Pacientes no pré-transplante de fígado podem apresentar diversas complicações da cirrose. Uma importante complicação é o hiperesplenismo, que pode resultar no sequestro de leucócitos e na consequente diminuição de neutrófilos e linfócitos, tornando o paciente, teoricamente, mais suscetível a infecções pelos vírus BKV e JCV. Por outro lado, esses pacientes também podem apresentar comprometimento renal (síndrome hepatorrenal) e de sistema nervoso central (encefalopatia hepática). Com complicações como essas e como esses pacientes apresentam-se com a resposta celular imune circulante comprometida, pode ser interessante investigar a presença dos poliomavirus nesses pacientes. O objetivo deste trabalho foi identificar e quantificar os poliomavirus BKV e JCV em saliva, lavado bucal, sangue e urina, de pacientes no pré-transplante de fígado. Para o grupo de estudo foram selecionados, 21, pacientes cirróticos, em fila de transplante de fígado, de ambos os sexos, com idade superior a 18 anos e para o grupo controle foram selecionados 20 pacientes normorreativos, de ambos os sexos, maiores de 18 anos. Nesses pacientes foram realizadas coletas de fluídos, urina, saliva, lavado bucal, e sangue. Após a coleta e congelamento das amostras, foram realizadas pesquisas de detecção e quantificação do poliomavírus humano dos subtipos BK e JC através do método de PCR em tempo real. Quinze pacientes (71,42%) do GE apresentaram-se positivos para o BKV em pelo menos uma amostra, mas nenhum dos pacientes desse grupo apresentou síndrome hepatorrenal. Por outro lado, 66,66% desses pacientes apresentaram encefalopatia hepática, mas, apesar de 10 pacientes serem positivos para o JCV, em nenhum deles foi possível quantificar a carga viral ou associar a presença do vírus com a doença clínica. Quanto às amostras, no GE, 21 (25%) foram positivas para BKV e 10 (11,90%) para JCV e no GC 27 (34,61%) foram positivas para BKV e 6 (7,69%) para JCV. Não houve diferença estatisticamente significante entre os grupos (p=0,52 e p=0,25). No GE encontramos um panorama semelhante ao GC, referente a presença do vírus em lavado, sangue e urina. A maior diferença entre as amostras apresentou-se na identificação do BKV em saliva. Assim foi possível concluir que pacientes cirróticos em fila de transplante hepático não apresentam maior prevalência de BKV ou JCV do que pacientes normorreativos e as complicações da cirrose (encefalopatia hepática e síndrome hepatorrenal) não estão associadas à presença desses vírus. Em pacientes normorreativos os fluídos bucais são equivalentes a urina na detecção do BKV, mas a saliva não é um bom fluido para detecção do BKV em pacientes cirróticos / Patients in the liver pre-transplantation can present several complications of cirrhosis. A major complication is hypersplenism, which can result in sequestration of leukocytes and the consequent decrease of neutrophils and lymphocytes, making the patient theoretically more susceptible to infection by BKV and JCV virus. Furthermore, these patients may also have renal impairment (hepatorenal syndrome) and central nervous system (hepatic encephalopathy). With complications like these and how these patients present with impaired current cellular immune response, it may be interesting to investigate the presence of polyomavirus in these patients. The objective of this study was to identify and quantify BKV and JCV polyomavirus in saliva, oral lavage, blood and urine of patients before liver transplantation. For the study group were selected, 21 cirrhotic patients, liver transplant waiting list, of both sexes, aged over 18 and for the control group were selected 20 normorreativos patients of both sexes, older than 18 years. In these patients fluid collections were performed, urine, saliva, oral lavage and blood. After collecting and freezing the samples were carried out research of detection and quantification of human polyomavirus BK and JC subtypes using the PCR method in real time. Fifteen patients (71.42%) of GE were positive for BKV in at least one sample, but none of the patients in this group had hepatorenal syndrome. Moreover, 66.66% of patients had hepatic encephalopathy, but while 10 patients were positive for JCV, none of them has been able to quantify the viral load or the presence of virus associated with clinical disease. As regards the samples, the GE 21 (25%) were positive for BKV and 10 (11.90%) for JCV and CG 27 (34.61%) were positive for BKV and 6 (7.69%) for JCV . There was no statistically significant difference between groups (p = 0.52 and p = 0.25). The GE find an overview similar to GC concerning the presence of the virus in washed blood and urine. The major difference between samples submitted to the BKV ID saliva. Thus it was concluded that patients with cirrhosis on liver transplant waiting list do not have a higher prevalence of BKV and JCV than normorreativos patients and complications of cirrhosis (hepatic encephalopathy and hepatorenal syndrome) are not associated with the presence of these viruses. In the oral fluid normorreativos patients are equivalent to detection of BKV urine, saliva but is not a good fluid for detection of BKV in cirrhotics

Pacientes no pré-transplante de fígado podem apresentar diversas complicações da cirrose. Uma importante complicação é o hiperesplenismo, que pode resultar no sequestro de leucócitos e na consequente diminuição de neutrófilos e linfócitos, tornando o paciente, teoricamente, mais suscetível a infecções pelos vírus BKV e JCV. Por outro lado, esses pacientes também podem apresentar comprometimento renal (síndrome hepatorrenal) e de sistema nervoso central (encefalopatia hepática). Com complicações como essas e como esses pacientes apresentam-se com a resposta celular imune circulante comprometida, pode ser interessante investigar a presença dos poliomavirus nesses pacientes. O objetivo deste trabalho foi identificar e quantificar os poliomavirus BKV e JCV em saliva, lavado bucal, sangue e urina, de pacientes no pré-transplante de fígado. Para o grupo de estudo foram selecionados, 21, pacientes cirróticos, em fila de transplante de fígado, de ambos os sexos, com idade superior a 18 anos e para o grupo controle foram selecionados 20 pacientes normorreativos, de ambos os sexos, maiores de 18 anos. Nesses pacientes foram realizadas coletas de fluídos, urina, saliva, lavado bucal, e sangue. Após a coleta e congelamento das amostras, foram realizadas pesquisas de detecção e quantificação do poliomavírus humano dos subtipos BK e JC através do método de PCR em tempo real. Quinze pacientes (71,42%) do GE apresentaram-se positivos para o BKV em pelo menos uma amostra, mas nenhum dos pacientes desse grupo apresentou síndrome hepatorrenal. Por outro lado, 66,66% desses pacientes apresentaram encefalopatia hepática, mas, apesar de 10 pacientes serem positivos para o JCV, em nenhum deles foi possível quantificar a carga viral ou associar a presença do vírus com a doença clínica. Quanto às amostras, no GE, 21 (25%) foram positivas para BKV e 10 (11,90%) para JCV e no GC 27 (34,61%) foram positivas para BKV e 6 (7,69%) para JCV. Não houve diferença estatisticamente significante entre os grupos (p=0,52 e p=0,25). No GE encontramos um panorama semelhante ao GC, referente a presença do vírus em lavado, sangue e urina. A maior diferença entre as amostras apresentou-se na identificação do BKV em saliva. Assim foi possível concluir que pacientes cirróticos em fila de transplante hepático não apresentam maior prevalência de BKV ou JCV do que pacientes normorreativos e as complicações da cirrose (encefalopatia hepática e síndrome hepatorrenal) não estão associadas à presença desses vírus. Em pacientes normorreativos os fluídos bucais são equivalentes a urina na detecção do BKV, mas a saliva não é um bom fluido para detecção do BKV em pacientes cirróticos / Patients in the liver pre-transplantation can present several complications of cirrhosis. A major complication is hypersplenism, which can result in sequestration of leukocytes and the consequent decrease of neutrophils and lymphocytes, making the patient theoretically more susceptible to infection by BKV and JCV virus. Furthermore, these patients may also have renal impairment (hepatorenal syndrome) and central nervous system (hepatic encephalopathy). With complications like these and how these patients present with impaired current cellular immune response, it may be interesting to investigate the presence of polyomavirus in these patients. The objective of this study was to identify and quantify BKV and JCV polyomavirus in saliva, oral lavage, blood and urine of patients before liver transplantation. For the study group were selected, 21 cirrhotic patients, liver transplant waiting list, of both sexes, aged over 18 and for the control group were selected 20 normorreativos patients of both sexes, older than 18 years. In these patients fluid collections were performed, urine, saliva, oral lavage and blood. After collecting and freezing the samples were carried out research of detection and quantification of human polyomavirus BK and JC subtypes using the PCR method in real time. Fifteen patients (71.42%) of GE were positive for BKV in at least one sample, but none of the patients in this group had hepatorenal syndrome. Moreover, 66.66% of patients had hepatic encephalopathy, but while 10 patients were positive for JCV, none of them has been able to quantify the viral load or the presence of virus associated with clinical disease. As regards the samples, the GE 21 (25%) were positive for BKV and 10 (11.90%) for JCV and CG 27 (34.61%) were positive for BKV and 6 (7.69%) for JCV . There was no statistically significant difference between groups (p = 0.52 and p = 0.25). The GE find an overview similar to GC concerning the presence of the virus in washed blood and urine. The major difference between samples submitted to the BKV ID saliva. Thus it was concluded that patients with cirrhosis on liver transplant waiting list do not have a higher prevalence of BKV and JCV than normorreativos patients and complications of cirrhosis (hepatic encephalopathy and hepatorenal syndrome) are not associated with the presence of these viruses. In the oral fluid normorreativos patients are equivalent to detection of BKV urine, saliva but is not a good fluid for detection of BKV in cirrhotics

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), characterized by lesions formed due to demyelination. MS is a complex disease thought to be triggered by environmental factors in genetically predisposed individuals. The strongest associated susceptibility allele is HLA-DRB1*1501. Environmental factors include smoking, latitude and previous infection of Epstein-Barr virus (EBV), a common herpes virus. There is no cure for MS, but several inhibitor and symptomatic drugs. Tysabri® (natalizumab) is the most effective drug, but it may lead to progressive multifocal leukoencephalopathy (PML), a rare but often fatal disease caused by reactivation of JC virus. The aim of this thesis was to replicate previous findings from a genome-wide association study and to find host genetic factors influencing JCV seropositivity and EBNA1 IgG titers in Swedish MS patients and healthy controls. Samples from the EIMS and IMSE studies were genotyped by TaqMan® OpenArray™ PCR, an end-point SNP genotyping analysis. 1143 cases and 556 healthy controls were genotyped. Due to poor call rates, genotype data from an Immunochip study was added. A total of 3408 samples (1664 cases and 1744 controls) were analyzed. EBNA1 IgG antibodies were previously measured as a detection of EBV infection and increased MS risk, and JCV IgG antibodies were measured to find patients potentially at risk for PML. One significant result was found, gene 105 (p = 0.01674, OR 0.68, CI 95% 0.49-0.93), with a protective effect in MS. More significant results might have been found with better loading of the plate, or with a different genotyping method.

Background: Although subclinical persistent infections with the human polyomaviruses are ubiquitous worldwide, they are known to vary in relation to geographical location, diseases present and may associate with different human tumours, especially in immunocompromised patients. The current study hypothesised that there may be co-operativity between HPV and polyomaviruses, particularly in HIV positive women, that could influence the rate of progression to invasive cervical carcinoma. Patients and Methods: Novel PCR methods were developed for the detection of SV40, MCV, JCV and BKV polyomavirus DNA. These were used to test DNAs extracted from 220 cervical smears and 77 invasive cervical carcinomas (ICCs) from HIV positive and negative Kenyan women of known HPV status. An expression plasmid was constructed containing JCV Large T (LT) antigen and this, in addition to empty vector control, used to stably transfect HPV16 E6/E7 immortalised human keratinocytes. Expression of LT was analysed in transfected cell lines by PCR, immunocytology and Western blotting. These cells were then used to test for changes in Cell contact growth inhibition; Growth rate and Epithelial to Mesenchymal Transition (EMT). Screening of full transcriptome microarrays was carried out on vector and LT transfected cells and their sensitivity to the drug mefloquine tested by comparison of growth rates and live/dead cell assays. Results: PCR accurately detected ~18 copies of SV40, MCV, JCV and BKV DNA in addition to simultaneous detection of JCV and BKV. None of the clinical samples tested were positive for SV40, MCV, or BKV DNA. However, JCV DNA was detected in 24/297 (8%) of cervical specimens. Comparison of the incidence of JCV in cervical smears and ICCs showed a ~3-fold increase in samples from HIV positive women with ICC (P=0.025) whereas no significant difference was found between smears and ICCs from HIV negative women (P=0.553). Analysis of the consequences of ectopic expression of JCV LT in E6/E7 immortalised human keratinocytes showed no difference in either growth rates or contact inhibition and changes in the EMT marker vimentin were found to be related to cellular clonality. Microarray analysis showed LT related alterations in gene expression which could have bearing on its carcinogenic potential in addition to changes related to clonality. JCV LT expressing monoclonal cell were the most sensitive to mefloquine treatment. Conclusion: The simultaneous JCV/BKV detection method, described herein, is unique and has been evaluated by the WHO for this purpose. The results indicate the prevalence JCV and BKV with respect to the African geographical location and suggest that JCV may combine with high-risk HPV in a sub-set of HIV positive women to influence the rate of progression to invasive cervical carcinoma. In vitro JCV LT was found not to be an overt oncogene in the cell system used although cell cloning procedures clearly affected the assays. LT induced changes in total gene expression were consistent with neoplastic progression although a high proportion of genes with unknown function were dsyregulated with respect to clonality. The anti JCV drug mefloquine showed some selectivity for LT expressing cells and further investigation of this indication is warranted.

A convincing body of evidence suggests that ubiquitination and the ubiquitin proteasome degradation pathway play a key role in neoplastic transformation. Ubiquitination, as post-translation modification, is involved both in functional regulation and degradation of specific cellular targets known as proto-oncogenes and tumor suppressors. Oncogenic viral proteins interact both with proto-oncoproteins and tumor suppressors leading to the modulation of their cellular function by several mechanisms including ubiquitination. Interestingly, viral oncoproteins themselves can also be regulated by this post-translation modification. Additionally, viruses can assemble their own E3 ligases or regulate the activity of cellular E3 ligases. E3 ligases, involved in the final step of the ubiquitination process, are the enzymes that provide the specificity for the interaction with target substrates by the means of a large number of proteins. Recent studies on the potential correlation between viral infection and oncogenesis, have addressed the emerging role of the ubiquitination system as a possible mediator for cancer transformation. In this scenario we hypothesized that JCV T-antigen may interfere with the ubiquitination system and we investigated a possible interaction between JCV T-antigen and the E3 ligase Cul7. To prove our hypothesis we performed co-immunoprecipitation and co-immunofluorescence experiments using the glioblastoma cell lines HJC12, U87MG and HJC5. Our results indicate that JCV T-antigen and Cul7 interact in the cytoplasmic compartment. In addition, JCV T-antigen stabilizes Cul7. These observations suggest that JCV T-antigen can modulate Cul7 E3 ligase activity leading to oncogenesis. Further study addressing the biological significance of this interaction will decipher the cellular processes modulated by JCV T-antigen and Cul7 and will indicate new avenues for therapeutic intervention. / Biology

Glioblastomas are the most common brain malignancy occurring in adults with the worst prognosis. Several obstacles have prevented the development of efficacious treatment strategies. Due to the insidious nature of these malignancies, tumors are not typically detected until late in the disease. Further, the delicate nature of surrounding normal brain tissue makes surgery and treatment with cytotoxic chemotherapeutics detrimental to the patient's quality of life. Despite decades of research and aggressive therapeutic strategies, most patients will develop recurrent tumors and succumb to the disease within 1 year of diagnosis. An enhanced understanding of the molecular interplay among tumor suppressors and oncoproteins can greatly contribute to the development of novel therapeutics that will extend life expectancies. The most common abnormality in these tumors is mutation of the p53 gene (TP53). Due to the expansive network of p53-responsive genes, loss of functional p53 prohibits the cell to the ability to regain control of aberrant proliferation in response to oncogenic stresses. Accordingly, glioblastomas have developed several mechanisms to inactivate this potent tumor suppressor. Similar to oncoproteins, viral regulatory proteins utilize p53 to prevent cell cycle arrest. One such example is the protein associated with the human polyoma virus, JC Virus (JCV). JCV is the etiologic agent of the fatal demyelinating disorder, Progressive Multifocal Leukoencephalopathy (PML), seen in severely immunocompromised patients. Infection of oligodendrocytes with JCV leads to their lytic destruction and the development of white matter lesions in PML patients. Its main regulatory protein, large tumor antigen (T-antigen), targets p53 to retain cells in a virus-producing state, thereby conveying an accidental oncogenicity. JCV T-antigen transgenic mice develop a multitude of CNS tumors, including malignant peripheral nerve sheath tumors similar to patients with a form of Neurofibromatosis. Neurofibromatosis types 1 and 2 are inherited cancer disorders resulting from the inactivation of their specific tumor suppressor genes, NF1 and NF2, respectively. Inactivation of the NF2 gene, results in the development of several multiple benign nervous system tumors. Traditionally, NF2 is viewed as a scaffolding protein primarily located at the plasma membrane, where it prevents excessive signaling via several cell surface receptors and their cytoplasmic kinases. NF2 links receptors at the plasma membrane to their cytoplasmic kinases to facilitate contact inhibition. However, NF2 can also interact with an array of cytoplasmic and a few nuclear proteins. To date, little is known about the function of NF2 in tumors not associated with NF2 syndrome. Loss of functional NF2 protein has become a staple of several sporadic cancers including mesotheliomas, and meningiomas. In glial cells, NF2 depletion results in hyperproliferation and development of oncogenic features. In the only prior report addressing the role of NF2 inactivation in glioblastoma, another group demonstrated that NF2 is a potent inhibitor of glioblastoma growth. Previously, our group has identified JCV T-antigen as a nuclear binding partner for NF2 in tumors derived from JCV T-antigen transgenic mice. The association of NF2 with T-antigen in neuronal origin tumors led us to hypothesize that NF2 could regulate the expression of the JCV T-antigen. Here, we report that NF2 suppresses T-antigen protein expression in U-87 MG human glioblastoma cells, which subsequently reduces T-antigen-mediated regulation of the JCV promoter. When T-antigen mRNA was quantified, it was determined that increasing expression of NF2 correlated with an accumulation of T-antigen mRNA; however, a decrease in T-antigen at the protein level was observed. NF2 was found to promote degradation of ubiquitin-bound T-antigen protein via a proteasome dependent pathway concomitant with the accumulation of the JCV early mRNA encoding T-antigen. The interaction between T-antigen and NF2 maps to the FERM domain of NF2 domain of NF2, which has been shown previously to be responsible for its tumor suppressor activity. Co-immunoprecipitation assays performed on a glioblastoma cell line revealed a ternary complex consisting of NF2, T-antigen, and the tumor suppressor protein, p53. Furthermore, these proteins were detected in various degrees in tumor specimens from patients, suggesting that these associations may occur in vivo. Collectively, these results demonstrate that NF2 negatively regulates JCV T-antigen expression by proteasome-mediated degradation, and suggest a novel role for NF2 as a suppressor of JCV T-antigen-induced oncogenesis. Studies in mouse and human tumors have inferred a relationship between NF2 and the primary target for JCV T-antigen, p53. In mouse models of cancer, concurrent loss of NF2 and p53 genes generates a highly malignant phenotype. Other groups reported that loss of NF2 and p53 in human tumors correlated with enhanced tumor grade. In transformed fibroblasts, NF2 can enhance the expression of p53 and promote p53-mediated apoptosis. However, the molecular details of the NF2 and p53 relationship have not yet been elucidated. Based on our data and previous literature, we believed that there is a tumor suppressive synergy that exists between NF2 and p53. Contrary to our expectations, we discovered that NF2 overexpression in U-87 MG cells results in the decline in p53 expression. We observed this effect in the p53-null cell line, Saos2, and in the presence of proteasome inhibitors. Further, we determined that NF2 utilizes cysteine proteases as part of a post translational mechanism to suppress p53 expression. Mutant p53, present in many glioblastomas, was resistant to NF2-mediated degradation. Additionally, we determined that p53 can reciprocally repress NF2 expression, by a post translational mechanism, independent of the proteasome, lysosome, or cysteine proteases. NF2 conformation mutants, S518A and S518D, can both degrade p53 and localize to the cytoplasm. However, the constitutively inactive, open form of NF2, S518D is resistant to p53-mediated degradation. NF2 and p53 do not directly interact, yet we were able to detect these proteins in the same patient glioblastoma samples. Using a conformation-specific antibody, we speculate that the majority of our glioblastoma samples may contain mutated p53. This novel relationship between NF2 and p53 we believe will have strong implications for chemotherapeutic sensitization of these typically resistant tumors. Cumulatively, these studies will provide evidence for novel tumor suppressive roles for NF2 and a greater understanding of the molecular events that shape glioblastoma progression. / Biomedical Neuroscience