Background: Elucidating the role of intestinal drug transporter function in drug development is crucial, as transporter proteins can impact on drug absorption, efficacy and adverse events. In Vitro-In Vivo Extrapolation linked to Physiologically-Based Pharmacokinetic (IVIVE-PBPK) models aim to predict the in vivo impact of transporters from in vitro cell–based transport data and expression-based scaling factors. Currently, these models depend on relative measurements of transporter expression i.e., mRNA or immunoblotting. There is a critical need for physiologically relevant measures of transporter protein abundance to populate these biological frameworks. Objectives: The key objectives were to develop and validate a targeted proteomics workflow to quantify transporter protein abundances in human enterocytes and Caco-2 cells with a QconCAT technique. A cross-laboratory comparison on matched samples was also performed to assess between-laboratory bias in abundance determination. Together with abundance data from each laboratory, BCRP and P-gp transporter activities from Caco-2 cells were used to identify function-abundance relationships, to facilitate the potential development of abundance-function scaling factors. Results: Development of a differential centrifugation technique to obtain plasma membranes was undertaken using MDCK-II and Caco-2 cells. The plasma membrane fraction showed little enrichment from the preceding total membrane fraction and was contaminated with endoplasmic reticulum, as assessed by marker enzyme activities. There were also no differences in Na/K-ATPase, BCRP and P-gp abundances between plasma and total membrane fractions in Caco-2 cells. This may be due to losses of protein from the target membrane fraction, thus, a theoretical framework combining protein assay (BCA) and transporter abundance determinations was proposed. Pilot data on the generation of recovery correction factors using Villin and Na/K-ATPase abundances, to account for protein losses is also presented. The abundances of 6 transporters in jejunal enterocyte membranes (n=3), including the key efflux proteins BCRP (2.56±0.82 fmol/μg), P-gp (1.89±1.07 fmol/μg) and MRP2 (0.59±0.246 fmol/μg) were determined with precision. In addition, peptide losses during protein digestion stages were accounted for in abundance determinations. A cross laboratory comparison of transporter abundances from intestinal (n=4) and Caco-2 cells (n=7) measured in our laboratory and Bertin Pharma (BPh), showed that P-gp abundances were highly correlated (rs=0.72), yet BPh abundances were systematically lower than determined in our laboratory (2.0±2.08 versus. 4.8±3.51 fmol/μg, respectively). No differences or correlations were found for Na/K-ATPase and BCRP abundances between laboratories. A jejunal-Caco-2 cell relative expression factor (REF) for each protein for both laboratories was generated. The P-gp REF was similar for BPh and our laboratory (0.37 vs. 0.4, respectively) however, for BCRP there was a distinct difference (1.11 versus 2.22, respectively). These findings provide the first evidence that employing expression factors generated from abundances quantified in different laboratories may produce altered IVIVE-PBPK outcomes. Functional studies in Caco-2 cells using E-3-S and vinblastine as probes for BCRP and P-gp, respectively, show that protein abundance is more closely correlated to transporter activity than mRNA expression. In addition, it was only possible to verify that increasing P-gp abundances in Caco-2 cells were ranked alongside vinblastine intrinsic clearance, as there was little consistency when estimating Km between the different Caco-2 cell models expressing increasing P-gp abundances, which may be attributed to limited absorptive transport saturation. Thus, forming any conclusions with confidence on concentration dependent abundance-activity relationships was difficult. These data suggest the value of REF scaling factors based on protein abundances, but emphasises the need to generate these from both in vitro and in vivo samples, using the same proteomic workflow. Further work to verify abundance-function relationships is required. Conclusion: A targeted proteomic workflow has been developed allowing quantification of protein abundances for key drug transporters in human gut tissues and cell models. The study has highlighted important areas including losses of targeted proteins, contamination of plasma membrane fractions and standardisation between laboratories that need to be addressed before implementation of transporter abundances into PBPK models is undertaken. Nevertheless, the evidence for a close relationship between transporter abundance and function indicate the potential value of this data for generation of robust REF scaling factors.

Xanthomonas citri (X. citri), causador do cancro cítrico, afeta muitas áreas de cultivo de citros com impacto comercial. Transportadores ABC foram relatados como essenciais para a patogênese de X. citri. O gene nrtT codifica a proteína periplasmática responsável pelo suposto transporte de nitrato/nitrito/taurina. Neste trabalho, mostramos que a deleção de nrtT não afeta o crescimento de X. citri, porém reduz e atrasa os sintomas do cancro durante a infecção em folhas de citros. Observamos redução na produção de goma xantana e capacidade de aderência na linhagem mutante. A proteína NrtT foi expressa monomérica e monodispersa. Foram observadas alterações na estrutura secundária e aumento da estabilidade térmica de NrtT na presença de MOPS, indicando ser este um possível ligante de NrtT. A proteína PotF descrita como ligadora de putrescina não sofre alteração significativa na estabilidade térmica na presença de putrescina, entretanto, dados de SAXS mostram alterações na estrutura possivelmente decorrentes da ligação com putrescina. / Xanthomonas citri (X. citri), which causes citrus canker, affects many citrus growing areas with commercial impact. ABC transporters have been reported as essential for the pathogenesis of X. citri. The nrtT gene encodes the periplasmic protein responsible for the alleged transport of nitrate/nitrite/taurine. In this work, we show that nrtT deletion does not affect the growth of X. citri, but delays and reduces the symptoms of cancer during infection of citrus leaves. We observed a reduction in the production of xanthan gum and adhesion capacity in the mutant strain. The NrtT protein was expressed monomeric and monodisperse. There were changes in the secondary structure and increased thermal stability NrtT in the presence of MOPS, indicating that this was a possible binder NrtT. The PotF protein described as putrescine-binding is not significantly altered thermal stability in the presence of putrescine, however, SAXS data shows changes in the structure possibly resulting from connection with putrescine.

Xanthomonas citri (X. citri), causador do cancro cítrico, afeta muitas áreas de cultivo de citros com impacto comercial. Transportadores ABC foram relatados como essenciais para a patogênese de X. citri. O gene nrtT codifica a proteína periplasmática responsável pelo suposto transporte de nitrato/nitrito/taurina. Neste trabalho, mostramos que a deleção de nrtT não afeta o crescimento de X. citri, porém reduz e atrasa os sintomas do cancro durante a infecção em folhas de citros. Observamos redução na produção de goma xantana e capacidade de aderência na linhagem mutante. A proteína NrtT foi expressa monomérica e monodispersa. Foram observadas alterações na estrutura secundária e aumento da estabilidade térmica de NrtT na presença de MOPS, indicando ser este um possível ligante de NrtT. A proteína PotF descrita como ligadora de putrescina não sofre alteração significativa na estabilidade térmica na presença de putrescina, entretanto, dados de SAXS mostram alterações na estrutura possivelmente decorrentes da ligação com putrescina. / Xanthomonas citri (X. citri), which causes citrus canker, affects many citrus growing areas with commercial impact. ABC transporters have been reported as essential for the pathogenesis of X. citri. The nrtT gene encodes the periplasmic protein responsible for the alleged transport of nitrate/nitrite/taurine. In this work, we show that nrtT deletion does not affect the growth of X. citri, but delays and reduces the symptoms of cancer during infection of citrus leaves. We observed a reduction in the production of xanthan gum and adhesion capacity in the mutant strain. The NrtT protein was expressed monomeric and monodisperse. There were changes in the secondary structure and increased thermal stability NrtT in the presence of MOPS, indicating that this was a possible binder NrtT. The PotF protein described as putrescine-binding is not significantly altered thermal stability in the presence of putrescine, however, SAXS data shows changes in the structure possibly resulting from connection with putrescine.

Autosomal dominant epidermolytic ichthyosis (EI) is a rare disease characterized by intra-epidermal blistering due to mutations in either of two keratin genes, KRT1 and KRT10, expressed by suprabasal keratinocytes. Autosomal recessive congenital ichthyosis (ARCI) is a non-blistering, hyperkeratotic disease caused by mutations in one of the following genes: ABCA12, ALOX12B, ALOXE3, TGM1, CYP4F22, NIPAL4 and SLC27A4, which are all essential for skin barrier homeostasis. ARCI and EI often respond well to treatment with retinoids, but the mechanism of action is unclear. The aim of this thesis was to increase the knowledge of pathogenic pathways in ichthyosis and to find new explanations to the effect of retinoids. In vitro studies of immortalized keratinocytes from EI patients showed an abnormal keratin aggregation after heat stress, that could be partially inhibited by pre-treatment with all-trans retinoic acid (ATRA) or retinoic acid receptor α-agonists. ATRA treatment also reduced the relative expression of mutated vs wildtype KRT10. The clearance of ATRA in human keratinocytes was found to be mediated by CYP26B1. In skin biopsies from ARCI patients, immunofluorescence analysis of 12R-LOX, eLOX-3, TGM1, ichthyin and FATP4 showed altered expression, not only of the mutated protein, but also of the other proteins. These observations are consistent with a feedback regulatory mechanism by which the loss of one protein results in an up-regulation of other proteins. Furthermore, 12R-LOX, eLOX-3 and TGM1 were intimately co-localized in stratum corneum, as were ichthyin and FATP4, suggesting that the proteins are linked to the same metabolic pathway. When treated with a CYP26 inhibitor known to raise the endogenous ATRA level of the skin, two patients with NIPAL4 mutations, initially exhibiting increased co-localization signals for 12R-LOX and eLOX-3, displayed normalized lipoxygenase expressions and showed clinical improvement. In conclusion, mechanisms are proposed by which pathogenic keratin aggregations in EI and epidermal protein deficiencies in ARCI patients may be mitigated by retinoids. Furthermore, the vivid crosstalk between proteins incriminated in ARCI suggests that these enzymes operate along a common metabolic pathway essential for producing barrier lipids in stratum corneum. Any abrogation of this production may cause barrier failure, hence resulting in a compensatory hyperkeratosis characteristic of congenital ichthyosis.

Exercise has been considered a cornerstone of diabetes prevention and treatment for decades, but the benefits of resistance training are less clear. Nineteen non-diabetic subjects (10 metabolic syndrome, 9 sedentary controls) underwent 8 weeks of supervised resistance training. After training, strength and V̇ O2max increased by 10% in both groups. Percent body fat decreased in subjects with the metabolic syndrome. Additionally, lean body mass increased in both groups (p<0.05). Expression of glucose transporter protein-4 (GLUT4), the principle insulin-responsive glucose transporter, increased significantly in both groups. 5-adenosine monophosphateactivated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) expression increased in both groups, indicating increased protein synthesis and mitochondrial biogenesis. Markers of insulin resistance measured by a euglycemic hyperinsulinemic clamp did not improve in subjects with the metabolic syndrome but increased significantly in control subjects (13%). Resistance training upregulates intracellular signaling pathways that may be beneficial for ameliorating the metabolic syndrome.

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