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PurposeThe brain is protected from circulating metabolites and xenobiotics by the blood–brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier. Previous studies report that P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) are expressed apically or subapically at the blood-CSF barrier (BCSFB), implying a paradoxical function to mediate blood-to-CSF transport of xenobiotics. As evidence of P-gp and Bcrp activity at the BCSFB is limited, the goal of this study is to investigate functional activity of P-gp and Bcrp at the murine BCSFB using a live tissue imaging approach.MethodsThe choroid plexuses (CP) forming the BCSFB were freshly isolated from mouse brain ventricles and incubated with fluorescent probes calcein-AM and BODIPY FL-Prazosin. Using quantitative fluorescence microscopy, the functional contributions of Bcrp and P-gp were examined using inhibitors and mice with targeted deletion of the Abcb1a/b or Abcg2 gene.ResultsApical transport of calcein-AM in choroid plexus epithelial (CPE) cells is sensitive to inhibition by elacridar and Ko143 but is unaffected by P-gp deletion. In wild-type mice, elacridar increased CPE accumulation of BODIPY FL-Prazosin by 220% whereas deletion of Bcrp increased BODIPY FL-Prazosin accumulation by 43%. There was no change in Mdr1a/1b mRNA expression in CP tissues from the Bcrp−/− mice.ConclusionsThis study demonstrated functional activity of Bcrp at the BCSFB apical membrane and provided evidence supporting an additional contribution by P-gp. These findings contribute to the understanding of transport mechanisms that regulate CSF drug concentrations, which may benefit future predictions of CNS drug disposition, efficacy, and toxicity.

Purpose Transporters at the blood-cerebrospinal fluid (CSF) barrier (BCSFB) play active roles in removing drugs and toxins from the CSF. The goal of this study is to develop a fluorescence microscopy approach to quantitatively study the transepithelial transport processes at the murine BCSFB in real time. Methods Choroid plexus (CP) tissues were isolated from mouse lateral ventricles and incubated with anionic (fluorescein-methotrexate, 8-fluorescein-cAMP) or cationic (IDT307) fluorescent probes. The CSF-to-blood transport was imaged and quantified using compartmental segmentation and digital image analysis. Real time images were captured and analyzed to obtain kinetic information and identify the rate-limiting step. The effect of transporter inhibitors was also evaluated. Results The transport processes of fluorescent probes can be captured and analyzed digitally. The intra- and inter- animal variability were 20.4% and 25.7%, respectively. Real time analysis showed distinct transport kinetics and rate-limiting step for anionic and cationic probes. A CP efflux index was proposed to distinguish between transepithelial flux and intracellular accumulation. Rifampin and MK571 decreased the overall transepithelial transport of anionic probes by more than 90%, indicating a possible involvement of organic anion transporting polypeptides (Oatps) and multidrug resistance-associated proteins (Mrps). Conclusions A CP isolation method was described, and a quantitative fluorescence imaging approach was developed to evaluate CSF-to-blood transport in mouse CP. The method is consistent, reproducible, and capable of tracking real time transepithelial transport with temporal and spatial resolution. The approach can be used to evaluate transport mechanisms, assess tissue drug accumulation, and assay potential drug-drug interactions at the BCSFB.

Advances in structural engineering and updates to building codes have rendered older buildings in seismic regions obsolete. Evans Hall, a hub for the economics, mathematics, and statistics departments at UC Berkeley, faces demolition due to its poor seismic rating. The future of buildings like Evans Hall is guided by financial and engineering constraints, often leading to demolition or a strict seismic retrofit. The traditional seismic retrofit, largely ignored in the discipline of architecture, relies on structural engineering principles through mathematical analysis. How can architecture expand the spatial possibilities of the retrofit? This thesis challenges and expands on structural engineering techniques for seismic retrofits through architectural interventions such as spatial methods of twinning, overscaling, replacing, and subtracting structural and nonstructural elements. The dual use of structure restabilizes Evans Hall while also generating adaptability by introducing new public space, access to light, and flexible space for existing and new program.

The blood-cerebrospinal fluid barrier (BCSFB) is formed by the choroid plexus epithelial (CPE) cells, which express several polyspecific membrane transporters that contribute towards clearance of xenobiotic and endogenous compounds from the cerebrospinal fluid (CSF). However, BCSFB transporters are poorly characterized with respect to function, activity, and pharmacokinetic significance. Previous approaches using fluorescence microscopy to study BCSFB transport in intact choroid plexus tissues were poorly validated and more qualitative in nature due to the lack of real time quantitative methods for image analysis. This dissertation research is aimed to develop and validate an approach to study BCSFB transport activity through live tissue imaging and quantitative fluorescence microscopy, and subsequently utilize this approach and other biochemical approaches to elucidate the molecular mechanisms and functional significance of organic anion transporting polypeptides (OATPs), breast cancer resistance protein (Bcrp), and P-glycoprotein (P-gp) at the BCSFB. To better assess the transepithelial transport process at the blood-CSF barrier, I developed and validated a quantitative confocal microscopy approach to study CSF-to-blood organic anion and organic cation transport processes at the murine BCSFB in real time. Real time quantification of fluorescence occurring at different tissue compartments can enable the deconvolution of transport kinetics occurring at the apical (CSF-facing) and basolateral (blood-facing) membranes of the BCSFB. This approach was demonstrated to be consistent, reproducible, and capable of tracking transepithelial transport at the BCSFB with temporal and spatial resolution. I showed that large organic anion probes, 8-fluorescein-cAMP (fluo-cAMP) and fluorescein methotrexate (FL-MTX), are efficiently transported from the CSF compartment into the CPE cells and subsequently effluxed into the subepithelial space. Transport of the large organic anion probes were rate limited by the apical uptake transport, presumed to be mediated by OATPs. In contrast, the small organic cation and plasma membrane monoamine transporter (PMAT) substrate IDT307, was transported into CPE cells and retained. A novel parameter, choroid plexus efflux index (CPEI), was proposed to distinguish between transepithelial flux and CPE cell accumulation. The approach presented is valuable for the characterization of compartment specific accumulation of substrates, perpetrator drug interactions, and rate-determining steps in transepithelial transport at the BCSFB.To elucidate the molecular mechanisms of OATP-mediated organic anion clearance at the blood-CSF barrier, I utilized the recently developed quantitative fluorescence microscopy approach alongside other biochemical approaches to probe OATP1A expression, localization, and function. Using RT-PCR and supporting literature data, we found that OATP1A5 is the primary OATP1A isoform expressed on the apical, CSF-facing membrane Using quantitative fluorescence microscopy, I demonstrated that the fluorescent organic anions, sulforhodamine101 (SR101), FL-MTX, and fluo-cAMP were efficiently transported across the blood-CSF barrier. Transepithelial transport of these compounds across the CPE cells was abolished in Oatp1a/1b-/- mice, suggesting OATP1A5 is the primary contributor to large organic anion uptake in mice. Using transporter-expressing cell lines, the fluorescent probes were confirmed to be substrates of mouse OATP1A5 and its human homolog OATP1A2, corroborating our findings in the isolated CP and suggesting an overlap in function between mouse OATP1A5 and human OATP1A2. Immunofluorescence staining revealed the presence of OATP1A2 protein at the apical membrane in human CP tissues. Based on these data, we proposed that large organic anions in the CSF are actively transported into CPE cells by apical OATP1A2 (OATP1A5 in mice), then subsequently effluxed into the blood by basolateral multidrug resistance associated proteins (MRPs). As OATP1A2 transports a wide range of xenobiotics and endogenous compounds, the presence of this transporter at the BCSFB apical membrane may imply a novel route for removing neurohormones, drugs, and toxins from the CSF.Previous studies report that P-gp and Bcrp are expressed apically or subapically at the blood-CSF barrier, implying a paradoxical function to mediate blood-to-CSF transport of xenobiotics. To probe P-gp and BCRP function at the BCSFB, I utilized the approach described in Chapter 2 alongside selective inhibitors and knockout models to functionally evaluate the activity, mechanisms, and potential interplay of P-gp and Bcrp BCSFB transport. Using qRT-PCR I identified the relative mRNA expression of P-gp and Bcrp isoforms at the murine BCSFB. Through quantitative fluorescence microscopy in isolated CP tissues of wild-type and Bcrp-/- mice, I demonstrated BODIPY FL-prazosin was actively transported by Bcrp, indicating functional activity of apical Bcrp efflux. Furthermore, I detected an additional Bcrp-independent, elacridar-sensitive apical efflux mechanism at the BCSFB, suggested, but not confirmed, to be P-gp. As Bcrp and P-gp at the BCSFB apical (CSF-facing) membrane may contribute to the entry of endogenous compounds and nutrients into the CNS and may alter the disposition of drugs within the CNS, this study highlights the need for further research on characterizing the role of these transporters towards endobiotic and xenobiotic transport at the BCSFB. This dissertation research has contributed greatly to our understanding of the molecular mechanisms mediating drug transport at the blood-CSF barrier. Notably, I revealed a functional role of OATP1A5 in clearing large organic anions at the BCSFB apical membrane from the CSF in mice and suggested a similar role for OATP1A2 at the human BCSFB. In addition, I have provided supporting evidence towards Bcrp and P-gp functional efflux activity at the BCSFB apical membrane in mice. Taken together, this research established an uptake transport mechanism of large organic anions at the BCSFB and provides novel mechanistic insights into several poorly defined transport pathways at the BCSFB. Knowledge gained from this dissertation research contributes to a mechanistic understanding of several major BCSFB drug transporters in regulating CSF drug concentrations and suggests potential roles of these transporters in modulating CNS disposition of drugs and endogenous substances. This knowledge can benefit our understanding of the relationship between CSF and unbound brain concentrations for transported drug substrates, which can subsequently improve the predictions of CNS drug disposition, efficacy, and toxicity in humans.