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Transformation by human adenovirus type 5 requires the cooperation of gene products from both the E1a and E1B early transcription units. Our major goal was to better understand the individual roles that the E1B proteins play in the transformation process. In order to determine the specific contribution made by the two major E1B proteins, 19K and 58K, mutants were constructed which were defective in the synthesis of each protein. Analysis with these mutants suggested that 58K appeared to be necessary for efficient plaque formation on human HeLa cells whereas 19K was not required. Mutants which failed to produce 19K or made a truncated 19K product displayed the cyt/deg phenotype characterized by production of large plaques and degradation of DNA These properties were not apparent with point mutants at methionine 120 or serine 164 of 19K or with mutants defective for 58K production. All E1B mutants produce E1A at levels comparable to wild type adenovirus 5, suggesting that neither E1B protein affects the regulation of E1A expression. Of interest was the observation that in combination with E1A, both 19K and 58K were able to induce transformation of baby rat kidney cells. However, the efficiency of transformation was greatly increased if both these E1B products were present. It seems likely that the mechanism of transformation involving each of these E1B proteins utilizes different pathways, but these pathways appear to be additive. / Thesis / Master of Science (MS)

Viruses infecting bacteria (phages) are the most abundant and ubiquitous entities on Earth and likely critical to any ecosystem, as they influence nutrient cycling, mortality and evolution. Ultimately, their impact depends on whether phage—host interactions lead to intracellular phage coexistence (temperate phage) or cell death (lytic phage). Temperate phages in the lysogenic cycle replicate their genome (either integrated into the host chromosome or extrachromosomally), until induced to become lytic, when they create and release progeny via cell lysis. While knowledge on lytic versus lysogenic outcomes is vast, it largely derives from few model systems that underrepresent natural diversity. Further, less is known about the efficiency of phage—host interactions and the regulation of optimal versus sub-optimal lytic infections, which are predicted as relevant under environmental (nutrients, temperature) and host (availability, density) conditions that are common in the ocean. In this dissertation I characterize the phage—host interactions in a new marine model system, phage ϕ38:1 and its Cellulophaga baltica bacterial host, member of the ubiquitous Bacteroidetes phylum. First, I show ϕ38:1’s ability to infect numerous, genetically similar strains of the C. baltica species, two of which display contrasting infection outcomes–lytic versus sub-optimally lytic or lysogenic on the original versus alternative hosts, respectively. Second, I collaboratively apply new gene marker-based approaches (phageFISH and geneELISA) to study ϕ38:1’s infection at the single-cell level and show that it is sub-optimal on the alternative host, rather than lysogenic. Third, I collaboratively develop whole-genome transcriptome datasets for ϕ38:1 infecting both, the optimal and sub-optimal hosts, to characterize the cellular response to infection and hypothesize potential transcriptional and post-transcriptional regulation of the sub-optimal infection. Together, these findings advance our knowledge of naturally-occurring phage—host interactions with a focus on nearly-unstudied sub-optimal infections.

Orientador: Helia Harumi Sato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-01T14:14:34Z (GMT). No. of bitstreams: 1 Ferro_LilianAparecida_D.pdf: 39343182 bytes, checksum: 85dd4ff7fee79e9940cf96d45fae535d (MD5) Previous issue date: 2002 / Resumo: Este trabalho objetivou o isolamento de microrganismos capazes de produzir enzimas que lisam a parede celular de leveduras, a produção, purificação e caracterização de 13-1,3-glucanase lítica. As bactérias líticas foram isoladas de Iodo da usina de açúcar e álcool Santa Helena, Piracicaba, SP. As bactérias líticas isoladas aderiram-se às células viáveis de Saccharomyces cerevisiae 701 e lisaram-nas. A linhagem YLM-B191-1, selecionada para o estudo, foi identificada através das características bioquímicas e fisiológicas como Cellulomonas cellulans. Para a produção da 13-1,3-glucanase lítica, a linhagem de C. cellulans YLM-B191-1 foi cultivada em meio composto de 15 9 de levedura seca; 2,0 9 de (NH4hSO4; 13,6 9 de KH2PO4; 4,2 9 de KOH; 0,2 9 de MgSO47H2O; 0,001 9 de Fe2(SO4h 6H2O; 1 mg de biotina e 1 mg de tiamina por litro. A 13-1,3-glucanase foi purificada do sobrenadante do meio de cultura através de ultrafiltração e cromatografia em coluna de CM-Sepharose CL-6B. A enzima purificada apresentou atividade ótima a 55°C e na faixa de pH entre 4,5 a 6,5. A 13-1,3- glucanase purificada apresentou estabilidade na faixa de pH 5,5 a 6,5 e foi inativada em temperaturas superiores a 55°C. A massa molecular da 13-1,3- glucanase purificada foi estimada em 17,1 kDa através de SDS-PAGE. A 13-1,3- glucanase purificada hidrolisou as ligações 13-1,3-glicosídicas da laminarina atuando como uma endoenzima. Através de microscopia eletrônica de varredura, observou- se que as enzimas líticas da linhagem de C. cellulans YLM-B191-1 foram capazes de alterar a superfície celular de leveduras / Abstract: The objective of this research was to isolate microorganisms which produced yeast cell walllytic enzymes and to study the production, purification and characterization of a Iytic ~-1,3-glucanase. The yeast-Iytic bacterium was isolated from the sludge of the Santa Helena sugar and alcohol factory in Piracicaba, SP. The isolated yeast-Iytic bacterium adhered to viable cells of Saccharomyces cerevisiae701 and Iysed them.The yeast-IyticbacteriumYLM-8191-1, selected for this study, was identified as Cellulomonas cellulans, from its biochemical and physiological characteristics.The strain C. cellulansYLM 8191-1 was cultivated in a medium containing (per liter) 2.0 9 of (NH4hSO4; 13.6 9 of KH2PO4;4.2 9 of KOH; 0.2 9 of MgSO47H2O; 0.001 9 of Fe2(SO4h6H2Oand 1 mg each of biotin and thiamin being supplemented with 15 9 of dried yeast as the carbon source for the production of ~-1,3-glucanase. The ~1 ,3-glucanase was purified from the culture fluid of C. cellulans YLM-8191-1 by ultrafiltration and CM-Sepharose CL-68 column chromatography. The purified enzyme showed greatest activity at 55°C and between pH 4.5 - 6.5. The purified ~-1,3-glucanase was stable in the range from pH 5.5 to 6.5 and was inactivated by heating at temperatures above 55°C. The molecular weight of purified ~-1,3-glucanasewas estimated at about 17.1 kDa by SDS-PAGE. The ~-1,3-glucanase hydrolyses the ~-1,3-glucosidic linkages of the laminarin acting as an endoenzyme. Scanning electron microscopy showed that Iytic enzymes from C. cellulans YLM-8191-1 were able to modify the cellular surface of yeast / Doutorado / Doutor em Ciência de Alimentos

Over the past few years, researchers have focused their attention on the development of targeted cancer therapies to minimize the side effects associated with non-targeted treatments such as chemotherapy. Specifically, these approaches have focused on blocking growth factor receptors (GFR) that are overexpressed in cancer cells. In this thesis, we also focus on targeting overexpressed GFR; however, instead of blocking the GFR, our novel approach aims at using them to selectively enhance the endocytotic process of macropinocytosis to deliver peptides that either disrupts the mitochondria or inhibits glycolysis. Herein, we show the selective enhancement of macropinocytosis by the fusion protein comprised of the keratinocyte growth factor (KGF) fused to elastin like polypeptide (ELP), KGF-ELP. Furthermore, we report the synthesis of the fusion protein consisting of mitochondriotoxic peptide (KLAKLAK)2 with ELP, (KLAKLAK)2-ELP. We show that (KLAKLAK)2-ELP forms nanoparticles (NPs) that are internalized via macropinocytosis and their internalization is facilitated by the interaction between the ELP domain and heparan sulfate proteoglycan (HSPG) on the cell surface. This internalization results in mitochondrial swelling, depolarization and subsequent cell death. Moreover, we show that heterogeneous NPs comprising of the two fusions KGF-ELP and (KLAKLAK)2-ELP selectively kill lung cancer cells expressing the keratinocyte growth factor receptor (KGFR). We also report the synthesis of the fusion consisting of peptides derived from a phosphorylated domain of the glycolytic enzyme phophoglycerate mutase (PGM) and ELP, PGM-ELP. We demonstrate that this fusion inhibits the step in glycolysis that converts 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG); the results show that cell death occurred preferentially in lung cancer cells compared to normal cells. Additionally, the heterogeneous NPs comprising of KGF-ELP and PGM-ELP selectively enhanced killing in lung cells with high levels KGFR. Finally, the synthesis of a fusion proteins consisting of four PGM domains fused to ELP, (PGM)4-ELP, exhibits higher cytotoxic effect and efficiency when compared to the single PGM domain fusion, PGM-ELP. Overall, we conclude that targeting overexpressed growth factor receptors to stimulate macropinocytosis can be a tremendously selective therapy for the treatment of lung cancer. This can result in attenuating side effects and improvement of the patient’s prognosis

An increasing appreciation for the role of small RNA regulators prompted us to investigate the scope of RNA regulation in the bacterium, Streptomyces coelicolor. Our search revealed an antisense RNA that corresponds to the upstream region of four genes encoding cell wall cleavage enzymes (cell wall hydrolases), and a previously uncharacterized population of transfer RNA (tRNA) cleavage products. Further characterization of the 'tRNAs led to the discovery that S. coelicolor tRNAs are cleaved into 'tRNA halves' in a developmentally regulated fashion. All tRNAs seem to be susceptible to tRNA cleavage, although a bias was detected for tRNAs specifying highly used codons. To date, our work is the sole description of 'tRNA half production in a bacterium, and recent studies suggest that it is a widespread phenomenon among eukaryotic organisms. In a separate line of investigation, we noticed that a previous study had predicted that the genes associated with the antisense RNA are under the control of a riboswitch- a regulatory RNA element that directly controls gene expression in response to specific conditions. Our multifaceted characterization of this system began with the construction and phenotypic analyses of deletion mutant strains for several of the cell wall hydrolase-encoding genes. We demonstrate that S. coelicolor cell wall hydrolases are involved in germination, vegetative growth, and sporulation. Finally, we studied the potential for riboswitch regulation of one of the cell wall hydrolase-encoding genes, rpfA. RpfA is a resuscitation: Qromoting factor protein that is important for the revival of dormant bacteria, including the human pathogen and S. coelicolor relative - Mycobacterium tuberculosis. Our investigation uncovered evidence suggesting that the riboswitch region is involved in the regulation of rpfA, and we identified specific conditions under which it is repressed. This work represents a novel paradigm in the regulation of cell wall hydrolase expression. / Thesis / Doctor of Philosophy (PhD)

Bacillus anthracis is a spore-forming bacterium that is included on the list of select agents compiled by the Centers for Disease Control. When a B. anthracis spore germinates, a protective layer of peptidoglycan known as the cortex must be depolymerized by germination-specific lytic enzymes (GSLEs) before the bacterium can become a metabolically active vegetative cell. By exploiting cortex lytic enzymes it may be possible to control germination. This could be beneficial in elucidating ways to enhance current decontamination methods. In this work we created in-frame deletion mutants to study not only the role of one GSLE, SleL, but by creating multi-deletion mutants, we were able to analyze how the protein cooperates with other lytic enzymes to efficiently hydrolyze the cortical PG. We determined that SleL plays an auxiliary role in complete peptidoglycan hydrolysis, secondary to cortex lytic enzymes CwlJ1, CwlJ2, and SleB. The loss of sleL results in a delay in the loss of optical density during germination. However, spores are capable of completing germination as long as CwlJ1 or SleB remains active. HPLC analysis of muropeptides collected from B. anthracis sleL strains indicates that SleL is an N-acetylglucosamidase that acts on cortical PG to produce small muropeptides which are quickly released from the germinating spore. By analyzing the in vitro and in vivo activities of SleL we confirmed the enzymatic activity of the protein, characterized its substrates, and studied the roles of its putative LysM domains in substrate binding and spore-protein association. We were able to show that purified SleL is capable of depolymerizing partially digested spore PG resulting in the production of N-acetylglucosaminidase products that are readily released as small muropeptides. In vitro, loss of the LysM domain(s) decreases hydrolysis effectiveness. The reduction in hydrolysis is likely due to LysM domains being involved in substrate recognition and PG binding. When the SleL derivatives are expressed in vivo those proteins lacking one or both LysM domains do not associate with the spore, suggesting that LysM is involved in directing protein localization. / Ph. D.

The Bacillus anthracis spore is highly resistant to environmental stresses, but cannot cause anthrax until it successfully germinates. An essential step of germination, degradation of the cortex peptidoglycan layer, is carried out by germination-specific lytic enzymes (GSLEs). While the GSLEs of several other Bacillus species have been investigated, they have not been characterized in the pathogen B. anthracis. In this work three GSLEs, CwlJ1, CwlJ2, and SleB are identified in B. anthracis and are investigated in order to better understand their functions. Genetic manipulation of cwlJ1, cwlJ2, and sleB was fundamental to this work. First, reporter gene fusions revealed that all three are expressed during spore formation and that CwlJ1 is likely the most abundant GSLE in the spore. Second, gene deletions eliminating each GSLE enabled the observation of mutant phenotypes during spore cortex degradation. CwlJ1 and SleB were identified as the most critical GSLEs for successful germination. High-performance liquid chromatography and mass spectroscopy revealed that SleB is required for lytic transglycosylase activity, but CwlJ1's mode of action was unclear. Multiple mutations of all of the GSLEs revealed that CwlJ2 is the least active GSLE, but that it participates in germination in response to Ca-Dipicolinic acid; a role it shares with the more dominant CwlJ1. Purification of the CwlJ1 and SleB proteins permitted in vitro assays of enzymatic activity as measured by changes in substrate optical density, solubility, and product formation. While CwlJ1 was recalcitrant to these methods, it was observed to cause cortex hydrolysis independently. SleB was more amenable and it was discovered to contain a peptidoglycan-binding domain that is primarily responsible for substrate binding, and a lytic transglycosylase domain that facilitates cortex-specific hydrolysis by recognizing muramic-δ-lactam. Future research will include determining the structure of SleB through x-ray crystallography and the identification of CwlJ1 activity by refining the protein purification method. The results of this and future research into CwlJ1, CwlJ2, and SleB may lead to a means to initiate spore germination prior to host infection. This will greatly ease spore decontamination measures, lower risk of infection, and discourage the use of B. anthracis spores as a biological weapon. / Ph. D.

Les cellules de mélanome humain expriment différents antigènes tumoraux qui sont reconnus par les lymphocytes T cytotoxiques CD8 + (CTL) induisant des réponses spécifiques de la tumeur in vivo. Cependant, chez les patients atteints de mélanome l'efficacité de la réponse naturelle des CTL ou stimulée par thérapie est limitée. Les mécanismes sous-jacents de l'échec de la phase effectrice des CTL contre les mélanomes sont encore largement méconnus. Notre hypothèse est que l'efficacité limitée des CTL dans leur combat contre les tumeurs est le résultat d'une balance défavorable entre la capacité des CTL à tuer les tumeurs et une résistance tumorale intrinsèque à l'activité cytolytique des CTL. Au cours de ma thèse je me suis concentrée sur la dynamique moléculaire qui se produit à la synapse lytique afin de pouvoir identifier un mécanisme précoce mis en place par les cellules de mélanome face à l'attaque des CTL. En combinant l'utilisation d'approches de microscopie de pointe et des outils moléculaires, j'ai pu montrer que, lors de l'interaction avec les CTL, les cellules de mélanome humain subissent une activation de leur trafic vésiculaire endosomal et lysosomal, lequel est intensifié à la synapse lytique et corrèle avec la dégradation par la cathepsine de la perforine et un défaut de pénétration d'entrée du granzyme B. De plus, j'ai démontré que le blocage du trafic lysosomal dépendant de SNAP23, la modification du pH (intra-vésiculaire) et l'inhibition de l'activité lysosomale protéotlytique des cellules de mélanome permet de restaurer leur sensibilité à l'attaque des CTL. Nos résultats révèlent une stratégie sans précédent d' " auto-défense " des cellules de mélanome à la synapse immunologique basée sur une sécrétion lysosomale massive et sur la dégradation de la perforine sécrétée par les CTL. Ainsi pouvoir interférer avec cette stratégie synaptique d'auto-défense des cellules de mélanome pourrait contribuer à potentialiser les réponses des CTL et les immunothérapies chez les patients atteints de mélanome. / Human melanoma cells express various tumor antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL) and elicit tumor-specific responses in vivo. However, natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying the failure of CTL effector phase against melanomas are still largely elusive. Our hypothesis is that the limited efficacy of CTL in their fight against tumors is the result of an unfavorable balance between CTL ability to kill tumors and an intrinsic tumor resistance to CTL cytolytic activity. During my thesis I focused on the molecular dynamics occurring at the lytic synapse in order to identify possible "early response-mechanism" of melanoma cells to CTL attack. Using a combination of cutting edge microscopy approaches and molecular tools, I showed that upon conjugation with CTL, human melanoma cells undergo an exacerbated late endosome/lysosome trafficking, which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking, pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Our results reveal an unprecedented strategy of melanoma cell "self-defense" at the immunologic synapse based on a lysosome secretory burst and perforin degradation at the lytic synapse. Interfering with this synaptic self-defense strategy might be instrumental to potentiate CTL-mediated therapies in melanoma patients.