 day co-cultures at different times with varying MPTP and rotenone concentrations and found 14 day old cultures were more vulnerable than 7 day old co-cultures to the effects of either neurotoxin with TH-ir cell numbers significantly lower in 14 day cultures compared to 7 day cultures. Both neurotoxins induced a dose-dependent TH-ir cell reduction in the co-cultures. In addition we compared the toxicity of MPTP and its active metabolite 1-methyl-4- phenylpyridinium (MPP+) as the neurotoxic effects of MPTP on DAergic cells depends on its conversion to MPP+ by astrocytes. We found no significant difference in TH-ir cell reduction in co-cultures treated with MPTP and MPP+. Rotenone was more toxic than MPTP with less TH-ir cell survival in the weeks post treatment. GDNF exposure produced increased cell size and significant increases in TH-ir cell branching in cocultures in a dose-dependent manner. Post treatment of GDNF against MPTP and rotenone provided significant neuroprotection as TH-ir cell survival was at the lower neurotoxin doses and not at the higher doses. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1346931 / Thesis (Ph.D.) - University of Adelaide, School of Medical Sciences, 2008

Chez la souris, la néphrogenèse débute par l'apparition du blastème metanéphrogène à 9.5 dpc. Une transition mésenchymo-épithéliale, comportant 5 étapes, débute a 11.5 dpc et aboutit au rein mature, composé de 3 structures : glomérules, tubules, et capillaires glomérulaires. Les étapes initiales du développement rénal peuvent être récapitulées en culture ex vivo; toutefois, l'organogenèse terminale et la maturation rénale sont incomplètes, et les structures rénales obtenues ex vivo ne sont pas fonctionnelles. Une étude du développement vasculaire in vivo au cours du développement rénal montre une angiogenèse (cellules Pecam-1 positives) et une vasculogenèse (cellules VEGFR-1 positives) précoces, dès 10.5 dpc. Une analyse quantitative par qRT-PCR confirme le rôle de Hif1α et VEGF dans la vasculogenèse rénale. En outre, la voie PGC1α, inductrice de VEGF indépendante de HIF, est activée en conditions hypoxiques. Pour améliorer le développement vasculaire rénal ex vivo, nous proposons un modèle de culture avec micro-perfusion rénale. L'étude morphologique par immunofluorescence des reins après culture micro-perfusée montre une survie tissulaire normale (TUNEL), et une intégrité anatomique (Néphrine, Cytokératine, WT1), en particulier vasculaire (Pecam-1). Une perfusion de vivo-morpholinos WT1 aboutit à une perte d'expression de WT1, confirmant le caractère fonctionnel de notre modèle. En conclusion, nous montrons le rôle précoce de l'angiogenèse et de la vasculogenèse au cours du développement rénal ; nous identifions le rôle de PGC1α dans la vasculogenèse rénale en conditions hypoxiques, et nous proposons une nouvelle technique de culture rénale ex vivo. / In mice, nephrogenesis starts with the formation of the metanephric mesenchyme, at e9.5 dpc. A mesenchymal epithelial transition, consisting of 5 steps, starts at e11.5 dpc, and leads to a mature kidney, composed of 3 main structures: glomeruli, tubules, and capillaries. The initial steps of renal development can be recapitulated ex vivo; however, terminal organogenesis and maturation are impaired, and the explants are not functional. A study of vascular development in vivo during renal development shows that angiogenesis (Pecam-1 positive cells) and vasculogenesis (VEGF-R1 positive cells) occur early, at e10.5 dpc. A quantitative analysis, by qRT-PCR, shows that Hif1α and VEGF play a major role in renal vasculogenesis. Moreover, the PGC1α signaling pathway, a HIF independent VEGF inductor, is activated under hypoxic conditions. To improve ex vivo vascular development, we propose a novel culture technique, with micro-perfusion of the explant. A morphologic analysis of the kidneys obtained by micro-perfused cultures shows no apoptosis (TUNEL), a conserved parenchymal structure (Nephrin, Cytokeratin, WT1), and a proper vascular development (Pecam-1). A micro-perfusion of WT1 vivo-morpholinos leads to a decrease in WT1 expression, thus validating our model. In conclusion, we showed the early role of angiogenesis and vasculogenesis in renal development, we analyzed PGC1α role in hypoxic kidney cultures, and we proposed a novel kidney culture model.

Os tumores da cérvice-uterina, que representam uma das principais doenças ginecológicas em mulheres na idade reprodutiva em todo o mundo, estão etiologicamente associados com a infecção pelo papilomavírus humano (HPV). A progressão de uma lesão intraepitelial escamosa de baixo-grau (LSIL) a um carcinoma invasivo de cérvix uterina está acompanhada da degradação da matriz extracelular (MEC) devido à ação progressiva das metaloproteinases de matriz (MMP-2, MMP-9 e MMP-14) no processo de invasão e metástase. Entretanto, o balanço entre as MMPs e seus reguladores como RECK e TIMPs é necessário para controlar esta invasão. O objetivo deste projeto consiste em avaliar a atividade e a expressão das metaloproteinases 2, 9, e 14, e caracterizar a expressão do gene supressor de metástase RECK e do inibidor tecidual de metaloproteinases (TIMP-2), em modelo de queratinócitos humanos infectados com retrovírus recombinantes que expressam os oncogenes E6 e/ou E7 de HPV 16, em culturas cultivadas em monocamada e organotípicas. Para isso, utilizamos ensaios de real-time PCR, zimografia, western blot, imunocitoquímica, ensaio de ELISA e imunohistoquímica. Em culturas em monocamada observamos que as células que expressam as oncoproteínas E6E7 de HPV16 apresentaram menores níveis protéicos de RECK e TIMP-2 em relação ao controle pXLSN. Quando analisamos as culturas organotípicas, também observamos esta diminuição dos níveis de RNAm e protéicos de RECK em rafts que expressam E6E7, acompanhado pelo aumento da atividade de MMP-9, em relação ao controle. Também observamos que o tratamento das culturas com a citocina TNF aumenta a expressão gênica, protéica e atividade de MMP-9 em todas as linhagens analisadas. Além disso, os oncogenes E6 e/ou E7 não afetam a expressão e/ou atividade de MMP-2, MT1-MMP. Nossos dados demonstraram que a expressão das oncoproteínas E6E7 de HPV16 estão relacionadas com o desequilíbrio entre MMPS e seus inibidores, sugerindo que em uma fase pré-invasiva do carcinoma cervical, não somente as MMPs, mas, principalmente seus inibidores são críticos para início da progressão tumoral. / Cervical cancer is etiologically associated with to high-risk human papillomavirus (HPV) infection. It has been observed that matrix metalloproteinases (MMPs) -2, -9, and MT1-MMP are required for basement membrane degradation during cervical carcinoma progression. Moreover, a counterbalancing among MMPs and their regulators, such as TIMPs and RECK, is necessary to modulate invasion. In order to study the effect of HPV oncogenes on MMPs expression, primary human keratinocytes (PHKs) were infected with recombinant retroviruses expressing wild-type HPV16 E6 and/or E7 oncogenes and were used to seed monolayers and organotypic cultures. Quantitative real-time PCR (Q-PCR), western blot, zimography, immunocitochemistry, ELISA assay and immunohistochemistry were used to determine the expression level and activity of MMP-2, MMP-9, MT1-MMP and their inhibitors RECK and TIMP-2. We observed that cultures expressing E6E7 presented lower RECK and TIMP-2 protein levels than control keratinocytes. In addition, rafts cultures presented the same lower RECK levels additionally presenting higher MMP-9 activity than control. Furthermore, we observed that expression of E6 and/or E7 proteins do not affect MMP-2 and MT1-MMP protein levels and/or activity. We also observed that TNF treatment enhance the MMP-9 gene and protein expression and activity in all studied cell lines. Taken together, our results demonstrate that HPV16E6E7 expression is related with the unbalance between MMPs and their inhibitors, suggesting that in the initial steps of HPV-related cervical disease, not only MMPs but also RECK and TIMP-2 are critical for tumor progression.