Melanoma is rapidly increasing in incidence throughout the world. Early stages are curable with surgical approaches with excellent prognosis. However, a substantial proportion of patients progress to metastatic disease with survival rates of less than 5% making melanoma the culprit for over 65% of all skin-cancer related deaths. Novel agents targeting the immune system and the signaling pathways of melanoma are generating new promise, but chemotherapy remains an important therapeutic alternative, despite low response rates. The resistance of melanoma to chemotherapy is in part due to DNA repair mechanisms that allow cells to survive alkylation damage. Several novel agents targeting the abrogation of DNA repair pathways alone and in combination with cytotoxic agents have been developed with varying measures of success. In this dissertation, we first identified the epigenetic silencing of the DNA mismatch repair (MMR) gene MLH1 as a determinant of response and survival for melanoma patients treated with alkylator-based chemotherapy (dacarbazine/ temozolomide). We then determined the safe dosage of the epigenetic agent decitabine that can be administered in combination with temozolomide. The safety, tolerability and efficacy of the combination of decitabine and temozolomide were evaluated in a Phase II population. We finally determined the pharmacokinetic and pharmacodynamic effects of treatment with the combination of decitabine and temozolomide in the blood and tumor tissues of metastatic melanoma patients.

Immunotherapy utilizing cytokines or immune regulatory check point blockade has consistently demonstrated superior clinical efficacy in melanoma when compared to tumor peptide immunization strategies reported to date. In this project, I conducted 2 model studies representing alternative immunotherapeutic approaches (non-antigen specific combination of interferon-á2b and an anti-CTLA4 monoclonal antibody, IFN-Treme compared to a tumor antigen specific multi-epitope vaccine given in adjuvant with the potent combination of a TLR-9 agonist and GM-CSF) designed to overcome tumor immune evasion and conducted separately in a similar patient population. In addition to evaluating safety and clinical efficacy, I tested the following hypotheses: (1) Clinical benefits are likely to be associated with markers of reversal of immune tolerance (autoimmunity). (2) Clinical benefits may be predicted by baseline peripheral biomarkers of immune tolerance/suppression (C-reactive protein, CRP and absolute lymphocyte count, ALC). (3) Superior antitumor efficacy is likely to be associated with more effective downregulation of the host suppressor immune response (circulating T regulatory cells, T-reg and myeloid derived suppressor cells, MDSC). My findings supported superior clinical efficacy that was associated with more significant modulation of immune tolerance by the combination of IFN-Treme. Autoimmunity correlated with improved clinical outcome among the recipients of IFN-Treme (but not the vaccine) and suggested more significant reversal of immune tolerance. Baseline CRP and ALC were significantly predictive of therapeutic benefit with the IFN-Treme combination and may serve as variables for stratification of future trials, as these are validated in larger studies. Finally, my findings supported more significant downregulation of the host suppressor immune response by the nonspecific IFN-á/Treme regimen as compared to the vaccine-TLR agonist/GM-CSF combination. There was apparent increase in CD4+CD25hi+ CD39+ Treg but this was associated with an increase in the overall CD4+ T cell population suggesting that direct inhibition of CTLA4 suppressive effects on T effector cells leading to their expansion and prolonged activation is likely more important than the regimens effect on T-reg. In addition, I saw parallel downregulation in several populations of MDSC following treatment with IFN-Treme which may have had a role in the reduction of immune suppression and superior clinical outcome observed.

There is a critical gap in translating scientific discoveries to public health benefit. For example, despite a multitude of efficacious physical activity interventions, only one in four adults in the United States meets the Physical Activity Guidelines for Americans. To bridge the research-practice gap, Dissemination and Implementation (DandI) Science has emerged as the study of how evidence-based interventions, programs, and policies are integrated in typical settings. Recent research illustrates barriers to conducting DandI Science and the need for methods that open the black box of implementation. Therefore, the overarching goal of this dissertation was to explore novel approaches for advancing DandI Science. This exploration is presented in three manuscripts and one report. The first manuscript presents a pragmatic, observational study applying the RE-AIM (Reach, Effectiveness, Adoption, Implementation, Maintenance) Framework to evaluate a perinatal health fair. Results include that the health fair reached 42 attendees and that 23 educators and seven organizations hosted booths and educational sessions. Mom Expo required 292 implementation hours with 71% of those hours devoted to building relationships. We generated 30 actionable strategies for implementing a health fair. The health fair developed into a non-profit organization, and the participatory approach used can be replicated in other communities to establish connections between local women, educators, and researchers. The second manuscript reports a one-year autoethnography (i.e., first-person narrative) of a perinatal health integrated research practice partnership (IRPP). Findings include three themes: (1) permeable work boundaries, (2) blind spots toward philosophical underpinnings of paradigms, and (3) maladaptive behaviors seemingly reinforced by the research culture. We concluded that autoethnography is an effective novel method to leverage researcher situatedness and capture implementation contexts, processes, and outcomes. The third manuscript presents the longitudinal pilot test of FUEL (focus, unplug, exercise, love), a one-on-one coaching program to promote human flourishing among DandI researchers. Results include that the coach spent 12.96+2.82 hours per participant (N= 16) implementing individually-tailored sessions, and that participants reported multiple, sustained benefits related to productivity, happiness, and health. We concluded that the program is a feasible, well-received approach with preliminary positive effects. Future work is needed to investigate physiological or performance outcomes and, ultimately, impact on DandI. The final report is a literature review and critical analysis of phenomenology within behavioral and community health research. Conclusions include that (1) physical activity is rooted in a scientific paradigm that prioritizes quantifiable mechanism over personal meaning, and (2) phenomenology, as a complement to basic science, is a compelling method, paradigm, and practice to improve research translation. Based on this research, I conclude that three pathways for advancing DandI Science are methods that capture first-person meaning, paradigms that incorporate phenomenological human experience as an essential dimension of health research, and practices that fuel researchers' capacity for generating transformative work. In all three pathways, the heart of elevating DandI Science is to embrace process, person, and presence. / Doctor of Philosophy / Scientific evidence does not automatically translate to real-world behavior change. For example, despite considerable research about the health benefits of physical activity, only one in four American adults meets the national physical activity recommendations. To bridge the research-practice gap, Dissemination and Implementation (DandI) Science has emerged as the study of how scientific findings are integrated in typical settings such as schools and communities. Recent research illustrates multiple barriers to DandI Science and a need for methods that capture hard-to-measure, chaotic implementation processes and outcomes. Therefore, the overarching goal of this dissertation was to explore novel approaches to DandI Science and bridging the research-practice gap. This exploration is presented in three manuscripts and one report. The first manuscript describes a perinatal health fair intended to connect local parents to community resources. The second manuscript is a 12-month autoethnography (i.e., first-person narrative) about the culture of DandI Science, including the role, impact, and practices of researchers themselves. The third manuscript presents the development and preliminary testing of FUEL (focus, unplug, exercise, love), a one-on-one coaching program for DandI researchers. The final report includes the history of randomized controlled trials as the gold standard for physical activity research, as well as critical analysis of using phenomenology to reduce the research-practice gap. Findings from the first manuscript suggest that (1) authentic relationship building was the key to launching a perinatal health fair that developed into a non-profit organization and (2) the RE-AIM (Reach, Effectiveness, Adoption, Implementation, Maintenance) Framework is a user-friendly DandI tool for implementing and evaluating a health fair. Findings from the second manuscript include descriptions of (1) blind spots within the DandI Science culture, (2) potential of autoethnography as a novel DandI method, and (3) strategies to optimize DandI researchers' capacity to thrive amid challenges. Findings from the third manuscript suggest that the FUEL coaching program is a promising and feasible approach to support researchers in leading "a more productive, healthier, and happier life," as one participant wrote. Future research on the program is needed to evaluate causation and whether organizations would adopt it. Conclusions in the final report include that (1) the applicability of physical activity research to daily life may be limited by deeply held scientific ideologies and (2) phenomenology, as the study of human meaning, may facilitate the translation of research to real-world behavior change. Based on the research presented in this dissertation, three pathways for advancing DandI Science are methods for how we conduct research studies, paradigms for how we collectively approach health science, and practices for how we manage our energy and awareness. In all three pathways, the heart of elevating DandI Science is to embrace process, person, and presence.

Objective: The purpose of this study was to assess the influence of three single nucleotide polymorphisms (SNP) previously associated with Alzheimer's disease on specific domains of cognition, when controlling for Apolipoprotein E gene (APOE), in a sample of individuals with Alzheimer's disease. Methods: The data were drawn from the Alzheimer's Disease Neuroimaging Initiative database, a comprehensive, longitudinal database of controls, persons with mild cognitive impairment, and persons with mild Alzheimer's disease. Each subject has a full neuropsychological assessment, neuroimaging, genetic sequencing, and physical evaluation. For the purposes of this study, individuals were selected based on the presence of the three SNPs of interest: CR1 (rs3818361_T), CLU (rs11136000_T), and PICALM (rs3851179_A). Each SNP was then measured against the available tests of the ADNI neuropsychological battery that measured immediate and long delay memory, semantic fluency, and confrontation naming. Results: Only the CR1 SNP (rs3818361_T) had significant findings. The presence of the CR1 SNP associated with lower performance on logical memory recall total score, AVLT immediate recall trials 2 and 4, AVLT delayed recall, and confrontation naming in the 12-month control group. Logical memory and AVLT delayed recall were also negatively associated with CR1 in the 12-month AD case group. Discussion: These results support previous findings that the CR1 SNP rs3818361_T is a risk factor for cognitive impairment in individuals with and without AD. Such findings can aid in the earlier detection of Alzheimer's disease, risk for domain specific cognitive impairment, and novel targets for personalized pharmacotherapy.

<div><b>Background:</b> Bupropion, an atypical antidepressant and smoking cessation aid, is associated with wide inter-subject variability in its efficacy and safety. Variability in response to bupropion therapy is thought to be driven by variability in metabolism. Bupropion undergoes complex phase 1 and 2 stereoselective metabolism. Though bupropion`s pharmacology is not fully understood, much of it is thought to be due to its metabolites, specially, S, S-hydroxybupropion. In vitro studies (functional assays measuring IC50 at dopamine transporter-DAT, norepinephrine transporter-NET, various subtypes of nicotinic receptors-nAChR) and mouse models (forced swim test to assess antidepressant effect, antinociceptive models to assess antagonism of nicotine effects) indicate S, S-hydroxybupropion to contribute more towards efficacy as an antidepressant and smoking cessation aid than racemic bupropion and R, R-hydroxybupropion, respectively. Both pharmacokinetics (PK) and pharmacodynamics (PD) of bupropion and its metabolites are complex and reported to be stereoselective. As bupropion is known to act on multiple central nervous system (CNS) targets (DAT, NET nAChR), understanding CNS disposition (target site) is critical to explain variability in bupropion`s therapeutic and toxic effects. </div><div><b>Objective: </b>The objective of our study was to characterize the exposure of bupropion enantiomers and corresponding phase 1 metabolite diastereomers in plasma and brain in a surrogate non-clinical species, and to subsequently develop animal-to-human-translational population-PK and Physiologically Based PK (PBPK) models to predict human brain concentrations of bupropion and its active metabolite S, S-hydroxybupropion. Application of these PK modeling approaches to map the time course of unbound brain concentration can then be compared to in vitro potency measures at DAT, NET and nAChRs to predict target engagement over time (PD). Establishing relationships between plasma PK, target site PK along with PD would elucidate possible cause(s) of inter-patient variability to bupropion therapy. </div><div><b>Methods: </b>The first step towards development of a CNS model was to identify a nonclinical species with phase 1 metabolism closest to humans. To accomplish this, hepatic microsomal incubations of four species-rat, mouse, non-human primates (NHPs) and humans were conducted separately for the R- and S-bupropion enantiomers, and the formation of enantiomer-specific metabolites was determined using LC-MS/MS. Intrinsic formation clearance (CLint) of metabolites across the four species (rats, mice, NHPs, humans) was determined from the formation rate versus substrate concentration relationship. </div><div>Racemic bupropion (10 mg/kg) and preformed S, S-hydroxybupropion (2 mg/kg) were administered subcutaneously to adult male Sprague Dawley rats (n = 24/compound). Brain and plasma were collected from rats (n = 3) at eight time points for 6 hours and analyzed using a chiral LC-MS/MS method. Rat plasma protein and brain homogenate binding studies were conducted for all analytes to correct for unbound fraction using equilibrium dialysis method.</div><div>A plasma-brain compartmental pharmacokinetic approach was used to describe the blood–brain-barrier transport of both bupropion and S, S-hydroxybupropion. Also, a 2-compartment permeability-limited brain model consisting of brain blood, brain mass compartments was developed and incorporated into a whole body physiologically-based pharmacokinetic (PBPK) parent-metabolite model for bupropion and S, S-hydroxybupropion. Both population PK and PBPK modeling approaches were subsequently translated to humans to predict human plasma and brain site exposure and its relationship to DAT and NET IC50 potencies.</div><div><b>Results: </b>The total clearance of S-bupropion was higher than that of R-bupropion in monkey and human liver microsomes. The contribution of hydroxybupropion to the total racemic bupropion clearance was 38%, 62%, 17%, and 96% in human, monkey, rat, and mouse, respectively. In the same species order, threohydrobupropion contributed 53%, 23%, 17%, and 3%, and erythrohydrobupropion contributed 9%, 14%, 66%, and 1.3%, respectively, to racemic bupropion clearance. Hepatic microsomal incubation studies indicated non-human primates to be the appropriate species to model CNS disposition. However, the cost and limited pharmacokinetic and pharmacodynamic data in NHPs were insurmountable barriers to conducting in vivo studies in NHPs. After considering multiple factors, such as the formation of reductive metabolites (higher in rats than mice), which are also thought to contribute to bupropion`s therapeutic efficacy, availability of microdialysis data measuring bupropion and dopamine, norepinephrine levels in brain extracellular fluid (ECF) and other in vitro potency evaluations in rats, rat was chosen as the surrogate species to model bupropion`s disposition.</div><div>In rats, unbound plasma and brain exposures and plasma clearances of both R and S-bupropion were similar. The exposure to parent was higher (50 to 100-fold) than to metabolites. The exposure of oxidative metabolites (R, R- and S, S-hydroxybupropion) was 2 to 3-fold higher in brain and plasma than reductive metabolites (R, R- and S, S-threohydrobupropion, S, R- and R, S-erythrohydrobupropion). Hepatic clearances of R- and S-bupropion scaled from in vitro rat hepatic microsomal incubation studies were 3-fold and 25-fold lower than their respective in vivo unbound apparent clearances. This could possibly be due to substantial contribution of metabolic pathways not characterized in this in vivo study and/or possible extrahepatic disposition in the rat. The unbound brain to unbound plasma AUC0-6h ratio (Kp,uu) of R- and S-bupropion were 0.43 and 0.38 respectively. Kp,uu of oxidative metabolites (R, R- and S, S-hydroxybupropion) and reductive metabolites (R, R- and S, S-threohydrobupropion) were close to 1. Kp,uu of S, R-erythrohydrobupropion was 0.43 and that of pre-formed S, S-hydroxybupropion was 5.</div><div>With respect to population PK modeling of both bupropion and S, S-hydroxybupropion, a plasma-brain compartmental model structure with time dependent change in brain influx clearance was required to adequately characterize the BBB transport of parent and this active metabolite. Using a physiologically-based pharmacokinetic model (PBPK) approach too, incorporation of active efflux and carrier mediated uptake terms in addition to passive permeability was necessary to adequately characterize brain disposition of bupropion and S, S-hydroxybupropion. Both modeling approaches (population-PK and PBPK) when translated to humans indicated that the predicted human brain exposures fall below the reported DAT and NET IC50 measures of bupropion and S, S-hydroxybupropion. </div><div><b>Conclusion: </b>Specific to our work in the rat, the discrepancy between in vitro scaled hepatic clearance and in vivo plasma clearance of R and S-bupropion suggests alternative non-CYP mediated clearance pathways and/or extra hepatic disposition of bupropion. Both translational PK models indicate active process such as efflux transporter or carrier mediated uptake could be involved in bupropion`s disposition in the brain. Variability in expression of these speculated active/carrier mediated transporters could possibly cause variability in response. Also, other CNS targets could contribute to bupropion`s therapeutic efficacy, elucidation of which would require further investigation.</div><div><br></div>

Psychiatric genetics is a basic science field that has potential for practical application and effective translation. To date, translational frameworks utilized by this field have been linear (e.g., sequential) in nature, focusing on molecular genetic information. It is proposed that non-linear (e.g., socio-ecological) frameworks are a better way to immediately translate non-molecular genetic information. This dissertation explored the translation of psychiatric genetic information in two ways. First, a survey was sent to academic stakeholders to assess the state of the science regarding the translation of genetic information to the clinical care of mental health disorders. Findings from this indicate a translation-genetic competence gap whereby genetic knowledge reinforces linear frameworks and genetic competence is needed to achieve effective translation in this content area. Second, a new risk factor model for social anxiety was created that incorporated genetic, environmental, and neurophysiological risk factors (behavioral inhibition, parental bonding, emotion reactivity). Findings indicate that genetic etiology is more informative knowledge that can influence risk factor models and possibly prevention and intervention efforts for social anxiety. Overall this dissertation paves the way for examining the translational capacity of psychiatric genetics in a clinical setting. It constitutes the first examination of barriers to and a potential solution for the most effective translation of psychiatric genetic information.

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