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• 1. [Article] Growth Phase-Dependent Gene Regulation in Vivo in Sulfolobus Solfataricus

  Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene ...

  Citation × Citation Citation Abstract Copy Title: Growth Phase-Dependent Gene Regulation in Vivo in Sulfolobus Solfataricus Author: DeYoung, Melissa, Thayer, Michelle, Van der Oost, John, Stedman, Kenneth M. Year: 2011 Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene regulation in Archaea. SSV1- based lacS reporter gene constructs containing the Sulfolobus 16S/23S rRNA gene core promoter, the TF55a core promoter, or the native lacS promoter were tested in Sulfolobus solfataricus cells lacking the lacS gene. The 42-bp 16S/23S rRNA gene and 39-bp TF55a core promoters are sufficient for gene expression in S. solfataricus. However, only gene expression driven by the 16S/23S rRNA gene core promoter is dependent on the culture growth phase. This is the smallest known regulated promoter in Sulfolobus. To our knowledge, this is the first study to show growth phase-dependent rRNA gene regulation in Archaea. Title: Growth Phase-Dependent Gene Regulation in Vivo in Sulfolobus Solfataricus Author: DeYoung, Melissa, Thayer, Michelle, Van der Oost, John, Stedman, Kenneth M. Year: 2011 Ribosomal genes are strongly regulated dependent on growth phase in all organisms, but this regulation is poorly understood in Archaea. Moreover, very little is known about growth phase-dependent gene regulation in Archaea. SSV1- based lacS reporter gene constructs containing the Sulfolobus 16S/23S rRNA gene core promoter, the TF55a core promoter, or the native lacS promoter were tested in Sulfolobus solfataricus cells lacking the lacS gene. The 42-bp 16S/23S rRNA gene and 39-bp TF55a core promoters are sufficient for gene expression in S. solfataricus. However, only gene expression driven by the 16S/23S rRNA gene core promoter is dependent on the culture growth phase. This is the smallest known regulated promoter in Sulfolobus. To our knowledge, this is the first study to show growth phase-dependent rRNA gene regulation in Archaea. Close

• 2. [Article] Coxiella burnetii and Leishmania Mexicana Residing Within Similar Parasitophorous Vacuoles Elicit Disparate Host Responses

  Coxiella burnetii is a bacterium that thrives in an acidic parasitophorous vacuole (PV) derived from lysosomes. Leishmania mexicana, a eukaryote, has also independently evolved to live in a morphologically ...

  Citation × Citation Citation Abstract Copy Title: Coxiella burnetii and Leishmania Mexicana Residing Within Similar Parasitophorous Vacuoles Elicit Disparate Host Responses Author: Millar, Jess A., Valdés, Raquel, Kacharia, Fenil, Landfear, Scott M., Cambronne, Eric D., Raghavan, Rahul, Soong, Lynn, Malchiodi, Emilio Luis Year: 2015 Coxiella burnetii is a bacterium that thrives in an acidic parasitophorous vacuole (PV) derived from lysosomes. Leishmania mexicana, a eukaryote, has also independently evolved to live in a morphologically similar PV. As Coxiella and Leishmania are highly divergent organisms that cause different diseases, we reasoned that their respective infections would likely elicit distinct host responses despite producing phenotypically similar parasite-containing vacuoles. The objective of this study was to investigate, at the molecular level, the macrophage response to each pathogen. Infection of THP-1 (human monocyte/macrophage) cells with Coxiella and Leishmania elicited disparate host responses. At 5 days post-infection, when compared to uninfected cells, 1057 genes were differentially expressed (746 genes up-regulated and 311 genes downregulated) in C. burnetii infected cells, whereas 698 genes (534 genes up-regulated and 164 genes down-regulated) were differentially expressed in L. mexicana infected cells. Interestingly, of the 1755 differentially expressed genes identified in this study, only 126 genes (∼7%) are common to both infections. We also discovered that 1090 genes produced mRNA isoforms at significantly different levels under the two infection conditions, suggesting that alternate proteins encoded by the same gene might have important roles in host response to each infection. Additionally, we detected 257 micro RNAs (miRNAs) that were expressed in THP-1 cells, and identified miRNAs that were specifically expressed during Coxiella or Leishmania infections. Collectively, this study identified host mRNAs and miRNAs that were influenced by Coxiella and/or Leishmania infections, and our data indicate that although their PVs are morphologically similar, Coxiella and Leishmania have evolved different strategies that perturb distinct host processes to create and thrive within their respective intracellular niches. Title: Coxiella burnetii and Leishmania Mexicana Residing Within Similar Parasitophorous Vacuoles Elicit Disparate Host Responses Author: Millar, Jess A., Valdés, Raquel, Kacharia, Fenil, Landfear, Scott M., Cambronne, Eric D., Raghavan, Rahul, Soong, Lynn, Malchiodi, Emilio Luis Year: 2015 Coxiella burnetii is a bacterium that thrives in an acidic parasitophorous vacuole (PV) derived from lysosomes. Leishmania mexicana, a eukaryote, has also independently evolved to live in a morphologically similar PV. As Coxiella and Leishmania are highly divergent organisms that cause different diseases, we reasoned that their respective infections would likely elicit distinct host responses despite producing phenotypically similar parasite-containing vacuoles. The objective of this study was to investigate, at the molecular level, the macrophage response to each pathogen. Infection of THP-1 (human monocyte/macrophage) cells with Coxiella and Leishmania elicited disparate host responses. At 5 days post-infection, when compared to uninfected cells, 1057 genes were differentially expressed (746 genes up-regulated and 311 genes downregulated) in C. burnetii infected cells, whereas 698 genes (534 genes up-regulated and 164 genes down-regulated) were differentially expressed in L. mexicana infected cells. Interestingly, of the 1755 differentially expressed genes identified in this study, only 126 genes (∼7%) are common to both infections. We also discovered that 1090 genes produced mRNA isoforms at significantly different levels under the two infection conditions, suggesting that alternate proteins encoded by the same gene might have important roles in host response to each infection. Additionally, we detected 257 micro RNAs (miRNAs) that were expressed in THP-1 cells, and identified miRNAs that were specifically expressed during Coxiella or Leishmania infections. Collectively, this study identified host mRNAs and miRNAs that were influenced by Coxiella and/or Leishmania infections, and our data indicate that although their PVs are morphologically similar, Coxiella and Leishmania have evolved different strategies that perturb distinct host processes to create and thrive within their respective intracellular niches. Close

• 3. [Article] The regulation of alternative splicing associated with Myotonic Dystrophy

  xiv, 78 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number.

  Citation × Citation Citation Abstract Copy Title: The regulation of alternative splicing associated with Myotonic Dystrophy Author: Warf, Michael Bryan Year: 2010, 2009 xiv, 78 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. Title: The regulation of alternative splicing associated with Myotonic Dystrophy Author: Warf, Michael Bryan Year: 2010, 2009 xiv, 78 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. Close

• 4. [Article] Control of alkane monooxygenase activity and expression in Pseudomonas butanovora

  The regulation of monooxygenase enzymes has been demonstrated in bacteria that grow on methane and long chain-length alkanes (>C10). Less is known about monooxygenase regulation in short chain alkane ...

  Citation × Citation Citation Abstract Copy Title: Control of alkane monooxygenase activity and expression in Pseudomonas butanovora Author: Doughty, David M. The regulation of monooxygenase enzymes has been demonstrated in bacteria that grow on methane and long chain-length alkanes (>C10). Less is known about monooxygenase regulation in short chain alkane oxidizing bacteria that grow on intermediate chain-length alkanes C2-C10. This dissertation focuses on the regulation of butane monooxygenase (BMO) expression and activity that occurred in response to alcohols, aldehydes, organic acids and O₂, in a short chain-length alkane oxidizing bacterium, Pseudomonas butanovora. Propionate and butyrate were able to inhibit and inactivate BMO while lactate and acetate had no effect on BMO activity. Inactivation of BMO by propionate was both time- and O₂-dependent, suggesting that the catalytic cycle of BMO is involved in the mechanism of propionate inactivation. Since treatment with [2-¹⁴C] propionate did not label BMO polypeptides, propionate inactivation of BMO does not follow the model of the suicide substrate, acetylene. Propane and butane were strikingly dissimilar in their ability to induce BMO in lactate-grown cells. Further studies revealed that BMO is induced by the primary alcohols (C2-C8) that are intermediate products in alkane metabolism, rather than by the alkane substrates, and that BMO is repressed by propionate, a product of odd but not even alkane metabolism. Interestingly, propionate repression of BMO was released following the induction of propionate consumption in P. butanovora suggesting that BMO expression, and alkane oxidation, are closely coupled to the ability to consume the products formed via monooxygenase activity. Because O₂ is a necessary cosubstrate for BMO catalytic turnover, and yet alcohol inducers of BMO are often formed under anoxic conditions, I hypothesized that the functional expression of BMO should be linked to the cell's ability to sense O₂. Induction of the BMO promoter was repressed during denitrification under anoxic growth on 1-butanol, and during incubation with 1-butanol at low O₂ concentrations provided that Cu was supplied in the media. These data suggest that Cu is involved in the mechanism to prevent BMO expression under conditions unfavorable for enzyme function. I propose a model in which Cu is involved in a redox linked mechanism to prevent alcohol induced BMO expression under anoxic conditions. Title: Control of alkane monooxygenase activity and expression in Pseudomonas butanovora Author: Doughty, David M. The regulation of monooxygenase enzymes has been demonstrated in bacteria that grow on methane and long chain-length alkanes (>C10). Less is known about monooxygenase regulation in short chain alkane oxidizing bacteria that grow on intermediate chain-length alkanes C2-C10. This dissertation focuses on the regulation of butane monooxygenase (BMO) expression and activity that occurred in response to alcohols, aldehydes, organic acids and O₂, in a short chain-length alkane oxidizing bacterium, Pseudomonas butanovora. Propionate and butyrate were able to inhibit and inactivate BMO while lactate and acetate had no effect on BMO activity. Inactivation of BMO by propionate was both time- and O₂-dependent, suggesting that the catalytic cycle of BMO is involved in the mechanism of propionate inactivation. Since treatment with [2-¹⁴C] propionate did not label BMO polypeptides, propionate inactivation of BMO does not follow the model of the suicide substrate, acetylene. Propane and butane were strikingly dissimilar in their ability to induce BMO in lactate-grown cells. Further studies revealed that BMO is induced by the primary alcohols (C2-C8) that are intermediate products in alkane metabolism, rather than by the alkane substrates, and that BMO is repressed by propionate, a product of odd but not even alkane metabolism. Interestingly, propionate repression of BMO was released following the induction of propionate consumption in P. butanovora suggesting that BMO expression, and alkane oxidation, are closely coupled to the ability to consume the products formed via monooxygenase activity. Because O₂ is a necessary cosubstrate for BMO catalytic turnover, and yet alcohol inducers of BMO are often formed under anoxic conditions, I hypothesized that the functional expression of BMO should be linked to the cell's ability to sense O₂. Induction of the BMO promoter was repressed during denitrification under anoxic growth on 1-butanol, and during incubation with 1-butanol at low O₂ concentrations provided that Cu was supplied in the media. These data suggest that Cu is involved in the mechanism to prevent BMO expression under conditions unfavorable for enzyme function. I propose a model in which Cu is involved in a redox linked mechanism to prevent alcohol induced BMO expression under anoxic conditions. Close

• 5. [Article] Chemical Screening and Antibacterial Testing of Fungal Endophytes

  Endophytes are bacteria or fungi that live within a host plant. The plant host provides a home for endophytes, and in return endophytes produce primary and secondary metabolites, of which many of the secondary ...

  Citation × Citation Citation Abstract Copy Title: Chemical Screening and Antibacterial Testing of Fungal Endophytes Author: Pham, Andrew Endophytes are bacteria or fungi that live within a host plant. The plant host provides a home for endophytes, and in return endophytes produce primary and secondary metabolites, of which many of the secondary metabolites possess biological activities. When microorganisms are grown under various media conditions, they produce different natural products as gene regulation is affected by environmental clues. Biologically active natural products are of huge biomedical interest as they can improve human health: over 50% of FDA approved clinically used drugs are natural products or natural products derived. We have been working with endophytic fungi isolated from marine algae. We tested their metabolic expression of natural products under various culture conditions and tested their extracts against common human pathogens like Bacillius subtilis, P. aeruginosa, Candida albicans and Staphlacoccus aureus. This chemical screening allows us to select strains for further purification of the secondary fungal metabolites as drug leads. The use of High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Mass Spectrometry (LCMS) and Nuclear Magnetic Resonance (NMR) allows us to first purify a compound, determine its molecular weight and then elucidate its chemical structure. Title: Chemical Screening and Antibacterial Testing of Fungal Endophytes Author: Pham, Andrew Endophytes are bacteria or fungi that live within a host plant. The plant host provides a home for endophytes, and in return endophytes produce primary and secondary metabolites, of which many of the secondary metabolites possess biological activities. When microorganisms are grown under various media conditions, they produce different natural products as gene regulation is affected by environmental clues. Biologically active natural products are of huge biomedical interest as they can improve human health: over 50% of FDA approved clinically used drugs are natural products or natural products derived. We have been working with endophytic fungi isolated from marine algae. We tested their metabolic expression of natural products under various culture conditions and tested their extracts against common human pathogens like Bacillius subtilis, P. aeruginosa, Candida albicans and Staphlacoccus aureus. This chemical screening allows us to select strains for further purification of the secondary fungal metabolites as drug leads. The use of High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Mass Spectrometry (LCMS) and Nuclear Magnetic Resonance (NMR) allows us to first purify a compound, determine its molecular weight and then elucidate its chemical structure. Close

• 6. [Article] Cullin-3 Targets Cyclin E for Ubiquitination and Controls S Phase in Mammalian Cells

  Cyclin E is an unstable protein that is degraded in a ubiquitin- and proteasome- dependent pathway. Two factors stimulate cyclin E ubiquitination in vivo: when it is free of its CDK partner, and when it ...

  Citation × Citation Citation Abstract Copy Title: Cullin-3 Targets Cyclin E for Ubiquitination and Controls S Phase in Mammalian Cells Author: Singer, Jeffrey D., Gurian-West, Mark, Clurman, Bruce, Roberts, James M. Year: 1999 Cyclin E is an unstable protein that is degraded in a ubiquitin- and proteasome- dependent pathway. Two factors stimulate cyclin E ubiquitination in vivo: when it is free of its CDK partner, and when it is phosphorylated on threonine 380. We pursued the first of these pathways by using a two-hybrid screen to identify proteins that could bind only to free cyclin E. This resulted in the isolation of human Cul-3, a member of the cullin family of E3 ubiquitin–protein ligases. We found that Cul-3 was bound to cyclin E but not to cyclin E-Cdk2 complexes in mammalian cells, and that overexpression of Cul-3 increased ubiquitination of cyclin E but not other cyclins. Conversely, deletion of the Cul-3 gene in mice caused increased accumulation of cyclin E protein, and had cell-type-specific effects on S-phase regulation. In the extraembryonic ectoderm, in which cells undergo a standard mitotic cycle, there was a greatly increased number of cells in S phase. In the trophectoderm, in which cells go through endocycles, there was a block to entry into S phase. The SCF pathway, which targets cyclins for ubiquitination on the basis of their phosphorylation state, and the Cul-3 pathway, which selects cyclin E for ubiquitination on the basis of its assembly into CDK complexes, may be complementary ways to control cyclin abundance. Title: Cullin-3 Targets Cyclin E for Ubiquitination and Controls S Phase in Mammalian Cells Author: Singer, Jeffrey D., Gurian-West, Mark, Clurman, Bruce, Roberts, James M. Year: 1999 Cyclin E is an unstable protein that is degraded in a ubiquitin- and proteasome- dependent pathway. Two factors stimulate cyclin E ubiquitination in vivo: when it is free of its CDK partner, and when it is phosphorylated on threonine 380. We pursued the first of these pathways by using a two-hybrid screen to identify proteins that could bind only to free cyclin E. This resulted in the isolation of human Cul-3, a member of the cullin family of E3 ubiquitin–protein ligases. We found that Cul-3 was bound to cyclin E but not to cyclin E-Cdk2 complexes in mammalian cells, and that overexpression of Cul-3 increased ubiquitination of cyclin E but not other cyclins. Conversely, deletion of the Cul-3 gene in mice caused increased accumulation of cyclin E protein, and had cell-type-specific effects on S-phase regulation. In the extraembryonic ectoderm, in which cells undergo a standard mitotic cycle, there was a greatly increased number of cells in S phase. In the trophectoderm, in which cells go through endocycles, there was a block to entry into S phase. The SCF pathway, which targets cyclins for ubiquitination on the basis of their phosphorylation state, and the Cul-3 pathway, which selects cyclin E for ubiquitination on the basis of its assembly into CDK complexes, may be complementary ways to control cyclin abundance. Close

• 7. [Article] Mechanisms for the interaction of environmental mycobacteria with host cells

  Environmental mycobacteria are important opportunistic pathogens for many hosts, including humans, cattle, and fish. Two well-studied species are Mycobacterium avium subsp. avium, a significant cause of ...

  Citation × Citation Citation Abstract Copy Title: Mechanisms for the interaction of environmental mycobacteria with host cells Author: Harriff, Melanie J. Environmental mycobacteria are important opportunistic pathogens for many hosts, including humans, cattle, and fish. Two well-studied species are Mycobacterium avium subsp. avium, a significant cause of disseminated bacterial disease in patients with AIDS, and Mycobacterium avium subsp. paratuberculosis, the cause of Johne’s disease in cattle. Many other species that are considerable sources of infections in fish, such as Mycobacterium chelonae and Mycobacterium marinum, also have zoonotic potential. To gain knowledge about the invasion of epithelial cells by environmental mycobacteria, selected genes and proteins involved in the uptake of M. avium by HEp-2 cells were analyzed by a variety of methods. Two transcriptional regulators (MAV_3679 and MAV_5138) were identified as being involved in invasion. A mycobacterial protein (CipA) with an amino acid sequence suggestive of an ability to be a part of the scaffolding complex that forms during cell signaling leading to actin polymerization was found to putatively interact with host cell Cdc42. Fusion of CipA to GFP, expressed in Mycobacterium smegmatis, revealed that CipA localizes to a structure on the surface of bacteria approaching HEp-2 cells. To establish whether species of environmental mycobacteria isolated from different hosts use similar mechanisms to M. avium for interaction with the mucosa, and for survival in macrophages, assays to determine invasion and replication were performed in different cell types, and a custom DNA microarray containing probes for known mycobacterial virulence determinants was developed. Bacteria cultured from macrophages indicated differences between the ability of M. avium and other environmental species to replicate in human, mouse, and carp cell lines. Hybridization of genomic DNA isolated from EM species against the sequenced MAC104 strain of M. avium showed that there are genes and regions of the chromosome absent from a subset of those species that may be important in determining host specificity and the in differences in virulence observed in vitro and in vivo. Finally, the intestinal epithelium was determined to be the primary route of infection for mycobacteria in zebrafish, similar to M. avium infection in humans. M. avium does not infect zebrafish, but the comparative virulence and genomics of some species isolated from fish suggest that they could be used as a surrogate model to continue progress in understanding the interaction between environmental mycobacteria and epithelial cells. Title: Mechanisms for the interaction of environmental mycobacteria with host cells Author: Harriff, Melanie J. Environmental mycobacteria are important opportunistic pathogens for many hosts, including humans, cattle, and fish. Two well-studied species are Mycobacterium avium subsp. avium, a significant cause of disseminated bacterial disease in patients with AIDS, and Mycobacterium avium subsp. paratuberculosis, the cause of Johne’s disease in cattle. Many other species that are considerable sources of infections in fish, such as Mycobacterium chelonae and Mycobacterium marinum, also have zoonotic potential. To gain knowledge about the invasion of epithelial cells by environmental mycobacteria, selected genes and proteins involved in the uptake of M. avium by HEp-2 cells were analyzed by a variety of methods. Two transcriptional regulators (MAV_3679 and MAV_5138) were identified as being involved in invasion. A mycobacterial protein (CipA) with an amino acid sequence suggestive of an ability to be a part of the scaffolding complex that forms during cell signaling leading to actin polymerization was found to putatively interact with host cell Cdc42. Fusion of CipA to GFP, expressed in Mycobacterium smegmatis, revealed that CipA localizes to a structure on the surface of bacteria approaching HEp-2 cells. To establish whether species of environmental mycobacteria isolated from different hosts use similar mechanisms to M. avium for interaction with the mucosa, and for survival in macrophages, assays to determine invasion and replication were performed in different cell types, and a custom DNA microarray containing probes for known mycobacterial virulence determinants was developed. Bacteria cultured from macrophages indicated differences between the ability of M. avium and other environmental species to replicate in human, mouse, and carp cell lines. Hybridization of genomic DNA isolated from EM species against the sequenced MAC104 strain of M. avium showed that there are genes and regions of the chromosome absent from a subset of those species that may be important in determining host specificity and the in differences in virulence observed in vitro and in vivo. Finally, the intestinal epithelium was determined to be the primary route of infection for mycobacteria in zebrafish, similar to M. avium infection in humans. M. avium does not infect zebrafish, but the comparative virulence and genomics of some species isolated from fish suggest that they could be used as a surrogate model to continue progress in understanding the interaction between environmental mycobacteria and epithelial cells. Close