Bunyamwera (BUN) virus multiplied readily in mosquitoes following intrathoracic injection and also after imbibing'-an infective blood meal. This agent also multiplied following inoculation of human and avian cells in tissue cultures, with production of cytopathic effects. Both in arthropod and vertebrate tissues, enveloped virions 84 nm diameter were visualized by electron microscopic observation of tissues collected after maximum viral proliferation was attained. Following intrathroacic injection of 10 ²•² mouse LD₅₀ of BUN virus into groups of wild-caught mosquitoes comprising both Aedes canadensis and A. vexans, increments of infectivity were first detected in salivary glands and gut at 3 days, and maximum titres of 10 ⁵•² mouse LD₅₀ per organ were attained in salivary glands at 10 days. However, 50 the virus content of legs, which provided a convenient means of sampling hemocelic fluid, increased at 2 days. Virus transmission by biting mice was demonstrated with mosquitoes injected 10 days previously, but not after shorter intervals. No virus replication was demonstrated following ingestion of 10⁴•⁰ mouse LD₅₀ of BUN virus in a blood meal. Aedes aegypti mosquitoes readily supported the replication of BUN virus following injection with 10 ³•³ mouse LD₅₀ or imbibing of 10 ⁴•⁶ mouse LD₅₀. After injection, virus titres of whole mosquitoes declined to 10 ¹•⁷ mouse LD₅₀ at 12 hours, followed by an increase to a peak amount of 10 ⁵•⁰ mouse LD₅₀ at 2 days. After feeding, virus was first detected in legs and salivary glands at 4 days, and attained maximum titres of 10 ⁵•⁰ mouse LD₅₀ in salivary glands at 10 days. Transmission of virus to mice was effected by A. aegypti following feeding and injection 10 days previously, but not at earlier intervals. Following exposure of whole gut cultures of adult A. aegypti mosquitoes to 10 ³•⁷mouse LD₅₀ maximum yields of 10 ⁶•⁰ mouse LD₅₀ per ml. were observed after k days incubation at 29°C, after an initial decline of infectivity to 10 ¹•⁸ mouse LD₅₀ at 12 hours. Enveloped virions with cores 45 nm diameter and total diameters 80 to 100 nm were observed within vacuoles and lining vacuolar membranes of salivary glands and gut cells of A. aegypti mosquitoes 10 days or more after infection with BUN virus. No particles were observed earlier, despite high virus titres 4 days or more after injection. After inoculation of continuous live tissue cultures of human epidermoid corcinoma cells (H.Ep. 2) with 10 ⁶•⁵ mouse LD₅₀, the highest amount of virus produced was 10 ⁷•⁰ mouse LD₅₀ per ml. cell suspension after 24 hours incubation at 37°C. Maximum yields of BUN virus (10 ⁶•² mouse LD₅₀ per ml. cell suspension) were attained 24 hours after inoculation of primary chick embryo fibroblast monolayers with 10 ⁵•² mouse LD₅₀ following incubation at 37 C . However, a peak titre of 10 ⁵•⁰ mouse LD₅₀ was attained 3 days after inoculation with 10 ³•⁷mouse LD₅₀ in cultures incubated at 29°C. Before an increment of virus titre was observed infectivity declined to zero during the initial 4 hours after inoculation of cultures incubated at 37°C, and a tenfold decline of infectivity was noted in cultures incubated at 29°C. Enveloped virions with total diameter 84 nm which contained electron-dense nucleoids 44 nm diameter were observed extracellularly in thin sections of chick embryo fibroblasts infected 12 hours previously with BUN virus. These particles were released by budding. Precursor particles 41 nm diameter were associated with intracellular membranes in occasional cells sectioned at 4 hours. Extracellular virions released one day after inoculation of H.Ep. 2 cultures were tagged by ferritin-labelled anti-BUN antibody. Enveloped virions with mean diameters 100 nm were observed in suspensions of suckling mouse brain infected with BUN virus and stained negatively with phosphotungstic acid. These results show clearly that BUN virus exhibits the essential biological and morphological characteristics of a mosquito-borne arbovirus. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Herpes Simplex Virus (HSV) requires the host cell secretory apparatus for the maturation and egress of newly synthesized viral particles. Not only do viral glycoproteins rely on the host ER and Golgi compartments for their proper processing, it is believed that enveloped particles are transported through these same organelles for their export out of the cells. Brefeldin A (BFA) is a compound that induces retrograde movement of material from the Golgi apparatus to the ER and causes the disassembly of the Golgi complex. In this study, the effects of BFA on the propagation of HSV-1 in infected cells were examined. Release of viral particles from infected cells was inhibited by as little as 1 µg/ml BFA. Further analysis revealed that BFA did not affect the normal assembly of viral nucleocapsids, but did block the movement of newly-enveloped particles from the nucleus into the cytoplasm. Naked nucleocapsids were found in the cytoplasm of infected cells treated with BFA, however, these particles were neither infectious, nor were they released from the cells. Although BFA altered the distribution of viral glycoproteins in infected cells, this alteration was reversed within 2 hours after the removal of BFA. In contrast, the BFA-induced blockage to viral release was not fully reversed after BFA was removed and cells were allowed to recover in fresh medium for 3 hours. These findings indicate that the BFA-induced retrograde movement of material from the Golgi complex to the ER early in infection arrests the ability of the host cell to support the maturation and egress of enveloped viral particles. Furthermore, exposure of infected cells to BFA during the exponential release phase of the viral life cycle can cause irreversible damage to the egressing particles. This suggests that productive growth of HSV-1 in infected cells relies on a series of events that, once disrupted by agents such as BFA, cannot be easily reconstituted. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Complications of rubella virus infection, including congenital rubella syndrome and the association of rubella virus with joint inflammation, emphasize the need for continued research on rubella virus. The finding that the association of rubella virus infection with joint manifestations is more pronounced with wild strains than with vaccine strains suggested the possibility of strain variation. Several different techniques have been employed in order to compare six rubella virus strains and identify variations in their structural proteins. Differences in biological activities were detected, including the extent of virus production and the ability of various cell types to support replication of rubella virus (tissue tropism). However, the strains were shown to have remarkably similar electrophoretic patterns. Variation appeared to result from alterations in glycosylation. Efforts to isolate the protein components of the two envelope glycoproteins were unsuccessful, and it was therefore not possible to localize variation to either the protein or the carbohydrate components. Future work employing more sensitive methods for examination of fine molecular structure and the correlation of these structures with biological activity will further our understanding of the pathogenesis of rubella virus infection. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Rubella virus is a small enveloped positive strand RNA virus. Two species of viral RNA are found in infected cells: a full-length genomic RNA and a subgenomic species corresponding to the 3' one third of the genomic RNA molecule. The 24S subgenomic RNA specifies a polyprotein which is cotranslationally processed by endoproteolytic cleavage by host signal peptidase to yield three structural proteins, El, E2 and capsid. El and E2 are membrane glycoproteins forming the virion spikes, and C protein binds to 40S genomic RNA to form a nucleocapsid. El and E2 proteins contain N-linked oligosaccharide as a consequence of their passage through the endoplasmic reticulum (ER) and Golgi apparatus. According to the signal hypothesis, translocation of secretory and membrane proteins into the ER is mediated by a hydrophobic signal peptide. The signal peptides for E2 and El have been localized by in vivo expression of El and E2 cDNAs. Oligonucleotide-directed mutagenesis was used to define the cleavage sites between C, E2, and El, as well as the effect of the cleavages on the transport and processing of E2 and El. The expression of the cleavage site mutants was studied in vitro and in vivo. It was found that uncleaved precursor polypeptides were retained in the ER and very little E2 or El polypeptide was observed at either the Golgi apparatus or the plasma membrane. The E2 and El polypeptides can cross the ER membrane without the cleavage of the signal peptide while the transport of E2 and El beyond the ER requires the cleavage of E2 from C and El from E2. The C-termini of the C and E2 proteins, which were not previously defined, have been partially characterized. Capsid protein does not appear to undergo further proteolytic processing after it is cleaved from E2 by signal peptidase, but E2 may be processed at a second cleavage site at its C-terminus by a trypsin-like enzyme. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The mechanism of membrane assembly for Semliki Forest Virus, a Group A Togavirus, was investigated through a series of radioactive pulse chase experiments. Initially a time course for the appearance of virus specified proteins in the microsomal fraction of infected BHK (baby hamster kidney) cells and mature virions was performed. Infected cells were harvested and fractionated at 0, 1, 2, 4, 6, 8 and 11 hours post-infection. Plaque assays were performed on the virus released into the growth medium at these times. It was found that virus production was maximal between 4 and 6 hours. Nucleocapsid was clearly evident at 6, 8 and 11 hours when the microsomal proteins were separated by SDS polyacrylarnide gel electrophoresis. In the next set of experiments infected cells were pulsed 3 for 20 minutes at 5 hours post-infection with H-Leu. Microsomes were prepared from the cells at 0 min., 20 min., 40 min. and 60 min. after removal of H-Leu and subjected to SDS polyacrylarnide gel electrophoresis. Virus was also isolated from the cell medium by sucrose gradient centrifugat-ion. Nucleocapsid protein radioactivity was at levels much greater than the combined peaks of radioactivity due to the membrane proteins E₁ and E₂. Little if any radioactive virus was released into the media during this time of chase. A similar experiment to the one just outlined was performed except that the radioactive chase was extended over the range of 0 hrs., 0.75 hrs., 1.50 hrs., 2.25 hrs. and 3.00 hrs. Levels of ³H- labelled nucleocapsid were again initially higher' than those of the combined E₁ and E₂ radioactive peak. The radioactivity of E₁ E₂ plateaus between 0.75 hrs. to 3.00 hrs. while that in the nucleocapsid continued to increase. This data appears to support the contention that nucleocapsid is synthesized prior to the viral membrane proteins. In hope of chasing the ³H-Leu label into and then out of the microsomes, infected BHK cells were pulsed at 3 hours and chased for 0, 1, 2, 3, 4, 5, and 6 hours after removal of labelled medium. Levels of ³H-Leu increased in both the nucleocapsid and E₁ E₂ protein bands of the SDS acrylamide gels until about 2 hours and then declined over the following time range. Loss of ³H-Leu in the microsomes appeared to correlate with the increase of label incorporated into the virus. Finally, after devising a method for separating plasma membrane (PM) ghosts and endoplasmic reticulum (ER) fragments, another pulse chase experiment was performed. Infected BHK cells were again radioactively pulsed at 3 hours infection and the level chased for 0, 1, 2, 4, 6, 8 and 11 hours following removal of the ³H-Leu. At the various time points labelled cells were harvested and fractionated into PM and ER. The samples of ER and PM were applied to SDS acrylamide gels and the radioactivity incorporated into the virus protein band was quantitated. Virus released into the medium was purified by sucrose gradient sedimentation, assayed for ³H-Leu, and also fractionated by SDS electrophoresis. Label was initially very high in the ER in the form of precursor proteins (NVPI65, NVP97, PE₂), envelope proteins (E₁, E₂), and nucleocapsid protein. This radioactivity was chased from the ER to the PM and then into mature virus. These results appear to indicate that Semliki Forest Virus nucleocapsid does indeed "bud" from the host cell membrane, thus obtaining its envelope. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Studies on purification and properties of potato virus M (PVM) were carried out using an isolate found in British Columbia. The narrow host range of the virus was confirmed, and no new susceptible species was discovered. Potato cultivars failed to develop symptomps even in plants produced by tubers of inoculated plants, but none was immune. An attempt to demonstrate transmissibility of the virus by plant contact was unsuccessful. In undiluted potato sap the virus had a thermal inactivation point (TIP) of 65 to 70°C, and a longevity in vitro (LIV) of 2 to 4 days. The dilution end point (DEP) was 10⁻⁴. The LIV and DEP of the virus in tomato sap were similar to those in potato sap. Crude sap diluted to 10⁻¹ induced more lesions on Red Kidney bean than undiluted sap. An efficient purification procedure for PVM was developed. The virus was purified from leaves of potato (Solanum tuberosum L.), by extraction with 0.5 M borate buffer, pH 7.8, clarification with ammonium sulfate (20%), and concentration with ammonium sulfate (30%). Further concentration was carried out by high speed centrifugation followed by polyethylene glycol (PEG 6000) precipitation and high speed centrifugation. Final purification was by sucrose density gradient centrifugation. The yield obtained from this procedure was 3.7 to 4.1 mg per Kg of infected leaves. The purified preparations contained rod-shaped particles 651 nm normal length and 13.4 nm average width. The particles had an A₂₆₀/A₂₈₀ratio of ]^3' an A max/A min ratio of 1.24, a maximum ultraviolet light absorption at 260 nm, a minimum absorption at 245 nm, and a buoyant density in CsCl of 1.304 (suggesting an RNA content of 6.2%). The molecular weight of the protein subunit was about 39,300 daltons. / Land and Food Systems, Faculty of / Graduate

The mechanism of viral penetration for Semliki Forest Virus into BHK-21 cells was investigated through a series of radioactive pulse-chase experiments. Entry of an enveloped virus such as SF Virus can be visualized to enter host cells both by pinocytosis (viropexis) or by fusion of the viral envelope and plasma membrane. Preliminary experiments were performed to obtain optimum conditions of viral adsorption to host cells. The conditions considered included temperature, time of infection, multiplicity of infection and ionic strength of the inoculum. In subsequent experiments BHK-21 cells were infected one half 35 hour with ³⁵S-Methionine-labeled Semliki Forest Virus. At various time points after removal of unadsorbed ³⁵Met-SF Virus, cells were harvested and fractionated into plasma membrane and endoplasmic reticulum fractions. The fractions were subjected to SDS polyacrylamide gel electrophoresis and analyzed for component proteins of SF Virus. Maximum levels of radioactivity corresponding to the envelope proteins (E₁, E₂ and nucleocapsid protein (NC) were found in the PM fraction at zero minutes of chase. Both E₁, E₂ and NC were found to decline during the chase period (approximately 90% within 60 minutes of chase). On the other hand, high levels of only nucleocapsid protein were observed associated with the endoplasmic reticulum fraction although no general pattern of incorporation was indicated furing the experiment. (There were high levles of NC present in the ER fraction throughout the chase period). The results of these studies are generally inconclusive as they can be rationalized both by the viropexis and fusion mechanisms. The loss of both E₁, E₂ and NC from the PM suggest that viropexis is the mechanism of entry, however, fusion is not eliminated as an alternative since the envelope proteins in the PM may be degraded after internalization of the virus core by host proteases. Although no conclusions have been drawn, this study has demonstrated that radioactive pulse-chase experiments can be performed to augment electron microscopy data concerning the mechanism of viral penetration into animal cells. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The data from chemical studies and electron microscopy-suggest that Semliki Forest virus obtains its envelope by budding into the medium from the plasma membrane of the host cell. Biochemical evidence for this phenomenon, however, has not been published. Therefore, we undertook a series of pulse-chase studies so that we might quantitatively evaluate the importance of the budding mechanism in the morphogenesis of Semliki Forest virus. Baby hamster kidney cells (clone 13) were grown in culture and infected with Semliki Forest virus. The cells were exposed to [4.5-³H] leucine for 20 min and the subsequent incorporation of the label into virus proteins associated with cytoplasmic membrane and extracellular virus was determined. Initial experiments had been conducted previously with microsomes and a precursor-product relationship demonstrated between viral proteins in the microsomes and extracellular virus (CD. Richardson and D.E. Vance, J. Biol. Chem. 251, 5544-5550). Further studies were performed with endoplasmic reticulum and plasma membrane preparations. Maximum incorporation of [³H]leucine was observed in the viral proteins located in the endoplasmic reticulum at the end of a 20-min pulse period; greater than 50% of this radioactivity had disappeared within 2 hr. The plasma membrane fraction contained no radioactivity at the end of the pulse period; subsequently, maximal labeling of the viral proteins in the plasma membrane occurred 3 hr into the chase period, and these labeled proteins disappeared from this membrane 8.5 hr after the pulse. At 8.5 hr chase, maximum incorporation of the labeled proteins into extracellular virus was observed. These data are consistent with a precursor-product relationship between viral proteins in the endoplasmic reticulum, plasma membrane, and extracellular media. Viral proteins migrate to the plasma membrane and are subsequently incorporated into extracellular virus. All the radioactivity in the extracellular virus appears to have been derived from viral proteins associated with the plasma membrane of the cell. Therefore, mechanisms for the morphogenesis of Semliki Forest virus (in baby hamster kidney cells), other than budding from the plasma membrane, are unlikely to be of quantitative importance. The possibility that an intact cytoskeletal system might be required for the assembly of Semliki Forest virus was investigated. The microtubules and microfilaments of baby hamster kidney cells (BHK-21) were disassembled with specific drugs and the effect on production of extracellular virus was determined. Colchicine, Nocodazole, dibucaine, and cytochalasin B reduced the production of extracellular virus by 75-90%. Lumicolchicine had no effect on virus growth. Other control experiments showed no effect by these drugs on the incorporation of [³H]leucine of [³⁵S] methionine. At various times after addition of one of these drugs, the incorporation of the labeled precursors into viral proteins associated with endoplasmic reticulum or plasma membrane of the cell was evaluated. The results clearly show that the envelope and nucleocapsid proteins of the virus move to the plasma membrane of the cell where they accumulated. These studies strongly suggested that the cytoskeletal system was involved in the final stages of membrane assembly of Semliki Forest virus at the plasma membrane. Studies were also performed with the cross-linking agents -dimethylsuberimidate (DMS), dithiobis(succinimidyl propionate) (DSP), and dimethylthiobi's(propionimidate) (DTBP) . The proteins of purified virus and infected cells reacted with these agents and the cross-linked proteins were evaluated by one- and two-dimensional SDS electrophoresis. Nucleocapsid protein cross-linked to form up to pentameric complexes, and envelope proteins reacted to yield dimeric species. Nucleocapsid protein did not crosslink with envelope proteins. Cross-linking agents were also utilized to determine the effects of colchicine and dibucaine on the proximity of viral proteins to each other in the plasma membrane of the host cell. Colchicine (which disrupts microtubules) appeared to have no effect on the degree to which [³⁵S]-labeled virus proteins reacted with the agents, while dibucaine (which supposedly disrupts both microtubules and microfilaments) abolished envelope protein dimers dramatically. This result was interpreted to mean that microtubules may not be required for the formation of patches of virus proteins in the plasma membrane prior to budding, while microfilaments may play a more dominant role in this process. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Memoria para optar al título profesional de Bioquímico / El virus HTLV-I (Virus Linfotrópico humano Tipo I) es el agente etiológico de dos patologías principales, la Mielopatía asociada al HTLV-I/Paraparesia Espástica Tropical (HAM/TSP), una axonopatía neurodegenerativa, donde existe falla en el transporte axonal y la Leucemia de células T del adulto (ATL). No se ha detectado infección por HTLV-I en neuronas, desconociéndose el mecanismo por medio del cual se produce la neurodegeneración en HAM/TSP. Puesto que el reservorio principal del virus son los linfocitos T CD4+, algunos productos virales secretados por estos linfocitos infectados infiltrados en el sistema nervioso central, podrían afectar vías de transducción de señales relacionadas con la reorganización de citoesqueletos y transporte axonal, produciendo el daño axonal de distal a proximal observado en pacientes HAM/TSP. Los productos secretados por células MT2 (linfocitos infectados con HTLV-I) provocan retracción en células de neuroblastoma SH-SY5Y diferenciadas y disminuyen la velocidad de crecimiento neurítico en células PC12 durante su diferenciación a tipo neuronal. Entre las proteínas virales secretadas, se encuentra Tax, la disminución de la velocidad de crecimiento neurítico en células PC12, podría estar mediada por esta proteína viral a través de la vía transduccional que involucra a semaforinas y sus receptores Plexinas. Como un ejemplo, en el caso de Sema3A-Plexin A, un aumento de la actividad de Cdk5 causa hiperfosforilación de Tau y CRMP-2. El fallo en el transporte axoplásmico, podría explicarse por una desregulación de la fosforilación de las subunidades de los motores moleculares kinesina KIF5 y dineína citoplasmática Dync en este escenario. En este trabajo, se propuso estudiar el efecto de los productos secretados desde linfocitos infectados que incluyen Tax sobre el proceso de diferenciación neuronal. Para esto, se utilizó la línea celular de feocromocitoma de rata, células PC12 diferenciables a tipo neuronal por acción de NGF. La Hipótesis planteada es la siguiente: “La exposición a productos secretados de linfocitos infectados por HTLV-I provoca un aumento en el estado de fosforilación de proteínas motoras en modelo neuronal de células PC12” El primer objetivo desarrollado, fue determinar si Tax es uno de los productos virales secretados desde medio de cultivo de células MT-2 responsable del efecto de retardo de crecimiento neurítico de PC12. La adición al cultivo celular de anticuerpos monoclonales anti-Tax mostró que se bloqueó este efecto de retardo de crecimiento, lo que apunta a que este efecto se debe a la acción de Tax presente en el medio de secreción de las células MT-2. El segundo objetivo, contempló estandarizar protocolos de inmunodetección selectivos para determinar las subunidades asociadas al control de funcionalidad de kinesina KIF5 y dineína citoplasmática en células PC12. Los análisis por Western blot mostraron que las cantidades de las subunidades de kinesina KIF5 (cadena pesada y liviana) y dineína citoplasmática (cadena intermedia) presentes en las células PC12, no varían durante el período de diferenciación estudiado. Esto significa, que Tax no estaría alterando su expresión proteica. El tercer objetivo planteado fue comparar el grado de fosforilación de las subunidades de proteínas motoras en células expuestas a productos secretados desde células MT-2 con la condición control con medio de cultivo de la línea celular K562. Al no disponer de anticuerpos contra las formas fosforiladas de estas proteínas motoras, se procedió a separarlas mediante la técnica de electroforesis de geles en dos dimensiones. Este objetivo requirió inicialmente estandarizar esta técnica y luego se emplearon las muestras de lisados de PC12 provenientes del tercer día de diferenciación que habían sido sometidas al medio condicionado MT-2 y al de K562. Estos análisis no mostraron diferencias significativas en las migraciones electroforéticas, que podrían dar cuenta de cambios del estado de fosforilación de estas subunidades motoras. No obstante, estos resultados no se puede descartar que existan diferencias en el grado de fosforilación de las subunidades de estos motores moleculares, puesto que las diferencias en el grado de fosforilación pueden ser muy menores para ser observadas como cambios en la migración electroforética. Se debiera usar un método inmunológico más sensible empleando anticuerpos que reconozcan fosforilaciones determinadas estas proteínas / Effect of secretable proteins from HTLV-I infected lymphocytes on phosphorylation of motor proteins in PC12 cells as a model of neuronal culture HTLV-I virus (Human T-cell lymphotropic virus type I) is the etiologic agent of two main pathologies, the Myelopathy associated to HTLV-I/Tropical Spastic Paraparesis (HAM/TSP), a neurodegenerative axonopathy with impairment of axonal transport, and the Leukemia of adult T-cells (ATL). No infection in neurons has been detected, being unknown the mechanisms of the neurodegeneration in HAM/TSP. Lymphocytes T-CD4+ represent the main reservoir of the virus. Therefore, some secreted products from these infected lymphocytes infiltrated into the central nervous system could produce alteration of signal transduction pathways related with cytoskeletons and axonal transport, thus accounting for the distal to proximal axonal damage observed in HAM/TSP patients. Secreted products from MT-2 cells (HTLV-I infected lymphopcytes cell line) produce retraction in differentiated neuroblastoma SH-SY5Y cells and reduced the rate of neurite growing during PC12 cell differentiation to neuronal type. Among the viral secretable proteins is Tax, hence, the reduction of PC12 neurite growing could be mediated by this viral protein through transduction signalings that involves semaphorins and their receptor Plexins. As an example, in the case of Sema3A-Plexin A, an increase in Cdk5 activity causes Tau and CRMP-2 hyperphoshorylations. The failure of axonal transport could be explained by deregulation of phosphorylation of molecular motor subunits kinesin KIF5 and cytoplasmatic dynein Dync. In this work it was proposed to study the effect of secreted products, including Tax, from infected lymphocytes on the neuronal differentiation process. We employed the rat pheochromocytoma cell line, PC12 cells during NGF-differentiation to neuronal type. The Hypothesis stated expresses that: “Exposure to secreted products from HTLV-I infected lymphocytes causes an increase in the phosphorylation state of motor proteins in a neuronal model of PC12 cells” The first goal developed was to determine if Tax was one of the viral secreted products to the culture medium of MT-2 cell line responsible of retardation of PC12 neurite growing. Addition to the cell culture monoclonal antibodies anti-Tax blocked the effect on retardation rate of neurite growing, this pointed to the action of Tax present in the secretion medium of MT-2 cells. The second goal consisted in the standardization of selective protocols for immunodetection of subunits associated with the functional control of kinesin KIF5 and cytoplasmic dynein present in PC12 cells. Western blots analyses showed that levels of kinesin KiF5 (heavy and light chains) and cytoplasmic dynein (intermediate chain) present in PC12 cells did not change during the followed period of differentiation. This result suggests that their protein expression is not altered by Tax. The third goal included the comparison of phosphorylation levels of the subunits of the motor proteins exposed to MT-2 cell secreted products with the control condition with K562 cell line medium. Due to non-availability of the antibodies against phosphorylated forms of motor proteins, we separated them by two-dimensional gel electrophoresis. This goal initially required the standardization of the technique, and then PC12 lysates obtained at third day of differentiation in the presence of either MT-2 or K562 cell culture medium were used. These analyses did not show significant differences in the electrophoresis migrations of KiF5 and dynein that could account for changes in the phosphorylation levels of these subunits of motor proteins. In spite of our results, it cannot be discarded the existence of differences in the phosphorylation degree of the molecular motor subunits because the differences in the phosphorylation degree could be minors and undistinguishable by electrophoretic migration changes. Thereby, it should be used more sensitive immunological methods employing antibodies capable of distinguish motor proteins phosphorylations

Orientador: Jorge Vega / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-23T23:57:17Z (GMT). No. of bitstreams: 1 Goncalves_MarcosCesar_M.pdf: 5087409 bytes, checksum: 9ec4108068877391da39bff0cb2ca417 (MD5) Previous issue date: 1998 / Resumo: A síndrome do amarelecimento foliar da cana-de-açúcar constitui hoje uma preocupação mundial para a produtividade da cultura. Contudo, controvérsias ainda são levantadas sobre a sua origem e principalmente sobre a natureza do agente causal da moléstia. Visando contribuir para o estabelecimento da etiologia da doença, o presente trabalho apresenta a purificação do luteovirus identificado em plantas com sintomas e a tentativa de produção de um anti-soro policlonal para o estabelecimento de um método seguro de diagnóstico. As alterações na fisiologia da planta provocadas pelo patógeno, refletindo ou não em sintomas visíveis, foram estudadas por monitoramento da eficiência fotossintética e dos conteúdos de pigmentos fotossintetizantes e carboidratos fotoassimilados. O método de purificação avaliado ofereceu uma boa eficiência, sendo obtidas razões A260/A280=1, 8 e concentrações de 100µg/ml de partículas virais de formato isométrico de ca. 25nm. Os anti-soros policlonais produzidos, porém, não foram úteis para detecção serológica do vírus, sendo constatada uma forte reação cruzada com plantas sadias em testes PTA-ELISA, DASI-ELISA e "Tissue-Blotting". Foram encontradas reduções nos teores de pigmentos fotossintetizantes e na razão Cla/Clb, indicando alterações na estrutura e função dos cloroplastos em plantas infectadas. Ocorreu diminuição da atividade fotossintética, em termos da taxa de troca líquida de CO2, principalmente sob altos níveis de radiação fotossinteticamente ativa. As plantas infectadas também apresentaram fotoinibição e alterações em algumas etapas do transporte eletrônico no PSII durante o preenchimento do "pool" de PQ. Houve acúmulo de açúcares nas folhas de plantas infectadas, provavelmente devido à presença do vírus nas células do floema foliar. O açúcar de maior acúmulo foi a sacarose, devido à interferência no seu carregamento e/ou transporte no floema. Essas alterações nas relações fonte dreno aliadas à queda na eficiência fotossintética foram interpretadas como os principais responsáveis pela quebra de produção da cultura / Abstract: PARTIAL CHARACTERIZATION OF SUGARCANE LEAF YELLOWING ASSOCIATED LUTEOVIRUS AND PHYSIOPATHOLOGICAL ASPECTS OF THE HOST PLANT. The sugarcane yeIIow Ieaf disease has become a serlous problem in most sugarcane growing countries. In recent studies, Iuteovirus-like particIes present in the phIoem companion cells have been found in symptomatic paints (VEGA et al., 1997). This work was intended to obtain adequate amounts of purified particIes to immunize animaIs against the virus, aIong with additional studies for its characterization. Polyclonal antisera were obtained by injecting rabbits and chickens with purified virus. Physiological aspects of the infected plants were also studied by monitoring photosynthetic processes using chIorophyll fIuorescence and gas exchange analysis, changes in photosynthetic pigment levels and carbohydrate determinations in the Ieaves. The best purifications were obtained using a final centrifugation for 3 hours at 35000 rpm in density gradients of 5 and 10% sucrose, 5,10,15 and 20% CS2SO4 in a 10% sucrose solution. Concentration of particles were around 100µg/ml and the 260/280 ratio of 1,8. Absorbance spectra at UV, after fractionating, presented curves characteristic of polyhedral particles with peaks at 260nm. Electron microscopy of fractions with higher A260 showed a great number of isometric isometric particles of ca. 25nm. Antisera obtained did not offer a good tool for diagnosis, since cross-reaction against healthy plants were frequently found in PTA-ELISA, DASI-ELISA and Tissue-Blotting. Levels of chlorophylls a and b, and carotenoids were reduced in infected plants as well as the ratio chla/chlb. It seems likely that these alterations are partially responsible for the reductions found in net rates of gas exchanges, by the photoinhibition and modifications on redox state of primary and secondary electron acceptors of photosystem II, QA and QB, during the filling up of the plastoquinone pool , (PQ) . Accumulations of carbohydrates, especially sucrose, were found in the leaves. The presence of the virus in phloem cells is thought to impair the loading and/or transport of sugars in the phloem, inducing their accumulations in the leaves / Mestrado / Mestre em Ciências Biológicas