Background: Foamy viruses (FVs) have developed a unique budding strategy within the retrovirus family. FV release requires co-expression and a highly specific interaction between capsid (Gag) and glycoprotein (Env), which cannot be complemented by heterologous Env proteins. The interaction domain in FV Env has been mapped in greater detail and resides mainly in the N-terminal tip of the cytoplasmic domain of the Env leader peptide subunit. In contrast, the corresponding domain within Gag is less well defined. Previous investigations suggest that it is located within the N-terminal part of the protein. Results: Here we characterized additional Gag interaction determinants of the prototype FV (PFV) isolate using a combination of particle release, GST pull-down and single cycle infectivity analysis assays. Our results demonstrate that a minimal PFV Gag protein comprising the N-terminal 129 aa was released into the supernatant, whereas proteins lacking this domain failed to do so. Fine mapping of domains within the N-terminus of PFV Gag revealed that the N-terminal 10 aa of PFV Gag were dispensable for viral replication. In contrast, larger deletions or structurally deleterious point mutations in C-terminally adjacent sequences predicted to harbor a helical region abolished particle egress and Gag – Env protein interaction. Pull-down assays, using proteins of mammalian and prokaryotic origin, support the previous hypothesis of a direct interaction of both PFV proteins without requirement for cellular cofactors and suggest a potential direct contact of Env through this N-terminal Gag domain. Furthermore, analysis of point mutants within this domain in context of PFV vector particles indicates additional particle release-independent functions for this structure in viral replication by directly affecting virion infectivity. Conclusions: Thus, our results demonstrate not only a critical function of an N-terminal PFV Gag motif for the essential capsid - glycoprotein interaction required for virus budding but also point out additional functions that affect virion infectivity.

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No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Cl?udio_Queiroz_TESE.pdf: 3430647 bytes, checksum: cd4fc13e7f5364c58653d2f2c6808b34 (MD5) Previous issue date: 2005 / As epilepsias do lobo temporal s?o as que apresentam maior refratariedade ao tratamento farmacol?gico e perfazem 2/3 das interven??es cir?rgicas de epilepsia, sendo portanto de grande custo social, econ?mico e psicol?gico. Assim, modelos animais de epilepsia do lobo temporal s?o de grande relev?ncia n?o s? para o entendimento das bases neurais dessa patologia, mas tamb?m para o desenvolvimento de abordagens terap?uticas capazes de evitar a instala??o da doen?a. Esses s?o os objetivos da presente disserta??o de doutorado. Ap?s um evento traum?tico (no caso deste trabalho, um estado de mal epil?ptico), diversas altera??es morfol?gicas e fisiol?gicas acontecem, caracterizando a g?nese da s?ndrome epil?ptico (epileptog?nese). Dentre as altera??es podemos destacar a intensa fosforila??o de prote?nas em res?duos de tirosina e a ativa??o de diferentes segundos mensageiros. Os dois primeiros cap?tulos desta tese descrevem a tentativa de bloquear os processos de epileptog?nese por meio da inibi??o da fosforila??o de res?duos de tirosina atrav?s do tratamento farmacol?gico com inibidores das tirosina cinases, a herbimicina A e o K-252a. O terceiro cap?tulo analisa eletrofisiologicamente o circuito neural do giro denteado em animais que apresentavam uma muta??o em um s?tio inibit?rio da prote?na c?lcio/calmodulina cinase do tipo II (CaMKII). No primeiro cap?tulo, mostramos que o tratamento agudo com herbimicina A (348?M, 5?L, icv), ? capaz de bloquear a potencia??o duradoura (LTP) induzida por um est?mulo tet?nico bem como de atenuar (~40%) a ativa??o neuronal (express?o de c-Fos) decorrente de um estado de mal epil?ptico induzido pela administra??o sist?mica de pilocarpina (SE). Apesar dos significativos efeitos agudos, este tratamento mostrou-se incapaz de atenuar a freq??ncia de crises espont?neas, bem como o padr?o de morte neuronal observado ap?s o estado de mal epil?ptico induzido pela pilocarpina. Entretanto, o tratamento com herbimicina A alterou o padr?o de marca??o de metais pesados (Zn+2) no hilo do giro denteado e na regi?o de CA3 do hipocampo, por?m n?o apresentou efeito sobre o padr?o de brotamento das fibras musgosas observado na camada molecular do giro denteado. No segundo cap?tulo, mostramos que a herbimicina e o K- 252a modificam a atividade epileptiforme induzida pela administra??o intra-hipocampal de ?cido ca?nico, sem alterar o padr?o de morte neuronal. Esses resultados sugerem que o tratamento com inibidores de prote?nas tirosina cinases ? capaz de modificar o padr?o de ativa??o agudo do hipocampo ap?s um est?mulo (i.e., o estado de mal epil?ptico ou a LTP), por?m sem qualquer efeito sobre o processo de epileptog?nese. No terceiro cap?tulo, estudamos a excitabilidade e a plasticidade do giro denteado ? estimula??o da via perfurante (principal afer?ncia da forma??o hipocampal) em animais que apresentam uma CaMKII geneticamente modificada. Essa prote?na uma vez ativada n?o pode ser inibida. A caracteriza??o eletrofisiol?gica demonstrou que esses animais apresentam potenciais de campo evocados no giro denteado aparentemente semelhante aos animais controle (wild-type), por?m sua responsividade a padr?es de estimula??o em salvas e sua plasticidade apresentaram clara altera??o. Essa modifica??o foi caracterizada por uma maior variabilidade nas respostas ? trens de estimula??o (freq??ncias de 1 e 2 Hz) e maior inibi??o do pulso pareado em trens de estimula??o (para pulsos pareados aplicados a freq??ncia de 5 Hz). Al?m disso, conforme j? descrito na literatura, mostramos que a susceptibilidade a atividade epileptiforme depende do padr?o de estimula??o utilizado para os diferentes animais (mutantes vs. wild-type). Assim, utilizando o modelo cl?ssico do abrasamento demonstramos que a muta??o n?o altera a evolu??o da epileptog?nese. Entretanto, ao utilizarmos duas variantes de um padr?o de estimula??o similar ? freq??ncia teta (5Hz, Intermittent vs. Continuous theta-burst stimulations), demonstramos a import?ncia da muta??o na manuten??o da excitabilidade do giro denteado. Esses resultados destacam a import?ncia da CaMKII na atividade epileptiforme al?m de sugerir novas abordagens experimentais (i.e., sensibilidade ? padr?es de estimula??o eletrofisiol?gica) no estudo da epileptog?nese. Em resumo, os resultados apresentados nessa tese contribuem para um melhor entendimento dos fen?menos subjacentes aos processos de plasticidade neuronal e da contribui??o destes para o fen?meno de epileptog?nese, al?m de sugerir / Temporal lobe epilepsies are highly refractory to pharmacological treatment. Up to 70% of these patients undergo chirurgical resection of temporal region, procedure with important consequences for the social, economic and psychological spheres. Experimental animal models that mimic temporal lobe epilepsy provide an insightful approach to study the neural basis of epilepsy as well as create opportunities to test promising therapeutic drugs. The present thesis tests the antiepileptogenic activity of two protein tyrosine kinase inhibitors and the relevance of Ca+2/calmodulin kinase type II (CaMKII) mutants. Multiples morphological and physiological alterations take place after a traumatic brain injury (in this thesis, the status epilepticus) leading the animal to an epileptic conditions. During this period, the epileptogenesis process, there is strong tyrsine phosphorylation with the activation of many second messengers. The first two chapters of the thesis describe experiments in which herbimycin A and K-252a, two protein tyrosine kinase inhibitors, were used to attenuate synaptic plasticity and epileptogenesis. The third chapter, the dentate gyrus network was studied after angular bundle stimulation in animals presenting one punctual mutation at the autoinhibitory phosphorylation site of the CaMKII. In the first chapter, we showed that one single herbimycin A injection (348?M, 5?L, icv) was able to attenuate long-term potentiation (LTP) in the commissural CA3 neurons and also, to decrease status epilepticus- (SE-) induced neuronal activation (c-Fos expression) in almost 40%. Although markedly acute effects, the present herbimycin A treatment was not able to diminish spontaneous seizure frequency, cell death or aberrant mossy fiber sprouting observed after the pilocarpine-induced SE. Curiously, herbimycin-treated animals presented decreased neo-Timm staining in the hilus and CA3 region despite the epileptic condition. In the second chapter, we confirmed the ability of protein tyrosine kinase inhibitors to decrease SE-induced neuronal activation. Herbimycin A icv treatment altered the kainic acid-induced epileptiform profile in EEG recordings. Cell death pattern was not altered by any pharmacological treatment. These results suggest that protein tyrosine kinase inhibitiors are able to modify the acute neuronal activation and plasticity (ictogenesis or LTP) but is ineffective in attenuating the epileptogenesis process. In the third chapter, we studied the dentate gyrus excitability and plasticity after angular bundle stimulation in CaMKII mutant animals. Once in its self-sustained mode, this mutation does not allow the reduction of the catalytic activity of the kinase. These animals present normal electrophysiological profiles (similar to wild-type animals) but with reduced amplitude. Shortterm plasticity was clearly altered. Mutant animals presented increased variability in the responses to trains of stimulation at 1 and 2 Hz, and at at 5Hz stronger paired-pulse inhibition. Accordingly to the literature, we also showed that the epileptiform susceptibility depends on the stimulation pattern used in both animals (mutants vs. wild-type). Thus, although the mutation did not altered the behavior and the electrographic kindling evolution, we showed that mutant animals were prone to afterdischarges when stimulate by an intermittent theta-burst stimulation. On the other hand, the same animals needed more bursts to induce afterdischarges when the stimulation was set in the continuos mode. Taken together, the present results contribute to a better understanding of the protein tyrosine kinase and CaMKII function in neuronal plasticity underlying the epileptogenesis process and sum efforts in searching for a clinic antiepileptogenic drug.

A identificação histopatológica dos agentes fúngicos é um método excelente de diagnóstico, devido ao fato de que as estruturas são facilmente identificadas por meio das técnicas histoquímicas. Até o momento, não dispõem-se de métodos aceitáveis para quantificação da atividade da infecção. Esta investigação protocola um método de estimativa para atividade biológica da criptococose através da determinação dos índice de brotamentos e carminofílico do Cryptococcus. Objetivos: Descrever os aspectos histopatológicos da criptococose através das técnicas histoquímicas básicas e especiais da micologia. Métodos: Foram avaliados 33 pacientes com diagnóstico histopatológico prévio da criptococose. Resultados: Houve predominância do sexo masculino. A idade variou entre 10 a 81 anos, com média de 45,6 anos. A criptococose é doença definidora dos casos de Aids, sendo considerado seu principal fator predisponente, seguido de transplantes. O trato respiratório é o mais envolvido. O microrganismos tem tropismo para o sistema nervoso central e apresenta disseminação para outros órgãos. Resultados falso-negativos é reflexo da deficiência de material capsular. A mortalidade foi de 36%, sendo o maior índice obervado até os 3 primeiros meses após o diagnóstico. A criptococose apresenta-se sob duas formas, reativa e paucireativa. Na infecção reativa, os organismos foram menos abundantes e predominantemente intracelulares aos histiócitos e às células gigantes. Na infecção paucireativa, há um grande número de leveduras, que proliferam-se extracelularmente e estão associadas a destruição do tecido afetado. Ambos infecções, reativas e paucireativas mostraram grande variação no índice de brotamento. O índice carminofílico foi menor nas infecção reativas, quando comparados a infecção paucireativa. Discussão: A coloração de Hematoxilina-Eosina é usada para visualizar as alterações estruturais das lesões, bem como da reação tecidual. A coloração da prata é a mais utilizada para identificação dos organismos fúngicos como o Cryptococcus. A coloração de Mucicarmim de Mayer detecta a cápsula mucopolissacarídica circundante corada na cor magenta. A coloração de FM oferece diagnóstico diferencial nos casos inconclusivos à coloração de Mucicarmim de Mayer. A quantificação de IB e IC é uma escala útil na interpretação da resposta inflamatória do hospedeiro e atividade biológica da criptococose. / The histopathologic identification from the fungal agents that's a method excellent of diagnostic, due the fact of what the structures são easily identified for histochemical techniques. So far, there is no methods you accepted about to measurement of biologic activity. This investigation aponta to an method of estimate of the activity biologic of cryptococcal infection by determination of the Budding Index and Carminophilic Index of the Cryptococcus. Objectives: Describes the histologic features of cryptococcosis by basic and special histochemical techniques of mycology. Methods: Have been evaluated 33 patients with previous cryptococcosis histopathologic diagnostic. Results: There is an predominance of the males. The age of the patients ranged from 10 to 81 years with a median value 45,4 years. The HIV infection was the main risk factor for disease, followg of transplants. The respiratory tract is the most frequently involved among the organ systems organs. The false-negative latex test are due to capsular deficiency. The moratlity rate was 36%, The high rate of 50% was observed between of 1 to 3 months. The cryptococcal infections is divided into two major histologic categories, reactive and paucireactive, based upon the host reaction. In reactive infection, the organisms were lessa abundant and were predominantly intracellular within histiocytes and giant cells. In paucireactive infection, thre is large numbers of yeats in the lesions, cryptococci proliferate extracellarly within the involved tissues, associated histologically with mucoid degeneration of the surrounding tissue. Both reactice and paucireactive infections showed great variation in Budding Index. The Carminophilic Inded was lower in the reactive infections, when compared with the paucireactive infection. In this Carminophilic Index presented higher measurements.. Discussion: The Hematoxilin-Eosin stain is used to look for strucutural changes of the infectd lesion, as well tissues reactions. The Gomori’s methenamine-silver stain is the more commonly used in identificatifying these organisms. The mucicarmine stain detecting the surrounding mucopolisacharides capsule of the magenta color. The Fontana-Masson staining offers differential diagnostic. The determination of IB and IC is an scale that relied, because it provides an interpretation of the host response and biological activity of the cryptoccosis.

A identificação histopatológica dos agentes fúngicos é um método excelente de diagnóstico, devido ao fato de que as estruturas são facilmente identificadas por meio das técnicas histoquímicas. Até o momento, não dispõem-se de métodos aceitáveis para quantificação da atividade da infecção. Esta investigação protocola um método de estimativa para atividade biológica da criptococose através da determinação dos índice de brotamentos e carminofílico do Cryptococcus. Objetivos: Descrever os aspectos histopatológicos da criptococose através das técnicas histoquímicas básicas e especiais da micologia. Métodos: Foram avaliados 33 pacientes com diagnóstico histopatológico prévio da criptococose. Resultados: Houve predominância do sexo masculino. A idade variou entre 10 a 81 anos, com média de 45,6 anos. A criptococose é doença definidora dos casos de Aids, sendo considerado seu principal fator predisponente, seguido de transplantes. O trato respiratório é o mais envolvido. O microrganismos tem tropismo para o sistema nervoso central e apresenta disseminação para outros órgãos. Resultados falso-negativos é reflexo da deficiência de material capsular. A mortalidade foi de 36%, sendo o maior índice obervado até os 3 primeiros meses após o diagnóstico. A criptococose apresenta-se sob duas formas, reativa e paucireativa. Na infecção reativa, os organismos foram menos abundantes e predominantemente intracelulares aos histiócitos e às células gigantes. Na infecção paucireativa, há um grande número de leveduras, que proliferam-se extracelularmente e estão associadas a destruição do tecido afetado. Ambos infecções, reativas e paucireativas mostraram grande variação no índice de brotamento. O índice carminofílico foi menor nas infecção reativas, quando comparados a infecção paucireativa. Discussão: A coloração de Hematoxilina-Eosina é usada para visualizar as alterações estruturais das lesões, bem como da reação tecidual. A coloração da prata é a mais utilizada para identificação dos organismos fúngicos como o Cryptococcus. A coloração de Mucicarmim de Mayer detecta a cápsula mucopolissacarídica circundante corada na cor magenta. A coloração de FM oferece diagnóstico diferencial nos casos inconclusivos à coloração de Mucicarmim de Mayer. A quantificação de IB e IC é uma escala útil na interpretação da resposta inflamatória do hospedeiro e atividade biológica da criptococose. / The histopathologic identification from the fungal agents that's a method excellent of diagnostic, due the fact of what the structures são easily identified for histochemical techniques. So far, there is no methods you accepted about to measurement of biologic activity. This investigation aponta to an method of estimate of the activity biologic of cryptococcal infection by determination of the Budding Index and Carminophilic Index of the Cryptococcus. Objectives: Describes the histologic features of cryptococcosis by basic and special histochemical techniques of mycology. Methods: Have been evaluated 33 patients with previous cryptococcosis histopathologic diagnostic. Results: There is an predominance of the males. The age of the patients ranged from 10 to 81 years with a median value 45,4 years. The HIV infection was the main risk factor for disease, followg of transplants. The respiratory tract is the most frequently involved among the organ systems organs. The false-negative latex test are due to capsular deficiency. The moratlity rate was 36%, The high rate of 50% was observed between of 1 to 3 months. The cryptococcal infections is divided into two major histologic categories, reactive and paucireactive, based upon the host reaction. In reactive infection, the organisms were lessa abundant and were predominantly intracellular within histiocytes and giant cells. In paucireactive infection, thre is large numbers of yeats in the lesions, cryptococci proliferate extracellarly within the involved tissues, associated histologically with mucoid degeneration of the surrounding tissue. Both reactice and paucireactive infections showed great variation in Budding Index. The Carminophilic Inded was lower in the reactive infections, when compared with the paucireactive infection. In this Carminophilic Index presented higher measurements.. Discussion: The Hematoxilin-Eosin stain is used to look for strucutural changes of the infectd lesion, as well tissues reactions. The Gomori’s methenamine-silver stain is the more commonly used in identificatifying these organisms. The mucicarmine stain detecting the surrounding mucopolisacharides capsule of the magenta color. The Fontana-Masson staining offers differential diagnostic. The determination of IB and IC is an scale that relied, because it provides an interpretation of the host response and biological activity of the cryptoccosis.

Among the various external signals perceived by yeast cells, nutrient availability is a condition to which these cells show a highly diverse biological response. Diploid cells in response to different nutritional stress conditions shows different developmental outcomes. On nitrogen starvation, cells undergo dimorphic transition whereby a unicellular yeast form transforms to a multicellular pseudohyphal form. While in the complete absence of a nitrogen source and a fermentable carbon source, yeast cells enter into a complex developmental program termed sporulation which culminates in haploid spores. The main objective of this work was to understand the role played by S. cerevisiaeSUB1 in starvation-induced meiotic program of diploid cells, decipher its target in sporulation specific gene expression cascade, study the domain architecture of Sub1 and examine its functional homology to mammalian PC4. Role of Sub1 in induction of sporulation and other stress responses in S. cerevisiae In a previous whole-genome screen for mutants with altered sporulation efficiency in the Saccharomyces cerevisiae S288c strain, SUB1 locus was identified as a negative regulator of sporulation (Deutschbaueret al., 2002). Moreover, genome-wide gene expression analysis in SK1 strain had shown that SUB1 transcript levels are repressed during sporulation (Chu et al., 1998). Many studies in different yeast strain backgrounds implicate more than 1,000 genesout of 6,200 genes in yeast genome as being differentially expressed during the sporulation process (Chu et al., 1998; Primiget al., 2000; Deutschbaueret al., 2002). Interestingly, these studies show the number of regulatory genes that negatively affect sporulation is far lower than those that are activators of sporulation and moreover their mechanism of action is poorly studied. S. cerevisiae.SUB1 is one among negative regulators of sporulation(Deutschbaueret al., 2002). Global transcriptome of diploid yeast cells undergoing sporulation showed SUB1 transcripts are greatly reduced with time progression (Chu et al., 1998). To understand the role of SUB1 in sporulation, we generated deletion of both SUB1 alleles in the diploid S288c strain background and compared the kinetics of asci formation in this strain with that of the wild-type. We observed that cells lacking SUB1 exhibit ~5-fold increase in tetrad asci. Based on Eosin Y and Calcoflour White staining assays, we find no change in spore morphology in the mutant. Thus the increase in sporulation efficiency in sub1/sub1diploids is not accompanied by formation of defective spores. We validated the reduction in SUB1 transcript levels during sporulation in wild-type SK1 strain background. We also examined the Sub1 protein levels by epitope-tagging of the chromosomal SUB1 open reading frame and determining protein levels in this strain. We find that consistent with the data on transcript levels, Sub1-TAP tagged protein levels too decreased gradually on shift to sporulation medium. We created sub1alleles in diploids in the SK1 strain background and using this strain background we investigated Sub1 target genes and chose IME2 (early), SMK1, SPS2 (middle), DIT1, DIT2 (mid-late) and SPS100 (late) genes as representative sporulation genes. We observed that sub1∆/sub1∆cells have a significantly elevated expression of middle genes (SPS2 and SMK1) that followed normal induction kinetics i.e., 5 hours post transfer to sporulation medium. However, the expression levels or timing for other class of sporulation genes did not change in sub1∆strain as compared with the wild-type. In order to confirm these observations, we also studied the effects of over-expression of SUB1 from the GAL1 promoter by transforming the high copy plasmid. This was done in wild-type SK1 cells and the expression of sporulation genes were analyzed. We observed that expression of SMK1 and SPS2middle sporulation genes was reduced on over-expression of SUB1.We used the Sub1-TAP protein to assess if Sub1 directly regulates these genes by Chromatin immunoprecipitation assays. For these studies, we examined the recruitment of Sub1 to these loci through the time course of sporulation. In wild-type SK1 cells, Sub1 was to bound to middle sporulation genes and this was striking in cells at 5th hour post-induction of sporulation. These data establish that Sub1 directly associates with chromatin at these loci co-incident with the time points where expression levels of these changes is altered in cells lacking Sub1. Furthermore, to assess the role of Sub1 in other stress responses, such as pseudohyphae formation in response to nitrogen starvation, pheromone-induced agar invasion and secretory stress, we employed a genetic approach. Genetic interaction studies of SUB1 with RPB4, a subunit of RNA polymerase with functions in stress response and HOS2, a subunit of Set3 complex and a close homolog of mammalian HDAC3, reported to be involved in sporulation and secretory stress, were performed. Based on sporulation frequency and pseudohyphal formation in the double mutants we conclude that SUB1 is downstream of both these genes. Moreover, our results from assays of schmoo formation and pheromone-induced agar invasion suggest that SUB1 functionally interacts with HOS2. Study of domain architecture of Sub1 and homology to human PC4 Comparison of the S. cerevisiae Sub1 protein with its higher eukaryotic homologs showed that the N-terminal region of yeast Sub1 (32-105 aa) is highly conserved (Knauset al., 1996; Henry et al., 1996) with the 106-292 C -terminal amino acids being yeast-specific. We employed deletion analysis to generate partial Sub1 proteins and used them to understand the roles played by these domains in different phenotypes associated with Sub1. Our analysis of the localization of various Sub1-GFP fusion proteins shows that 146-172 aa in the C-terminal domain of Sub1 confers nuclear localization. Sporulation frequency analysis of the different domains of Sub1 suggests that both the N and C terminal domains are necessary for sporulation function of Sub1. The N terminal domain of yeast Sub1 shares homology with human PC4 and not surprisingly possesses ssDNA binding ability first attributed to human PC4 (Kaiser et al., 1995). In order to investigate whether the effects of SUB1 on kinetics of sporulation require its ssDNA binding function, we generated the sub1(Y66A) ssDNA binding mutant (Sikorskiet al., 2011) and over-expressed it in the S288c genetic background. We assessed sporulation efficiency of sub1∆/sub1∆cells over-expressing sub1(Y66A) mutant allele as compared to cells over-expressing wild-type SUB1. Interestingly, cells with over-expression of sub1(Y66A) have reduced sporulation efficiency that is equivalent to the levels achieved on over-expression of wild type SUB1. This data suggests that the ssDNA-binding ability of Sub1 is not important for its role in sporulation. Furthermore, we examined the ability of human PC4 to contribute to yeast sporulation process by complementation analysis. We observed that over-expression of PC4 complemented the phenotypes of sub1∆strain, suggesting that the function of Sub1/PC4 family is evolutionarily conserved. Studies on biochemical interactions of Sub1 with histone proteins Human PC4 is a chromatin-associated protein, present on metaphase chromosomes (Das et al., 2006). The short C-terminal domain of PC4(62-87 aa) interacts with core histones H3 and H2B in vitro and in vivo and this interaction mediates chromatin condensation. The homology between S. cerevisiaeSub1 (32-105 aa) and human PC4 (62-127 aa)is in the domain required for their DNA binding properties and coactivator functions, suggesting possible conservation in their interactions. We tested the interactions of yeast Sub1 with histone proteins by adopting both in vitro and in vivo interaction assays. We find recombinant Sub1 had strong interactions with rat and yeast histone H3in vitro. Moreover,Sub1 was found to interact with histone H2B, but not with H2A, in vivo, a binding specificity also shown by human PC4.Thus, we demonstrate conservation in the interaction of Sub1 with histone proteins.

Background: Foamy viruses (FVs) have developed a unique budding strategy within the retrovirus family. FV release requires co-expression and a highly specific interaction between capsid (Gag) and glycoprotein (Env), which cannot be complemented by heterologous Env proteins. The interaction domain in FV Env has been mapped in greater detail and resides mainly in the N-terminal tip of the cytoplasmic domain of the Env leader peptide subunit. In contrast, the corresponding domain within Gag is less well defined. Previous investigations suggest that it is located within the N-terminal part of the protein. Results: Here we characterized additional Gag interaction determinants of the prototype FV (PFV) isolate using a combination of particle release, GST pull-down and single cycle infectivity analysis assays. Our results demonstrate that a minimal PFV Gag protein comprising the N-terminal 129 aa was released into the supernatant, whereas proteins lacking this domain failed to do so. Fine mapping of domains within the N-terminus of PFV Gag revealed that the N-terminal 10 aa of PFV Gag were dispensable for viral replication. In contrast, larger deletions or structurally deleterious point mutations in C-terminally adjacent sequences predicted to harbor a helical region abolished particle egress and Gag – Env protein interaction. Pull-down assays, using proteins of mammalian and prokaryotic origin, support the previous hypothesis of a direct interaction of both PFV proteins without requirement for cellular cofactors and suggest a potential direct contact of Env through this N-terminal Gag domain. Furthermore, analysis of point mutants within this domain in context of PFV vector particles indicates additional particle release-independent functions for this structure in viral replication by directly affecting virion infectivity. Conclusions: Thus, our results demonstrate not only a critical function of an N-terminal PFV Gag motif for the essential capsid - glycoprotein interaction required for virus budding but also point out additional functions that affect virion infectivity.

El presente trabajo de investigación tiene el propósito de indagar en la relevancia de las startups como agentes dinamizadores para el desarrollo de las economías incipientes. Por tal razón, se busca determinar el valor útil de las startups en el desarrollo financiero de las economías incipientes a través de la investigación bibliográfica de artículos académicos. En nuestro estudio, revisamos algunas definiciones y alcances de las startups. Asimismo, determinamos cuáles son las principales características de las economías incipientes y explicamos los principales aportes de las startups. Además, determinamos la relevancia financiera e impacto de las startups en este tipo de economías. El alcance de nuestro estudio aborda el avance del uso de la tecnología y la innovación donde se observa un importante crecimiento de las startups como alternativas de negocio. En los últimos años, las startups han desarrollado modelos comerciales para la resolución de problemas sociales. No obstante, las economías incipientes enfrentan algunos desafíos a estos cambios, debido a insuficientes políticas de gobierno, falta de apoyo empresarial, entre otros, para la creación de startups. El presente trabajo contribuye con la limitada literatura actual sobre el rol de las startups en las economías en desarrollo y abre un debate sobre el impacto real de las startups en el desarrollo económico de los países. Finalmente, se propone que futuras investigaciones profundicen más en esta discusión y con ello construir un marco teórico que brinde valor a la toma de decisiones de futuros emprendedores. Otra contribución es aportar información relevante a los responsables de las políticas gubernamentales para impulsar el emprendimiento en las economías incipientes. / This research work has the purpose of investigating the relevance of startups as dynamic agents for the development of incipient economies. For this reason, we seek to determine the useful value of startups in the financial development of incipient economies through bibliographic research of academic articles. In our study, we review some definitions and scopes of startups. We also determine the main characteristics of incipient economies and explain the main contributions of startups. In addition, we determine the financial relevance and impact of startups in these types of economies. The scope of our study addresses the advancement of the use of technology and innovation where an important growth of startups as business alternatives is observed. In recent years, startups have developed business models for solving social problems. However, incipient economies face some challenges to these changes, due to insufficient government policies, lack of business support, among others, for the creation of startups. This paper contributes to the limited current literature on the role of startups in developing economies and opens a debate on the real impact of startups on the economic development of countries. Finally, it is proposed that future research deepen this discussion and thereby build a theoretical framework that provides value to the decision-making of future entrepreneurs. Another contribution is to provide relevant information to those responsible for government policies to promote entrepreneurship in incipient economies. / Trabajo de Suficiencia Profesional

<p dir="ltr">The Ebola virus (EBOV) is a filamentous RNA virus which causes severe hemorrhagic fever. It is one of the most dangerous known pathogens with a high fatality rate. Multiple outbreaks of EBOV have occurred since the 1970s with the most widespread outbreak starting in December 2013. This outbreak continued through May of 2016 and had a fatality rate of approximately 50%. EBOV outbreaks are recurrent because the virus is still present in animal reservoirs. Despite multiple EBOV outbreaks we still lack a clear understanding of how new viral particles are formed and spread through virus assembly and release. Given the widespread global travel, EBOV now poses a threat to the entire world. EBOV encodes for the matrix protein, VP40, which is one of the most conserved viral proteins. VP40 can form different structures leading to different functions of the protein in different stages of the EBOV life cycle. The VP40 dimer traffics to the inner leaflet of the plasma membrane to facilitate assembly and budding. The VP40 octameric ring has been implicated in transcriptional regulation. This thesis focuses on understanding in further detail the determinates of VP40 plasma membrane assembly and exit from an infected cell.</p><p dir="ltr">The assembly and trafficking of VP40 to the plasma membrane requires a network of protein-protein and lipid-protein interactions (PPIs and LPIs). Studying these interfaces is important for understanding how VP40 structure and function regulates trafficking and assembly and can shed light on therapeutic strategies to target EBOV. The alteration of host cell Ca²⁺ levels is one of the strategies that viruses use to perturb the host cell signaling transduction mechanism in their favor. Evidence has emerged demonstrating that Ca²⁺ is important for the assembly and budding of EBOV in a VP40-dependent manner. The relationship between intracellular Ca²⁺ levels and EBOV matrix protein VP40 function is still unknown. In this work we utilize biophysical techniques to study the role of LPIs and intracellular Ca²⁺ on VP40 dynamics at the plasma membrane and key residues for assembly and budding. This work highlights the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding and a critical interaction between Ca²⁺ and the VP40 dimer that are important for lipid binding at the plasma membrane.</p>

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