HMS Theses and Dissertations

Two billion people world-wide use kerosene-burning lamps for household lighting. These lamps produce large quantities of soot. In Chapter 2, I describe our field study examining 230 people in rural Uganda. I show that kerosene lamps are a major source of smoke exposure in the developing world, and that replacing such lamps with solar-powered lights reduces indoor soot levels 17-fold, leading to significant improvements in health within months. This finding is particularly notable because respiratory disease is the #1 cause of death in children under 5 worldwide.

Because solar cells are a challenge to manufacture in the developing world, I next examined the potential of harvesting electrons from soil-based microbes as a source of clean energy. Such devices are known as microbial fuel cells (MFCs); because soil is available everywhere, MFCs can, in principle, be locally constructed all over the world. In Chapter 3, I describe our exploration of the biology of MFCs, using high-throughput DNA sequencing to demonstrate a role for genus Pseudomonas in energy production. I also examine numerous agricultural products available throughout the developing world to determine whether any could serve as a suitable ‘feed’ for MFC soil. I find that dried animal blood increases MFC energy production 10-fold. In Chapter 4, I describe our design of a modular, stackable MFC, demonstrate that it can be easily constructed in rural Africa, and use it to power lights and to charge a cell phone battery.

Background and Aims: Several single nucleotide polymorphisms (SNPs), including rs4444903 in the epidermal growth factor (EGF) gene, rs12979860 near the interleukin-28B (IL28B) gene, and rs738409 in the patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene, have been linked to treatment response, steatosis, fibrosis, and development of hepatocellular carcinoma (HCC) in chronic hepatitis C (HCV). No study has comprehensively examined the effects of these SNPs on the natural history of HCV-related cirrhosis.

Methods: We performed a retrospective cohort study of 169 subjects with chronic HCV and biopsy-proven cirrhosis who had long term followup for clinical events. Formalin-fixed, paraffin-embedded liver biopsy specimens were genotyped for EGF, IL28B, and PNPLA3 using a TaqMan assay with commercial probes and primers. Cox proportional hazards modeling was used to determine the hazard ratio for clinical decompensation, defined as the development of ascites, encephalopathy, variceal hemorrhage, HCC, or cirrhosis-related death.

Results: During a median followup of 6.6 years, 66 patients (39%) experienced clinical decompensation. On univariate analysis, EGF non-A/A, PNPLA3 non-C/C, and IL28B non-C/C genotypes were each associated with increased risk of decompensation. In multivariable Cox regression modeling, EGF non-A/A genotype was independently associated with an increased rate of clinical decompensation (HR = 3.00, p = 0.005).

Conclusions: HCV cirrhotics with the EGF A/G and G/G genotypes at rs4444903, a functional polymorphism associated with higher intrahepatic EGF levels, have an increased risk of clinical decompensation. Further study of the predictive value of EGF genotyping in patients with earlier stages and other etiologies of liver disease is warranted.

Background: The United States health care system faces two fundamental challenges: a high growth rate of health care spending and deficiencies in quality of care. The growth rate of health care spending is the dominant driver of our nation’s long-term federal debt, while the inconsistent quality of care hinders the ability of the health care system to maximize value for patients. To address both of these challenges, public and private payers are increasingly changing the way they pay providers—moving away from fee-for-service towards global payment contracts for groups of providers coming together as accountable care organizations. This thesis evaluates the change in health care spending and in quality of care associated with moving to global payment for accountable care organizations in Massachusetts in the first 4 years. This thesis studies the Blue Cross Blue Shield of Massachusetts Alternative Quality Contract (AQC), a global payment contract that provider organizations in Massachusetts began to enter in 2009. The AQC pays provider organizations a risk-adjusted global budget for the entire continuum of care for a defined population of enrollees insured by Blue Cross Blue Shield of Massachusetts. It also awards substantial pay-for-performance incentives for organizations meeting performance thresholds on quality measures. This work assesses its effect on spending and quality through the first 4 years of the contract. Methods: Enrollee-level claims data from 2006-2012 were used with a difference-in-differences design to evaluate the changes in spending and quality associated with the Alternative Quality Contract over the first 4 years. The study population consisted of enrollees in Blue Cross Blue Shield of Massachusetts plans (intervention group) and enrollees in commercial employer-sponsored plans across 5 comparison states (control group). Unadjusted and adjusted results are reported for each comparison between intervention and control. Changes in spending for all 4 AQC cohorts relative to control were evaluated. In adjusted analyses of spending, I used a multivariate linear model at the enrollee-quarter level, controlling for age, sex, risk score, indicators for intervention, quarters of the study period, the post-intervention period, and the appropriate interactions. For analyses of quality, an analogous model at the enrollee-year level was used. Process and outcome quality were evaluated. Results: Seven provider organizations joined the AQC in 2009, with a total of 490,167 individuals who were enrolled for at least 1 calendar year in the study period. The control group had 966,813 unique individuals enrolled for at least 1 year during the study period. Average age, sex, and risk scores before and after the AQC were similar between the two groups. In the 2009 cohort, claims spending grew on average $62.21 per enrollee per quarter less than control over 4 years (p<0.001), a 6.8% savings. Analogously, the 2010, 2011, and 2012 cohorts had average savings of 8.8% (p<0.001), 9.1% (p<0.001), and 5.8% (p=0.04), respectively, by the end of 2012. Savings on claims were concentrated in the outpatient facility setting, specifically procedures, imaging, and tests (8.7%, 10.9%, and 9.7%, respectively, p<0.001). Organizations with and without risk-contracting experience saw similar average savings of 6.3% and 7.7%, respectively, over 4 years (p<0.001). About 40% of savings were explained by lower volume. Pre-intervention trends were not statistically different between intervention and control (-$4.57, p=0.86), suggesting savings were not driven by inherently different trajectories of spending. No differences in coding intensity were found. In sensitivity analyses, estimates were robust to alterations in the model, variables, and sample. Notably, claims savings were exceeded by incentive payments to providers (shared savings and quality bonuses) in 2009-2011, but exceeded incentives payments in 2012, generating net savings. Improvements in quality among intervention cohorts generally exceeded New England and national comparisons. Quality performance on chronic care measures increased from 79.6% pre-intervention to 84.5% post-intervention in the 2009 cohort, compared to 79.8% to 80.8% for the HEDIS national average, a 3.9 percentage-point relative increase over the 4 years. Analogously, preventive care and pediatric care measures increased 2.7 and 2.4 percentage points relative to control, respectively. On outcome measures, achievement of hemoglobin A1c, LDL cholesterol, and blood pressure control grew by 2.1 percentage points per year in the 2009 cohort after the AQC, while HEDIS averages remained largely unchanged (Figure). Conclusion: After 4 years, physician organizations in the AQC had lower spending growth relative to control and generally outperformed national averages on quality measures. Shared savings coupled with quality bonuses can exceed savings on claims in initial years, but over time, savings on claims may outgrow incentive payments. Incentive payments themselves may serve meaningful purposes, as quality measures may protect against stinting and shared savings may help ease providers into risk contracts. Changes in utilization suggest that this payment model can help modify underlying care patterns, a likely prerequisite for sustainable reform. The AQC experience may be useful to policymakers, insurers, and providers embarking on payment reform. Combining global budgets with pay-for- performance may encourage organizations to embark on the delivery system reforms necessary to slow spending and improve quality.

Tuberous sclerosis complex (TSC) is an autosomal-dominant neurocutaneous disease caused by loss of the TSC1 (encoding hamartin) or TSC2 (encoding tuberin) genes. Neurologic symptoms are common and varied in TSC and include epilepsy and behavioral conditions like autism spectrum disorders (ASD). Between 17 and 61% of children with TSC exhibit symptoms of ASD.

The purpose of this study was to investigate a potential correlate of poor neurological outcome in TSC by assessing the integrity of brain language pathways and the relationship to ASD.

42 patients with TSC and 42 age-matched control subjects were scanned with advanced diffusion-weighted MRI. White matter language pathways were identified with a validated automatic method and analyzed for microstructural characteristics, including fractional anisotropy (FA) and mean diffusivity (MD). Well-defined white matter pathways in the brain are characterized by high FA and low MD. During normal development, brain white matter pathways increase in FA and decrease in MD.

Out of 42 patients with TSC, 12 had ASD (29%). After controlling for age, TSC patients without ASD showed a small decrease in FA of the arcuate fasciculus compared to control subjects, and TSC patients with ASD had much lower FA than both control subjects and TSC patients without ASD. Similarly, while TSC patients without ASD had only a small increase in MD compared to control subjects in the arcuate fasciculus, TSC patients with ASD had much higher MD than control subjects and TSC patients without ASD. A new method for assessing the microstructure of young patients showed similar results with decreased compactness in language pathways of TSC patients with ASD. Another new method designed to better analyze regions with crossing pathways showed modifications in language pathway microstructure that correlated with ASD diagnosis in the TSC patients. Preliminary analysis of neuropsychiatric data also showed a trend toward an association of arcuate fasciculus MD with verbal IQ, although the result was not significant after multiple comparisons correction.

It remains unclear why some patients with TSC develop ASD, while others have better language outcomes. Our results suggest that aberrant development of language pathways may act as a marker for poor neurological outcome in TSC patients. The impaired microstructure in language pathways of TSC patients may be responsible for the development of ASD, although prospective studies examining the development of language pathways and subsequent ASD diagnosis in this patient population remain essential. It is also possible that a primary problem with language leads to decreased use and subsequent poor development of language pathways. Early diagnosis of ASD is crucial for improving the outcomes of affected children.

Background: Although palliative chemotherapy is the standard of care for patients diagnosed with stage IV NSCLC, patients with a small metastatic burden, “oligometastatic” disease, may benefit from definitive local therapy.

Methods: We identified 186 patients (26% of Stage IV patients) prospectively enrolled in our institutional database from 2002-2012 with oligometastatic disease, which we defined as five or fewer distant metastatic lesions at diagnosis. Univariable and multivariable Cox proportional hazards models were used to identify patient and disease factors associated with improved survival. Using propensity score methods, we investigated the effect of definitive local therapy to the primary site on overall survival.

Results: Median age at diagnosis was 61 years, 51% of patients were female, 12% had squamous histology, and 33% had N0-1 disease. On multivariable analysis, ECOG performance status ≥2 (hazard ratio [HR] 2.43), nodal status N2-3 (HR 2.16), squamous pathology, and metastases to multiple organs (HR 2.11) were associated with a greater hazard of death (all p<0.01). Number of metastatic lesions and size of primary were not significantly associated with overall survival. Definitive local therapy to the primary tumor was associated with prolonged survival (HR 0.65, p=0.043).

Conclusions: Definitive local therapy to the primary tumor appears to be associated with improved survival in patients with oligometastatic NSCLC. Select patient and tumor characteristics, including good performance status, non-squamous histology, and limited nodal disease, may predict for improved survival in these patients.

Background: While pain is essential for physiological functioning, chronic or pathologic pain is responsible for a major burden of disease in society. Novel approaches to treating acute and chronic pain have employed neuromodulatory tools to target the central and peripheral neural structures that mediate pain. Transcranial direct current stimulation (tDCS), for example, is a safe, non-invasive brain stimulation technique that has been shown in preliminary studies to reduce chronic pain when applied to the primary motor cortex. In contrast to this exogenous neuromodulatory approach, diffuse noxious inhibitory controls (DNIC) refers to endogenous pain regulatory mechanisms that decrease pain following introduction of heterotopic noxious stimuli. This thesis explores whether combining these exogenous and endogenous pain modulation approaches synergistically increases the threshold at which pain is perceived. Methods: We conducted a double-blinded, randomized, placebo-controlled trial with a crossover design to investigate the effects of tDCS and DNIC on pain thresholds in 15 healthy human subjects. Pain thresholds were assessed prior to and following administration of active tDCS, sham tDCS, cold-water-induced DNIC, and combined active tDCS and DNIC. Using magnetic resonance spectroscopy, we examined whether baseline concentrations of brain metabolites such as N-acetylaspartate in pain-related regions of interest were associated with responses to the varying neuromodulatory conditions. Results: Pain thresholds significantly increased following both active tDCS and the DNIC paradigm. These modulatory approaches appeared to have additive effects when combined. Pain threshold increases after active tDCS were positively correlated with baseline levels of N-acetylaspartate, a marker of good neural function, in the anterior cingulate cortex and negatively correlated with baseline levels of glutamine in the thalamus. Conclusions: Combining endogenous pain regulatory mechanisms with exogenous stimulation of the motor cortex can more effectively increase pain thresholds in healthy humans. Future studies should examine whether existing pain therapies may be enhanced with noninvasive brain stimulation and activation of DNIC. They should also assess whether brain metabolite levels can be utilized to predict clinical response to therapeutic interventions.

Since academic literature indicates that emotional intelligence (EI) is tied to work performance, there is increasing interest in understanding physician EI. We studied the EI of resident physicians in surgery, pediatric, and pathology residency programs at three academic centers to describe the EI profiles of residents in different specialties and determine whether gender differences in resident physician EI profiles mirror those in the general population. 325 residents were electronically invited to complete the validated Trait Emotional Intelligence Questionnaire (TEIQue), a tool comprised of 153 items that cluster to 15 independent facets, 4 composite factors, and 1 global EI score. The overall response rate was 42.8% (n=139, 84 women). Global EI of all residents (mean=101.0, SD=8.0) was comparable to the general population sample and was not statistically different between specialties or genders. EI profiling demonstrated distinct strengths and opportunities for development between specialty groups with an effect of specialty on sociability factor (p=0.005) and five TEIQue facets including optimism, stress management, emotion management, assertiveness, and social awareness (p=0.008-0.043). Women scored higher than men in emotionality factor (p=0.044) and the TEIQue facets impulse control, empathy, relationships, and self-motivation (p=0.004-0.049). Men scored higher than women in sociability factor (p=0.034) and 2 facets including stress management and emotion management (p=0.008-0.023). Linear regression demonstrated that age had a statistically significant predictive relationship with Global EI, though the effect was small (B=0.033, p=0.014). These findings suggest that similar to the general population, male and female residents may benefit from specific training of different EI domains to enhance well-rounded development. EI profiling may also inform future educational programming decisions for each specialty. Future research should focus on the functional relationship between educational interventions that promote targeted EI development and enhanced clinical performance.

Given the recent concern from multiple healthcare stakeholders that the pipeline of medical innovation is slowing, this thesis provides insights on how to spur breakthrough medical innovation in present day. The findings and recommendations are derived from one of the largest collections of interview transcripts from biomedical innovators (n=143) responsible for developing critical devices, drugs and diagnostics used in medicine today. An exemplary case (coronary artery stent) was selected for an in-depth analysis, which included a detailed recounting of stent development and an exhaustive analysis of the patent literature. External factors either catalyzed (e.g., supportive institutions, strong underlying science and collaboration) or hindered (e.g., technology transfer challenges, lack of funding and onerous conflict of interest rules) the development process. Strategies aimed towards promoting transformative medical innovation should focus on institutional-level policies targeting early stages of innovation. This includes providing individuals with unique expertise with the capacity to pursue innovative work. Technology transfer processes should be simplified to enable collaboration for individuals between institutions with disparate expertise. Policymakers should continue to support basic science research, which underlies future innovations. In contrast, policies that increase reimbursement or reduce taxes for industry or extend patent terms are less likely to impact transformative innovation.

Objective: I sought to determine the systolic intensification threshold, time-to-intensification and time-to-follow-up associated with the lowest risk of cardiovascular events or death in primary care patients with hypertension. Methods: A retrospective cohort study of 88,756 patients was performed. Systolic intensification threshold, time-to-intensification and time-to-follow-up were analyzed with respect to risk of acute cardiovascular event or death. The Cox model was adjusted for age, sex, smoking status, socioeconomic deprivation, history of diabetes, cardiovascular disease or CKD, Charlson Comorbidity Index, BMI, medication possession ratio, and baseline blood pressure. Results: During median follow-up of 37.4 months, 9,985 participants experienced acute cardiovascular event or death (11.3%). Systolic intensification thresholds of 130-150 mmHg were associated with no difference in risk, while higher thresholds were associated with progressively greater risk. Risk increased progressively from the lowest (0-1.4 months) to the highest quintile of time to medication intensification. The highest quintile of time to-follow-up (>2.7 months) was also associated with increased risk. Conclusions: Systolic intensification threshold higher than 150 mmHg, delays of greater than 1.4 months before medication intensification following systolic blood pressure elevation, and delays of greater than 2.7 months before blood pressure follow-up following medication intensification were associated with increased risk for acute cardiovascular events or death.

Glaucoma is the leading cause of irreversible blindness worldwide, and is a major cause of blindness in the United States. It affects approximately 5% of Caucasians and 10% of African- Americans over the age of 60 years. Elevated intraocular pressure (IOP) is currently the only modifiable risk factor for glaucoma. Impaired outflow of aqueous humor from the eye is thought to be the cause of pathologically elevated IOP. However, the etiology of outflow impairment is unknown. Anatomically, the aqueous humor drains into the iridocorneal angle of the eye, where the iris inserts at the transition between the cornea and sclera. In humans, approximately 80-90% of the aqueous traverses through the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), Schlemm’s canal, collector channels and empties into episcleral veins. Abnormalities at these sites are thought to cause impaired outflow. Abnormal accumulation of extracellular matrix (ECM) in the TM or JCT, abnormal endothelial function in Schlemm’s canal, or a combination of these components have been strongly implicated. Our laboratory has focused on the role of Secreted Protein Acidic and Rich in Cysteine (SPARC) in regulating outflow. SPARC is the prototypical matricellular protein that mediates ECM organization and turnover in numerous human tissues. Our lab was first to demonstrate that SPARC is highly expressed in the TM and JCT regions of the eye, and that the SPARC knockout (KO) mouse has a significant decrease in IOP of 15-20%. SPARC may affect the degree of segmental flow, a theory that states that variable aqueous outflow occurs around the circumference of the eye; only certain portions of the TM are thought to display active outflow at any particular moment. The cytokine transforming growth factor-ß2 (TGFß2) has been shown to modulate multiple ECM proteins, including SPARC. TGFß2 is significantly upregulated by 2 to 3-fold in the aqueous humor of glaucoma patients compared to controls. In addition, when TGFß2 is overexpressed in rodent eyes, increased ECM deposition is observed within the trabecular meshwork leading to IOP elevation. SPARC is one of the most highly upregulated proteins by TGFß2, and is downstream of TGFß2. We hypothesized that wild-type (WT) mice would demonstrate segmental flow, while SPARC KO mice would display a more continuous pattern of outflow around the eye. We also believed that IOP would be inversely correlated with outflow area. We also hypothesized that SPARC is essential to the process of TGFß2-mediated ocular hypertension, and that the lack of SPARC would impair IOP elevation.

We conducted a tracer study utilizing fluorescent microbeads to determine the location of outflow circumferentially around the mouse TM. Microbeads were injected intracamerally into the eyes of WT and KO mice. After a 45-minute incubation period, the mice were euthanized and eyes were processed for confocal, light, and electron microscopy. During the second group of experiments, empty or TGFß2-containing adenovirus was injected intravitreally into WT and SPARC KO mice and IOP was measured for 2 weeks. Immunohistochemistry was completed on all tissues to assess for changes in major ECM proteins.

Percentage effective filtration length (PEFL), or area of the TM labeled by tracer, was significantly increased in SPARC KO mice (70.61% ± 11.36%, p<0.005; N=11) compared to WT mice (54.68% ± 9.95%; N=11). In addition, the pressures between the two sets of eyes were significantly different with mean pressures of 16.3 mm Hg in WT mice and 12.6 mm Hg in KO mice (p<0.005, N=11 pairs). In addition, PEFL and IOP were inversely correlated with R2 = 0.72 (N=10 pairs); in eyes with higher IOP, PEFL was reduced. Electron microscopy demonstrated that high-tracer TM areas had a greater separation between trabecular beams. Collagen fibril diameter was found to be smaller in the KO (28.272 nm) compared to WT (34.961 nm; p<0.0005, N=3 pairs). These data provided structural correlations to the functional data regarding segmental flow.

In the second set of experiments, IOP was found to be significantly elevated in TGFß2- injected WT mice compared to empty vector-injected WT mice during days 4-11 (p<0.05, N=8). However, IOP was not significantly elevated in TGFß2-injected KO mice compared to controls. Immunohistochemistry demonstrated that TGFß2 increased expression of collagen IV, fibronectin, plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), and SPARC within the TM of WT mice, but only PAI-1 and CTGF in KO mice (p<0.05, N=3 pairs).

These data support our hypotheses, indicating that SPARC plays an integral role in the modulation of aqueous humor outflow. In addition, it appears as though SPARC is essential to the regulation of TGFß2-mediated ocular hypertension. Aside from providing further evidence of the importance of ECM in IOP regulation, our work presents the novel discovery of segmental flow in the mouse. Given the potential role of SPARC in TGFß2-mediated ocular hypertension, SPARC may not only play an integral role in ECM homeostasis within the trabecular meshwork, but may be a valuable target for pharmacologic therapy in treating primary open-angle glaucoma.

Objectives: Technological innovation in medicine is a significant driver of healthcare spending growth in the United States. Factors driving adoption and utilization of new technology is poorly understood, however market forces may play a significant role. Vascular surgery has experienced a surge in development of new devices and serves as an ideal case study. Specifically, the share of total abdominal aortic aneurysm (AAA) repairs performed by endovascular aneurysm repair (EVAR) increased rapidly from 32% in 2001 to 65% in 2006 with considerable variation between states. This paper hypothesizes that that hospitals in competitive markets were early EVAR adopters and had improved AAA repair outcomes.

Methods: The Nationwide Inpatient Sample (NIS) and linked Hospital Market Structure (HMS) data was queried for patients who underwent repair for non-ruptured AAA in 2003. In HMS the Herfindahl Hirschman Index (HHI, range 0-1) is a validated and widely accepted economic measure of competition. Hospital markets were defined using a variable geographic radius that encompassed 90% of discharged patients. Bivariable and multivariable linear and logistic regression analyses were performed for the dependent variable of EVAR use. A propensity score-adjusted multivariate logistic regression model was used to control for treatment bias in the assessment of competition on AAA-repair outcomes.

Results: A weighted total of 21,600 patients was included in the analyses. Patients at more competitive hospitals (lower HHI) were at increased odds of undergoing EVAR vs. open repair (Odds Ratio 1.127 per 0.1 decrease in HHI, P<0.0127) after adjusting for patient demographics, co-morbidities and hospital level factors (bed size, teaching status, AAA repair volume and ownership). Competition was not associated with differences in in-hospital mortality or vascular, neurologic or other minor post-operative complications.

Conclusion: Greater hospital competition is significantly associated with increased EVAR adoption at a time when diffusion of this technology passed its tipping point. Hospital competition does not influence post-AAA repair outcomes. These results suggest that adoption of novel technology is not solely driven by clinical indications, but may also be influenced by market forces.

Poly(ADP-ribose)polymerase-1 and -2 (PARP1/2) are nuclear proteins involved in DNA repair. Tumors with defects in homologous recombination, including BRCA1- and BRCA2-deficient cancers, have been shown to be sensitive to PARP inhibition. The Weissleder group has synthesized fluorescent and radioactive derivatives of the PARP1/2 inhibitor AZD2281. We hypothesized that fluorescent and radioactive AZD2281-based imaging agents would quantify PARP1/2 expression in vitro and in vivo. To test this hypothesis, a panel of pancreatic ductal adenocarcinoma and ovarian carcinoma cell lines were characterized by immunocytochemistry for PARP1/2 expression. AZD2281-derived fluorescence signal correlated with anti-PARP antibody fluorescence signal strength in vitro. Four cell lines representing a range of PARP1/2 expression levels were then xenografted into Nu/Nu mice. Mice bearing four tumor types each were imaged with AZD2281-derived imaging agents, sacrificed, and their tumors excised for stand-alone imaging and Western blot. AZD2281-derived signal correlated with tumor PARP1/2 expression determined by Western blot, indicating that PARP1/2 expression level is a determinant of fluorescent signal strength and SUVs of AZD2281-derived agents in vivo. These data indicate that AZD2281-derived agents are useful tools for quantifying intracellular PARP1/2 both in vitro and in vivo, which could one day enable prospective identification of tumors likely to respond to PARP inhibitors.

Short interfering RNA (siRNA) is a class of nucleotide drugs with a profound potential to improve patient health through its ability to silence the expression of specific genes at the post-transcriptional level. However, the clinical application of siRNA therapeutics remains hindered by a lack of efficient delivery systems that deposit siRNA into the cytoplasm of cells, a step necessary for siRNA’s silencing effect. Much research has focused on the development of siRNA delivery agents to overcome this challenge. There are no standard pre-clinical models for testing of siRNA delivery agents, and investigators have chosen to evaluate efficacy in a variety of systems including in vitro tissue culture and animal models. These systems have vastly different cellular microenvironments which may modulate cellular behavior and affect the response of cells to siRNA, thus altering the apparent efficacy of siRNA delivery agents. The substrate on which cells adhere is one aspect of the microenvironment that has been previously shown to alter cellular behavior. In this work, we tested the hypothesis that changing the properties of cellular adhesion substrates can change the apparent efficacy of a siRNA delivery agent. Specifically, we used a commonly employed in vitro cationic lipid siRNA delivery vector and evaluated siRNA silencing efficacy in U251 cells seeded on alginate hydrogel surfaces. These surfaces were synthesized to have systematic variation in integrin ligand arginine-glycine-aspartate (RGD) density and elastic modulus. We found that an eightfold increase in RGD content of the alginate grown substrate increased siRNA knockdown efficacy from 25 ± 12% to 52 ± 10%, with constant concentrations of siRNA and delivery agent. We found no difference in siRNA mediated knockdown efficacy over the elastic modulus range tested (53-133 kPa). These results indicate that the cell-adhesion substrate interaction can modulate siRNA protein silencing efficacy, a finding important for evaluation of siRNA therapeutics in the in vitro setting.

Great disparities exist in women’s health outcomes in rural indigenous communities in Guatemala; indigenous women are less likely to utilize family planning and cervical cancer screening services. This pilot study assessed baseline women’s health knowledge and the effects of a comprehensive women’s health course on women’s health knowledge and behaviors. From February to November 2013, 61 participants in a seven-week language-appropriate women’s health curriculum were evaluated before and after the course using a 10-item knowledge assessment. Cervical cancer screening and contraceptive utilization rates were assessed by self-report before and after the course. Several women’s health knowledge deficits were noted: belief that contraception causes cancer, inability to name symptoms of STIs, lack of understanding of pap smears, and lack of familiarity with condoms. The average pre-test score was 54.6%; increasing to 83.7% on the post-test (p<0.0001). 79% had received a pap smear prior to the course; this increased to 92% at the post-test (p=0.013). 53% had utilized contraception prior to the course; at the time of the post-test the percentage was 54% (p=1). This study reveals important patterns in women’s health beliefs in rural Guatemala, and shows that a seven-week curriculum was effective in improving scores on a knowledge assessment.

The ability to precisely manipulate the genome in a targeted manner is fundamental to driving both basic science research and development of medical therapeutics. Until recently, this has been primarily achieved through coupling of a nuclease domain with customizable protein modules that recognize DNA in a sequence-specific manner such as zinc finger or transcription activator-like effector domains. Though these approaches have allowed unprecedented precision in manipulating the genome, in practice they have been limited by the reproducibility, predictability, and specificity of targeted cleavage, all of which are partially attributable to the nature of protein-mediated DNA sequence recognition. It has been recently shown that the microbial CRISPR-Cas system can be adapted for eukaryotic genome editing. Cas9, an RNA-guided DNA endonuclease, is directed by a 20-nt guide sequence via Watson-Crick base-pairing to its genomic target. Cas9 subsequently induces a double-stranded DNA break that results in targeted gene disruption through non-homologous end-joining repair or gene replacement via homologous recombination. Finally, the RNA guide and protein nuclease dual component system allows simultaneous delivery of multiple guide RNAs (sgRNA) to achieve multiplex genome editing with ease and efficiency.

The potential effects of off-target genomic modification represent a significant caveat to genome editing approaches in both research and therapeutic applications. Prior work from our lab and others has shown that Cas9 can tolerate some degree of mismatch with the guide RNA to target DNA base pairing. To increase substrate specificity, we devised a technique that uses a Cas9 nickase mutant with appropriately paired guide RNAs to efficiently inducing double-stranded breaks via simultaneous nicks on both strands of target DNA. As single-stranded nicks are repaired with high fidelity, targeted genome modification only occurs when the two opposite-strand nicks are closely spaced. This double nickase approach allows for marked reduction of off-target genome modification while maintaining robust on-target cleavage efficiency, making a significant step towards addressing one of the primary concerns regarding the use of genome editing technologies.

The ability to multiplex genome engineering by simply co-delivering multiple sgRNAs is a versatile property unique to the CRISPR-Cas system. While co-transfection of multiple guides is readily feasible in tissue culture, many in vivo and therapeutic applications would benefit from a compact, single vector system that would allow robust and reproducible multiplex editing. To achieve this, we first generated and functionally validated alternate sgRNA architectures to characterize the structure-function relationship of the Cas9 protein with the sgRNA in DNA recognition and cleavage. We then applied this knowledge towards the development and optimization of a tandem synthetic guide RNA (tsgRNA) scaffold that allows for a single promoter to drive expression of a single RNA transcript encoding two sgRNAs, which are subsequently processed into individual active sgRNAs.

Background: Despite multi-modality therapy, the majority of patients with metastatic or recurrent Ewing sarcoma (ES), the second most common pediatric bone malignancy, will die of their disease. ES tumors express aberrantly activated ETS transcription factors through translocations that fuse the EWS gene to ETS family genes FLI1 or ERG. The aberrant activation of ETS transcription factors promotes malignant transformation and proliferation. While, FLI1 or ERG cannot be readily targeted, there is an opportunity to deploy functional genomics screens, to develop novel therapeutic approaches by identifying targetable liabilities in EWS/FLI1 dependent tumors. Materials and Methods: We performed a near whole-genome pooled shRNA screen in a panel of five EWS/FLI1 dependent Ewing sarcoma cell lines and one EWS/ERG cell line to identify essential genes. Essential genes were defined as those genes whose loss resulted in reduced viability selectively in ES cells compared to non-Ewing cancer cell lines. Essential hits were subsequently validated with genomic knockdown and chemical inhibition in vitro, followed by validation of the on-target effect of chemical inhibition. Next, we determined the in vivo effects of small-molecule inhibition on survival and tumor growth in NOD scid gamma (NSG) mice with established subcutaneous ES xenografts. Results: Top hits in our screen that could be readily targeted by small-molecule inhibitors, and thus have potential for rapid clinical validation, were selected for further investigation. These hits included IKBKE, CCND1 and CDK4. IKBKΕ, a non-canonical IKK with an oncogenic role in breast cancer, was one of the top kinase hits in the screen. IKBKΕ shares significant homology to TBK1, another non-canonical IKK that is essential in k-RAS dependent lung cancer. We validated IKBKE through small-molecule inhibition of IKBKE/TBK1 and shRNA based knockdown. Ewing sarcoma cell lines are sensitive to low micromolar concentrations of two IKBKE/TBK1 inhibitors (CYT387 and MRT67307). Additionally, in a panel of ES cell lines, knockdown of IKBKE resulted in decreased growth and impaired colony formation. These observations, paired with impairment of NF-κB nuclear localization following CYT387 treatment suggests that non-canonical IKK mediated signaling may be essential in Ewing sarcoma. We further validated these results through inhibition of IKBKE/TBK1 in in vivo xenograft models treated with 100 mg/kg/day of CYT387. Treatment over the course of twenty-nine days resulted in a significant increase in survival (p-value = 0.0231) and a significant decrease (p-value = 0.036) in tumor size after fifteen days of treatment. CDK4 and CCND1 are highly expressed in Ewing sarcoma as compared to other tumor types. shRNA mediated knockdown of CDK4 and CCND1 resulted in impaired viability and anchorage independent growth. Furthermore, treatment of Ewing sarcoma cell lines with a highly selective CDK4/6 inhibitor, LEE011, resulted in decreased viability (IC50 range of 0.26-18.06 μM), potent G1 arrest in six of eight EWS/FLI1 containing Ewing sarcoma lines tested and apoptosis in a panel of four highly sensitive lines. Administration of 75 mg/kg/day and 250 mg/kg/day of LEE011 in NSG mice with Ewing xenografts resulted in significant impairment of tumor growth, (p-value <0.001 for both treatment arms), as compared to vehicle control.
Conclusions: These studies suggest a role for the targeting of IKBKE and CDK 4/6 in Ewing sarcoma, findings with immediate clinical relevance for patients with this malignancy, because small-molecule inhibitors of these proteins have already entered clinical trial for other disease indications.

By buffering cytosolic calcium, mitochondria can shape the magnitude and duration of intracellular calcium transients, which in turn govern key physiological events. Although controlled uptake of calcium into the matrix influences the rate of ATP production, excess calcium within the matrix triggers non-specific permeabilization of the mitochondrial inner membrane, resulting in cell death. Despite its importance in cellular physiology, the molecular identity of the mitochondrial calcium uniporter remained a mystery for nearly five decades. Recently, an approach inspired by comparative genomics was used to identify two proteins required for high-capacity mitochondrial calcium uptake. These include MICU1, an EF-hand protein that may function as a regulatory component by sensing calcium, and MCU, the channel-forming subunit of the uniporter. In this work, I explore two distinct areas within the growing field of molecular mitochondrial calcium biology. First, I discuss the identification of a new protein, MICU1-paralog EFHA1, and present data that implicates it in mitochondrial calcium uptake. Subsequently, I describe efforts to establish an in vitro system to characterize the channel activity of MCU, including my contribution to the development of a liposome-based assay for calcium transport and preliminary work aimed at reconstituting MCU transport activity in proteoliposomes.

Repair of peripheral nerve injury often results in poor functional motor recovery. This deficit has previously been attributed to the failure of axons to regenerate into the muscle. However, we have recently reported that following nerve injury in mice, axons have regenerated to the motor end plate in animals with poor recovery. We proposed that following axonal injury, there is a critical period during which the axon must reach the muscle in order to form a functional neuromuscular junction. We have developed a mouse model of prolonged denervation, in which the sciatic nerve is crushed repeatedly every few days, preventing regenerating axons from reaching the muscle. This multiple crush model allows us to vary the period of denervation by modifying the number of crushes. Motor recovery as assessed using the toe-spreading score occurs after 3 or 4 multiple crushes every 7 days (24 or 31 days of denervation) but not after 5 crushes (38 days). Immunostaining for alpha-bungarotoxin and neurofilament confirmed end plate reinnervation. Thus following denervation > 38 days, a motor deficit persists despite end plate reinnervation. Although the mechanism for the deficit requires investigation, these results suggest that functional neuromuscular junction reestablishment more than end plate reinnervation and that there is a time limit for functional synapse reformation.

Glioblastoma is the most common primary brain tumor in adults, and it is associated with a dismal prognosis with a median survival of 15 months. Despite treatment with chemotherapy, radiation therapy and surgery, patients inevitably have disease recurrence. Bevacizumab is a monoclonal humanized antibody that inhibits vascular endothelial growth factor signaling, and it has been shown to be effective in recurrent glioblastoma with respect to prolonging progression-free survival (PFS). The use of bevacizumab and other anti-angiogenic agents in recurrent glioblastoma have created novel challenges in interpreting magnetic resonance imaging (MRI) of patients. Furthermore, since only some patients appear to have a durable benefit from bevacizumab, there is a need for imaging biomarkers that can reliably identify this subgroup of patients.

Partly due to the challenges created by anti-angiogenic agents, the Response Assessment in Neuro-Oncology (RANO) was proposed to address some of the limitations with traditional response assessment criteria. In the first part of this project, we attempted to validate the RANO criteria by performing a comparative analysis of the RANO criteria vs. the Macdonald criteria using imaging from the phase II BRAIN trial. As we hypothesized, the RANO criteria yielded a significantly decreased PFS by identifying a subset of patients who had progression of nonenhancing tumor evident on T2-weighted imaging. Additionally, response and progression as defined by the RANO criteria correlated with subsequent overall survival (OS) in landmark analyses. While this supports the implementation of RANO criteria for response assessment in glioma clinical trials, future research will be necessary to further improve response assessment by incorporating advanced techniques such as volumetric anatomic assessment, perfusion-weighted MR (PWI-MR), diffusion-weighted MR (DWI-MR), MR spectroscopy (MRS) and positron emission tomography (PET).

Advanced imaging techniques are becoming increasingly recognized for their ability to provide objective, non-invasive assessment of treatment response but also to serve as predictive and prognostic biomarkers allowing for stratification of patient subgroups with better treatment outcome. In the second part of the project, we attempted to perform volumetric analysis of tumor size based on conventional MRI, as well as a histogram analysis of apparent diffusion coefficients (ADC) derived from diffusion-weighted MRI, to evaluate imaging parameters as predictors for PFS and OS in a single institution database of recurrent glioblastoma patients initiated on bevacizumab. Volumetric percentage change and absolute early post-treatment volume (3-6 weeks after initiation) of enhancing tumor can stratify survival for patients with recurrent glioblastoma receiving bevacizumab therapy. ADC histogram analysis using a multi-component curve-fitting technique within both enhancing and nonenhancing components of tumor prior to the initiation of bevacizumab can also be used to stratify OS in recurrent glioblastoma patients. While prospective studies are necessary to validate findings, future studies will increasingly incorporate multiparametric approaches to elucidate biomarkers that combine the value of conventional MRI with advanced techniques such as DWI-MR, PWI-MR, MRS and PET to obtain better predictors for PFS and OS in recurrent glioblastoma.

Introduction: During the last decade, our institution saw a 260% increase in bilateral breast reconstruction cases, consistent with national trends. We reported a drop in average age of prophylactic mastectomy from 57 to 51 years. There is limited data on the likelihood of histological abnormalities in this population. This study measures the prevalence of occult histological findings in prophylactic mastectomy patients. Given the current healthcare reform climate, we estimate the lifetime cost implications of prophylactic mastectomy with immediate reconstruction vs. surveillance.

Methods: A retrospective database of breast reconstructions at the Massachusetts General Hospital was searched from 2004 to 2011 for prophylactic mastectomy patients. Breasts with prior biopsy-proven LCIS, DCIS, or cancer were excluded. Patient demographics, risk factors, and pathology reports were collected. Lifetime treatment reimbursements were estimated with 2013 rates from the Center for Medicare and Medicaid Services using Medicare billing codes. Reimbursements were estimated for 45-year-old patients undergoing contralateral prophylactic mastectomy and 40-year-old patients undergoing bilateral prophylactic mastectomies, and then were compared to women opting for surveillance. Conversion rates to cancer in these patients were used to estimate the percentage patients in the surveillance groups that would need therapeutic mastectomy. Sensitivity analyses were done to test the robustness of the models.

Results: 495 prophylactic mastectomy specimens were identified, of which 2.0% had invasive cancer, 4.4% had ductal carcinoma in situ (DCIS), and 10.9% had lobular carcinoma in situ (LCIS) as the highest-risk lesion. Only age group was predictive of finding DCIS or cancer (P=0.02). The likelihood of finding LCIS, DCIS, or cancer increased with age group (P<0.001) and decreased with prior bilateral salpingo-oophorectomy (BSO)(P=0.02). In almost all scenarios, lifetime reimbursements were lower for pursuing either contralateral or bilateral prophylactic mastectomy, with immediate single-stage implant, expander, or abdominal perforator free flap (DIEP) reconstruction, as compared to surveillance.

Conclusions: Prophylactic mastectomy patients have a significant rate of occult histological findings, increasing with age group and decreasing with prior BSO. Lifetime cost estimates suggest a cost-saving role in bilateral and contralateral prophylactic mastectomies. Ultimately, such a critical decision needs to be made individually, but should not be hindered by cost concerns. This study addresses a gap in knowledge with broad interest, contributing evidence of oncologic risk and cost to help guide decision-making in prophylactic mastectomy.