DNA-mediated oxidation of transcription factor p53

Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs with a shallow distance dependence over long distances through the π-stacked DNA bases, leading to the oxidation and dissociation of DNA-bound p53. The extent of p53 dissociation depends upon the redox potential of the response element DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using radiolabeled oligonucleotides containing both synthetic and human p53 response elements with an appended anthraquinone photooxidant. Greater p53 dissociation is observed from DNA sequences containing low redox potential purine regions, particularly guanine triplets, within the p53 response element. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites, which correspond to locations of preferred electron hole localization, were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets within the response element. Oxidative DNA damage is inhibited in the presence of p53, however, only at DNA sites within the response element, and therefore in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress, as well as possible biological implications, have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA-mediated oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell.

To determine whether the change in p53 response element occupancy observed in vitro also correlates in cellulo, chromatin immunoprecipition (ChIP) and quantitative PCR (qPCR) were used to directly quantify p53 binding to certain response elements in HCT116N cells. The HCT116N cells containing a wild type p53 were treated with the photooxidant [Rh(phi)2bpy]³⁺, Nutlin-3 to upregulate p53, and subsequently irradiated to induce oxidative genomic stress. To covalently tether p53 interacting with DNA, the cells were fixed with disuccinimidyl glutarate and formaldehyde. The nuclei of the harvested cells were isolated, sonicated, and immunoprecipitated using magnetic beads conjugated with a monoclonal p53 antibody. The purified immounoprecipiated DNA was then quantified via qPCR and genomic sequencing. Overall, the ChIP results were significantly varied over ten experimental trials, but one trend is observed overall: greater variation of p53 occupancy is observed in response elements from which oxidative dissociation would be expected, while significantly less change in p53 occupancy occurs for response elements from which oxidative dissociation would not be anticipated.

The chemical oxidation of transcription factor p53 via DNA CT was also investigated with respect to the protein at the amino acid level. Transcription factor p53 plays a critical role in the cellular response to stress stimuli, which may be modulated through the redox modulation of conserved cysteine residues within the DNA-binding domain. Residues within p53 that enable oxidative dissociation are herein investigated. Of the 8 mutants studied by electrophoretic mobility shift assay (EMSA), only the C275S mutation significantly decreased the protein affinity (KD) for the Gadd45 response element. EMSA assays of p53 oxidative dissociation promoted by photoexcitation of anthraquinone-tethered Gadd45 oligonucleotides were used to determine the influence of p53 mutations on oxidative dissociation; mutation to C275S severely attenuates oxidative dissociation while C277S substantially attenuates dissociation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide labeled, while oxidized cysteines participating in disulfide bonds were ¹³C₂D₂-iodoacetamide labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed using a QTRAP 6500 LC-MS/MS system, quantified with Skyline, and directly compared. A distinct shift in peptide labeling toward ¹³C₂D₂-iodoacetamide labeled cysteines is observed in oxidized samples as compared to the respective controls. All of the observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds potentially among the C124, C135, C141, C182, C275, and C277. Based on these data it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.

Constraining the interpretation of 2-methylhopanoids through genetic and phylogenetic methods

Hopanoids are a class of sterol-like lipids produced by select bacteria. Their preservation in the rock record for billions of years as fossilized hopanes lends them geological significance. Much of the structural diversity present in this class of molecules, which likely underpins important biological functions, is lost during fossilization. Yet, one type of modification that persists during preservation is methylation at C-2. The resulting 2-methylhopanoids are prominent molecular fossils and have an intriguing pattern over time, exhibiting increases in abundance associated with Ocean Anoxic Events during the Phanerozoic. This thesis uses diverse methods to address what the presence of 2-methylhopanes tells us about the microbial life and environmental conditions of their ancient depositional settings. Through an environmental survey of hpnP, the gene encoding the C-2 hopanoid methylase, we found that many different taxa are capable of producing 2-methylhopanoids in more diverse modern environments than expected. This study also revealed that hpnP is significantly overrepresented in organisms that are plant symbionts, in environments associated with plants, and with metabolisms that support plant-microbe interactions; collectively, these correlations provide a clue about the biological importance of 2-methylhopanoids. Phylogenetic reconstruction of the evolutionary history of hpnP revealed that 2-methylhopanoid production arose in the Alphaproteobacteria, indicating that the origin of these molecules is younger than originally thought. Additionally, we took genetic approach to understand the role of 2-methylhopanoids in Cyanobacteria using the filamentous symbiotic Nostoc punctiforme. We found that hopanoids likely aid in rigidifying the cell membrane but do not appear to provide resistance to osmotic or outer membrane stressors, as has been shown in other organisms. The work presented in this thesis supports previous findings that 2-methylhopanoids are not biomarkers for oxygenic photosynthesis and provides new insights by defining their distribution in modern environments, identifying their evolutionary origin, and investigating their role in Cyanobacteria. These efforts in modern settings aid the formation of a robust interpretation of 2-methylhopanes in the rock record.

Diet and condition of American Alligators (Alligator mississippiensis)in three central Florida lakes

Understanding the diet of crocodilians is important because diet affects condition, behavior, growth, and reproduction. By examining the diet of crocodilians, valuable knowledge is gained about predator-prey interactions and prey utilization among habitats. In this study, I examined the diet and condition of adult American alligators (Alligator mississippiensis) in three central Florida lakes, Griffin, Apopka, and Woodruff. Two hundred adult alligators were captured and lavaged from March through October 2001, from April through October 2002, and from April through August 2003. Alligators ate a variety of vertebrate and invertebrate prey, but vertebrates were more abundant and fish dominated alligator diets in the lakes. Species composition of fish varied among the lakes. The majority of the diet of alligators from Lakes Apopka and Woodruff was fish, 90% and 84% respectively. Lake Apopka alligators consumed a significantly (P = 0.006) higher proportion of fish in their diet. Fish were 54% of the diet of Lake Griffin alligators and the infrequent occurrence of reptiles, mammals, birds, and amphibians often resulted in a large biomass. Differences in alligator diets among lakes may be due to differences in sample size (higher numbers of samples from Lake Griffin), prey availability, habitat, prey vulnerability, or prey size. Alligator condition (Fulton’s Condition Factor, K) was significantly (P < 0.001) different among the lakes. Alligators from Lake Apopka had the highest condition, followed by those from Lake Griffin, and alligators from Lake Woodruff had the lowest condition. Composition of fish along with diversity and equitability of fish in alligator diets may have contributed to differences in condition among lakes. Condition was probably also due to factors other than diet such as alligator hunting behavior, alligator density, or year-round optimal temperature that prolongs feeding. The observed diet and condition differences probably reflect both habitat differences and prey availability in these three lakes.

Alligator Diet in Relation to Alligator Mortality on Lake Griffin, FL

Alligator mississippiensis (American Alligators) demonstrated low hatchrate success and increased adult mortality on Lake Griffin, FL, between 1998 and 2003. Dying Lake Griffin alligators with symptoms of poor motor coordination were reported to show specific neurological impairment and brain lesions. Similar lesions were documented in salmonines that consumed clupeids with high thiaminase levels. Therefore, we investigated the diet of Lake Griffin alligators and compared it with alligator diets from two lakes that exhibited relatively low levels of unexplained alligator mortality to see if consumption of Dorosoma cepedianum (gizzard shad) could be correlated with patterns of mortality. Shad in both lakes Griffin and Apopka had high levels of thiaminase and Lake Apopka alligators were consuming greater amounts of shad relative to Lake Griffin without showing mortality rates similar to Lake Griffin alligators. Therefore, a relationship between shad consumption alone and alligator mortality is not supported.

Herpetofaunal Inventories of the National Parks of South Florida and the Caribbean: Volume III. Big Cypress National Preserve

Amphibian declines and extinctions have been documented around the world, often in protected natural areas. Concern for this trend has prompted the U.S. Geological Survey and the National Park Service to document all species of amphibians that occur within U.S. National Parks and to search for any signs that amphibians may be declining. This study, an inventory of amphibian species in Big Cypress National Preserve, was conducted from 2002 to 2003. The goals of the project were to create a georeferenced inventory of amphibian species, use new analytical techniques to estimate proportion of sites occupied by each species, look for any signs of amphibian decline (missing species, disease, die-offs, and so forth.), and to establish a protocol that could be used for future monitoring efforts. Several sampling methods were used to accomplish these goals. Visual encounter surveys and anuran vocalization surveys were conducted in all habitats throughout the park to estimate the proportion of sites or proportion of area occupied (PAO) by each amphibian species in each habitat. Opportunistic collections, as well as limited drift fence data, were used to augment the visual encounter methods for highly aquatic or cryptic species. A total of 545 visits to 104 sites were conducted for standard sampling alone, and 2,358 individual amphibians and 374 reptiles were encountered. Data analysis was conducted in program PRESENCE to provide PAO estimates for each of the anuran species. All of the amphibian species historically found in Big Cypress National Preserve were detected during this project. At least one individual of each of the four salamander species was captured during sampling. Each of the anuran species in the preserve was adequately sampled using standard herpetological sampling methods, and PAO estimates were produced for each species of anuran by habitat. This information serves as an indicator of habitat associations of the species and relative abundance of sites occupied, but it will also be useful as a comparative baseline for future monitoring efforts. In addition to sampling for amphibians, all encounters with reptiles were documented. The sampling methods used for detecting amphibians are also appropriate for many reptile species. These reptile locations are included in this report, but the number of reptile observations was not sufficient to estimate PAO for reptile species. We encountered 35 of the 46 species of reptiles believed to be present in Big Cypress National Preserve during this study, and evidence exists of the presence of four other reptile species in the Preserve. This study found no evidence of amphibian decline in Big Cypress National Preserve. Although no evidence of decline was observed, several threats to amphibians were identified. Introduced species, especially the Cuban treefrog (Osteopilus septentrionalis), are predators and competitors with several native frog species. The recreational use of off-road vehicles has the potential to affect some amphibian populations, and a study on those potential impacts is currently underway. Also, interference by humans with the natural hydrologic cycle of south Florida has the potential to alter the amphibian community. Continued monitoring of the amphibian species in Big Cypress National Preserve is recommended. The methods used in this study were adequate to produce reliable estimates of the proportion of sites occupied by most anuran species, and are a cost-effective means of determining the status of their populations.

Photonic and Device Design Principles for Ultrahigh-Efficiency (>50%), Spectrum-Splitting Photovoltaics

The sun has the potential to power the Earth's total energy needs, but electricity from solar power still constitutes an extremely small fraction of our power generation because of its high cost relative to traditional energy sources. Therefore, the cost of solar must be reduced to realize a more sustainable future. This can be achieved by significantly increasing the efficiency of modules that convert solar radiation to electricity. In this thesis, we consider several strategies to improve the device and photonic design of solar modules to achieve record, ultrahigh (> 50%) solar module efficiencies. First, we investigate the potential of a new passivation treatment, trioctylphosphine sulfide, to increase the performance of small GaAs solar cells for cheaper and more durable modules. We show that small cells (mm2), which currently have a significant efficiency decrease (~ 5%) compared to larger cells (cm2) because small cells have a higher fraction of recombination-active surface from the sidewalls, can achieve significantly higher efficiencies with effective passivation of the sidewalls. We experimentally validate the passivation qualities of treatment by trioctylphosphine sulfide (TOP:S) through four independent studies and show that this facile treatment can enable efficient small devices. Then, we discuss our efforts toward the design and prototyping of a spectrum-splitting module that employs optical elements to divide the incident spectrum into different color bands, which allows for higher efficiencies than traditional methods. We present a design, the polyhedral specular reflector, that has the potential for > 50% module efficiencies even with realistic losses from combined optics, cell, and electrical models. Prototyping efforts of one of these designs using glass concentrators yields an optical module whose combined spectrum-splitting and concentration should correspond to a record module efficiency of 42%. Finally, we consider how the manipulation of radiatively emitted photons from subcells in multijunction architectures can be used to achieve even higher efficiencies than previously thought, inspiring both optimization of incident and radiatively emitted photons for future high efficiency designs. In this thesis work, we explore novel device and photonic designs that represent a significant departure from current solar cell manufacturing techniques and ultimately show the potential for much higher solar cell efficiencies.

Eu é um outro: manifestações do duplo em Luiz Alfredo Garcia-Roza

Verificamos na ficção brasileira contemporânea uma reescritura do gênero policial, que deixa de ter como principal meta a retirada das máscaras que encobrem a verdade. Nesta era de ambiguidades e incertezas, em que a verdade passa a ser substituída pelas versões, o tema do duplo encontrará a condição ideal para sua revitalização. É o que podemos observar na obra de Luiz Alfredo Garcia-Roza, objeto desta dissertação, cuja finalidade principal é a de identificar e analisar as manifestações do duplo em alguns romances do autor, situando-os no contexto contemporâneo. Para tal, foram selecionados como corpus de análise cinco de seus nove romances: Vento Sudoeste (1999), Perseguido (2003), Espinosa sem saída (2006), Na multidão (2007) e Céu de origamis (2009). Traçamos, primeiramente, um breve panorama da representação literária do tema do duplo, além de alguns conceitos psicanalíticos, buscando introduzir o tema, utilizando como base teórica os estudos de Otto Rank, Freud, Nicole Bravo, Ana Maria Lisboa de Mello, entre outros. Num segundo momento, identificamos as manifestações do duplo nas narrativas policiais de enigma, noir e pós-utópica, empregando o termo criado por Haroldo de Campos. Verificamos que a narrativa policial brasileira, desde os seus primórdios, já revela uma incompatibilidade com as certezas preconizadas pela escola de enigma. Dessa forma, abrimos o caminho para atingirmos o foco deste estudo: a análise do duplo na narrativa de Luiz Alfredo Garcia-Roza, articulando o quadro teórico à leitura dos textos. O duplo aparece na ficção do autor como representação dos antagonismos humanos que levam à fragmentação do eu e como estratégia para ressaltar as ambiguidades e incertezas da narrativa policial pós-utópica, em contraposição às verdades absolutas do gênero policial em sua forma clássica. Daí deduzimos que, contrariando a principal norma do gênero, a narrativa de Garcia-Roza coloca máscaras, em vez de retirá-las

A presença do duplo em Lygia Bojunga

Verificamos, na ficção brasileira infanto-juvenil contemporânea, uma série de aspectos envolvendo personagens, narradores, cenários que favorecem uma abordagem crítica baseada na presença do duplo. Podemos observar a presença do duplo na obra de Lygia Bojunga, objeto desta dissertação, que tem por finalidade principal identificar e analisar algumas das manifestações do duplo em nove de suas vinte e duas obras: A bolsa amarela (1976), Tchau (1984), Nós Três (1987), Livro: um encontro com Lygia Bojunga (1988), Fazendo Ana Paz (1991), Seis vezes Lucas (1995), O abraço (1995), Dos vinte 1 (2007), Querida (2009). Como base teórica, utilizamos textos de Otto Rank, Nicole Bravo, Berenice Sica Lamas, entre outros. Num segundo momento, identificamos as manifestações do duplo nas narativas de Lygia Bojunga. O duplo aparece na ficção da autora como representação das incompatibilidades do ser humano, que levam à fragmentação do eu. O encontro do eu e o outro, nem sempre amistoso, nos traz questões que servem de base para a pesquisa do duplo na obra de Lygia Bojunga. Por fim, o trabalho metaficcional presente na obra desta autora também foi analisado como forma de manifestação do duplo

Tracking Pacific bluefin tuna (Thunnus thynnus orientalis) in the northeastern Pacific with an automated algorithm that estimates latitude by matching sea-surface-temperature data from satellites with temperature data from tags on fish

Data recovered from 11 popup satellite archival tags and 3 surgically implanted archival tags were used to analyze the movement patterns of juvenile northern bluefin tuna (Thunnus thynnus orientalis) in the eastern Pacific. The light sensors on archival and pop-up satellite transmitting archival tags (PSATs) provide data on the time of sunrise and sunset, allowing the calculation of an approximate geographic position of the animal. Light-based estimates of longitude are relatively robust but latitude estimates are prone to large degrees of error, particularly near the times of the equinoxes and when the tag is at low latitudes. Estimating latitude remains a problem for researchers using light-based geolocation algorithms and it has been suggested that sea surface temperature data from satellites may be a useful tool for refining latitude estimates. Tag data from bluefin tuna were subjected to a newly developed algorithm, called “PSAT Tracker,” which automatically matches sea surface temperature data from the tags with sea surface temperatures recorded by satellites. The results of this algorithm compared favorably to the estimates of latitude calculated with the lightbased algorithms and allowed for estimation of fish positions during times of the year when the lightbased algorithms failed. Three near one-year tracks produced by PSAT tracker showed that the fish range from the California−Oregon border to southern Baja California, Mexico, and that the majority of time is spent off the coast of central Baja Mexico. A seasonal movement pattern was evident; the fish spend winter and spring off central Baja California, and summer through fall is spent moving northward to Oregon and returning to Baja California.

Assessment of natural resource conditions in and adjacent to Dry Tortugas National Park

This project characterized and assessed the condition of coastal water resources in the Dry Tortugas National Park (DRTO) located in the Florida Keys. The goal of the assessment was to: (1) identify the state of knowledge of natural resources that exist within the DRTO, (2) summarize the state of knowledge about natural and anthropogenic stressors and threats that affected these resources, and (3) describe strategies being implemented by DRTO managers to meet their resource management goals. The park, located in the Straits of Florida 113 km (70 miles) west of Key West, is relatively small (269 square kilometers) with seven small islands and extensive shallow water coral reefs. Significant natural resources within DRTO include coastal and oceanic waters, coral reefs, reef fisheries, seagrass beds, and sea turtle and bird nesting habitats. This report focuses on marine natural resources identified by DRTO resource managers and researchers as being vitally important to the Tortugas region and the wider South Florida ecosystem. Selected marine resources included physical resources (geology, oceanography, and water quality) and biological resources (coral reef and hardbottom benthic assemblages, seagrass and algal communities, reef fishes and macro invertebrates, and wildlife [sea turtles and sea-birds]). In the past few decades, some of these resources have deteriorated because of natural and anthropogenic factors that are local and global in scale. To meet mandated goals (Chapter 1), resource managers need information on: (1) the types and condition of natural and cultural resources that occur within the park and (2) the stressors and threats that can affect those resources. This report synthesizes and summarizes information on: (1) the status of marine natural resources occurring at DRTO; and (2) types of stressors and threats currently affecting those resources at the DRTO. Based on published information, the assessment suggests that marine resources at DRTO and its surrounding region are affected by several stressors, many of which act synergistically. Of the nine resource components assessed, one resource category – water quality – received an ecological condition ranking of "Good"; two components – the nonliving portion of coral reef and hardbottom and reef fishes – received a rating of "Caution"; and two components – the biotic components of coral reef and hardbottom substrates and sea turtles – received a rating of "Significant concern" (Table E-1). Seagrass and algal communities and seabirds were unrated for ecological condition because the available information was inadequate. The stressor category of tropical storms was the dominant and most prevalent stressor in the Tortugas region; it affected all of the resource components assessed in this report. Commercial and recreational fishing were also dominant stressors and affected 78% of the resource components assessed. The most stressed resource was the biotic component of coral reef and hardbottom resources, which was affected by 76% of the stressors. Water quality was the least affected; it was negatively affected by 12% of stressors. The systematic assessment of marine natural resources and stressors in the Tortugas region pointed to several gaps in the information. For example, of the nine marine resource components reviewed in this report, the living component of coral reefs and hardbottom resources had the best rated information with 25% of stressor categories rated "Good" for information richness. In contrast, the there was a paucity of information for seagrass and algal communities and sea birds resource components.

Integration of submersible transect data and high-resolution multibeam sonar imagery for a habitat-based groundfish assessment of Heceta Bank, Oregon

In the face of dramatic declines in groundfish populations and a lack of sufficient stock assessment information, a need has arisen for new methods of assessing groundfish populations. We describe the integration of seafloor transect data gathered by a manned submersible with high-resolution sonar imagery to produce a habitat-based stock assessment system for groundfish. The data sets used in this study were collected from Heceta Bank, Oregon, and were derived from 42 submersible dives (1988–90) and a multibeam sonar survey (1998). The submersible habitat survey investigated seafloor topography and groundfish abundance along 30-minute transects over six predetermined stations and found a statistical relationship between habitat variability and groundfish distribution and abundance. These transects were analyzed in a geographic information system (GIS) by using dynamic segmentation to display changes in habitat along the transects. We used the submersible data to extrapolate fish abundance within uniform habitat patches over broad areas of the bank by means of a habitat classification based on the sonar imagery. After applying a navigation correction to the submersible-based habitat segments, a good correlation with major boundaries on the backscatter and topographic boundaries on the imagery were apparent. Extrapolation of the extent of uniform habitats was made in the vicinity of the dive stations and a preliminary stock assessment of several species of demersal fish was calculated. Such a habitat-based approach will allow researchers to characterize marine communities over large areas of the seafloor.

Modelling the long-term sustainability of indigenous hunting in Manu National Park, Peru: landscape-scale management implications for Amazonia

P> Widespread hunting throughout Amazonia threatens the persistence of large primates and other vertebrates. Most studies have used models of limited validity and restricted spatial and temporal scales to assess the sustainability. We use human-demographi

Zebu Cattle Are an Exclusive Legacy of the South Asia Neolithic

Animal domestication was a major step forward in human prehistory, contributing to the emergence of more complex societies. At the time of the Neolithic transition, zebu cattle (Bos indicus) were probably the most abundant and important domestic livestock species in Southern Asia. Although archaeological evidence points toward the domestication of zebu cattle within the Indian subcontinent, the exact geographic origins and phylogenetic history of zebu cattle remains uncertain. Here, we report evidence from 844 zebu mitochondrial DNA (mtDNA) sequences surveyed from 19 Asiatic countries comprising 8 regional groups, which identify 2 distinct mitochondrial haplogroups, termed I1 and I2. The marked increase in nucleotide diversity (P < 0.001) for both the I1 and I2 haplogroups within the northern part of the Indian subcontinent is consistent with an origin for all domestic zebu in this area. For haplogroup I1, genetic diversity was highest within the Indus Valley among the three hypothesized domestication centers (Indus Valley, Ganges, and South India). These data support the Indus Valley as the most likely center of origin for the I1 haplogroup and a primary center of zebu domestication. However, for the I2 haplogroup, a complex pattern of diversity is detected, preventing the unambiguous pinpointing of the exact place of origin for this zebu maternal lineage. Our findings are discussed with respect to the archaeological record for zebu domestication within the Indian subcontinent.

Językowe środki wyrażania postawy mówiącego w łotewskich debatach sejmowych

Wydział Neofilologii: Instytut Językoznawstwa