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京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23818号 / 医科博第139号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 萩原 正敏, 教授 藤渕 航, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Phosphate (PO4-3 or Pi) is an essential molecule necessary for sustaining life and it plays important roles in nucleic acid and cell membrane integrity. However, phosphate is found in growth-limiting concentrations in most environments. Bacteria have developed a diverse set of transport systems to uptake and scavenge phosphate from their environment for use in cellular processes. In the soil bacterium, Sinorhizobium meliloti, one such Pi transport system is the Pap-Pit system. Pit is a membrane transporter for Pi and is associated with a cytosolic protein of unknown function known as Pap (Pit-accessory protein). Interestingly, the stop codon of pap overlaps with the start codon of pit by a single nucleotide. In previous work, the pap gene appeared to be required immediately upstream of pit in an operon for functional Pi transport. Thus, in a pap deletion mutant, when pap is present in trans, there is no Pi transport. This suggests a possible translational coupling mechanism between Pap and Pit, in which the translation of Pap is required for the translation of Pit. Here, an alkaline phosphatase (phoA/lacZ) and a β-glucuronidase (gusA) translational reporter were fused to Pit as a measure of its translation and to understand the role of translational coupling in the Pap-Pit system. Growth complementation experiments with a conditionally Pi transport deficient S. meliloti mutant carrying various mutations in both pap and pit have also been performed in an attempt to determine the function of Pap in Pi uptake. The results presented here provide evidence that pap and pit are translationally coupled, and this is necessary for functional Pi transport via Pap-Pit. / Thesis / Master of Science (MSc) / Microbes require phosphorus in the form of inorganic phosphate (Pi) as an essential nutrient, but it is often found in growth-limiting concentrations in the environment. Bacteria have developed a diverse set of Pi transport systems to scavenge and take up phosphate from the environment. In the soil bacterium, Sinorhizobium meliloti, one such Pi transport system is the Pap-Pit system. Pit is a membrane transporter for Pi and is associated with a cytosolic protein of unknown function known as Pap. Various mutations in both pap and pit have been constructed in an attempt to determine the function of Pap in Pi uptake via Pit. The pap gene appears to be required immediately upstream of pit in an operon for functional Pi transport. The pap and pit genes overlap by a single nucleotide and this may suggest a translational coupling mechanism that is required for functional Pi transport via Pap-Pit.

Molecular chaperones play a key role in maintaining a healthy cellular proteome by performing protein quality control. Heat shock protein 70s (Hsp70s) are a diverse class of evolutionarily conserved chaperones that interact with short hydrophobic sequences presented in unfolded proteins, promoting productive folding, and preventing proteins from aggregation. Most of the extensive research on chaperone examines mechanism, substrate promiscuity, and engagement with many co-chaperones. Only recently were chaperones recognized to be frequent targets of post-translational modifications (PTMs). Despite the recent rise in PTMs identified, the impact of these modifications on chaperone function, whether singular or in concert with other modifications, remains elusive. To investigate the impact of PTMs on chaperone function, we chose to characterize two sites of phosphorylation on the linker of HspA1, the stress inducible human Hsp70. To mimic these phosphoserines, we used aspartate as a phosphomimetic substitution for all experiments. Interdomain allostery ties together chaperone structure and function. Therefore, the impact of phosphorylation on interdomain allostery is probed using biophysical and biochemical techniques. Altogether, data suggest that phosphorylation of the linker and SBD destabilizes the chaperone, while shifting the population towards the docked state. This result alludes to a previously described region of the protein that uncouples domain docking from conformational changes in the substrate-binding domain. The cross-communication between these phosphorylation sites reveals a novel, synergistic effect on chaperone structure and function.

Hemophilia B is a debilitating and life-threatening disorder caused by a deficiency in or dysfunction of factor IX (FIX), a complex plasma glycoprotein required for the formation and maintenance of blood clots. Treatment of hemophilia B involves infusion of replacement FIX currently derived from two sources: FIX purified from pools of human plasma (pd-FIX) and a single recombinant FIX product generated in genetically engineered Chinese hamster ovary (CHO) cells. Both of these FIX products are prohibitively expensive, limiting of the treatment options of hemophiliacs worldwide. As a result, a more abundant and affordable FIX product would greatly improve the life prospects for hemophiliacs. The biological activity of FIX is dependent upon its numerous post-translational modifications (PTMs), including gamma-carboxylation, proteolytic maturation, phosphorylation, sulfation, and glycosylation. Of these PTMs, those known to be vital for activity are gamma-carboxylation of multiple glutamate residues near the N-terminus and proteolytic cleavage of the FIX propeptide. When expressed at a high rate in exogenous expression systems, however, the ability of current systems to effect the necessary PTMs is severely rate limited, restricting the production of active FIX. The transgenic pig bioreactor represents a promising source for the production of large quantities biologically active FIX due to its demonstrated ability to perform the required FIX PTMs. It was the goal of this study to characterize the PTM structure and the resulting function of recombinant FIX when expressed at 1-3 mg/ml in the transgenic pig mammary epithelium (tg-FIX). It was found that the expressed tg-FIX is comprised of a heterogeneous mixture of FIX PTM isoforms. This mixture represents a spectrum of tg-FIX molecules of varying gamma-carboxyglutamic acid (Gla) and propeptide content, indicating that rate limitations in effecting these PTMs are present. A purification process was developed utilizing heparin-affinity chromatography to purify the total population of tg-FIX from pig milk, a complex multi-phase feedstock. Subsequently, a process was developed to fractionate the total population of tg-FIX into subpopulations based upon the extent of post-translational modification. Q ion-exchange chromatography was utilized to fractionate tg-FIX based upon molecular acidity which was found to be correlated to both biological activity and Gla content. The resulting biologically active tg-FIX population contained an average of 7 of the 12 Gla residues found in pd-FIX. Immuno-affinity chromatography was subsequently utilized to further fractionate tg-FIX into mature tg-FIX and propeptide-containing tg-FIX populations. The isolated FIX PTM populations were subjected to functional analysis by investigating in vitro clotting activity, activation by factor XIa, and in vivo pharmacokinetics. From this analysis it was found that mature tg-FIX with an average 7 Gla residues, representing approximately 9% of the total tg-FIX produced, exhibits wild-type in vitro clotting activity and normal activation by factor XIa. The remainder of the tg-FIX produced, characterized by either a lower Gla content or the presence of the propeptide, was found to be inactive and displayed less efficient activation by factor IXa. In an in vivo pharmacokinetic study in the hemophilia B mouse model, biologically active tg-FIX was found to possess altered circulating properties. Tg-FIX was characterized by a lower recovery, approximately one-sixth that of pd-FIX, but an extended circulation half-life. From this study it was found that the mean residence time of tg-FIX after injections is approximately twice that observed for pd-FIX. These altered pharmacokinetic properties are likely linked to the unique tg-FIX PTM structure, perhaps through altered endothelial cell binding characteristics caused by the reduced Gla content. / Ph. D.

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.