Made available in DSpace on 2014-06-12T18:07:24Z (GMT). No. of bitstreams: 2 arquivo6719_1.pdf: 1718714 bytes, checksum: bd74bdae33e28d1e9514c43b983a8308 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Faculdade de Amparo à Ciência e Tecnologia do Estado de Pernambuco / A regulação da expressão gênica nos tripanossomatídeos é predominantemente póstranscricional, ocorrendo através de mecanismos como controle da estabilidade e tradução dos RNAs mensageiros (mRNAs). Em eucariotos a tradução é controlada principalmente no recrutamento dos ribossomos para os mRNAs, sendo mediada pelos fatores eucarióticos de iniciação da tradução (eIFs). Neste contexto, o reconhecimento dos mRNAs é realizado principalmente pelo complexo eIF4F, formado pelas subunidades eIF4E, eIF4A e eIF4G. O eIF4G é a proteína estruturadora do complexo, interagindo com o eIF4E e o eIF4A, além de outras proteínas, pois apresenta em sua estrutura inúmeros domínios de ligação, entre eles, o MIF4G e o MA3, responsáveis pela ligação ao eIF4A. A proteína eIF4G é o protótipo de uma família de polipeptídeos que apresentam estes domínios, onde foi incluída recentemente a proteína NOM1, proteína com localização nucleolar, identificada em pacientes que apresentavam Leucemia Mielóide Aguda, sendo encontrados ortólogos em diferentes espécies, incluindo os tripanossomatídeos. Em trabalhos prévios, os genes da NOM1 de T.brucei e L.major foram clonados em vetores de expressão e a proteína recombinante TbNOM1 foi utilizada para imunização de coelhos. No presente trabalho, iniciamos a caracterização funcional da TbNOM1, demonstrando que a proteína é capaz tanto de interagir com os homólogos do eIF4A de T.brucei, como de formar dímeros ou estruturas mais complexas de ligação TbNOM1-TbNOM1. Sua expressão é constante durante todo o ciclo celular na forma procíclica e na forma sanguínea é expressa em maior concentração entre 0 e 6 horas, não sendo regulada por fosforilação em suas formas nativas. A localização subcelular da proteína TbNOM1 é citoplasmática, tendo sido confirmada em experimentos de imunofluorescência, fracionamento celular e superexpressão de proteína fluorescente. Outro aspecto relevante avaliado foi que a TbNOM1 é uma proteína não essencial, uma vez que sua depleção mediada por interferência de RNA, não afeta de forma significativa a viabilidade celular. Neste trabalho iniciou-se a caracterização funcional da TbNOM1, sendo necessário a realização de outras abordagens metodológicas para dar prosseguimento a este processo de investigação

The biological function of RNA is often linked to an ability to adopt one or more mutually exclusive conformational states or isomers, a characteristic that distinguishes this biomolecule from proteins. Two examples of conformationally inconverting RNAs were structurally investigated. The first is found in the 3' untranslated region (UTR) of the coronavirus mouse hepatitis virus (MHV). A proposed molecular switch between mutually exclusive stable stem loop and pseudoknot conformations was investigated using thermal unfolding methods, NMR spectroscopy, sedimentation velocity ultracentrifugation and fluorescence resonance energy transfer (FRET) spectroscopy. Utilizing a "divide and conquer" approach we establish that the independent subdomains are folded as predicted by the proposed model and that a pseudoknotted conformation is accessible. Using the subdomains as spectral markers for the investigation of the intact 3' UTR RNA, we show that the 3' UTR is indeed a superposition of a double stem conformation and a pseudoknotted conformation in the presence of KCl and MgCl2. In the absence of added salt however, the 3' UTR adopts exclusively the double stem conformation. Analysis of the pseudoknotted stem reveals only a marginally stable folded state (deltaG25 = 0.5 kcal mol-1, tm = 31 oC) which makes it likely that a viral or host encoded protein(s) is required to stabilize the pseudoknotted conformation. A second conformationally interconverting RNA system investigated is an RNA element that stimulates -1 programmed ribosomal frameshifting in the human Ma3 gene. Structural analysis of the frameshifting element reveals a dynamic equilibrium between a functionally inactive double stem loop conformation and the active pseudoknotted conformation. Thermal melting and NMR spectroscopy reveal that the double stem loop is the predominant conformation in the absence of added KCl or MgCl2. The addition of KCl and MgCl2 results in the formation of a pseudoknot conformation. This conformation is dominant in solution only when the competing double stem loop conformation is abrogated by mutation. Functional studies of the Ma3 pseudoknot reveal that abrogation of double stem conformation increases frameshift stimulation by 2-fold and indicates that the pseudoknot is the active conformation.

Murine leukemia virus (MLV), classified as a gammaretrovirus, has been studied extensively to enhance our understanding of the biology and replication of retroviral infection. Typically referred to as a simple retrovirus, its usefulness as a model is highlighted owing to its minimal genome. The genetic material for MLV was thought to only code the basic and essential defining features of a retrovirus. Through the understanding developed from the use of simple retroviruses, the clinical and research communities were immeasurably more prepared to combat the more complex and decidedly infamous human immunodeficiency virus (HIV). Interestingly, a scenario of convergent evolution has directed MLV to encode an accessory protein, termed Glycosylated Gag (gGag), that shares functionality reminiscent of several HIV proteins. Herein, I present a dissection of a novel function of this enigmatic protein, paired with an improved understanding of the biology of MLV that was revealed by the development of small particle flow cytometry performed on viruses, also known as flow virometry. Initially, we elucidated that gGag is responsible for the resistance of MLV towards the restriction factor murine APOBEC3 (mA3). I showed that even endogenous mA3 from primary cells exhibited an enhanced enzymatic activity towards MLV with mutant gGag proteins which have lost glycosylation sites. In our following study, I illustrated that these mutants displayed a reduced viral core stability, the severity of which was correlated directly with susceptibility to mA3. These results are in line with the hypothesis that viral core stability and APOBEC3-susceptibility are directly linked. Furthermore, I showed for the first time that unprocessed gGag was associated with viral particles released from producer cells in the orientation of a type I membrane protein, with the structural regions directed within the viral core. This may be the direct evidence of how gGag improves capsid stability, a mechanism which is still unresolved. On the flip side, gGag as a type II membrane protein was observed exclusively on virus-like particles devoid of detectable envelope glycoprotein (Env). This marks a potential new function for gGag in the context of infection. Given the ubiquitous necessity of an optimized core stability for any virus, combined with the overlapping function of gGag with HIV accessory proteins, continuation of this work represents an as of yet clinically unexplored avenue for the development of HIV therapeutics. At the same time, in order to characterize individual viral particles, I played an instrumental role in developing the technique of flow virometry within our core facility. I illustrated that the Env of MLV does not significantly accumulate on extracellular vesicles (EVs) and acts as an effective marker for viral particles. With this evidence in hand, the enumeration of MLV virions was made possible. By correlating this information with an absolute viral genome determination, I was able to estimate the packaging efficiency for MLV in a quantitative manner. This information suggests that roughly 80-85% of MLV particles are missing their essential genetic information. These findings may implicate the disease progression of MLV infection may be enhanced by the use of defective-interfering particles, a theory that has been suggested for HIV. This work highlighted the fact that flow virometry is uniquely capable to discriminate viral particles from other cell-derived membraned vesicles in a highly sensitive manner. Overall, my work has unveiled new complexities of a simple retrovirus, while laying the groundwork towards both diagnostics and therapeutics for the ongoing battle with HIV.

This thesis deals with the R-material use in asphalt mixtures. In the theoretical part, different kinds of flexible pavement recycling and ways of adding R-material into asphalt mixtures are discussed. The practical part deals with asphalt layers design and manufacture, specifically for binder course ACL 16+. R-material content in the blends is 0%, 20%, 25% and 30%. EVOTHERM MA3 rejuvenator is used as additive for softening of R-material. Various mixtures are compared based on tests - resistance to permanent deformation, resistance to the effects of water and low temperature properties.

APOBEC3 (A3) proteins are a family of host-encoded cytidine deaminases that protect against retroviruses and other viral intruders. Retroviruses, unlike other viruses, are able to integrate their genomic proviral DNA within hours of entering host cells. A3 proteins hinder retroviral infectivity by editing retroviral replication intermediates, as well as by inhibiting retroviral replication and integration through deamination-independent methods. These proteins thus constitute the first line of immune defense against endogenous and exogenous retroviral pathogens. The overall goal of my Master's project was to better understand the critical role A3 proteins play in restricting inter- and intra-host transmission of retroviruses. There are two specific aspects that I focused on: first, investigating the role of mouse APOBEC3 (mA3) in limiting the zoonotic transmission of murine leukemia retroviruses (MLVs) in a rural environment; second, to identify the molecular features in MLVs that confer susceptibility or resistance to deamination by mA3. For the first part of my project, we collected blood samples from dairy and production cattle from four different geographical locations across Canada. We then designed a novel PCR screening strategy targeting conserved genetic regions in MLVs and Mouse Mammary Tumor Virus (MMTV) and MMTV-like betaretroviruses. Our results indicate that 4% of animals were positive for MLV and 2% were positive for MMTV. Despite crossing the species barrier by gaining entry into bovine cells, our study also demonstrates that the bovine A3 protein is able to potently inhibit the spread of these murine retroviruses in vitro. The next question we asked was whether mA3 could also mutate and restrict murine endogenous retroviruses and thereby partake in limiting zoonotic transmission. Moloney MLV and AKV MLV are two highly homologous murine gammaretroviruses with opposite sensitivities to restriction by mA3: MoMLV is resistant to restriction and deamination while AKV is sensitive to both. Design of MoMLV/AKV hybrid viruses enabled us to map the region of mA3 resistance to the region encoding the glyco-Gag accessory protein. Site-directed mutagenesis then allowed us to correlate the number of N-linked glycosylation sites with the level of resistance to deamination by mA3. Our results suggest that Gag glycosylation is a possible viral defence mechanism that arose to counteract the evolutionary pressure imposed by mA3. Overall, my projects show the important role A3 proteins play in intrinsic immunity, whether defending the host from foreign retroviral invaders or endogenous retroviral foes.