The relationship between the adenovirus early region 1B55K (E1B55K) protein and the cellular DNA damage response

Adenoviruses are members of the small DNA tumour virus family; there are more than 100 identified Human Adenovirus (HAdV) types which are assigned into 7 groups designated A to G. The adenoviral early region 1B55K (E1B55K) protein is essential for complete HAdV- mediated transformation of cells in culture and for efficient adenovirus replication. During infection, E1B55K forms a complex with early region 4 open reading frame 6 (E4orf6) protein; together they bind and target several cellular proteins, many of which are involved in the DNA damage response (DDR), for proteasomal-mediated degradation. This is the case for HAdV5 and HAdV12 although other HAdV types such as HAdV16 (group B), HAdV9 (group D), HAdV4 (group E), and HAdV40 (group F) do not cause degradation of many of the same proteins. Most previous research has focused on the activities of the HAdV5-E1B55K protein during viral infection while in collaboration with E4orf6 and functioning as part of the E3 ubiquitin ligase. Here, we examined effects of E1B55K both as part of the E3 ligase and in isolation. Furthermore, we have concentrated on the properties of the group A, B, D, E, and F E1B55K proteins rather than HAdV5. Initially, we confirmed the interactions between HAdV5-E1B55K, and proteins previously identified as part of mass spectroscopy screens and showed that some, but not all, are degraded during viral infection. We have further shown binding of the same proteins to HAdV12-E1B55K, although degradation during infection was more limited. Novel association of HAdV12-E1B55K with replisome components has also been demonstrated. In the second part of the study, we have confirmed that HAdV12-E1B55K can induce genome instability in human fibroblasts in the absence of any other viral proteins. We investigated the effects of HAdV12-E1B55K protein on the DNA damage response (DDR) and on genomic stability, using human skin fibroblasts (HSF) expressing only HAdV12-E1B55K (55K+HSF). Our results show that HAdV12-E1B55K interferes with DNA replication dynamics, seen as a significant increase in stalled forks and R-loops, both of which are indicative of increased replication stress. We have also demonstrated that HAdV12-E1B55K expression sensitises cells to genotoxic agents, as shown by an increase in DNA damage foci formation and ATM activation. Furthermore, our findings are in line with previous observations which indicated that HAdV12 induces genomic instability in human cells, represented by an increase in chromosomal breaks. We hypothesise that interactions of HAdV12-E1B55K with different DDR and replication complex proteins is responsible for majority of these effects. Similarly, the expression of E1B55K protein from groups B, D, E and F HAdVs increased genomic instability in human tumour cells, seen as an increase in micronuclei formation. The viral proteins from these HAdV types associate with several homologous recombination DNA damage repair components, although with a limited effect, compared to HAdV12. Additionally, we have shown that HAdV-E1B55K from group B, D, E and F inhibits DNA double strand break repair as seen by the increased DNA damage repair foci after induced DNA damage. Using proteomic analysis, we have also identified novel E1B55K-interacting cellular substrates that bind these viral proteins, although the significance of these interactions is yet to be determined

Identifying Potential Exposure Pathways and Estimating Risk from Marcellus Shale Gas Development

Concern over natural gas extraction across the U.S. and particularly from the Marcellus Shale formation, which underlies approximately two-thirds of the state of Pennsylvania, has been growing in recent years as natural gas drilling activity has increased. Identifying sources of concern and risk from shale gas development, particularly form the hydraulic fracturing process, is an important step in better understanding sources of uncertainty within the industry. Hydraulic fracturing is a well stimulation technique used in the production of natural gas from shale. While hydraulic fracturing has been in use for decades as a method for oil and gas recovery, recent advances in horizontal drilling techniques and fracturing fluid production have made previously unattainable natural gas reservoirs accessible and economically recoverable. In the years after hydraulic fracturing came into widespread use in Pennsylvania, a large amount of data on flowback characteristics became available due to public and regulatory attention to the process. Chapter 3 examines and analyzes the constituents that make up flowback waters collected from drilling sites in the states of Pennsylvania, New York, and West Virginia. Flowback sampling data were collected from four different sources and compiled into one database with a total of 35,000 entries. Descriptive statistical analysis revealed high concentrations of chlorinated solvents, disinfectants, dissolved metals, organic compounds, radionuclides and TDS. Relative prioritization scores were developed for 58 constituents by dividing observed mean concentrations by the Maximum Contamination Level (MCL) guidelines for drinking water. The following constituents were found to have mean concentrations over 10 times greater than the MCL: Barium, Benzene, Benzo(a)pyrene, Chloride, Dibromochloromethane, Radium, and Thallium. Regulatory inspection and violation reports also provide insight into the impact of natural gas extraction on the surrounding environment, human health, and public safety. Inspection reports for natural gas wells in Pennsylvania were collected from the Pennsylvania DEP Compliance Report from 2000 to 2014. Logistic regression analysis of 215,444 inspection records for 70,043 conventional and unconventional wells was conducted in order to compare the odds of violations occurring under different circumstances. The results in Chapter 4 revealed that, when inspected, conventional wells had 40% higher odds of having a violation, but unconventional wells had higher odds for environmental violations related to waste discharge as well as cementing and casing failures. From there, a list of twelve failure scenarios of concern was developed focusing on specific events that may occur during the shale gas extraction process involving an operational failure or a violation of regulations to identify and prioritize potential failure scenarios for natural gas drilling operations through an elicitation of people who work in the industry. Illegal dumping of flowback water, while rated as the least frequently occurring scenario, was considered the scenario least protected by safety controls and the one of most concern to the general public. In terms of worker safety, the highest concern came from improper or inadequate use of personal protective equipment. While safety guidelines appear to be highly protective regarding PPE usage, inadequate PPE is the most directly witnessed failure scenario. Spills of flowback water due to equipment failure are of concern both in regards to the welfare of the general public and worker safety as they occur more frequently than any other scenario examined in this study. In Chapter 6 of this study, the flowback data collected and the violation and failure scenario analyses conducted are used to develop potential exposure scenarios to wastewater from shale gas development. A risk assessment of occupational and residential exposure pathways to flowback water as carried out. Constituents of concern in flowback water were identified from the previous prioritization. The occupational cancer risk estimate for median concentrations did not exceed the target lifetime cancer risk of 10-6 except for benzo(a)pyrene, which exceeds the target risk level even at the 2.5 percentile value. The upper limit of cancer risk form exposure to heptachlor also exceeds 10-6 in this model. Hazard quotient for barium in the same model exceeds 1 (1.7) and results in a total hazard index of 2. The residential risk assessment revealed that several carcinogenic compounds found in flowback water exceed target limits and significantly increase the risk of an individual developing cancer following chronic exposure. In general, exposure from the dermal pathway posed the greatest risk to human health. Considering non-carcinogenic effects, only barium and thallium exceed target limits, where the ingestion pathway seems to be of greater concern than dermal exposure. Exposure to radionuclides in flowback water, particularly through the inhalation pathway as they volatilize from the water to the air, poses a greater threat to human health than other contaminants examined in this assessment.Ph.D., Civil Engineering -- Drexel University, 201

The Promotion of Genomic Instability in Human Fibroblasts by Adenovirus 12 Early Region 1B 55K Protein in the Absence of Viral Infection

The adenovirus 12 early region 1B55K (Ad12E1B55K) protein has long been known to cause non-random damage to chromosomes 1 and 17 in human cells. These sites, referred to as Ad12 modification sites, have marked similarities to classic fragile sites. In the present report we have investigated the effects of Ad12E1B55K on the cellular DNA damage response and on DNA replication, considering our increased understanding of the pathways involved. We have compared human skin fibroblasts expressing Ad12E1B55K (55K+HSF), but no other viral proteins, with the parental cells. Appreciable chromosomal damage was observed in 55K+HSFs compared to parental cells. Similarly, an increased number of micronuclei was observed in 55K+HSFs, both in cycling cells and after DNA damage. We compared DNA replication in the two cell populations; 55K+HSFs showed increased fork stalling and a decrease in fork speed. When replication stress was introduced with hydroxyurea the percentage of stalled forks and replication speeds were broadly similar, but efficiency of fork restart was significantly reduced in 55K+HSFs. After DNA damage, appreciably more foci were formed in 55K+HSFs up to 48 h post treatment. In addition, phosphorylation of ATM substrates was greater in Ad12E1B55K-expressing cells following DNA damage. Following DNA damage, 55K+HSFs showed an inability to arrest in cell cycle, probably due to the association of Ad12E1B55K with p53. To confirm that Ad12E1B55K was targeting components of the double-strand break repair pathways, co-immunoprecipitation experiments were performed which showed an association of the viral protein with ATM, MRE11, NBS1, DNA-PK, BLM, TOPBP1 and p53, as well as with components of the replisome, MCM3, MCM7, ORC1, DNA polymerase δ, TICRR and cdc45, which may account for some of the observed effects on DNA replication. We conclude that Ad12E1B55K impacts the cellular DNA damage response pathways and the replisome at multiple points through protein–protein interactions, causing genomic instability

Assessing Residential Exposure Risk from Spills of Flowback Water from Marcellus Shale Hydraulic Fracturing Activity

Identifying sources of concern and risk from shale gas development, particularly from the hydraulic fracturing process, is an important step in better understanding sources of uncertainty within the industry. In this study, a risk assessment of residential exposure pathways to contaminated drinking water is carried out. In this model, it is assumed that a drinking water source is contaminated by a spill of flowback water; probability distributions of spill size and constituent concentrations are fit to historical datasets and Monte Carlo simulation was used to calculate a distribution of risk values for two scenarios: (1) use of a contaminated reservoir for residential drinking water supply and (2) swimming in a contaminated pond. The swimming scenario did not produce risks of concern from a single exposure of 1 h duration, but 11 such 1-h exposures did produce risks of 10−6 due to radionuclide exposure. The drinking water scenario over a 30-year exposure duration produced cancer risk values exceeding 10−6 for arsenic, benzene, benzo(a)pyrene, heptachlor, heptachlor epoxide, pentachlorophenol, and vinyl chloride. However, this extended exposure duration is probably not realistic for exposure by a spill event. Radionuclides produced risks in the residential drinking water scenario of 10−6 in just 8 h, a much more realistic timeline for continual exposure due to a spill event. In general, for contaminants for which inhalation exposure was applicable, this pathway produced the highest risks with exposure from ingestion posing the next greatest risk to human health followed by dermal absorption (or body emersion for radionuclides). Considering non-carcinogenic effects, only barium and thallium exceed target limits, where the ingestion pathway seems to be of greater concern than dermal exposure. Exposure to radionuclides in flowback water, particularly through the inhalation route, poses a greater threat to human health than other contaminants examined in this assessment and should be the focus of risk assessment and risk mitigation efforts