| 51. |
- Tripathi, Rekha, PhD student, 1985-
(author)
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Unlocking the Role Of Orphan Solute Carrier SLC38A10 In Brain Metabolism : The SLC38A10 transporter in nutrient and metabolic regulation
- 2021
-
Doctoral thesis (other academic/artistic)abstract
- Membrane transporters are the primary gatekeepers of cells and regulate the transport of nutrients, metabolites, ions, water, and neurotransmitters into and across the human cells. The solute carrier transporters (SLCs) are the most prominent transporters, comprising 430 members divided into 65 subfamilies. SLCs are located on the plasma membrane and organelles such as mitochondria, vesicles, peroxisomes, endoplasmic reticulum, Golgi, and lysosomes. This thesis aimed to study SLCs of the SLC38 family under nutrient stress, focused particularly on the orphan SLC38A10 transporter.In Paper I, regulation of members of SLC38 family transporter, after amino acid starvation in mouse hypothalamic cells and primary cortex cells, was studied using microarrays and qPCR. We found several members of the SLC38 family that were strongly affected under amino acid starvation and showing a potential role in amino acid signaling in the brain. In Paper II, we performed a cellular and tissue localization and functional study of SLC38A10 transporter and revealed that SLC38A10 was expressed in both excitatory and inhibitory neurons in the mouse brain and has a unique subcellular localization in the ER and Golgi membrane. Furthermore, knockdown of the SLC38A10 gene resulted in reduced nascent protein synthesis in PC12 cells. Further, to unlock the biological function of the SLC38A10 transporter, in Paper III and Paper IV, we used SLC38A10 knockout mouse model.In Paper III, the goal was to uncover the role of SLC38A10 in acute glutamate and oxidative stress. Here, we found that a loss of SLC38A10 KO resulted in changes in the p53 levels and affected the mitochondrial function. Thus, this study established a possible role of SLC38A10 in cell survival, linked with p53, in mouse primary cortex cells. In Paper IV, we examined the role of SLC38A10 in amino acid metabolism and nutrient sensing in the mTOR signaling pathway. We performed complete amino acid starvation and refeed experiment on SLC38A10 knockout primary cortex cells. We concluded that SLC38A10 acts as a transceptor and regulates mTOR-dependent protein and lipid synthesis in brain cells, corroborating the findings from Paper II. To summarize, the present work has uncovered the function of SLC38A10 in the brain. It also provides knowledge of SLC38A10’s role in amino acid metabolism and signaling pathway(s). The findings of this thesis will enhance an understanding of SLC38A10 transporter and provide insight into future disease targeted drug studies focused on metabolic disorder and neurodegenerative disease.
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| 52. |
- Tyrefors, Niklas, et al.
(author)
-
Two new types of assays to determine protein concentrations in biological fluids using mass spectrometry of intact proteins with cystatin C in spinal fluid as an example
- 2014
-
In: Scandinavian Journal of Clinical & Laboratory Investigation. - : Informa UK Limited. - 1502-7686 .- 0036-5513. ; 74:6, s. 546-554
-
Journal article (peer-reviewed)abstract
- There is no reference method that is generally acknowledged to be unbiased for the determination of the concentration of any protein in biological fluids. This is probably because mass spectrometry (MS) methods acknowledged as reference methods for determination of low molecular mass substances in biological fluids, e.g. creatinine, have been difficult to adapt for proteins. Here we suggest two tentative MS methods, which might be used as reference methods for the determination of protein concentrations in biological fluids. One is based upon the addition to the fluid of a non-proteome reference protein, very similar to the one to be measured, and analyzing the ratio between the corresponding peaks in a selected ion monitoring (SIM) chromatogram. We call this method LC-MS-NPRP (NPRP, Non-Proteome Reference Protein). The other method is based upon the classical standard addition assay for low molecular mass substances. The results of these assays for cystatin C in spinal fluid were compared to those obtained by an immunoassay. Both methods indicated lower concentration than the immunoassay. This was found to be due to the presence of a significant fraction of monohydroxylated cystatin C in spinal fluid. It turned out that the sum of the unhydroxylated and hydroxylated cystatin C concentrations, determined by either of the two MS methods, were close to the results obtained by the immunoassay. These MS-based methods analyze intact proteins and therefore seem more suitable for the determination of protein concentrations in biological fluids than other MS-based methods requiring proteolytic degradation with its inherent lack of precision.
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| 53. |
- van West, Dirk, et al.
(author)
-
Glucocorticoid receptor gene-based SNP analysis in patients with recurrent major depression
- 2006
-
In: Neuropsychopharmacology. - : Nature Publishing Group. - 0893-133X .- 1740-634X. ; 31:3, s. 620-627
-
Journal article (peer-reviewed)abstract
- Dysregulation of the hypothalamic-pituitary-adrenal axis, one of the stress-response systems, is one of the key neurobiological features of major depression (MDD). Data supporting the notion that glucocorticoid-mediated feedback inhibition is impaired in MDD come from a multitude of studies demonstrating nonsuppression of cortisol secretion following administration of the synthetic glucocorticoid dexamethasone. We examined whether genetic variations in the glucocorticoid receptor gene (Nuclear Receptor Subfamily 3, Group C, Member 1; NR3C1) could be associated with increased susceptibility for MDD using a whole gene-based association analysis of single nucleotide polymorphisms (SNPs). Four SNPs were identified in NR3C1 and genotyped in two well-diagnosed samples of patients with MDD ascertained in Belgium and northern Sweden, and matched control samples. In total, 314 MDD patients and 354 control individuals were included in the study. In the Belgian sample, we observed significant allele (p=0.02) and genotype (p=0.02) association with an SNP in the promoter region (NR3C1-1); in the Swedish sample, we observed significant allele (p=0.02) and genotype (p=0.02) association with the R23K SNP. The haplotype association studies showed modest evidence for an involvement of the 5' region of the NR3C1 gene in the genetic vulnerability for MDD. This study suggests that polymorphisms in the 5' region of the NR3C1 gene may play a role in the genetic vulnerability for MDD.
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| 54. |
- Yau, Estelle, et al.
(author)
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Global Sensitivity Analysis of the Rodgers and Rowland Model for Prediction of Tissue: Plasma Partitioning Coefficients: Assessment of the Key Physiological and Physicochemical Factors That Determine Small-Molecule Tissue Distribution
- 2020
-
In: AAPS Journal. - : Springer Nature. - 1550-7416. ; 22:2, s. 1-13
-
Journal article (peer-reviewed)abstract
- In physiologically based pharmacokinetic (PBPK) modelling, the large number of input parameters, limited amount of available data and the structural model complexity generally hinder simultaneous estimation of uncertain and/or unknown parameters. These parameters are generally subject to estimation. However, the approaches taken for parameter estimation vary widely. Global sensitivity analyses are proposed as a method to systematically determine the most influential parameters that can be subject to estimation. Herein, a global sensitivity analysis was conducted to identify the key drug and physiological parameters influencing drug disposition in PBPK models and to potentially reduce the PBPK model dimensionality. The impact of these parameters was evaluated on the tissue-to-unbound plasma partition coefficients (Kpus) predicted by the Rodgers and Rowland model using Latin hypercube sampling combined to partial rank correlation coefficients (PRCC). For most drug classes, PRCC showed that LogP and fraction unbound in plasma (fup) were generally the most influential parameters for Kpu predictions. For strong bases, blood:plasma partitioning was one of the most influential parameter. Uncertainty in tissue composition parameters had a large impact on Kpu and Vss predictions for all classes. Among tissue composition parameters, changes in Kpu outputs were especially attributed to changes in tissue acidic phospholipid concentrations and extracellular protein tissue:plasma ratio values. In conclusion, this work demonstrates that for parameter estimation involving PBPK models and dimensionality reduction purposes, less influential parameters might be assigned fixed values depending on the parameter space, while influential parameters could be subject to parameters estimation.
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| 55. |
- Yusof, Siti R, et al.
(author)
-
Rate and extent of mitragynine and 7-hydroxymitragynine blood-brain barrier transport and their intra-brain distribution : the missing link in pharmacodynamic studies
- 2019
-
In: Addiction Biology. - : Wiley. - 1355-6215 .- 1369-1600. ; 24:5, s. 935-945
-
Journal article (peer-reviewed)abstract
- Mitragyna speciosa is reported to be beneficial for the management of chronic pain and opioid withdrawal in the evolving opioid epidemic. Data on the blood-brain barrier (BBB) transport of mitragynine and 7-hydroxymitragynine, the active compounds of the plant, are still lacking and inconclusive. Here, we present for the first time the rate and the extent of mitragynine and 7-hydroxymitragynine transport across the BBB, with an investigation of their post-BBB intra-brain distribution. We utilized an in vitro BBB model to study the rate of BBB permeation of the compounds and their interaction with efflux transporter P-glycoprotein (P-gp). Mitragynine showed higher apical-to-basolateral (A-B, i.e. blood-to-brain side) permeability than 7-hydroxymitragynine. 7-Hydroxymitragynine showed a tendency to efflux, with efflux ratio (B-A/A-B) of 1.39. Both were found to inhibit the P-gp and are also subject to efflux by the P-gp. Assessment of the extent of BBB transport in vivo in rats from unbound brain to plasma concentration ratios (Kp,uu,brain ) revealed extensive efflux of both compounds, with less than 10 percent of unbound mitragynine and 7-hydroxymitragynine in plasma crossing the BBB. By contrast, the extent of intra-brain distribution was significantly different, with mitragynine having 18-fold higher brain tissue uptake in brain slice assay compared with 7-hydroxymitragynine. Mitragynine showed a moderate capacity to accumulate inside brain parenchymal cells, while 7-hydroxymitragynine showed restricted cellular barrier transport. The presented findings from this systematic investigation of brain pharmacokinetics of mitragynine and 7-hydroxymitragynine are essential for design and interpretation of in vivo experiments aiming to establish exposure-response relationship.
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| 56. |
- Zhang, Cheng, et al.
(author)
-
Discovery of therapeutic agents targeting PKLR for NAFLD using drug repositioning
- 2022
-
In: eBioMedicine. - : Elsevier BV. - 2352-3964. ; 83
-
Journal article (peer-reviewed)abstract
- Background Non-alcoholic fatty liver disease (NAFLD) encompasses a wide spectrum of liver pathologies. However, no medical treatment has been approved for the treatment of NAFLD. In our previous study, we found that PKLR could be a potential target for treatment of NALFD. Here, we investigated the effect of PKLR in in vivo model and performed drug repositioning to identify a drug candidate for treatment of NAFLD. Methods Tissue samples from liver, muscle, white adipose and heart were obtained from control and PKLR knock-out mice fed with chow and high sucrose diets. Lipidomics as well as transcriptomics analyses were conducted using these tissue samples. In addition, a computational drug repositioning analysis was performed and drug candidates were identified. The drug candidates were both tested in in vitro and in vivo models to evaluate their toxicity and efficacy. Findings The Pklr KO reversed the increased hepatic triglyceride level in mice fed with high sucrose diet and partly recovered the transcriptomic changes in the liver as well as in other three tissues. Both liver and white adipose tissues exhibited dysregulated circadian transcriptomic profiles, and these dysregulations were reversed by hepatic knockout of Pklr. In addition, 10 small molecule drug candidates were identified as potential inhibitor of PKLR using our drug repositioning pipeline, and two of them significantly inhibited both the PKLR expression and triglyceride level in in vitro model. Finally, the two selected small molecule drugs were evaluated in in vivo rat models and we found that these drugs attenuate the hepatic steatosis without side effect on other tissues. Interpretation In conclusion, our study provided biological insights about the critical role of PKLR in NAFLD progression and proposed a treatment strategy for NAFLD patients, which has been validated in preclinical studies. Copyright (C) 2022 The Authors. Published by Elsevier B.V.
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| 57. |
- Isola, M., et al.
(author)
-
Dynamics of the melatonin MT1 receptor in the rat parotidgland upon melatonin administration
- 2016
-
In: Journal of Physiology and Pharmacology. - 0867-5910. ; 67:1, s. 111-119
-
Journal article (peer-reviewed)abstract
- Our recent ultrastructural study of human parotid glands revealed that the melatonin receptors, MT1 and MT2, are localised in the plasma cell membranes of acinar and ductal cells but also, and intriguingly, predominantly in acinar secretory granules, giving rise to the working hypothesis that secretory granules are a part of a transcytotic transport system for melatonin. To put this hypothesis to the test in rat parotid glands, anaesthetised animals were exposed to a high melatonin dose (3 mg/kg per hour), infused intravenously over two hours and aiming to stimulate a glandular melatonin-receptor-dependent intracellular transport system, if any. Thirty minutes later, the right parotids were removed. Pre-stimulation, left parotid gland tissue was removed to serve as (untreated) controls. Gland tissues were processed for the gold post-embedding technique and for western blot analysis. In untreated glands, on transmission electron microscope images, melatonin receptors displayed a distribution pattern similar to that in human parotids, i.e. here, too, the receptors were principally associated with the acinar secretory granules. In melatonin-treated glands, the number of granules associated with the MT1 receptor was twice that in untreated glands, despite the same total granule number in the two glands. Moreover, the density of gold particles showing MT1-receptor immunoreactivity associated with granules in melatonin-treated glands was 2.5 times that in untreated glands. The number of MT1 receptors associated with the granule membrane was about three times higher in melatonin-treated glands than in untreated glands, while the number of MT1 receptors inside the granules was about twice that in untreated glands. The immunoblotting of membrane-enriched samples showed that the MT1-receptor expression was about three times that of untreated glands. When it came to the MT2 receptor, no changes were observed. Melatonin itself thus exerts dynamic effects on its MT1 receptor, which may reflect an adaptive receptor-linked carrier system for melatonin, delivering - upon gland stimulation - melatonin to the saliva by exocytosis. © 2016, Polish Physiological Society. All right reserved.
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| 58. |
- Nylander, Erik, 1986-
(author)
-
The effects of growth hormone on opioid-induced toxicity in vitro
- 2019
-
Doctoral thesis (other academic/artistic)abstract
- There is an ongoing opioid crisis in the United States that is portrayed by a large number of opioid-related deaths. Many of these cases involve commonly used prescription opioids, such as morphine, oxycodone, fentanyl, and methadone. This is concerning and highlights the problems associated with long-term opioid treatment. In addition to opioid-related deaths, long-term opioid use may impact higher brain functions, such as cognitive function. The cause of cognitive decline following opioid treatment may be associated with increased neuronal cell death, inhibited neurogenesis, and altered volumes of specific brain regions important for cognition. Growth hormone (GH), a pituitary hormone regulated by the hypothalamic somatotropic axis, may counteract several of these effects. The hormone, alongside with its mediator insulin-like growth factor-1 (IGF-1), is associated with pro-cognitive effects and display promising neuroprotective actions in the CNS. The main aim for this thesis was to examine the impact of opioids on cell viability and the potentially protective, restorative, and effects linked to pro-cognitive properties of GH in mixed neuronal cell cultures and cell lines. The results clearly display that specific opioids, such as methadone, decrease cell viability, possibly via negative effects on mitochondrial morphology. GH treatment alleviated the negative effects of methadone in cortical cell cultures as well as successfully restored mitochondrial and membrane integrity past injury. Moreover, GH treatment to primary hippocampal cell cultures increased the number of dendritic spines, which are linked to higher cognitive functions, indicating that the hormone act as a cognitive enhancer in the CNS. In conclusion, this thesis provides further evidence that opioids negatively impact cell viability, an effect that may underlie reduced cognitive function as seen in several patients consuming opioids-long term. GH was able to counteract these effects and also able to restore damaged cellular functions. This thesis further confirms the essential role of GH in acting as a cognitive enhancer in the CNS, highlighting the potential role of GH as a treatment for cognitive dysfunctions.
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| 59. |
- Vicente Carrillo, Alejandro, 1989-
(author)
-
Sperm Membrane Channels, Receptors and Kinematics : Using boar spermatozoa for drug toxicity screening
- 2016
-
Doctoral thesis (other academic/artistic)abstract
- Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.
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| 60. |
- Ekqvist, David, et al.
(author)
-
Safety and pharmacokinetics-pharmacodynamics of a shorter tuberculosis treatment with high-dose pyrazinamide and rifampicin : a study protocol of a phase II clinical trial (HighShort-RP)
- 2022
-
In: BMJ Open. - : BMJ Publishing Group Ltd. - 2044-6055. ; 12:3
-
Journal article (peer-reviewed)abstract
- Introduction: Increased dosing of rifampicin and pyrazinamide seems a viable strategy to shorten treatment and prevent relapse of drug-susceptible tuberculosis (TB), but safety and efficacy remains to be confirmed. This clinical trial aims to explore safety and pharmacokinetics-pharmacodynamics of a high-dose pyrazinamide-rifampicin regimen.Methods and analysis: Adult patients with pulmonary TB admitted to six hospitals in Sweden and subjected to receive first-line treatment are included. Patients are randomised (1:3) to either 6-month standardised TB treatment or a 4-month regimen based on high-dose pyrazinamide (40 mg/kg) and rifampicin (35 mg/kg) along with standard doses of isoniazid and ethambutol. Plasma samples for measurement of drug exposure determined by liquid chromatography tandem-mass spectrometry are obtained at 0, 1, 2, 4, 6, 8, 12 and 24 hours, at day 1 and 14. Maximal drug concentration (C-max) and area under the concentration-time curve (AUC(0-24h)) are estimated by non-compartmental analysis. Conditions for early model-informed precision dosing of high-dose pyrazinamide-rifampicin are pharmacometrically explored. Adverse drug effects are monitored throughout the study and graded according to Common Terminology Criteria for Adverse Events V.5.0. Early bactericidal activity is assessed by time to positivity in BACTEC MGIT 960 of induced sputum collected at day 0, 5, 8, 15 and week 8. Minimum inhibitory concentrations of first-line drugs are determined using broth microdilution. Disease severity is assessed with X-ray grading and a validated clinical scoring tool (TBscore II). Clinical outcome is registered according to WHO definitions (2020) in addition to occurrence of relapse after end of treatment. Primary endpoint is pyrazinamide AUC(0-24h) and main secondary endpoint is safety.Ethics and dissemination: The study is approved by the Swedish Ethical Review Authority and the Swedish Medical Products Agency. Informed written consent is collected before study enrolment. The study results will be submitted to a peer-reviewed journal.
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