Prandial state and biological sex modulate clinically relevant efflux transporters to different extents in Wistar and Sprague Dawley rats

P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) are clinically relevant efflux transporters implicated in the oral absorption of many food and drug substrates. Here, we hypothesised that food intake could influence protein and mRNA intestinal expression of P-gp/abcb1a, BCRP/abcg2, and MRP2/abcc2 differently in male and female Wistar and Sprague Dawley rats. To test this hypothesis, we used enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR) to quantify the protein and mRNA intestinal expression of these transporters, respectively. Our study found food and sex differences in P-gp expression, whereby in the fed state P-gp expression decreased in male Wistar rats, but P-gp expression increased in females. In the fed state, BCRP expression increased in both male and female Wistar rats, compared with the fasted state. In contrast, no sex differences or food effect differences were seen in Sprague Dawley rats for P-gp and BCRP expression. On the other hand, in the fed state, MRP2 expression was higher in male and female Wistar and Sprague Dawley rats when compared with the fasted state. Sex differences were also observed in the fasted state. Overall, significant strain differences were reported for P-gp, BCRP and MRP2 expression. Strong to moderate positive linear correlations were found between ELISA and PCR quantification methods. ELISA may be more useful than PCR as it reports protein expression as opposed to transcript expression. Researchers must consider the influence of sex, strain and feeding status in preclinical studies of P-gp, BCRP and MRP2 drug substrates

Characterization of OATP1B3-1B7 (LST-3TM12) - a novel transporter of the OATP1B family

The effectiveness of a drug is determined by its pharmacodynamic and pharmacokinetic properties. While pharmacodynamics describes the interaction between the target structure and the drug, pharmacokinetics is an umbrella term for all the processes influencing the entry of a drug into the organism and eventually its elimination from the organism. An important mechanism that affects the pharmacokinetics of a drug is its transport across membranes by uptake and efflux transporters. Two uptake transporters that have been extensively studied for their impact on pharmacokinetics are OATP1B3 and OATP1B1. These two transporters are encoded on chromosome 12 by the genes SLCO1B3 and SLCO1B1, respectively. Between these genes lies another gene locus annotated as SLCO1B7. This gene is deemed to be a pseudogene as no function has been reported for its translational product. In 2005, an mRNA sequence named LST-3TM12 that is highly similar to SLCO1B7 was submitted to the National Center of Biotechnology (NCBI) by Mizutamari, H. and Abe, T. (NCBI#, AY257470). The aim of this thesis was to assess the function and transcriptional regulation of this mRNA. By aligning the transcripts of SLCO1B3, SLCO1B7, and LST-3TM12, we could show that the initial five exons of LST-3TM12 originate from SLCO1B3 and the remaining part of LST-3TM12 is encoded by SLCO1B7. Due to this finding, LST-3TM12 is referred to as OATP1B3-1B7 in this thesis. Because the OATP1B3-1B7 mRNA and OATP1B3 have the same 5’UTR it seemed likely that they share the same promoter, which was corroborated by our finding that silencing the exon 4 of SLCO1B3 significantly inhibited OATP1B3-1B7 mRNA transcription. Given that the gene SLCO1B3 is controlled by, among others, farnesoid X receptor (FXR), we tested and confirmed that FXR also regulates OATP1B3-1B7 transcription. Hence, OATP1B3-1B7 is part of the FXR regulated gene-network. Our functional assessments of OATP1B3-1B7 revealed that OATP1B3-1B7 transports exogenous and endogenous compounds. Endogenous substances were dehydroepiandrosterone sulfate (DHEAS), estradiol β-D-glucuronide (E2G), taurocholic acid (TCA), and lithocholic acid (LCA). Exogenous substances were the drugs ezetimibe and atorvastatin. Real-time PCR assessment of OATP1B3-1B7 mRNA showed that it is highly expressed in the liver and, to a lesser extent, in the small intestine. Consequently, the protein OATP1B3-1B7 is detectable in the liver and intestine. Strikingly, the cellular location of OATP1B3-1B7 is not sinusoidal, as is the case for OATP1B1 and OATP1B3, but it is located in the smooth endoplasmic reticulum (SER). Given that OATP1B3-1B7 has a broad substrate range and is expressed in tissues dedicated to metabolism, we hypothesized that OATP1B3-1B7 could have a function related to the high metabolic activity of these tissues. One enzyme class that is highly expressed in such tissues are uridine 5'-diphospho-glucuronosyltransferases (UGTs). UGTs are anchored in the SER membrane and have their active enzymatic site facing the SER lumen. However, it is still not clearly understood how UGT substrates reach and leave the active enzymatic site. As OATP1B3-1B7 is a SER transporter, we assessed whether it could provide access to or exit from the lumen and thus contribute to the metabolic activity of the UGTs. We have investigated this hypothesis with regard to ezetimibe, which is a substrate of OATP1B3-1B7 and is highly metabolized by UGTs. In this case, we were able to show that inhibition of OATP1B3-1B7 lowers the glucuronidation rate of ezetimibe. Hence, we propose that OATP1B3-1B7 is a drug transporter that is a gateway for the SER lumen

Self-assembling polycations for gene delivery: Effects of polymer structure and environmental pH

ZUSAMMENFASSUNG In der vorliegenden Arbeit wurden innovative polymerische Vektoren, die für die Durchführung von Gentransfers entwickelt gewesen sind, vorgestellt. Hierbei richtete sich der besondere Akzent auf den Zusammenhang zwischen Struktur und Funktion sowohl aus physikalisch-chemischen als auch biologischen Blickpunkten. Zuerst wurden die für eine Transfektion relevanten Unterschiede von klassischen Vektoren mit hohem und niedrigem Verzweigungsgrad - PEI und PLL - unter alternierenden pH-Bedingungen von Krebsgewebe untersucht. Weiter wurde strukturelles Design für siRNA-Transfer auf Basis von PEG-PCL-PEI, einem multi-funktionellen selbst-assoziierenden ABC-Konstrukt, mit Betonung der Wichtigkeit der gesamten Hydrophilie-Lipophilie-Bilanz für effiziente Stilllegung der Genfunktion vorgeschlagen. Für Transport und Zustellung von DNA wurde ein neuer niedermolekularer diblock pDMAEMA-Abkömmling synthetisiert und charakterisiert, wonach der Vektor sich als effizient und geringfügig toxisch erwiesen hat. Der Zusammenhang zwischen pHEMA-Gehalt und Flexibilität der Polymerkette konnte mittels Dynamischer Simulation (MD) aufgeklärt werden. Die Besonderheiten zu thermodynamischen Aspekten von Polymer-DNA Bindung, die an die Glaspunkttemperatur (Tg) gekoppelt zu sein scheinen, wurden mittels ITC beobachtet. Sowohl die MD als auch die ITC- Methodiken lieferten neue Informationen zum Cargo-Carrier- Selbstorganisationsprozess in Lösung, welche wichtig in Bezug auf die Transfektionsleistung der Diblock-Copolymere sind

The new technique for accurate estimation of the spinal cord circuitry:recording reflex responses of large motor unit populations

We propose and validate a non-invasive method that enables accurate detection of the discharge times of a relatively large number of motor units during excitatory and inhibitory reflex stimulations. HDsEMG and intramuscular EMG (iEMG) were recorded from the tibialis anterior muscle during ankle dorsiflexions performed at 5%, 10%, and 20% of the maximum voluntary contraction (MVC) force, in 9 healthy subjects. The tibial nerve (inhibitory reflex) and the peroneal nerve (excitatory reflex) were stimulated with constant current stimuli. In total, 416 motor units were identified from the automatic decomposition of the HDsEMG. The iEMG was decomposed using a state-of-the-art decomposition tool and provided 84 motor units (average of two recording sites). The reflex responses of the detected motor units were analyzed using the peri-stimulus time histogram (PSTH) and the peri-stimulus frequencygram (PSF). The reflex responses of the common motor units identified concurrently from the HDsEMG and the iEMG signals showed an average disagreement (the difference between number of observed spikes in each bin relative to the mean) of 8.2±2.2% (5% MVC), 6.8±1.0% (10% MVC), and 7.5±2.2% (20% MVC), for reflex inhibition, and 6.5±4.1%, 12.0±1.8%, 13.9±2.4%, for reflex excitation. There was no significant difference between the characteristics of the reflex responses, such as latency, amplitude and duration, for the motor units identified by both techniques. Finally, reflex responses could be identified at higher force (four of the nine subjects performed contraction up to 50% MVC) using HDsEMG but not iEMG, because of the difficulty in decomposing the iEMG at high forces. In conclusion, single motor unit reflex responses can be estimated accurately and non-invasively in relatively large populations of motor units using HDsEMG. This non-invasive approach may enable a more thorough investigation of the synaptic input distribution on active motor units at various force levels

Intelligent computational system for colony-forming-unit enumeration and differentiation

Accurate quantitative analysis of microorganisms is recognized as an essential tool for gauging safety and quality in a wide range of fields. The enumeration processes of viable microorganisms via traditional culturing techniques are methodically convenient and cost-effective, conferring high applicability worldwide. However, manual counting can be time-consuming, laborious and imprecise. Furthermore, particular pathologies require an urgent and accurate response for the therapy to be effective. To reduce time limitations and perhaps discrepancies, this work introduces an intelligent image processing software capable of automatically quantifying the number of Colony Forming Units (CFUs) in Petri-plates. This rapid enumeration enables the technician to provide an expeditious assessment of the microbial load. Moreover, an auxiliary system is able to differentiate among colony images of Echerichia coli, Pseudomonas aeruginosa and Staphylococcus aureus via Machine Learning, based on a Convolutional Neural Network in a process of cross-validation. For testing and validation of the system, the three bacterial groups were cultured, and a significant labeled database was created, exercising suited microbiological laboratory methodologies and subsequent image acquisition. The system demonstrated acceptable accuracy measures; the mean values of precision, recall and F-measure were 95%, 95% and 0.95, for E. coli, 91%, 91% and 0.90 for P. aeruginosa, and 84%, 86% and 0.85 for S. aureus. The adopted deep learning approach accomplished satisfactory results, manifesting 90.31% of accuracy. Ultimately, evidence related to the time-saving potential of the system was achieved; the time spent on the quantification of plates with a high number of colonies might be reduced to a half and occasionally to a third.A análise quantitativa de microrganismos é uma ferramenta essencial na aferição da segurança e qualidade numa ampla variedade de áreas. O processo de enumeração de microrganismos viáveis através das técnicas de cultura tradicionais é económica e metodologicamente adequado, conferindo lhe alta aplicabilidade a nível mundial. Contudo, a contagem manual pode ser morosa, laboriosa e imprecisa. Em adição, certas patologias requerem uma urgente e precisa resposta de modo a que a terapia seja eficaz. De forma a reduzir limitações e discrepâncias, este trabalho apresenta um software inteligente de processamento de imagem capaz de quantificar automaticamente o número de Unidades Formadoras de Colónias (UFCs) em placas de Petri. Esta rápida enumeração, possibilita ao técnico uma expedita avaliação da carga microbiana. Adicionalmente, um sistema auxiliar tem a capacidade de diferenciar imagens de colónias de Echerichia coli, Pseudomonas aeruginosa e Staphylococcus aureus recorrendo a Machine Learning, através de uma Rede Neuronal Convolucional num processo de validação cruzada. Para testar e validar o sistema, os três grupos bacterianos foram cultivados e uma significativa base de dados foi criada, recorrendo às adequadas metodologias microbiológicas laboratoriais e subsequente aquisição de imagens. O sistema demonstrou medidas de precisão aceitáveis; os valores médios de precisão, recall e F-measure, foram 95%, 95% e 0.95, para E. coli, 91%, 91% e 0.90 para P. aeruginosa, e 84%, 86% e 0.85 para S. aureus. A abordagem deep learning obteve resultados satisfatórios, manifestando 90.31% de precisão. O sistema revelou potencial em economizar tempo; a duração de tarefas afetas à quantificação de placas com elevado número de colónias poderá ser reduzida para metade e ocasionalmente para um terço

Analysing functional genomics data using novel ensemble, consensus and data fusion techniques

Motivation: A rapid technological development in the biosciences and in computer science in the last decade has enabled the analysis of high-dimensional biological datasets on standard desktop computers. However, in spite of these technical advances, common properties of the new high-throughput experimental data, like small sample sizes in relation to the number of features, high noise levels and outliers, also pose novel challenges. Ensemble and consensus machine learning techniques and data integration methods can alleviate these issues, but often provide overly complex models which lack generalization capability and interpretability. The goal of this thesis was therefore to develop new approaches to combine algorithms and large-scale biological datasets, including novel approaches to integrate analysis types from different domains (e.g. statistics, topological network analysis, machine learning and text mining), to exploit their synergies in a manner that provides compact and interpretable models for inferring new biological knowledge. Main results: The main contributions of the doctoral project are new ensemble, consensus and cross-domain bioinformatics algorithms, and new analysis pipelines combining these techniques within a general framework. This framework is designed to enable the integrative analysis of both large- scale gene and protein expression data (including the tools ArrayMining, Top-scoring pathway pairs and RNAnalyze) and general gene and protein sets (including the tools TopoGSA , EnrichNet and PathExpand), by combining algorithms for different statistical learning tasks (feature selection, classification and clustering) in a modular fashion. Ensemble and consensus analysis techniques employed within the modules are redesigned such that the compactness and interpretability of the resulting models is optimized in addition to the predictive accuracy and robustness. The framework was applied to real-word biomedical problems, with a focus on cancer biology, providing the following main results: (1) The identification of a novel tumour marker gene in collaboration with the Nottingham Queens Medical Centre, facilitating the distinction between two clinically important breast cancer subtypes (framework tool: ArrayMining) (2) The prediction of novel candidate disease genes for Alzheimer’s disease and pancreatic cancer using an integrative analysis of cellular pathway definitions and protein interaction data (framework tool: PathExpand, collaboration with the Spanish National Cancer Centre) (3) The prioritization of associations between disease-related processes and other cellular pathways using a new rule-based classification method integrating gene expression data and pathway definitions (framework tool: Top-scoring pathway pairs) (4) The discovery of topological similarities between differentially expressed genes in cancers and cellular pathway definitions mapped to a molecular interaction network (framework tool: TopoGSA, collaboration with the Spanish National Cancer Centre) In summary, the framework combines the synergies of multiple cross-domain analysis techniques within a single easy-to-use software and has provided new biological insights in a wide variety of practical settings