Browsing by Subject "Radiation"

Osteoporosis and low bone density affect an estimated 54 million adults of 50 years and over in the United States, resulting in $19 billion in costs for osteoporosis-related bone breaks. Current treatments include the use of antiresorptive and anabolic drugs to decrease the rate of bone resorption and increase the rate of bone formation, respectively. However, these current treatments are unable to completely normalize skeletal integrity. As bone diseases become increasingly prevalent, there is an urgent need to identify novel therapies to improve quality of life and reduce economic burden on the healthcare system.

To identify novel therapeutic targets, we must first begin to understand the cellular complexity of the bone marrow niche and how cellular function is regulated within the bone tissue. Bone-resident cells, such as skeletal progenitors and their descendants, are critically influenced by extrinsic signals derived from the local microenvironment. Previous studies have identified hypoxia as a key microenvironment factor in bone. Thus, the ability to target the hypoxic bone marrow niche presents an attractive and untapped potential for regenerative medicine.

Much of the work investigating the role of hypoxia and HIF signaling have focused on mature osteoblast and chondrocyte populations. In contrast, studies investigating the contribution of HIF signaling on skeletal progenitors and marrow adipocyte populations are scarce. In this dissertation, I investigate the role of hypoxia and HIF signaling in skeletal lineage cells, chiefly skeletal progenitor cells and marrow adipogenic lineage cells. Using cellular, genetic, and pharmacological-based approaches, I characterize the roles of HIF-1α and HIF-2α in both homeostatic and pathological contexts in the aforementioned cell populations.

First, I propose an optimized cell-based system to investigate the function of skeletal progenitors in vitro. Here, I highlight the limitations of current in vitro isolation techniques and introduce a relatively simple method of bone marrow stromal cell purification using hypoxia. Using this system, I assess how skeletal progenitors respond to hypoxic cues and interrogate skeletal progenitor cell differentiation and functional responses in my subsequent research. Next, using genetic and pharmacological approaches, I investigate the role of HIF-2α in bone formation following radiation-injury where I identify HIF-2α as a negative regulator of bone recovery. Additionally, with the assistance of my collaborators, I develop and characterize a bone-targeting nanocarrier to ameliorate radiation-induced bone loss. Lastly, I detail early work I conducted to investigate the role of HIF signaling in marrow adipogenic lineage cells. Here, I establish and characterize animal models to determine how hypoxia and HIF signaling influences adipogenic lineage commitment and expansion in an early and mature marrow adipogenic population.

In summary, this dissertation aims to expand our limited understanding on how the hypoxic bone microenvironment and HIF signaling regulate skeletal lineage cells in vivo, with a special focus on skeletal progenitor and marrow adipogenic populations. In terms of boarder impacts, understanding the signaling networks that regulate bone homeostasis and recovery processes will not only expand our basic understanding of the molecular mechanisms underlying skeletal development, but also provide novel insights for developing therapies to treat bone loss.

Purpose:

The overall purpose of this study was a comparison of radiation exposure for patients and staff during intra-operative imaging for orthopedic lumbar spine surgery. In order to achieve this, we: (1) Characterized each x-ray machine for physics performance, (2) Measured occupational radiation exposure inside the surgical suite for multiple intra-operative imaging devices utilizing currently in place clinical protocols for abdominal/spinal imaging, and (3) Measured specific organ doses for a phantom of three different Body Mass Indices (BMI) for each machine. We also compared the dose changes relative to changes in BMI as well as surgical image quality changes relative to BMI. This served as the majority of the first phase of a two phase project. The purpose of the second phase of the project will be to optimize scan parameters for surgical hardware placement in terms of image quality and organ dose for the devices that allow for modifications of scanner settings.

Materials and Methods:

(1) X-Ray quality control meters were used to verify particular beam characteristics and additional information was calculated from the beam data. Both a small volume ionization chamber as well as Metal-Oxide-Semiconductor Field Effect Transistor (MOSFET) dosimeters were used to validate linear response of new design X-Ray tubes. (2) Both handheld ionization chamber survey meters as well as Geiger-Muller based personal dose meters were used to measure stray radiation for room surveys in locations representative of typical radiation worker positions during intra-operative imaging. (3) MOSFET dosimeters were placed in an adult male anthropomorphic phantom representing a normal BMI. 20 MOSFETs were used in nine organs with two small volume ion chambers used for skin surface dosimetry. Two additional layers of adipose equivalent material were progressively added to the phantom to represent BMI values of overweight and obese.

Results:

(1) The maximum tube potential, half value layer (HVL), effective energy, and soft tissue f-factor for each machine is as follows: IMRIS VISIUS iCT: 118.4 kVp, 7.66 mm Al, 53.64 keV, and 0.934 cGy/R; Mobis Airo: 122.3 kVp, 7.21 mm Al, 51.31 keV, and 0.925 cGy/R; Siemens ARCADIS Orbic 3D: 83 kVp, 7.12 mm Al, 32.76 keV, and 0.914 cGy/R; GE OEC 9900 Elite: 75 kVp, 4.25 mm Al, 46.6 keV, and 0.920 cGy/R. (2) The highest exposure rates measured during clinically implemented protocols for each scanner are as follows: IMRIS VISIUS iCT: 800 mR/hr; Mobis Airo: 6.47 R/hr; Siemens ARCADIS Orbic 3D: 26.4 mR/hr. (3) The effective dose per scan of each device for a full lumbar spine scan are as follows, for normal, overweight, and obese BMI, respectively: IMRIS VISIUS iCT: 12.00 ± 0.30 mSv, 15.91 ± 0.75 mSv, and 23.23 ± 0.55 mSv; Mobius Airo: 5.90 ± 0.25 mSv, 4.97 ± 0.12 mSv, and 3.44 ± 0.21 mSv; Siemens ARCADIS Orbic 3D: 0.30 ± 0.03 mSv, 0.39 ± 0.02 mSv, and 0.28 ± 0.03 mSv; GE OEC 9900 Elite: 0.44 mSv, 0.77 mSv, and 1.14 mSv.

Conclusion:

(1) The IMRIS VISIUS iCT i-Fluoro capable CT scanner and Mobius Airo mobile CT scanner have similar beam characteristics with significantly different tube parameter modulation protocols. Siemens ARCADIS Orbic 3D and GE OEC 9900 offer comparable beam characteristics but different imaging methods. All scanners performed within factory specifications. (2) The IMRIS VISIUS iCT should not be used in i-Fluoro mode for surgical procedures active during scanning due to the 1.42 cGy/s point dose rate in the beam field. The high exposure rate from the Mobius Airo is offset by short scan times and can be mitigated by ensuring enforcement of currently established radiation protection regulations and policies. Minimal stray radiation is measured from the Siemens ARCADIS Orbic 3D. (3) The differences in tube modulation of the CT scanners means the Mobius Airo offers a significantly reduced effective dose with increasing patient BMI over the IMRIS VISIUS iCT. Effective dose from the CT scanners varies as much as one to two orders of magnitude higher than the C Arms, but the Siemens ARCADIS Orbic 3D offers unusable image quality for patients with higher than normal BMI. Based off of physician reported usable surgical image quality of Mobius Airo, this device is recommended for continued integration and implementation during routine surgical procedures for patients of all BMI in orthopedic lumbar spine surgery.

Since the 2011 meltdown, the health of “Fukushima children” has become a problem for parents, politics, and future imaginaries in post-nuclear Japan. What are the ethical and political implications of making life around a child imperiled by radiation when (re)productivity of life must be remade in a compromised environment? This dissertation investigates (re)production of life in the wake of the Fukushima Daiichi nuclear disaster in Japan by studying the strivings of families who seek to raise healthy children amidst radiation as a condition of living: what I call “anxious care.” By foregrounding the family as a site for environmental struggles in an emerging politics of life, I examine the work of making children live against and within radiation, looking to consider the radical implications of caring for children in radioactive uncertainty. In particular, this project focuses on inner cities of Fukushima Prefecture that have been on the frontline of radiation debates for having been exposed to disaster-induced radiation while not designated for evacuation. Shifting focus to the edges of delimited disaster zones, I examine the multifaceted aftermath of the nuclear disaster, ranging from differentially altered forms of life conditioned by radioactive uncertainty, the unequal distribution of radiation risk through public/private organizations such as the family form, and the everyday impact of post-Fukushima radiation. Theorizing the stakes of living with nuclear risk as situated political ecologies which generates tensions and possibilities for new forms of life, this dissertation argues that notions of life are undergoing a moment of reconfiguration in post-nuclear Japan by both real-life families and the family form. In doing so, it contributes to critiquing and broadening the anthropological horizons of life amid environmental uncertainty in and beyond Japan.

Purpose: (1) To verify the accuracy and linearity of the ThermoScientific Radeye G Personal Rate Meter with respect to exposure rate across the full dynamic range of the instrument. (2) Use a combination of empirical data and Monte Carlo methods to estimate dose distribution in a GE Discovery 690 PET/CT scanner room and adjacent hallway. (3) Quantify components of occupational dose to PET technologists.

Materials & Methods: (Project 1) The Radeye unit and a calibrated ion chamber were placed in the beam of a Cesium 137 calibrator. They were exposed from 46 μR/hr to 1 R/hr with the pulse of each beam lasting for 90 seconds. The Radeye made 15 exposure rate measurements during each pulse. The ion chamber was read in the mid-point of each pulse's duration. (Project 2) Six Radeye units were placed at key points within the Discovery 690 scan room and two were placed in the adjacent hallway. 1600 exposure rate measurements were made over eleven hours during each day of operation. Data was collected for seven days. The total integrated data from the detectors inside the room was used to develop a Monte Carlo model of the room using FLUKA software. This model was then able to estimate the contribution from radiation escaping the scan room to the detectors in the hallway. (Project 3) Three PET technologists wore Radeye units while performing their daily tasks. The detectors recorded a mean exposure rate over each 25 second sampling period. The technologists were also asked to maintain a written log of all their interaction with radioactive material as well as their interactions with injected patients. Each day the Radeye unit produced a plot of radiation exposure with respect to time. Each interaction with radioactivity from the logs was highlighted on the plot and integrated to obtain the exposure received while performing that task.

Results: (Project 1) The Radeye deviated from the known value of exposure by up to 9.3% and deviated from the ion chamber measurement by up to 8.6% for exposure rates of 1 mR/hr and greater. The Radeye measured up to 29.6% higher than the known rate and up to 33.6% higher than the ion chamber measurement for exposure rates less than 1 mR/hr. The variance in the Radeye measurements decreased as exposure rate increased. The standard deviation of the Radeye measurements were less than 4% of their respective mean values for exposure rates less than 1 mR/hr. This value increased for lower exposure rates, up to 14% at 0.046 mR/hr. (Project 2) Mean daily exposures to five points in the PET/CT scan room were measured for CT and PET emissions separately. A Monte Carlo model of the scan room was created to model the distribution, including an initial approximation for the scanner gantry. The simulations showed that the virtual scanner should be thinner (i.e. less attenuating), especially for the 511KeV PET photons. (Project 3) The mean exposure received per dose draw and accompanying injection was 0.70±0.23mR for the 113 injections recorded over the course of the study. No correlation was observed between the dosage injected and the exposure received. The percent contributed to the total exposure by each category and participant was as follows. Technologist #1: 68% from Dose Draw, 6% from Patient Positioning, 4% from Patient Transport, 1% from General Patient Care, 21% from nonspecific sources. Technologist #2: 34%, 32%, 14%, 6%, and 14%. Technologist #3: 32%, 32%, 16%, <1%, and 20%. The dose draws and accompanying injections account for between one and two thirds of daily exposure. This indicates it is likely a 30% daily dose reduction could be achieved with use of automated injection equipment.

Background

Methods

Results

Conclusions

There is a critical need to target tumor hypoxia as patients with hypoxic tumors have worse prognosis due to aggressive phenotypes and resistance to radiotherapy and chemotherapy. The overall goal of this work is to improve response to conventional cancer therapies by targeting tumor hypoxia. This has been carried out and evaluated through the use of polymersome-encapsulated myoglobin (PEMs) with the hypothesis that O2-releasing PEMs will increase tumor oxygenation, and thereby improve response to radiotherapy. Mb was chosen as an O2 carrying protein to deliver to tumors because it has a strong association to O2, providing a mechanism to deliver O2 only within the hypoxic regions of the tumor. Mb was loaded within nanoscale polymeric vesicles that were expected to accumulate within solid tumors due to the enhanced permeability and retention (EPR) effect. This hypothesis has been tested through the following aims:

1. Develop NIR imaging techniques for studying the biodistribution and pharmacokinetics of polymersomes

2. Establish the effects of Mb-containing polymersomes on tumor physiology

3. Modify tumor growth through delivery of Mb polymersomes in combination with a cytotoxic therapy specific to aerobic tumors

These aims have been evaluated through numerous in vivo studies. First, polymersomes of various polymer formulations and diameters ranging from 110-550 nm were prepared with a near-infrared (NIR) -emissive fluorophore. Using live animal fluorescence imaging, I was able to study the biodistribution of the polymersomes following i.v. administration, demonstrating significant polymersome accumulation in orthotopic 4T1 mammary carcinomas. In addition, a novel method for measuring pharmacokinetics was developed, using serial small volume blood draws from individual mice. The plasma fluorescence in microcapillary tubes was used to quantify polymersome concentrations, demonstrating long circulation half-lives that varied from 6-23 h. Toxicity of various polymersome formulations were also studied in vitro and in vivo, revealing negligible toxicities.

For the second aim, PEMs were administered i.v. in tumor-bearing mice. Unexpectedly, we observed a dramatic gross tumor effect within hours of treatment in both orthotopic 4T1 tumors and flank Renca renal cell carcinomas. Histological analysis revealed endothelial cell apoptosis as early as 1 h following treatment, with scattered tumor cell death throughout the tumor by 4 h. Hematoxylin and eosin staining showed significant necrosis 24 h following PEM treatment. Vascular effects and polymersome distribution were studied in 4T1 window chamber tumors. Following i.v. treatment with PEMs, intravital microscopy was used to image polymersome fluorescence, brightfield transmission was imaged for vessel morphology and blood flow, and a tunable filter was used for determining hemoglobin (Hb) oxygen saturation. Tumor hemorrhaging was observed within hours of PEM treatment, which was not seen with empty polymersomes. This was consistent with the gross tumor effects observed initially. Hb saturation decreased in both the PEM and empty polymersome groups, but not in saline-treated mice. While we expected to observe an increase in tumor oxygenation by using Mb as an oxygen carrier, we actually observed hemorrhage, decreased oxygenation, and central tumor necrosis. In vitro studies using human endothelial cells demonstrated dramatic changes in cell morphology and increased permeability due to Mb and PEM treatments, which appear to be enhanced in an oxidative environment. These in vitro and in vivo observations are similar to what is seen with tumor vascular disrupting agents.

For the third aim, I combined radiotherapy (RT) and PEM treatment with a new hypothesis. I originally expected the PEMs to increase tumor oxygenation, thus making the tumor more susceptible to RT. However, considering the results from the second aim, this hypothesis was modified: the PEMs would result in necrosis of the tumor core, while RT would target the more oxygenated rim of the tumor, thus leading to improved tumor growth delay compared with PEM or RT alone. This hypothesis was tested in both orthotopic, syngeneic 4T1 tumors as well as flank FaDu xenografts. 4T1 tumor cells were surgically implanted within the dorsal mammary fat pad of mice and grown until ~200 mm3. A CT microirradiator with a square collimator was used in order to locate and specifically irradiate the tumor. Within 1 h following RT, the PEMs were administered i.v.. Mice receiving PEMs with no RT showed a significant decrease in tumor growth compared with saline-treated mice (p = 0.0001 for time to 3x original tumor volume). In addition, the combination of RT plus PEMs reduced tumor growth compared with RT alone (p = 0.0144 for time to 3x original tumor volume). However, this effect was not seen with FaDu tumors. This may have been due to excessive radiation dose or other compounding factors: the timing between RT and PEM treatment was not optimized, and the number of mice per group was small (3-4).

Thus, the conclusions for each aim are as follows:

1. Develop NIR imaging techniques for studying the biodistribution and pharmacokinetics of polymersomes

NIR imaging techniques were optimized for studying polymersomes, demonstrating long plasma circulation times and accumulation within tumors.

2. Establish the effects of Mb-containing polymersomes on tumor physiology

While the hypothesis was that PEMs would accumulate within hypoxic tumors and subsequently increase O2 tension, we observed a rapid decrease in tumor oxygenation followed by a dramatic hemorrhagic effect of Mb polymersomes, which appear to be due to both endothelial cell apoptosis and morphological changes, resulting in central tumor necrosis.

3. Modify tumor growth through delivery of Mb polymersomes in combination with a cytotoxic therapy specific to aerobic tumors

Combination therapy of PEMs with RT results in enhanced tumor growth delay in aggressive 4T1 mammary carcinomas compared with RT or PEMs alone.

These studies have led to a proposed mechanism for the PEM anti-tumor effect in combination with RT. Prior to PEM administration, RT is administered, resulting in tumor cell kill of the well-oxygenated tumor periphery. Mb polymersomes are then injected i.v. and begin to accumulate within tumors due to the EPR effect. As shown in Aim 1, this accumulation occurs over a short time scale. Within 30 min of PEM treatment, the Mb is believed to act on tumor vessels, resulting in morphological changes and apoptosis of endothelial cells. These effects are expected to increase permeability of the vessels and expose the basement membrane, which leads to clotting and decreased blood flow. Both decreased perfusion and increased permeability are believed to have a catastrophic effect on interior tumor vessels. Hemorrhage results as the endothelial cells die, resulting in tumor core necrosis. Therefore, the result is tumor cell kill at the periphery due to RT and central tumor necrosis due to PEM treatment.

PEMs have potential in cancer therapy as a new class of VDAs. While the mechanism requires further investigation, this work has demonstrated that PEM treatment results in tumor vessel destruction and central necrosis. PEMs accumulate within tumors, thus minimizing the systemic toxicity of treatment commonly seen with VDAs. By combining PEMs with a therapy that kills the better perfused tumor periphery, PEMs show promise in improving tumor response. Future mechanistic studies will be needed in order to maximize vessel damage and optimize combination dosing schedules to improve outcome.

Recently, there has been renewed interest in decreasing radiation dose to patients from diagnostic imaging procedures. So far, efforts to decrease radiation dose have focused on the amount of radiation delivered from typical techniques and fail to capture the variation in radiation dose between patients. Despite the feasibility of estimating patient-specific radiation doses and the potential for this practice to aid in protocol optimization, it is not currently standard procedure for hospitals to monitor radiation dose for all patients. To address this shortcoming, we have developed an institution-wide patient-specific radiation dose monitoring program for computed tomography, digital radiography, and nuclear medicine.

Over half of all cancer patients receive radiation therapy, and it contributes to over 40% of cancer cures. Within the last decade, cancer immunotherapy has become a pillar of cancer therapy, along with surgery, chemotherapy, and radiation therapy. The most commonly used type of immunotherapy is immune checkpoint blockade, which can increase immune cell activity by blocking inhibitory signals. Preclinical studies with transplanted tumors demonstrate high cure rates with either immune checkpoint blockade, radiotherapy, or combination treatment. These studies have led to hundreds of clinical trials testing checkpoint blockade and radiotherapy alone or in combination with other therapies, but emerging results are disappointing. My thesis work seeks to develop novel mouse models of cancer that recapitulate human disease, understand mechanisms of tumor resistance to radiation and immunotherapy, and identify novel immunologic targets that can enhance patient responses to radiation therapy.

Using complex genetically engineered mouse models of cancer, we investigated the contributions of the tumor microenvironment to therapeutic resistance. First, we explored the role of neutrophils in mediating radiation response. We showed that elevated neutrophil levels were associated with poor local control and survival in cervical cancer patients treated with definitive chemoradiation. Furthermore, in a genetically engineered mouse model of sarcoma, we demonstrated that genetic and antibody-mediated depletion of neutrophils increases radiosensitivity and decreases a mitogen-activated protein kinase transcriptional program. These results demonstrate that neutrophils promote tumor resistance to radiotherapy.

In complementary work using novel genetically engineered mouse models of sarcoma, we found that cells with high tumor mutational burden transplanted into syngeneic mice were cured by immune checkpoint blockade and radiation therapy, but the identical treatment failed in autochthonous sarcomas. To understand the mechanisms by which primary tumors were resistant to tumor cure by radiation and immunotherapy, we generated a single cell atlas of tumor-infiltrating immune cells from transplant and primary sarcomas treated with radiation and immunotherapy, which revealed marked differences in their immune landscapes. We found that radiation therapy remodeled myeloid cell phenotypes in primary and transplant sarcomas, but only transplant tumors were enriched for the effector CD8+ T cells that mediate response to combination therapy. In contrast, mice with autochthonous sarcomas demonstrated tumor-specific tolerance. These results indicate that radiation and immunotherapy cooperate to promote immunity within the tumor microenvironment, but identify immune tolerance in autochthonous tumors that must be overcome for this promising combination treatment to cure cancers that co-evolve with the immune system.

ATRX is one of the most frequently altered genes in soft tissue sarcoma, with alterations occurring in 29% of these tumors. However, the role of ATRX in the development and response to cancer therapies in soft tissue sarcoma remains poorly understood. Here, we developed a primary mouse model of soft tissue sarcoma and studied the effect of Atrx deletion on tumor development and therapeutic response. Our findings demonstrate that Atrx deletion regulates tumor development and increases sarcoma sensitivity to radiation therapy. In the absence of Atrx, irradiated sarcomas have increased persistent DNA damage, telomere dysfunction, and mitotic catastrophe. We find that Atrx deleted tumors have impaired cGAS-STING signaling, with accompanying sensitivity to the novel clinical therapy oncolytic herpesvirus. Translation of these results to patients with ATRX mutant cancers could enable genomically-guided cancer therapeutic approaches that improve patient outcomes.

There is a scarcity of knowledge regarding the cardiovascular effects of low dose ionizing radiation (IR) such as the one experienced during medical tests, radiation therapy or space travel. This is becoming more of a pressing problem given the enormous increase in radiation exposure by the average American today, and the renewed interest in deep space travel. Multiple epidemiologic studies suggest a higher rate of delayed cardiovascular related morbidity and mortality after low dose acute radiation exposure. These studies are significantly limited by a number of confounders such as cancer comorbidity, poor follow up, and largely estimated radiation doses that might not be accurate. These limitations are also seen in studies on the effect of space radiation on long term cardiovascular mortality and accelerated atherosclerosis. Animal studies have been used to simulate the effect of terrestrial and space radiation scenarios on cardiac function. These studies have led to conflicting conclusions, and had important challenges related to methods of assessment of cardiac injury. Furthermore, available studies to date had limited follow up times, and no study has evaluated the effects of more complex radiation scenarios that are likely to be experienced in space such as Galactic Cosmic Rays (GCRs). Our overall hypothesis is that IR is associated with early damage to healthy cardiomyocytes and vascular cells that eventually leads to long term cardiovascular dysfunction.

In the first part of this work, we hypothesize that IR is associated with a delayed cardiovascular derangement phenotype late after initial exposure. To test this hypothesis, we use a mouse animal model to study the effect of different radiation scenarios on cardiovascular function. Animals were exposed to one of the following (a) Gamma Rays (50-200 cGy), (b) 56Fe (15-50 cGy), (c) 16O (15-50cGy) heavy ions, and (d) 150cGy Galactic Cosmic Rays all using the particle accelerator at Brookhaven National Labs (BNL). They were then followed up for 9-12 months, and underwent cardiac MRI, pressure volume loop assessments, transthoracic echocardiograms and other histological evaluations. These studies revealed that GCR exposed animals had a clinically meaningful decline in their cardiac function, with a significant change in their arterial elastance. These findings were further confirmed on histology with their aortas demonstrating elastic fiber destruction and disorganization.

These animal studies however could not fully differentiate between a primary cardiac injury or a secondary cardiac response to a primary vascular injury to the aorta. Our hypothesis for the second part of this work, was that IR is associated with a unique and differentiated injury to cardiomyocytes that contributes to the previously seen cardiovascular phenotype. We therefore conducted additional experiments on isolated rat ventricular cardiomyocyte in collaboration with the Bursac lab. These studies used both 2D cultured cells, as well as a novel cardiac patch system both exposed to Gamma rays and X-rays (0.1-2 Gy). These cells underwent proteomics analysis, as well as a number of different biological and functional assays. While the acute functional effect of these radiation doses on cardiomyocytes was small, these irradiated cells produced a significant amount of reactive oxygen species and exhibited a large effect of radiation on mitochondrial related proteins, including elements of oxidative phosphorylation. We also noted a number of different pathways involved at different doses of radiation. This was an important finding, given that despite no changes in early cell death, the effect of these important proteomics changes on long term cardiac function maybe important.

Finally, for the last part of this dissertation, our hypothesis was that IR uniquely affects endothelial cells (ECs) and smooth muscle cells (SMCs) by inducing early senescence that is primarily due to over production of mitochondrial specific reactive oxygen species. To test this hypothesis, we use primary coronary artery endothelial cells, primary human aortic endothelial cells and primary coronary artery smooth muscle cells. These relevant cell types were then examined for their response to a single dose of radiation exposure. Given the previous findings of important mitochondria involvement even at low radiation doses, we used a novel mitochondrial specific ROS scavenger, that blocks the release of mROS, mito-TEMPO. Cells treated with mito-TEMPO had a significant decrease in observed cellular senescence an important hallmark indicator of cellular dysfunction. This strategy might have a potential therapeutic role in the prolonged cardiovascular effects of radiation exposure.

In summary, data generated in this dissertation supports the overall hypothesis that IR is associated with long term cardiovascular dysfunction that can be explained by early injury to cardiac, endothelial and smooth muscle cells. Galactic Cosmic Rays appear to significantly effect long term cardiovascular function, which has important implications on deep space travel. This effect is likely multifactorial, involving a number of organs, including the aorta. Cardiomyocytes, despite being resilient to death from radiation as compared to other cells types, appear to undergo a number of proteomics alterations after low dose radiation exposure, with significant involvement of the mitochondrial machinery. Finally, human vascular ECs and SMCs are highly sensitive to radiation exposure, and strategies that target mitochondrial specific ROS production might play an important role in mitigating the long-term vascular effects after radiation exposure.

Endogenous retroviruses (ERVs) are ancient deactivated viral elements that have integrated into the human genome over the course of millennia of evolution. ERVs are normally kept transcriptionally repressed, but recent evidence has demonstrated that ERV transcription can be upregulated in response to a variety of stimuli and pharmacological treatments. Furthermore, ERV transcription in the form of double-stranded RNA (dsRNA) was shown to upregulate a host of immune signaling pathways, with implications for the future of cancer therapy. In this study, we utilize the transcriptional corepressor KRAB-Associated Protein 1 (KAP1), a chromatin modulator responsible for suppressing ERV transcription sites, as a tool to investigate ERV expression in cancer therapy.

We demonstrate that radiotherapy, historically believed to mediate tumor cell repression through direct cell killing, can also upregulate ERV transcription, which stimulates downstream interferon production and interferon-stimulated genes through the MDA5/MAVS innate antiviral immunity pathway, a separate pathway from cGAS/STING signaling. KAP1 depletion enhances the effect of radiation, as ERV and interferon transcription is significantly upregulated. Additionally, KAP1 depletion enhances the effect of radiation-induced anti-tumor response in vivo in two separate tumor models. Our findings indicate a novel and understudied pathway of radiotherapy-induced tumor control.

We also demonstrate that KAP1 depletion is sufficient to provoke interferon signaling and immune effects. KAP1 depletion upregulates ERV transcription, driving a similar effect to that seen with irradiation. Furthermore, KAP1 depletion is sufficient to inhibit tumor growth of B16F10 tumors in in vivo studies, but this effect is dependent on an intact host immune system. Tumor growth inhibition in vivo is driven by increased recruitment of immune cells to the tumor microenvironment, along with upregulated of interferon-stimulated genes. Patient data support our findings, as mRNA analysis of TCGA patient cohorts reveals that Trim28 expression is negatively associated with survival and immune recruitment. Taken as a whole, our work indicates KAP1 as a crucial modulator of ERVs, with significant consequences for cancer therapy.

Low-dose ionizing radiation (LDIR) exposure (under 10.0 centigray (cGy)) from man-made sources, such as diagnostic imaging, predominates in the US population and comprises nearly 50% of an average individual's yearly radiation exposure (Ullrich, Brooks et al. 2009). The increase in such exposures has led to public and government alarm about the impact of LDIR on human health (Ullrich, Brooks et al. 2009). Besides the mutational effects of radiation exposure, there is concern it might also result in modifications of the epigenome. Such aberrations can disrupt normal development and are involved in the progression of numerous diseases, including cancer (Gasser and Li 2011). High doses of radiation (>100.0 cGy) have been shown to cause epigenetic disruption (Kaup, Grandjean et al. 2006; Tamminga, Koturbash et al. 2008; Ilnytskyy, Koturbash et al. 2009), which is necessary for the persistence of radiation-induced genomic instability (Rugo, Mutamba et al. 2011); however, it is presently unclear to what extent LDIR in vivo alters the epigenome.

The viable yellow agouti (Avy) mouse was used here to characterize the dose-dependent epigenetic response to LDIR. The Avy mouse is a unique biological model that functions as a biosensor for environmentally induced epigenetic changes and disease susceptibility due to the presence of a metastable epiallele that modulates coat color (Waterland and Jirtle 2003). Pregnant dams were whole-body exposed to one of five doses of X-ray radiation ranging from 0-10.0 cGy on gestational day 4.5. Using a phantom mouse model, the intrauterine doses were estimated to be 0.0 cGy, 0.4 cGy, 0.7 cGy, 1.4 cGy, 3.0 cGy, and 7.6 cGy, respectively. At weaning, offspring coat colors were assessed and tissues were collected for methylation analysis. First, methylation changes at CpG sites in the Avy and Cdk activator binding protein (CabpIAP) metastable epialleles and at intracisternal a particle (IAP) elements across the genome were quantified using Sequenom technology. Second, three imprinted genes, Peg3, Nnat, and H19, were assessed for methylation changes in differentially methylated regions (DMRs) that regulate their parent-of-origin monoallelic expression using Sequenom technology. Lastly, it was postulated that the epigenetic changes at the Avy locus could be counteracted with dietary alterations. To test this hypothesis, female mice were placed on an antioxidant-supplemented diet prior to pregnancy and throughout gestation and lactation. Pregnant dams were irradiated with 3.0 cGy of whole-body X-rays. Offspring coat colors were assessed and methylation changes at the Avy allele were measured with the Sequenom platform.

Herein, I demonstrate that in utero LDIR exposure induced epigenetic changes in the Avy mouse in a dose-dependent and sex-specific manner. Acute, whole-body exposure to 0.7 cGy, 1.4 cGy, 3.0 cGy or 7.6 cGy X-rays significantly shifted offspring coat color distribution toward pseudoagouti. Acute exposure to 1.4 cGy, 3.0 cGy, and 7.6 cGy significantly increased methylation at multiple CpG sites in the Avy metastable epiallele in male offspring, but not female offspring. Methylation changes at DMRs in Nnat, Peg3, and H19 also occurred in a dose-dependent manner. Furthermore, inhibition of the phenotypic and Avy methylation changes with an antioxidant-supplemented diet suggests that the mechanisms to induce epigenetic changes are mediated by oxidative stress. These results demonstrate that relevant, low doses of radiation can elicit epigenetic changes that lead to a persistent phenotype, but can be mitigated with dietary supplementation. The successful completion of this project has resulted in the first in vivo epigenetic characterization of LDIR exposure and will contribute to the development of more relevant risk assessment strategies for protecting human populations.

Introduction: Stereotactic body radiation therapy (SBRT) is a common treatment techniquethat can be used to treat tumors for multiple cancer sites. Density heterogeneity in the target volume and beam path combined with small treatment fields has made dose calculation in lung SBRT difficult. Dose calculation algorithms used historically have difficulty modelling the extreme density heterogeneity present in lung SBRT and have been shown to overestimate the dose delivered to tumors situated in the lung parenchyma. Recently, more advanced algorithms that directly model heterogeneity have been implemented for clinical treatment planning. The limited accuracy of historically utilized dose calculation algorithms has raised questions about their effects on local control due to the possibility of tumor underdosing. The first part of this work establishes a proper dose normalization technique when implementing these advanced algorithms for treatment planning in order to keep consistent radiation beam settings and to quantify the dosimetric effect of various dose normalizations. The second aim is to quantify the effects dosimetric accuracy has on local control in lung SBRT.

Materials/Methods: 87 lung SBRT plans with doses originally calculated with the AnisotropicAnalytical Algorithm (AAA) had their doses recalculated with the new Acuros XB (AXB) algorithm, which is able to directly model the heterogeneity of the lungs and treatment volume. After recalculation, the plan was normalized to the planning target volume (PTV) D95%, internal target volume (ITV) D99%, and to match the original PTV coverage. The percentage change in total monitor units (MU) between the AXB renormalized plans and the original AAA plans were calculated to quantify how the delivered radiation would change when implementing the AXB algorithm for treatment planning. Percentage changes in relevant PTV and ITV dose metrics as well as absolute changes in relevant organ at risk. (OAR) dose metrics were quantified to compare plan dosimetry. OAR doses were also compared to the current institutional planning objectives to investigate the feasibility of meeting the current objectives with the new algorithm. 162 patients previously treated with SBRT were selected from a retrospective protocol comparing the efficacy of SBRT and surgery for treatment of early-stage non-small cell lung cancer. Plans had their doses originally computed with the Pencil Beam Convolution (PBC, n = 8) algorithm or AAA (n = 156). Each plan was recalculated with AXB with identical beam settings. A subset was also recalculated with Monte Carlo to validate the accuracy of the AXB calculations. Percentage changes in relevant PTV and ITV biologically effective doses (BED) were calculated between the original and AXB plans to quantify the magnitude of the dosimetric differences between the old and new algorithm. A multivariable linear regression was performed to investigate which patient and treatment parameters influenced the magnitude of these dosimetric changes. A competing risk analysis was performed to quantify the association between the magnitude of the dosimetric changes and local failure.

Results: Normalizing the AXB plan to the PTV D95% and keeping the original PTVcoverage typically resulted in a total MU increase (average increase of 7.0% and 7.9%, respectively) while normalizing to the ITV D99% resulted in similar total MU (average increase of 0.31%). When normalizing to the PTV D95%, the AXB plans had increased PTV and ITV D1%[Gy] (median increases of 3.4% and 3.2%, respectively) while normalizing to the ITV D99% showed a median 1.9% decrease. Normalizing the AXB plans to the PTV D95% typically resulted in increased OAR dose for all OARs and an inferior ability to meet the OAR planning constraints. Reoptimization of the renormalized plans showed the current OAR objectives to be manageable when using the AXB algorithm. The AXB dose calculations were much more consistent with Monte Carlo than were the original dose calculations. A large range of dosimetric decreases upon recalculation with AXB were observed for both patients who failed locally and those who were controlled. Higher beam energy was found to increase the magnitude of the dosimetric decreases (expected decrease in PTV mean BED of 3.6%, 5.9%, and 9.1% when using 6X, 10X, or 15X, respectively). Total lung volume was also associated with an increased magnitude of dosimetric decrease (expected decerease of 0.8% per 500 cc for the PTV mean BED). The median follow-up time of the cohort was 26 months. 15 patients experienced local failures. Upon univariate analysis, the dosimetric decreases in the PTV and ITV D1% BED were found to be associated with local failure (hazard ratio (HR) of 0.89 (p=0.04) and 0.87 (p=0.02), respectively). Upon multivariate analysis, the dosimetric decrease in the ITV D1% BED remained significant when controlling for PTV volume (HR=0.89 (p=0.04)).

Conclusions: More accurate dose calculation algorithms are beginning to be implementedfor clinical treatment planning. When implementing these new algorithms, issues arise with dose normalization due to the potential for vast differences between the dose distributions calculated with the different algorithms. Normalizing the dose to the PTV D95% in the AXB plan will result in a delivered dose increase relative to a AAA plan while normalizing to the ITV D99% will keep similar delivered doses between the plans. Dose metrics typically increase when normalizing to the PTV D95% (for targets and OARs) while normalizing to the ITV D99% typically decreased the reported dose metrics. The OAR planning objectives are manageable using the AXB algorithm. Many factors are related to the magnitude of the dosimetric decreases observed when recalculating plans with AXB, including but not limited to beam energy and lung volume. Most of the investigated dose metrics were not associated with local failure, but the change in the PTV and ITV D1% BEDs were found to be associated with local failure in the univariate analysis.

An increasing number of experimental studies attempt to maximize biomass production of trees in plantations by removing nutrient and water limitations. The results from these studies begin to inform operational managers. We investigated a Populus trichocarpa Torr. x P. deltoides Bartr. & Marsh plantation with a combined irrigation and nutrient supply system designed to optimize biomass production. Sap flux density was measured continuously over four of the six growing season months, supplemented with periodic measurements of leaf gas exchange and water potential. Measurements of tree diameter and height were used to estimate leaf area and biomass production using allometric relations. Sap flux was converted to canopy conductance, and analyzed based on an empirical model to isolate the effects of water limitation. Actual and soil water-unlimited potential CO2 uptakes were estimated using a Canopy Conductance Constrained Carbon Assimilation (4C-A) scheme, which couples actual or potential canopy conductance with vertical gradients of light distribution, leaf-level conductance, maximum Rubisco capacity (Vcmax) and maximum electron transport (Jmax). Net primary production (NPP) was ~0.43 of gross primary production (GPP); when estimated for individual trees, this ratio was independent of tree size. Based on the same ratio, we found that current irrigation reduced growth by ~18 % compare to growth with no water limitation. To achieve this maximum growth, however, would require 70% more water for transpiration, and would reduce water use efficiency by 27 %, from 1.57 to 1.15 g stem wood C kg-1 water. Given the economic and social values of water, plantation managers appear to have optimized water use.

Radiation therapy can cause acute toxicity and long-term side effects in normal tissues. Because part of the acute toxicity of radiation is due to p53-mediated apoptosis, blocking p53 during irradiation can protect some normal tissues from acute radiation injury and might improve the therapeutic ratio of radiation therapy. However, the mechanisms by which p53 regulates late effects of radiation are not well understood. Here, I utilized genetically engineered mouse models to dissect the role of p53 in regulating two of the most clinically significant late effects of radiation: radiation-induced carcinogenesis and radiation-induced myocardial injury.

It has been well characterized that mice with one allele of p53 permanently deleted are sensitized to radiation-induced cancer. Therefore, temporary inhibition of blocking p53 during irradiation could promote malignant transformation. Experiments with mice lacking functional p53 in which p53 protein can be temporarily restored during total-body irradiation (TBI) suggest that the radiation-induced p53 response does not contribute to p53-mediated tumor suppression. Here, I performed reciprocal experiments and temporarily turned p53 off during TBI using transgenic mice with reversible RNA interference against p53. I found that temporary knockdown of p53 during TBI not only ameliorated acute hematopoietic toxicity, but in both Kras wild-type and tumor-prone KrasLA1 mice also prevented lymphoma development. Mechanistic studies show that p53 knockdown during TBI improves survival of hematopoietic stem and progenitor cells (HSPCs), which maintains HSPC quiescence and prevents accelerated repopulation of surviving cells. Moreover, using an in vivo competition assay I found that temporary knockdown of p53 during TBI maintains the fitness of p53 wild-type HSPCs to prevent the expansion of irradiated mutant cells. Taken together, our data demonstrate that p53 functions during TBI to promote lymphoma formation by facilitating the expansion of irradiated HSPCs with adaptive mutations.

p53 functions in the heart to promote myocardial injury after multiple types of stress, including ischemic injury, pressure overload and doxorubicin-induced oxidative stress. However, how p53 regulates radiation-induced myocardial injury, which develops after radiation therapy, is not well understood. Here, I utilized the Cre-loxP system to demonstrate that p53 functions in endothelial cells to protect mice from myocardial injury after a single dose of 12 Gy or 10 daily fractions of 3 Gy whole-heart irradiation (WHI). Mice in which both alleles of p53 are deleted in endothelial cells succumbed to heart failure after WHI due to myocardial necrosis, systolic dysfunction and cardiac hypertrophy. Moreover, the onset of cardiac dysfunction was preceded by alterations in myocardial vascular permeability and density. Mechanistic studies using primary cardiac endothelial cells (CECs) irradiated in vitro indicate that p53 signals to cause a mitotic arrest and protects CECs against radiation-induced mitotic catastrophe. Furthermore, mice lacking the cyclin-dependent kinase inhibitor p21, which is a transcriptional target of p53, are also sensitized to myocardial injury after 12 Gy WHI. Together, our results demonstrate that the p53/p21 axis functions to prevent radiation-induced myocardial injury in mice. Our findings raise the possibility that when combining radiation therapy with inhibitors of p53 or other components of the DNA damage response that regulate mitotic arrest, patients may experience increased radiation-related heart disease.

Taken together, our results demonstrate crucial but distinct roles of p53 in regulating late effects of radiation: p53-mediated apoptosis promotes radiation-induced lymphomagenesis, but p53-mediated cell cycle arrest prevents radiation-induced myocardial injury. These findings indicate that p53 may generally play a protective role from radiation, particularly at high doses, in cells where p53 activation is uncoupled from the induction of the intrinsic pathway of apoptosis. Therefore, selectively inhibiting p53-mediated apoptosis may be a promising approach to ameliorate acute radiation toxicity without exacerbating late effects of radiation.

The lungs are vital organs whose airways are lined with a continuous layer of epithelial cells. Epithelial cells in the distal most part of the lung, the alveolar space, are specialized to facilitate gas exchange. Proximal to the alveoli is the airway epithelium, which provides an essential barrier and is the first line of defense against inhaled toxicants, pollutants, and pathogens. Although the postnatal lung is a quiescent organ, it has an inherent ability to regenerate in response to injury. Proper balance between maintaining quiescence and undergoing repair is crucial, with imbalances in these processes leading to fibrosis or tumor development. Stem and progenitor cells are central to maintaining balance, given that they proliferate and renew both themselves and the various differentiated cells of the lung. However, the precise mechanisms regulating quiescence and repair in the lungs are largely unknown. In this dissertation, ionizing radiation is used as a physiologically relevant injury model to better understand the repair process of the airway epithelium. We use in vitro and in vivo mouse models to study the response of a secretory progenitor, the club cell, to various doses and qualities of ionizing radiation. Exposure to radiation found in space environments and in some types of radiotherapy caused clonal expansion of club cells specifically in the most distal branches of the airway epithelium, indicating that the progenitors residing in the terminal bronchioles are radiosensitive. This clonal expansion is due to an increase in p53-dependent apoptosis, senescence, and mitotic defects. Through the course of this work, we discovered that p53 is not only involved in radiation response, but is also a novel regulator of airway epithelial homeostasis. p53 acts in a gene dose-dependent manner to regulate the composition of airway epithelium by maintaining quiescence and regulating differentiation of club progenitor cells in the steady-state lung. The work presented in this dissertation represents an advance in our understanding of the molecular mechanisms underlying maintenance of airway epithelial progenitor cells as well as their repair following ionizing radiation exposure.

The outcomes for both (i) radiation therapy and (ii) preclinical small animal radio- biology studies are dependent on the delivery of a known quantity of radiation to a specific and intentional location. Adverse effects can result from these procedures if the dose to the target is too high or low, and can also result from an incorrect spatial distribution in which nearby normal healthy tissue can be undesirably damaged by poor radiation delivery techniques. Thus, in mice and humans alike, the spatial dose distributions from radiation sources should be well characterized in terms of the absolute dose quantity, and with pin-point accuracy. When dealing with the steep spatial dose gradients consequential to either (i) high dose rate (HDR) brachytherapy or (ii) within the small organs and tissue inhomogeneities of mice, obtaining accurate and highly precise dose results can be very challenging, considering commercially available radiation detection tools, such as ion chambers, are often too large for in-vivo use.

In this dissertation two tools are developed and applied for both clinical and preclinical radiation measurement. The first tool is a novel radiation detector for acquiring physical measurements, fabricated from an inorganic nano-crystalline scintillator that has been fixed on an optical fiber terminus. This dosimeter allows for the measurement of point doses to sub-millimeter resolution, and has the ability to be placed in-vivo in humans and small animals. Real-time data is displayed to the user to provide instant quality assurance and dose-rate information. The second tool utilizes an open source Monte Carlo particle transport code, and was applied for small animal dosimetry studies to calculate organ doses and recommend new techniques of dose prescription in mice, as well as to characterize dose to the murine bone marrow compartment with micron-scale resolution.

Hardware design changes were implemented to reduce the overall fiber diameter to <0.9 mm for the nano-crystalline scintillator based fiber optic detector (NanoFOD) system. Lower limits of device sensitivity were found to be approximately 0.05 cGy/s. Herein, this detector was demonstrated to perform quality assurance of clinical 192Ir HDR brachytherapy procedures, providing comparable dose measurements as thermo-luminescent dosimeters and accuracy within 20% of the treatment planning software (TPS) for 27 treatments conducted, with an inter-quartile range ratio to the TPS dose value of (1.02-0.94=0.08). After removing contaminant signals (Cerenkov and diode background), calibration of the detector enabled accurate dose measurements for vaginal applicator brachytherapy procedures. For 192Ir use, energy response changed by a factor of 2.25 over the SDD values of 3 to 9 cm; however a cap made of 0.2 mm thickness silver reduced energy dependence to a factor of 1.25 over the same SDD range, but had the consequence of reducing overall sensitivity by 33%.

For preclinical measurements, dose accuracy of the NanoFOD was within 1.3% of MOSFET measured dose values in a cylindrical mouse phantom at 225 kV for x-ray irradiation at angles of 0, 90, 180, and 270˝. The NanoFOD exhibited small changes in angular sensitivity, with a coefficient of variation (COV) of 3.6% at 120 kV and 1% at 225 kV. When the NanoFOD was placed alongside a MOSFET in the liver of a sacrificed mouse and treatment was delivered at 225 kV with 0.3 mm Cu filter, the dose difference was only 1.09% with use of the 4x4 cm collimator, and -0.03% with no collimation. Additionally, the NanoFOD utilized a scintillator of 11 µm thickness to measure small x-ray fields for microbeam radiation therapy (MRT) applications, and achieved 2.7% dose accuracy of the microbeam peak in comparison to radiochromic film. Modest differences between the full-width at half maximum measured lateral dimension of the MRT system were observed between the NanoFOD (420 µm) and radiochromic film (320 µm), but these differences have been explained mostly as an artifact due to the geometry used and volumetric effects in the scintillator material. Characterization of the energy dependence for the yttrium-oxide based scintillator material was performed in the range of 40-320 kV (2 mm Al filtration), and the maximum device sensitivity was achieved at 100 kV. Tissue maximum ratio data measurements were carried out on a small animal x-ray irradiator system at 320 kV and demonstrated an average difference of 0.9% as compared to a MOSFET dosimeter in the range of 2.5 to 33 cm depth in tissue equivalent plastic blocks. Irradiation of the NanoFOD fiber and scintillator material on a 137Cs gamma irradiator to 1600 Gy did not produce any measurable change in light output, suggesting that the NanoFOD system may be re-used without the need for replacement or recalibration over its lifetime.

For small animal irradiator systems, researchers can deliver a given dose to a target organ by controlling exposure time. Currently, researchers calculate this exposure time by dividing the total dose that they wish to deliver by a single provided dose rate value. This method is independent of the target organ. Studies conducted here used Monte Carlo particle transport codes to justify a new method of dose prescription in mice, that considers organ specific doses. Monte Carlo simulations were performed in the Geant4 Application for Tomographic Emission (GATE) toolkit using a MOBY mouse whole-body phantom. The non-homogeneous phantom was comprised of 256x256x800 voxels of size 0.145x0.145x0.145 mm3. Differences of up to 20-30% in dose to soft-tissue target organs was demonstrated, and methods for alleviating these errors were suggested during whole body radiation of mice by utilizing organ specific and x-ray tube filter specific dose rates for all irradiations.

Monte Carlo analysis was used on 1 µm resolution CT images of a mouse femur and a mouse vertebra to calculate the dose gradients within the bone marrow (BM) compartment of mice based on different radiation beam qualities relevant to x-ray and isotope type irradiators. Results and findings indicated that soft x-ray beams (160 kV at 0.62 mm Cu HVL and 320 kV at 1 mm Cu HVL) lead to substantially higher dose to BM within close proximity to mineral bone (within about 60 µm) as compared to hard x-ray beams (320 kV at 4 mm Cu HVL) and isotope based gamma irradiators (137Cs). The average dose increases to the BM in the vertebra for these four aforementioned radiation beam qualities were found to be 31%, 17%, 8%, and 1%, respectively. Both in-vitro and in-vivo experimental studies confirmed these simulation results, demonstrating that the 320 kV, 1 mm Cu HVL beam caused statistically significant increased killing to the BM cells at 6 Gy dose levels in comparison to both the 320 kV, 4 mm Cu HVL and the 662 keV, 137Cs beams.

The vascular contribution to tumor radiation response is controversial, but may have profound clinical implications. This is especially true of a new class of radiation therapies which employ spatial fractionation techniques--high radiation doses delivered in a spatially modulated pattern across the tumor. Window chamber tumor models may prove useful in investigating vascular parameters due to their facilitation of non-invasive, serial measurements of living tumors. However, presently there do not exist automated and accurate algorithms capable of quantitatively analyzing window chamber data.

Here we attempt to address these two problems through (1) the generation of novel optical data processing techniques for the quantification of vascular structural and functional parameters, and (2) the application of these methods to the study of vascular radiation effects in window chamber models.

Results presented here demonstrate the versatility and functionality of the data processing methods that we have developed. In the first part of Aim 1, we have developed a vessel segmentation algorithm specifically designed for processing tumor vessels, which present a challenge to existing algorithms due to their highly branching, tortuous structure. This provides us with useful information on vascular structural parameters. In the second part of Aim 1, we demonstrate a complementary vascular functional analysis algorithm, which generates quantitative maps of speed and direction. We prove the versatility of this method by applying it to a number of different studies, including hemodynamic analysis in the dorsal window chamber, the pulmonary window, and after neural electro-stimulation. Both the structural and functional techniques are shown capable of generating accurate and unbiased vascular structural and functional information. Furthermore, that automated nature of these algorithms allow for the rapid and efficient processing of large data sets. These techniques are validated against existing techniques.

The application of these methods to the study of vascular radiation effects produced invaluable quantitative data which suggest startling tumor adaptations to radiation injury. Window chamber grown tumors were treated with either widefield, microbeam, or mock irradiation. After microbeam treatment, we observed a profound angiogenic effect within the radiation field, and no signs of vascular disruption. Upregulation of HIF-1, primarily in the tumor rim, suggested that this response may have been due to bystander mechanisms initiated by oxidative stress. This HIF-1 response may have also initiated an epithelial-mesenchymal transition in the cells of the tumor rim, as post-treatment observation revealed evidence of tumor cell mobilization and migration away from the primary tumor to form secondary satellite clusters. These data indicate the possibility of significant detrimental effects after microbeam treatment facilitated through a HIF-1 response.

Understanding the differences between normal and malignant tissue is required to find vulnerabilities in cancer that can be exploited. One of the hallmarks of cancer is its ability to sustain proliferative signaling, leading to unbridled cellular replication. This puts an increased pressure on the cell’s ability to maintain genome integrity and creates a potential vulnerability to be targeted by cancer therapies. Targeting how a cancer cell prevents or responds to DNA damage is one way to take advantage of this vulnerability.

My dissertation work aims to better understand this DNA damage response in cancer and tests two hypotheses: The first is whether inhibition of the transcriptional regulator BRD4 leads to an increase in transcription-replication conflicts, DNA damage, and cell death. The second is whether the tumor microenvironment alters the way cancer cells respond to DNA damage induced by radiation therapy in glioblastoma.

Effective spatio-temporal control of transcription and replication during S-phase is paramount to maintain genomic integrity and cell survival. My work shows that BRD4, a BET bromodomain protein and known transcriptional regulator, is important for preventing dysregulation of these systems leading to conflicts between the transcription and replication machinery in S-phase. We demonstrate that inhibition or degradation of BET bromodomain proteins leads to an accumulation of RNA:DNA hybrids, a known cause of transcription-replication conflicts, and causes increased DNA damage and cell death in cancer cells actively undergoing replication. Furthermore,

over-expression of full-length BRD4, which contains a P-TEFb interacting domain known to activate efficient transcription, is necessary and sufficient to rescuing this effect. These results give mechanistic insight into chemotherapeutics that target BRD4 currently in clinical trials.

In complementary work, we explored the effect that the extracellular environment of cancer plays in its response to DNA damage caused by radiation therapy. Standard methods of culturing cancer cells, which do not replicate the extracellular environment of a native tumor, have led to an incomplete understanding of response to therapies such as ionizing radiation in vivo. To understand the role that the tumor environment plays on the radiation response, we used both human and murine glioblastoma cells to show that organotypic brain slice culture was better able to recapitulate the expression profiles of in vivo tumors. Specifically, we saw that pathways involved in multicellular processes, cell morphogenesis, and the extracellular matrix were not only significantly upregulated in glioblastoma cells cultured on brain slices compared to in vitro culture but were also critically important to radiation survival.

Collectively, this dissertation provides novel understandings of how cancer cells prevent and respond to DNA damage as well as a framework for future work in cancer biology.