The interferon (IFN) system is a powerful antiviral defense system. Host cell pattern recognition receptors (PRRs) recognise pathogen-associated molecule patterns (PAMPs) which when activated, lead to the transcription of the IFN-β gene. As a consequence IFN is secreted from the cell and activates the JAK-STAT pathway to up-regulate the transcription of IFN-stimulated genes (ISGs). The products of many ISGs inhibit viral replication and cell proliferation. Viruses encode IFN antagonists that dampen down the IFN response, making it less effective. However, within a virus population, there are always likely to be naturally occurring mutant viruses that have lost the ability to circumvent the host IFN response, and if isolated, these viruses would be unlikely to cause severe disease in the host and may therefore be developed as live attenuated virus vaccine candidates. To develop a methodology to rapidly isolate IFN-inducing mutant viruses, we generated an A549 reporter cell-line in which expression of GFP was driven by the IFN-β promoter. Using this cell-line, we show that the number of cells that became positive for GFP correlated with the amount of IFN secreted by the infected cells and the number of defective interfering (DI) particles within the virus preparations. However, we were unable to isolate IFN-inducing mutant viruses using the A549/pr(IFN-β).GFP cell-line(s). Possible reasons for this may be either that, in cells infected by IFN-inducing mutant viruses, an antiviral state was established independent of IFN that prevented virus replication in the reporter cells in which the IFN-β promoter was activated; or the viruses that activated the IFN-β promoter were DIs only which were not be able to replicate without non-defective helper viruses. A549/pr(IFN-β).GFP cells are also being used for high throughput assays to screen chemical libraries for compounds that block IFN induction. Such compounds may be potential candidates for anti-inflammatory drugs.

published_or_final_version / abstract / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Botany / Master / Master of Philosophy

The Best PhD thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing prize,2006-2007 / published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Orientador: Rejane Maria Tommasini Grotto / Banca: Alexandre Naime Barbosa / Banca: André Martins / Resumo: A coinfecção HIV/VHC tornou-se um importante problema de saúde pública devido à possibilidade desses vírus agirem sinergicamente, acelerando a progressão da doença hepática relacionada ao VHC e podendo favorecer a proliferação do HIV. Apesar de estabelecido que a alta variabilidade genética do HIV gera mutações, conferindo ao vírus a capacidade de responder rapidamente as alterações da pressão seletiva exercida pelo sistema imunológico ou pela terapia antirretroviral (TARV), não é conhecido se a presença do VHC pode favorecer a emergência de variantes do HIV resistentes. Assim, este estudo teve como objetivo avaliar o perfil de mutações de resistência a inibidores de transcriptase reversa: Inibidores de Transcriptase Reversa análogo nucleosídeo ou nucleotídeo (ITRNs), Inibidores de Transcriptase Reversa não análogo nucleosídeo (ITRNNs); e inibidores de protease (IPs) em variantes de HIV circulantes em indivíduos coinfectados pelo VHC. Foram incluídos 19 pacientes coinfectados pelos vírus HIV/VHC, maiores de 18 anos, com carga viral plasmática do HIV de pelo menos 1.000 cópias de RNA/mL, atendidos no Ambulatório de Gastroenterologia da Faculdade de Medicina de Botucatu e, no Hospital dia "Domingos Alves Meira". A genotipagem e o sequenciamento foram realizados no Laboratório de Biologia Molecular do Hemocentro de Botucatu, Faculdade de Medicina, UNESP, utilizando o Trugene HIV-1 Genotyping Kit (Siemens HealthcareDiagnóstics, Inc. Tarrytown, NY, USA). Foram observados dois subtipos do HIV-1, B e F, sendo o subtipo B o mais freqüente, 94,74%. Para o VHC foram observados os genótipos 1 (94,74%) e 3 (5,26%). Todos os pacientes apresentaram mutações de resistência as classes avaliadas, tendo maior freqüência mutações associadas aos ITRNs como M184V em 57,89%. As mutações associadas aos ITRNNs mais freqüentes foram a K103N e G190A, presentes em 21,05% das amostras. As mutações principais ... / Abstract: HIV/HCV coinfection has become an important public health problem due to the possibility of these viruses act synergistically, which can accelerate the progression of liver disease related to HCV and may favor the spread of HIV. It is established that the high genetic variability of HIV generates mutations, giving the virus the ability to respond quickly to changes in selective pressure exerted by the immune system or by antiretroviral therapy (ART). It is not known whether the presence of HCV can promote the emergence of resistant variants of HIV. Thus, this study aimed to evaluate the profile of resistance mutations to classes of reverse transcriptase inhibitors: nucleoside and nucleotide analogue Reverse Transcriptase Inhibitors (NRTIs), non-nucleoside analogue Reverse Transcriptase Inhibitors (NNRTIs) and protease inhibitors (PIs), in circulating HIV variants in HCV coinfected individuals. In this study were included 19 HIV/HCV coinfected patients, over 18, with plasma HIV viral load of at least 1,000 RNA copies/mL. These patients were treated at the Ambulatory of Gastroenterology of the Faculty of Medicine of Botucatu and at the Day-Hospital "Domingos Alves Meira". Genotyping and sequencing were performed at the Laboratory of Molecular Biology of the Blood Center of Botucatu, Faculty of Medicine, UNESP, using the Trugene HIV-1 Genotyping Kit (Siemens HealthcareDiagnóstics, Inc. Tarrytown, NY, USA). Two subtypes of HIV-1, B and F, were observed, but the subtype B was observed more often, 94.74%. It was observed HCV genotypes 1 (94.74%) and 3 (5.26%). All patients presented resistance mutations to the evaluated classes, and the mutations associated to NRTIs as M184V were observed more often (57.89%). The most common mutations associated to NNRTIs were K103N and G190A, present in 21.05% of the samples. The main mutations associated to IPs more frequent were M46I, I54V and V82A, with 15.78% ... / Mestre

Enteroendocrine cells (EEs) are critical components in our bodies' ability to maintain homeostasis after eating a meal. Hormones released by EEs mediate processes ranging from triglyceride processing to glucose balance to hydration maintenance. Despite their importance, they remain relatively poorly understood in terms of development as well as function. Drosophila melanogaster is a promising model in which to study EEs. I performed a gene expression assay in Drosophila, and found 19 transcription factors likely to be specific to EEs. I am in the process of analyzing their mutant phenotypes in the fly midgut. Additionally, by a limited screen of the homologs to the fly EE-specific transcription factors, I was able to identify two candidates for novel transcriptional regulators involved in EE specification or functionality. I will be analyzing the mutant phenotypes for these two genes, Lmx1a and Lmx1b, in addition to a third mutant Prox1, chosen because of the strong phenotype of its homologous gene's knockdown in the fly. I am hoping I will be able to add to the ever-growing body of knowledge in reference to enteroendocrine development. Additionally, several assays were performed on flies lacking EEs. I found that flies without EEs lay significantly fewer eggs, and have apparent defects in oviposition and defecation. I will outline several experiments to continue the phenotype analysis of flies lacking EEs.

Vertebrate immune systems are armed with the ability to generate highly specific immune responses capable of responding to nearly any foreign molecular threat. One of the major mediators of this response is immunoglobulins (Igs) produced by B lymphocytes. The specificity of individual Igs is created through a tightly orchestrated series of somatic DNA manipulations at Ig encoding loci resulting in functional gene rearrangements and nucleotide substitutions. These events serve to create a pool of naive B cells expressing Igs with distinct specificities, capable of expansion in response to antigen specific selection. Affinity of Ig towards antigen is enhanced through nucleotide substitutions introduced at the antigen binding variable region gene segments through the enzyme activation induced cytidine deaminase (AID) during the process of somatic hypermutation (SHM). AID also generates point mutations within noncoding DNA segments of the Ig heavy chain locus that are processed into double strand breaks leading to constant region isotype switching during class switch recombination (CSR). The Ig diversification processes of SHM and CSR critically depend upon transcriptional activation of the relevant DNA segments. Transcription is thought to facilitate single strand DNA substrate recognition by AID during unwinding of the DNA duplex. The 3'-5' exoribonuclease RNA exosome serves as a transcription dependent cofactor of AID. RNA exosome is comprised of multiple structurally integral core subunits and associated nuclease subunits. In this work, RNA exosome core subunit Exosc3 and nuclease Exosc10 have been targeted for conditional mutagenesis and loss of function analysis in mouse cells. RNA exosome deficient B cells were significantly impaired in AID dependent SHM and CSR Ig diversification processes. Transcriptome analyses revealed a striking accumulation of promoter proximal antisense divergent noncoding transcripts (xTSS-RNA) at a subset of genes upon loss of RNA exosome function. xTSS-RNAs mark regions of chromatin containing RNA exosome activity. Multiple known AID target sites including IgH and Myc were observed to express xTSS-RNA. Furthermore, genomic sites of recurrent AID dependent chromosomal translocations were enriched for xTSS-RNA. In addition to promoter proximal xTSS-RNA, cryptic intragenic antisense noncoding transcripts were found to accumulate at many genomic loci. In fact, multiple translocation hotspots precisely overlap regions of RNA exosome sensitive antisense transcription. AID targeted divergently transcribed promoters containing RNA exosome substrates possessed greater amounts of RNA:DNA hybrids, indicative of frequent transcriptional arrest. Lastly, RNA exosome deficient transcriptomes have revealed a substantial number of novel long intergenic noncoding RNAs and enhancer RNAs, indicating a hidden layer of cellular transcriptional activity. A model of AID targeting utilizing transcriptional arrest is becoming increasingly apparent. Transcribed chromatin prone to undergo transcriptional arrest, such as Ig loci or xTSS-RNA expressing regions, frequently undergoes premature transcription termination coupled to RNA exosome mediated degradation of the nascent transcript. This process results in the creation of AID substrates and serves to stabilize its association with chromatin through multiple interactions involving RNA exosome and transcription complex subunits.