Dissertations / Theses on the topic 'Active biological transport'

Thesis (Ph. D.)--Illinois State University, 2005.
Title from title page screen, viewed on April 22, 2007. Dissertation Committee: Maarten E.A. Reith (chair), Hou Tak Cheung, Stephen M. Lasley, Robert L. Preston, Brian J. Wilkinson. Includes bibliographical references and abstract. Also available in print.

The cellular mechanisms responsible for rectal acidification in the desert locust, Schistocerca gregaria, were investigated in isolated recta mounted as flat sheets in modified Ussing chambers. In the absence of exogenous CO₂, HCO₃⁻, and phosphate, the isolated rectum (under both open- and short-circuit current conditions) was capable of rates of net acid secretion (J[subscript]H+) similar to those observed in vivo, demonstrating the viability of the preparation and suggesting that rectal acidification was due to proton secretion rather than selective movements of HCO₃⁻ or phosphate. The possibility that trace levels of metabolic CO₂ might be generating sufficient HCO₃⁻ to account for the observed rates of rectal acidification (via HCO₃⁻ reabsorption) was assessed by adding exogenous CO₂/HCO₃⁻ to the contraluminal bath. The small increases in J[subscript]H+ observed after addition of 2% or 5% CO₂ were shown to be due to simple hydration of CO₂ which had diffused into the lumen (from the contraluminal bath), rather than changes in rates of HCO₃⁻ reabsorption. Since measurable quantities of luminal HCO₃⁻ did not directly affect the apical acid/base transport mechanism per se, it was concluded that metabolic CO₂ could not generate sufficient HCO₃⁻ in the lumen to account for the rates of rectal acidification observed under nominally CO₂/HCO₃⁻-free conditions and that J[subscript]H+ must be due to a proton secretory rather than bicarbonate reabsorptive mechanism. Microelectrode measurements of intracellular pH (pHi) and apical and basolateral membrane potentials (Va and Vb respectively) indicated that luminal pH was not in equilibrium with either contraluminal pH or pHi and that the mechanism responsible for active luminal acid secretion resided on the apical membrane. Preliminary measurements of bath total ammonia (ie. NH₃ + NH₄+) levels in the previous experiments suggested that the rectum was actively secreting ammonia at significant rates across the apical membrane into the lumen. If the ammonia crossed the apical membrane as NH₃ rather than NH₄+, rates of luminal ammonia secretion (J[subscript]Amm) would have to be added to J[subscript]H+ to obtain corrected values of luminal proton secretion. In the absence of exogenously added ammonia and CO₂, ammonia was preferentially secreted into the lumen under both open- and short-circuit current conditions. J[subscript]Amm was dependent on the presence of luminal amino acids and was relatively unaffected by K[superscript]+ removal or changes in luminal pH from 7.00 to 5.00. Bilateral Na+ substitution or luminal addition of ImM amiloride reduced J[subscript]Amm by 63% and 65% respectively. The data consistently demonstrate that the rectum secretes significant quantities of endogenously produced ammonia preferentially into the lumen as NH₄+ rather than NH₃ via an apical Na[superscript]+/NH₄[superscript]+ exchange mechanism. Clearly, rates of net acid secretion estimated by titratable acidity do not have to include a correction for luminal ammonia secretion. Although J[subscript]H+ was completely unaffected by changes in contraluminal pH, it could be progressively reduced (and eventually abolished) by imposition of either transepithelial pH gradients (lumen acid) or transepithelial electrical gradients (lumen positive). Under short-circuit current conditions, the bulk of J[subscript]H+ was not dependent on Na[superscript]+, K[superscript]+, CI⁻, Mg₂+, or Ca+ and was due to a primary electrogenic proton translocating mechanism located on the apical membrane. A small component (10-16%) of J[subscript]H+ measured under these conditions could be attributed to an apical amiloride-inhibitable Na[superscript]+/H[superscript]+ exchange mechanism. Inhibition of JH+ by anoxia or reduction of luminal pH unmasked a significant proton diffusional pathway on the apical membrane in parallel with the active proton pump. The fact that J[subscript]H+ was significantly inhibited (42%-66%) by contraluminal addition of ImM cAMP and relatively unaffected by changes in contraluminal pCO₂ or pH suggests that net acid secretion in the locust rectum in vivo is modulated by circulating hormonal factors rather than haemolymph pH or pCO₂ per se.
Science, Faculty of
Zoology, Department of
Graduate

2015 - 2016
Purpose of the PhD thesis was to develop dedicated lipid nanostructured vectors with tailored features (in terms of size, surface charge, load capability, stimuli responsive ability and stability) through the design of novel production processes expressly developed for nutraceutical and therapeutic agents encapsulation. The preliminary performed review of the main processes used for liposomes production have underlined that the majority of the conventional and more innovative methods adopted show a number of drawbacks such as few product volumes in output (directly linked to the impossibility in scaling up the process), high energy consumption, long times of production together with the use of toxic solvents and other process drastic conditions. To the light of these literature findings and with the aim to produce nanostructured vectors through more sustainable processes, two novel techniques, sharing the ultrasound technology as process intensification tool used in particles size reduction and homogenization operations, were designed and developed to respond to the needs of a better process performance, improving its efficiency and cutting down energy consumption. At first, based on the use of ultrasound as alternative energy resource, a solid particles size reduction process was developed and coupled with the bench scale conventional Thin Film Hydration (TFH) method. This technique provides the generation of a lipid film which is formed after solvents evaporation through the use of a rotary evaporator. The dried film is then hydrated, spontaneously producing micrometric vesicles characterized by the presence of several bilayers. Then the method was revisited by adding the ultrasound assisted step developed in order to produce, in a versatile manner, structures with the desired dimension (on micro/nano scale), starting from the micrometric ones. Four are the main sections composing the set-up to apply this innovative protocol: a feeding section, a solvent evaporation section, a liposomes production/homogenization section and a recovery section. In particular, the homogenization section is composed of a 3 mm sonication tip (operative frequency 20 kHz) which acts on micrometric vesicles sample aliquots. Subsequently to the realization of the production bench scale apparatus, the phenomenology connected to the vectors constitution was investigated and a dynamic model able to describe the curvature of a lipid bilayer under the effect of ultrasonic energy was then proposed and tested. In that regard, starting from micrometric vesicles, the ultrasound energy is used to break the lipid bilayer into smaller pieces, then these pieces close themselves in spherical structures producing small vesicles. Moreover the role of several process parameters were also elucidated. Once established its reliability and due its great potential in reducing time spent, without compromising the integrity of the liposomal systems produced (in terms of structure and load), the ultrasound intensification tool was also used for liposomes homogenization operation during vesicles production through a similmicrofluidic approach. As a matter of fact, in order to produce higher volumes of lipid vectors, potentially on production scale, directly with nanometric size, a simil-microfluidic apparatus was expressly designed and fabricated, overcoming the limitations of the small output volumes typical of the conventional bench scale techniques. There are five main sections composing the realized apparatus: a feeding section, a pumping section, a production section, an homogenization section and a recovery section. In particular the homogenization section is composed of a 6 mm sonication tip (operative frequency 20 kHz) directly immersed in the entire hydroalcholic solution containing nanoliposomes. As previously done, the phenomenological aspects involved in vectors constitution were investigated for this new adopted set-up. In particular, the reproduction of the phenomenology connected to the vesicles formation through a microfluidic approach was achieved by the use of constructive expedients (millimetric diameter of tubes, peristaltic pumps, injection needle). Particularly, nanostructured vectors formation 3 happens at the interfaces between the alcoholic and water phases, when they start to interdiffuse in a direction normal to the liquid flow stream; changes in flow conditions result in size variations of the insertion section of the organic phase reflecting on the vesicles dimensional features. In that regards, taking into account that size and size distribution are key parameters determining liposomes performance as carrier systems in both pharmaceutical and nutraceutical applications, a control on the produced nanoliposomes dimensional features was demonstrated by tuning the volumetric flow rates and the lipids concentration process parameters. In particular, it was understood that increasing the ratio between the water volumetric flow rate to the lipids-ethanol volumetric flow rate the liposomes dimensional distibution increases; on contrary, ultrasonic energy enhances the homogenization of the hydroalcoholic bulk and, as expected on the bases of previous studies conducted on smaller volumes, its duty cycle application efficaciously promoted a better vesicles dimensional distribution. This result was also confirmed by working at equal flow rates but at different lipid concentrations. Finally, the developed similmicrofluidic apparatus, working at room conditions and in absence of toxic solvents, makes nanoliposomes production a safe and low cost process, highly productive due to the use of ultrasound which was demonstrated to be a scalable means for process intensification. By using the two developed experimental set-up, several classes of liposomal structures were formulated and produced to respond to specific requests of nutraceutical and pharmaceutical applications. Through the ultrasound assisted tool at first coupled with the conventional THF method and subsequently used as integrant part of the homogenization section of the simil-microfluidic apparatus, different active molecules were successfully encapsulated in lipid nanostructured vectors solving the critical issues linked to their naked administration and transport through biological membranes. In particular, nanoliposomes containing vitamins with different hydrophobicity (α-tocopherol, ergocalciferol, vitamin B12) and ferrous sulfate, with highly interesting features for nutraceutical market, were produced achieving stable loaded nanoliposomes with high encapsulation efficiencies and good dimensional features. In details, for vitamins-nanoliposomes productions, neuter vesicles with micrometric size, ranging from 2.9 μm to 5.7 μm, were produced, obtaining, after sonication in duty cycle, small vesicles in the average range of 40 nm to 51 nm in size. High encapsulation efficiency (e.e.) was obtained in both micrometric vesicles, with a e.e. % of 72.0 ± 00 % for vitamin B12, 95.0 ± 7.07 % for α-tocopherol and 81.5 ± 2.12 % for ergocalciferol, and small vesicles, with an e.e. % of 56.2 ± 8.51 % for vitamin B12, 76.3 ± 14.02 % for αtocopherol and 57.5 ± 13.9 % for ergocalciferol (the higher the vitamin hydrophobicity, the higher the encapsulation efficiency). Finally, a comparison between vitamin B12 load achievable with the developed technique and the vitamin load achievable by breaking unloaded preformed liposomes (conventional approach) showed an increase of encapsulation efficiency in small vesicles from 40% to 56.2 %, confirming the effectiveness of the pointed out technique. Regarding the ferrous sulfate-nanoliposomes, their massive production was possible due to the similmicrofluidic approach with a precise control on particles size and size distribution. In particular, the effect of different weight ratios of iron to the total formulation components (0.06, 0.035, 0.02 and 0.01 iron/total components weight ratio) on the final vesicles encapsulation efficiency was investigated obtaining with the last formulation an high encapsulation efficiency (up to 97%). In general, ferrous sulfate loaded nanoliposomes, negative charged, with good dimensional features (127135 nm for not sonicated and 48-76 nm for sonicated liposomes) were successfully produced through the use of the simil-microfluidic method developed, obtaining an elevated process yield if compared to the classical bench scale techniques (THF and Ethanol Injection). For pharmaceutical purposes, anionic nanoliposomes containing a new synthetized peptide (KRX29) for a not conventional heart failures therapy and new, cutting edge, nucleic acids based therapeutics agents (NABDs), used in gene therapy, were successfully produced. 4 Regarding KRX29-nanoliposomes production, micrometric particles of 7.2-11.7 μm were obtained and sized with the use of the developed ultrasound assisted process thus achieving 22 – 35 nm vesicles. The effect of liposomes charge on both peptide encapsulation and recovery efficiencies was at first studied, showing an higher encapsulation efficiency (about 100%) achieved (both in small and large vesicles) by using the higher charge ratio formulation (13:1 (-/+)). Viceversa, the ability to recover the entrapped peptide was obtained for loaded systems (both in small and large vesicles) at the lower charge ratio formulation (1:1 (-/+)). As the charge ratio, also the peptide concentration showed influence on the liposomes encapsulation efficiency. For NABDs complexes production, at first preliminary experiments in which dsDNA was used to simulate the structure of siRNA molecule were done by testing different dsDNA/DOTAP lipid charge ratio (3:1, 5:1 and 7:1 (+/-)) in order to achieve the higher dsDNA encapsulation efficiency in the smaller carrier possible. DOTAP phospholipid was used due to its positive charge. The performed activities have confirmed the versatility of the ultrasound assisted technique for producing micro (2.2 – 2.9 μm) and nano lipid vectors (28 - 56 nm) encapsulating NABDs. In particular, the charge ratio (+/-) variation from 3:1 to 7:1 (+/-) by changing the amount of positive lipid (DOTAP) used in liposome preparation have allowed to an improved e.e. wich was 64 % and 100 % respectively for small and large vesicles by using the 7:1 (+/-) charge ratio. Starting from these preliminary tests, siRNAs-nanoliposomes complexes were produced for the inhibition of E2F1 protein expression, studied as a potential way to treat colorectal cancer associated to Inflammatory Bowel Diseases. By the TFH/sonication technique nanoliposomes with 33-38 nm range size and 100% siRNA encapsulation efficiency were obtained. The produced loaded nanoliposomes demonstrated a very low excellent uptake in the cultured human colon mucosa tissues. A remarkable anti-E2F1 expression effect after siE2F1-1324-nanoliposome samples transfection has been demonstrated also in a dynamic human model such the colon tissue microenvironment (i.e. an 80.5% reduction of E2F1 expression respect to the basal tissue was achieved in patient 4), a clear tendency to respond in a patient-dependent way was observed. All the achieved results highlight the potentiality of the purposely designed nanoliposomes in deliver, in a controlled manner, different active molecules for both pharmaceutical and nutraceutical purposes. The formulative and the chemical engineering approaches adopted in this thesis for nanostructured vectors production respectively enhance the product quality (nanoparticles with tailored features) and make the process more attractive in terms of improved safety and reduced costs. [edited by Author]
XV n.s. ( XXIX ciclo)

This thesis examines the hypothesis that human erythrocyte net sugar transport is the sum of two serial processes: sugar translocation followed by interaction of newly imported sugar with an intracellular binding complex from which sugar dissociates into the bulk cytosol. This hypothesis suggests that steady-state transport measurements in the human erythrocyte do not accurately reflect the intrinsic catalytic features of the glucose transporter and unless correctly interpreted, may lead to apparent inconsistencies in the operational behavior of the human erythrocyte sugar transport system. Our results support this proposal by demonstrating that although sugar transport measurements in human red blood cells suggest that transport is catalytically asymmetric, ligand binding measurements indicate that transport must be symmetric. In order to examine the serial compartments hypothesis, we set out to determine the following: 1) identify the component(s) of the proposed sugar binding complex, 2) determine whether cytosolic ATP levels and transporter quaternary structure affect sugar binding to the sugar binding complex, and 3) determine whether the sugar binding site(s) are located within or outside the cell. We present findings which support the hypothesis that the sugar binding complex is in fact the sugar transport protein, GLUT1. The number of sugar binding sites and the release of sugar from the GLUT1 complex are regulated by ATP and by GLUT1 quaternary structure. The sugar binding sites are located on a cytoplasmic domain of the GLUT1 complex. We show how these observations can account for the apparent complexity of erythrocyte sugar transport and its regulation by ATP.

Phosphate is an essential but often growth-limiting nutrient for bacteria. At low concentrations of phosphate in the growth medium, bacteria induce high-affinity uptake systems for phosphate, and this is usually the ABC-type phosphate specific transport system Pst. In the fully sequenced genomes of pathogenic species of mycobacteria, several copies of the genes encoding for the Pst system (pstSCAB) have been identified and some of these genes have been shown to be virulence factors. The reasons for the presence of multiple copies of pst genes in pathogenic mycobacteria are not understood, and phosphate transport by these bacteria, as well as the gene regulation involved, is poorly characterised. The fast-growing M. smegmatis contains only a single copy of the pst operon, but we recently identified a gene locus containing three genes, phnDCE, which encode for a putative ABC-type phosphate/phosphonate transport system, and a gene, phnF, which encodes for a putative transcriptional regulator of the HutC subfamily of GntR like regulators. To identify a function for the PhnDCE transport system and to characterise high-affinity phosphate transport in M. smegmatis, we created allelic exchange mutants in phnD and pstS, as well as a phnD pstS double deletion mutant. All three mutants failed to grow in minimal medium containing 10 mM phosphate, while the wildtype was able to grow in the presence of micromolar phosphate concentrations. No differences were observed in complex growth medium. Steady-state levels of [��P]-phosphate uptake were approximately 25% lower in all mutant strains as compared to the wildtype. Kinetics of phosphate uptake in the wildtype strain when grown at low phosphate concentrations (50 [mu]M P[i]) were biphasic, suggesting the presence of two inducible transport systems with apparent K[m] values of 16 [mu]M P[i] and 64 [mu]M P[i], respectively. Analysis of the kinetics of phosphate transport in the mutant strains led us to the proposition that the Pst system has an apparent Km value of ca. 16 [mu]M P[i], and the Phn system has an apparent Km of ca. 60 [mu]M P[i]. A third inducible phosphate transport system, which was active in the double mutant strain, had an apparent K[m] of ca. 90 [mu]M P[i]. Uptake of phosphate in all strains was not inhibited by the presence of excess phosphonates or phosphite, suggesting that all three transport systems were specific for phosphate. The study of phosphate transport in the presence of various metabolic inhibitors revealed that uptake by the Phn and Pst systems is driven by ATP-hydrolysis, consistent with ABC-type transport, while the third, unidentified transport system may be driven by the proton motive force. We showed that phnDCE formed an operon, and that the promoter area of the operon lies within 200 bp of the start of phnD. To investigate the regulation of the phn and pst genes, β-galacosidase activities of strains carrying transcriptional lacZ-fusions of the pstSCAB, phnDCE and phnF promoter areas, and levels of mRNA of the phn and pst genes were studied. All genes were induced when phosphate concentrations fell below a threshold value of 30 [mu]M, which coincided with a shift in the growth characteristics of M. smegmatis. Expression of the pst operon appeared to be controlled directly by the PhoPR two-component regulatory system, while the phn operon may be under direct or indirect control by PhoPR. To identify a role for PhnF in the regulation of phn gene expression, we created a phnF deletion mutant. PhnF appeared to repress transcription of phnDCE and phnF under phosphate-replete conditions. We identified two putative binding sequences for PhnF in the intergenic region between phnD and phnF with the sequence TGGTATAGACCA, which is similar to the proposed recognition consensus for HutC-like transcriptional regulators. Using site-directed mutagenesis of these sequences, we demonstrated that they are required for the repression of phnDCE and phnF. To prove PhnF binding to these potential binding sites, we attempted to express the M. smegmatis PhnF protein in E. coli, but could not obtain soluble recombinant protein. Electrophoretic mobility shift assays of the phnDCE promoter fragment using cell-free crude extracts of M. smegmatis were not successful. We propose that Pst and Phn both constitute high-affinity phosphate specific transport systems of M. smegmatis, and that a third inducible phosphate transport system is present in this bacterium. PhnF is required for repression of phnDCE and phnF transcription under phosphate-replete conditions, while induction of the pst operon, and possibly the phn operon, under phosphate-limited conditions involves the PhoPR system.

Physical activity (PA) has been labelled the miracle drug (Pimlott, 2010) and participating in regular PA has ample physical and mental wellbeing benefits. However, physical inactivity remains a critical public health concern, particularly across adolescence. In England the proportion of adolescents aged 13-15 years meeting the recommended guidelines for PA decreased significantly from those at a younger age (Health and Social Care Information Centre, 2009; 2012; 2015). The adolescent years (13 18 years) have been identified as the age of greatest decline in PA, although it is possible that large declines can also be seen at younger ages (Sallis, 2000). Among girls the decline in PA is greater at younger ages (9 12 years old) and among boys it is greater at older ages (13 16 years old) (Dumith, Gigante, Domingues, & Kohl, 2011). Thus, examining behaviour of early adolescents (aged 11-13 years) is a primary focus of this thesis. Researchers have called for a more comprehensive grasp of PA correlates and determinants and their impact on behaviour (Biddle & Mutrie, 2001, 2008). This broader picture needs to incorporate longitudinal study designs to accurately portray developmental changes (Evenson & Mota, 2011). This thesis aims to work towards a better understanding of associations among variables across aspects of the ecological model in relation to PA behaviour during early adolescence. Early adolescents within their first year of secondary school (year 7, aged 11 12 years) were recruited through schools across the East Midlands, United Kingdom (UK). These participants completed various measures across an 18 month period to compile all data required for the thesis. The thesis begins with a focus on active transport as a means of commuting to school which can significantly contribute to overall PA levels (Aibar, Bois, Generelo, Bengoechea, & Paillard, 2015; Slingerland, Borghouts, & Hesselink, 2012). The distance from home to school is an important influence on the decision to use active transport; however, ecological perspectives would suggest this variable may interact with individual, interpersonal and environmental factors. Therefore, the first study of this thesis investigates whether the relationship between distance to school and active transport is moderated by (i) gender, (ii) biological maturation, (iii) perceived family support for PA and (iv) multiple deprivation. Cross-sectional results from the baseline data collected demonstrated that the relationship between distance to school and the likelihood to actively travel to school is moderated by biological maturation, multiple deprivation and family support of PA in adolescents. Further analysis revealed that late-maturing children, those from less socio-economically deprived backgrounds and children with low family support of PA are less likely to actively commute to school as distance to school increases. Due to the interaction between these variables described above, the second study focused on the variables collectively using a person-oriented approach, which aimed to classify distinct profiles of early adolescents based on correlates of PA. The outcome variables were also broadened to include active transport and overall PA across two time points. Findings from this second study illustrate that the highly supported, shortest commuters produced the highest levels of self-reported PA and that affluent, short commuters were the most likely to use active transport to travel to school. The affluent, short commuters lived a relatively short distance to school in areas of the lowest deprivation and had relative moderate family support of PA. The highly supported, shortest commuters were characterised by the highest family support of PA and lived the shortest distance to school in areas of low deprivation. Study 1 evidenced an association between biological maturation and PA behaviour; however, study 2 displayed that biological maturation did not meaningfully contribute to the class characteristics, and were not a predictor of PA. Previous evidence as to whether early, average or late maturing adolescents are more likely to disengage from PA is mixed and tends to focus on one gender only (Sherar, Cumming, Eisenmann, Baxter-Jones, & Malina 2010; Bacil, Mazzardo, Rech, Legnani, & Campos, 2015). Thus for the third study a more focused inspection of biological maturity was undertaken. Biological maturity status was investigated as a predictor of PA behaviour at two subsequent time points (6 9 months after baseline and 12 18 months after baseline) and whether there was variation across genders. Findings displayed that biological maturity status does not predict subsequent PA, with no distinction across genders. To conclude, the final study examined additional forms of PA behaviour. For children to develop and maintain healthy PA behaviours, their PA during the school day, particularly during physical education (P.E) classes is important (Owen, Smith, Lubans, Ng, & Lonsdale, 2014). Self-reported PA was divided into school-time PA (during P.E. lessons, break and lunchtimes) and leisure-time PA (after school, during evenings and weekends). The final study fully utilised the longitudinal data collected and utilised longitudinal growth modelling to describe the changes in PA behaviour across 12-18 months during early adolescence. Results displayed that school-time PA and leisure-time PA are distinct. Males; those from less deprived backgrounds and individuals with higher family support of PA all separately reported more school-time PA than their counterparts (females, those from higher deprived backgrounds and individuals with lower family support of PA) at baseline. Males and those with higher family support of PA also reported more leisure-time PA than their respective counterparts at baseline. On average, both genders decreased in school-time PA across 18 months yet for leisure-time PA, on average, there was no change over time and no significant difference in the rate of change between genders. There were no observed significant differences in the rate of change between multiple deprivation status and biological maturation across the 18 months for both behaviours. For family support, on average school-time PA decreased over time and results showed significant difference in the rate of change between individuals with lower or higher levels of family support of PA across the 18 months. On average, there was no change over time for leisure-time PA yet there was a significant difference in the rate of change between individuals and their family support of PA across 18 months. Further analysis demonstrated if an individual s family support increases, so does their leisure-time PA and vice versa. These overall key findings demonstrate the complexity of PA behaviour throughout early adolescence. This thesis works towards predicting individuals, correlates and determinants that may be susceptible to physical inactivity and/or a decrease in activity over time. Results can be used to target and direct PA intervention work.

Les membranes biologiques sont le centre de nombreux phénomènes hors équilibres qui sont essentiels pour les cellules. Pour une description physique plus complète des biomembranes, nous avons étudié, théoriquement et expérimentalement, les effets de deux processus hors équilibres sur les propriétés de membranes artificielles. Premièrement, nous avons développé une théorie, complètement covariante, des membranes qui sont soumises à des échanges de matériels biologiques (i.e. lipides, enzymes et protéines membranaires). Avec cette description, l'étude des fluctuations de telles membranes nous a montré que, sous certaines conditions, celles-ci peuvent devenir instables en développant un long et fin tubule qui présente certaines similitudes morphologiques avec les membranes des organelles. Nous avons appliqué ce modèle avec succès pour décrire le phénomène de fusion observé expérimentalement entre de petites et de grosses vésicules chargées mimant le phénomène d'endocytose. Deuxièmement, nous avons étudié les effets de l'activité des protéines membranaires sur le spectre des fluctuations. Un modèle théorique qui prend en compte l'activité hors-équilibre des protéines prévoit une amplification des fluctuations lorsqu'un bruit hors-équilibre s'ajoute au bruit thermique. Pour tester expérimentalement ces prévisions, nous avons reconstitué une pompe ATP-dépendante, l'ATPase-Ca2+ dans des vésicules géantes unilamellaires. Puis grâce à la technique d'aspiration par micropipette, nous avons mis en évidence à la fois une décroissance du module de courbure liée à la présence des protéines dans la membrane, et une augmentation des fluctuations dans les membranes actives.

Transport through biological environments that are densely crowded with obstacles is often classified as anomalous, rather than Fickian diffusion. Researchers often describe these transport processes using either a random walk model or a fractional order differential equation model. To explore these ideas, we simulate transport through a crowded environment that is populated by impenetrable immobile obstacles. Our work suggests that it may be inappropriate to model transport through a crowded environment using these standard approaches. We develop a new analytical method for modelling the transport of an agent through a crowded environment. Using our new method, we calculate the exact long-time diffusivity as well as the crossover time, which is the time scale required for the transport process to effectively become Fickian. Finally, we extend our new model to include interactions between the motile agent and the obstacles such as adhesion and repulsion.

Signal transduction by transforming growth factor β (TGF-β) cytokines is mediated by an evolutionarily conserved mechanism that depends on the Smad proteins to transduce an extracellular stimulus into the nucleus. In the unstimulated state, Smads spontaneously shuttle across the nuclear envelope and distribute throughout the cell. Upon TGF-β or bone morphogenetic protein (BMP) stimulation, the receptor-activated Smads are phosphorylated, assemble into complexes with Smad4, and become mostly localized in the nucleus. Such signal-induced nuclear translocation of activated Smads is essential for TGF-β–dependent gene regulation that is critical for embryonic development and homeostasis. The molecular machinery responsible for this process, especially how the activated Smads are imported as complexes, is not entirely clear. Thus, I became interested in investigating the molecular requirements for nuclear targeting of Smads upon stimulation. Recently, whole-genome RNAi screening offers a complementary cell-based approach to functionally identify molecules that mediate nuclear accumulation of Smads in response to TGF-β. In the first part of this dissertation, I performed a genome-wide RNAi screen that uncovered the importin moleskin (Msk) required in nuclear import of Dpp-activated MAD. Both genetic and biochemical studies further confirmed this finding. I also investigated Smad interactions with the Msk mammalian orthologues, Importin7 and 8 and validated that Smads are bona fide cargos of Imp7/8. Besides the importin Msk, the screen also uncovered a subset of nucleoporins as required factors in signal-induced nuclear accumulation of MAD. Thus in the second part of this thesis, I focused on how the NPC mediates this Msk-dependent nuclear import of activated MAD. Most of these nucleoporins, including Sec13, Nup75, Nup93 and Nup205, were thought to be structural nucleoporins without known cargo-specific functions. We, however, demonstrated that this subset of nucleoporins was specifically used in the Msk-dependent nuclear import of activated MAD but not the constitutive import of cargos containing a classic nuclear localization signal (cNLS). I also uncovered novel pathway-specific functions of Sec13 and Nup93. Regulation of TGF-β signaling can be achieved not only by modulating Smad nuclear translocation but also by modifying Smad phosphorylation status. Previously we identified a kinase, Misshapen (Msn), that caused the linker phosphorylation of MAD, resulting in negative regulation of Dpp signaling (Drosophila BMP). In the third part of this thesis, I investigated the biological relevance of Msn kinase to Dpp signaling in Drosophila wings. Both over-expression and RNAi studies suggest that Msn is a negative regulator of the Dpp/MAD pathway in vivo. As a whole, my findings delineated two critical requirements for MAD nuclear import: the importin Msk and a unique subset of nucleoporins. For the first time, structural Nups are implicated in the direct involvement of cargo import, providing a unique trans-NPC mechanism.

L'utilisation de l'acide para-chloromercuribenzene sulfonique (pcmbs) (reactif peu permeant des groupements thiols) comme marqueur differentiel du transporteur de saccharose des tissus foliaires de feve (vicio faba) revele une forte inhibition selective de l'absorption du saccharose. Cette inhibition est due au blocage du transporteur et non a celui de la pompe a protons fournissant l'energie necessaire au transport. Le point d'impact du pcmbs se situe au niveau du site actif de l'enzyme. Ce marquage differentiel a permis de mettre au point un test de reconnaissance des substrats par le transporteur de saccharose. Les resultats obtenus precisent les exigences stereochimiques du transporteur de saccharose et sont utilisables pour la synthese bioprogrammee de pesticides a systemie phloemienne

El principal objetivo de la presente Tesis es caracterizar la actividad de transporte de diferentes canales iónicos, así como regular dicha actividad de transporte. Por otro lado la Tesis abarca el estudio del transporte a través de bicapas de moléculas con alto potencial biológico. En este sentido la presente Tesis abarca diferentes sistemas de estudio que comparten la capacidad de interactuar con las membranas lipídicas dando como resultado el transporte iónico a través de la formación de poros, o bien por medio de la translocación a través de la propia membrana.

Pour ma thèse, j'ai étudié trois problèmes autour des propriétés de transport des suspensions actives. J'ai utilisé principalement des suspensions de bactéries Escherichia Coli mais aussi des systèmes de nageurs artificiels auto-propulsés. En premier lieu, j'ai étudié l'activation du mouvement Brownien de particules passives dans une suspension de bactéries, près d'une surface. En utilisant diverses solutions et diverses conditions expérimentales permettant de changer les conditions de nage des bactéries et le confinement, j'ai montré que la diffusivité des traceurs passifs augmente linéairement avec ce que j'ai défini comme le flux actif de la suspension; c'est à dire la concentration de nageurs actifs multipliée par leur vitesse moyenne de nage. De manière générale, le confinement entre deux parois ou par rapprochement d'une paroi, montre un meilleur transfert de la quantité de mouvement qui a pour conséquence une augmentation du facteur de couplage entre diffusivité et fluide actif. Le remplacement des bactéries par des nageurs artificiels comme des bâtonnets bi-métalliques en condition réductrice produit des résultats identiques. Deuxièmement, j'ai étudié la modification de la viscosité d'un fluide produite par la présence d'entités autopropulsées. Il a été montré théoriquement que la présence de nageurs du type "pousseurs" comme les bactéries, réduit la viscosité de la suspension à une valeur inférieure de celle du fluide porteur. Le manque de résultats expérimentaux qui mettent en évidence cet effet au sein d'une suspension (cela été montré pour des films liquides minces), nous a poussé à fabriquer un rhéomètre microfluidique en forme d'Y permettant d'étudier la réponse rhéologique d'une suspension d'E. Coli. Des résultats préliminaires révèlent un comportement non Newtonien de la suspension active avec une baisse de viscosité du liquide aux faibles taux de cisaillement et aux faibles fractions volumiques. Troisièmement, j'ai proposé d'étudier les effets de dispersion et de transport de solutions E. Coli dans un micro-canal rectangulaire possédant une constriction en son centre. Dans un tel milieu confiné, les interactions avec les parois ainsi que la géométrie du canal jouent un rôle essentiel sur les propriétés de transport. Mes résultats, de façon inattendue, montrent que l'écoulement dans un canal produit une re-concentration en bactéries après la constriction et que cet effet est contrôlé par l'écoulement même.

Nucleoside-derived drugs are used for the treatment of haematological malignancies, viral infections and autoimmune or inflammatory diseases. These drugs require membrane transporters to be internalised and be active. Transporters implicated in nucleoside and nucleoside derivatives internalisation are concentrative nucleoside transporters (CNTs) encoded by SLC28 and equilibrative nucleoside transporters (ENTs) encoded by SLC29. Other transporters such as organic cation transporters (OCTs) encoded by SLC22 have been implicated in the transport of some antiviral nucleoside derivatives, too. Nucleoside transporters as well as hOCT1 have been detected in immune cells, as well as in polarised epithelia where they are asymmetrically distributed mediating a net flux of the natural nucleoside or the derivative. Membrane transporters can present polymorphic variants which can alter its ability to interact with the substrate. Of all studied transporters, hOCT1 is the most polymorphic, being its polymorphic variants implicated in pharmacokinetics and pharmacodynamics of some drugs used in clinics such as metformine among others. In this thesis we show the importance polymorphic variants of hOCT1 have not only in lamivudine uptake but also in drug-drug interactions with other drugs co-administrated with lamivudine. Infected PBMCs showed an up-regulation of hOCT1 expression which would made it a better lamivudine target. We also discard the possibility that polymorphisms located at the extracellular loop might affect oligomerization. hOCT1 has been also identified as a bendamustine transporter and hOCT1 polymorphic variants modulated bendamustine sensitivity. Bendamustine sensitivity in CLL cells ex vivo can partially be explained by the hOCT1 polymorphic variants they are carrying. In case of DNMT inhibitors, nucleoside transporters are responsible for zebularine internalisation but not decitabine and mediate a apical-basolateral flux in polarised epithelia. hOCT1 can mediate zebularine efflux which is impaired by the presence of polymorphic variants. In this case polymorphic variants would confer sensitivity to zebularine treatment. In order to better understand the transporter-substrates interaction a hCNT3 model has been generated based on the recent crystallised V.cholerae nucleoside transporter. This model has been preliminary proved experimentally showing, so far, to be a good model. In summary, this thesis highlights the importance drug transporters and their polymorphic variants play in drug bioavailability and action
Los fármacos derivados de nucleósidos son ampliamente utilizados en el tratamiento de enfermedades hematológicas, infecciones virales y enfermedades autoinmunes o inflamatorias. Estos fármacos necesitan de transportadores para ser internalizados en la célula y así, ser activos. Los transportadores implicados en la internalización de los nucleósidos y sus derivados son: los transportadores de nucleósidos concentrativos (CNTs) codificados por SLC28 y, los transportadores de nucleósidos equilibrativos (ENTs) codificados por SLC29. Otros transportadores, como los transportadores de cationes orgánicos (OCTs) codificados por SLC22, están implicados también en el transporte de algunos análogos usados en el tratamiento del SIDA. Los transportadores de nucleósdios y hOCT1 han sido detectados en células de sistema inmune, así como también, en epitelio polarizado donde se encuentran distribuidos asimétricamente mediando un flujo neto de nucleósidos y de sus derivados. Los transportadores de membrana pueden presentar polimorfismos alterando su habilidad de interacción con el sustrato. De todos los transportadores estudiados, hOCT1 es el más polimórfico, estando relacionadas sus variantes con la farmacocinética y farmacodinámica de algunos fármacos, como la metformina. En esta tesis mostramos la importancia que tienen estas variantes polimórficas, no solamente en la internalización de lamivudina, sino también en las interacciones de ésta con otros fármacos co-administrados. La infección de PBMCs con VIH mostró un aumento en la expresión de hOCT1, convirtiéndolos en mejor diana. Se descartó también la posibilidad de que los polimorfismos localizados en el loop extracelular, pudieran afectar la oligomerización del transportador. hOCT1 se ha identificado también como transportador de bendamustina, observándose a su vez, que sus variantes polimórficas modificaban la sensibilidad al fármaco. La sensibilidad de células de CLL ex vivo puede ser debida, en parte, a las variantes polimórficas que presentan. En el caso de los inhibidores de la ADN metiltransferasa (DNMT), observamos que los transportadores de nucleósidos son los responsables de la captación de zebularina pero no de decitabina, mediando un flujo de esta apical-basolateral en un modelo de epitelio polarizado. hOCT1 es capaz de mediar la extrusión de zebularina, en cambio, no lo son las variantes polimórficas estudiadas. Estos resultados estarían indicando que los polimorfismos serían responsables de la sensibilidad al tratamiento con zebularina. Para poder entender mejor la interacción transportador-sustrato, se generó un modelo preliminar de hCNT3 basado en el cristal de CNT de V. cholerae. En resumen, esta tesis muestra la importancia que tienen los transportadores y sus variantes polimórficas, en la biodisponibilidad y acción de los fármacos.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in inflammatory bowel disease. For the in situ delivery of IL-10 by Escherichia coli as carrier chassis, a modified transporter was designed with the ability to secrete biologically active IL-10. De novo DNA synthesis comprised a 561-bp fragment encoding the signal sequence of the E. coli outer membrane protein F fused in frame to an E. coli codon-optimized mature human IL-10 gene under control of a T7 promoter. The construct was overexpressed in E. coli laboratory strains, E. coli BL21 (DE3) and E. coli MDS42:T7. The mean concentrations of human IL-10 in the periplasm and culture supernatant of E. coli BL21 (DE3) were 355.8 ± 86.3 and 5.7 ± 1.7 ng/ml, respectively. The molecular mass of the recombinant E. coli-derived human IL-10 was 19 kDa, while under non-reducing conditions the native IL-10 dimer could be demonstrated. Reduction of tumor necrosis factor-α secretion in lipopolysaccharide-stimulated mouse macrophages and detection of the activated form of the transcription factor signal transducer and activator of transcription protein 3 proved the biological activity of the bacteria-produced human IL-10
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in inflammatory bowel disease. For the in situ delivery of IL-10 by Escherichia coli as carrier chassis, a modified transporter was designed with the ability to secrete biologically active IL-10. De novo DNA synthesis comprised a 561-bp fragment encoding the signal sequence of the E. coli outer membrane protein F fused in frame to an E. coli codon-optimized mature human IL-10 gene under control of a T7 promoter. The construct was overexpressed in E. coli laboratory strains, E. coli BL21 (DE3) and E. coli MDS42:T7. The mean concentrations of human IL-10 in the periplasm and culture supernatant of E. coli BL21 (DE3) were 355.8 ± 86.3 and 5.7 ± 1.7 ng/ml, respectively. The molecular mass of the recombinant E. coli-derived human IL-10 was 19 kDa, while under non-reducing conditions the native IL-10 dimer could be demonstrated. Reduction of tumor necrosis factor-α secretion in lipopolysaccharide-stimulated mouse macrophages and detection of the activated form of the transcription factor signal transducer and activator of transcription protein 3 proved the biological activity of the bacteria-produced human IL-10.
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Les réactions photochimiques, dans leur ensemble, présentent de nombreux avantages en synthèse organique. Le photon, considéré comme un réactif à part entière, permet de limiter l'utilisation de composés onéreux et/ou risqués à la manipulation et pour l'environnement. La photochimie organique s'inscrit alors dans les recommandations de la chimie durable.Trois des plus importants domaines de la photochimie organique sont étudiés dans cette thèse. Premièrement une réaction de transfert d'atome d'hydrogène photoinduit par absorption directe de l'énergie lumineuse est étudiée dans le cas des imines, fonction chimique peu étudiée dans les photoréactions. Une cyclisation radicalaire induit par ce transfert d'hydrogène est présentée dans le cas des pseudo-oxazolones. Deuxièmement, la catalyse photorédox, qui a participé à la renaissance de la photochimie est utilisée dans une réaction de transfert d'atome d'hydrogène impliquant la Lévoglucosénone, une molécule issue de la biomasse lignocellulosique. Les différents dérivés obtenus peuvent être asujettis à des réactions futures et des applications dans des domaines variés de la chimie. Enfin, la photooxygénation du furfural, elle aussi molécule issue de la biomasse est étudiée dans la conception de colorants polyméthines, ces derniers ayant de vastes utilisations dans les domaines de la physico-chimie
Photochemical reactions, as a whole, display numerous benefits in organic synthesis. The photon, considered as a complete reagent, enables to lessen the use of expensive and/or hazardous compounds to handle and for the environment. Thus, organic photochemistry forms part of sustainable chemistry recommandations.Three of the most important fields in organic photochemistry are studied in this thesis. Firstly, photoinduced hydrogen atom transfer via direct absorption of light energy is presented in the case of imine moiety, a chemical function whose few studies have been done to date. A radical intramolecular cyclisation induced by a hydrogen atom transfer is introduced in the case of pseudo-oxazolone structures. Secondly, photoredox catalysis, involved in the revival of photochemistry, is used to make a hydrogen atom transfer engaging Levoglucosenone, a molecule coming from lignocellulosic biomass. Derivatives of this latter obtained from photoreactions could be subjected to further reactions and applications in various areas of chemistry. In closing, photooxygenation of furfural, a molecule also issued from biomass is studied to design polymethine dyes, these latter having extensive uses in the fields of physico-chemistry

L'objectif de ce travail est de comparer la liaison et les effets biologiques de l'hormone de croissance (gh) 1) dans les adipocytes du tissu inguinal de jeunes rats zucker obeses fa/fa et minces fa/fa; 2) dans les preadipocytes de rats zucker en culture primaire au cours de la differenciation adipocytaire. 1. Les etudes de liaison de la gh et les courbes dose-reponse pour le transport et le metabolisme du glucose sont realisees avec des adipocytes soit fraichement isoles (f) soit preincubes 3 h (p)

Les champignons phytopathogènes sont à l'origine d'importantes pertes économiques. Parmi ces champignons, Botrytis cinerea est particulièrement destructeur. Afin de protéger les plantes, des molécules anti-fongiques sont développées. Elles sont classées selon leur Mode d'Action dont la compréhension permet d'élucider la façon dont les composés actifs bloquent les fonctions métaboliques ou voie de signalisation intracellulaires du champignon. Certaines molécules peuvent induire chez ce champignon des changements morphologiques dramatiques, ou phénotypes, observables par microscopie et associés au mode d'action (connu ou non) de la molécule étudiée. Chaque molécule est testée à plusieurs concentrations car il existe une dépendance entre la concentration et le phénotype. A ce jour l'analyse des images de microscopie se fait manuellement. Elle représente donc un coût important qui peut être réduit drastiquement par une analyse informatique. Mon projet de thèse s'inscrit dans ce cadre et vise à mettre en évidence les relations « Famille de Molécules <=> Mode d'Action <=> Phénotype » pour de nouvelles molécules testées. Afin de caractériser les différents phénotypes de Botrytis cinerea, nous avons mis en place une analyse automatique des images de microscopie. Elle comprend des étapes de traitement d'images et d'extraction de paramètres morphométriques. Puis, une méthode de classification automatique des phénotypes incluant une classe de rejet pour les phénotypes encore inconnus a été développée. Elle propose une stratégie générale dans un contexte supervisé fondée sur trois étapes principales : apprentissage d'un modèle indépendamment pour chaque classe, apprentissage d'un seuil par modèle fondé sur les interactions entre classes, et procédure de prédiction s'appuyant sur les réponses des modèles par rapport à leur seuil. Un "système expert" proposant une hypothèse de Mode d'Action d'une molécule anti-fongique a également été développé pour prendre en compte l'ensemble des décisions aux différentes concentrations de la molécule. Outre la conclusion sur le mécanisme d'action, cette procédure permet d'obtenir une analyse de la molécule testée, notamment en fournissant des indications sur son degré d'efficacité. Nous avons également développé une approche de classification alternative fondée sur le transport optimal. A noter que cette approche offre en outre un moyen original d'estimer la fonction de densité de probabilité sous-jacente à une population. La force du transport optimal réside dans sa capacité à prendre en compte la géométrie de répartition des échantillons. Ainsi, nous avons proposé de transformer les données de sorte qu'elles suivent un modèle simple (en pratique gaussien), la complexité des données étant alors "cachée" dans la transformation de transport. Enfin, nous nous sommes intéressés à la croissance du champignon au cours du temps dans le but de comprendre voire de prédire l'apparition d'un phénotype. Pour chaque phénotype, différents paramètres morphométriques sont estimés d'après des séquences d'images de croissance. Pour cela, nous avons étudié l'évolution de la valeur de ces paramètres en fonction de la molécule testée, de sa concentration, et du temps d'incubation. Ensuite, nous avons conçu des modèles de croissances calibrés à partir de ces données réelles. Les modèles construits sont des processus stochastiques à temps discret utilisant des lois discrètes et continues pour piloter les différents événements (croissance, création d'une branche...) et leur ampleur. Nous avons alors simulé la croissance de champignons suivant les traitements testés, pour des phénotypes donnés. Ce travail a permis d'acquérir une meilleure compréhension de la croissance de botrytis cinerea en présence d'une molécule antifongique en fonction de son mode d'action
Phytopathogenic fungus are the cause of significant economic losses. Among them, botrytis cinerea is particularly destructive. Therefore, anti-fungal molecules are developed for crop protection. They are classified according to their Mode of Action, whose understanding is necessary to infer how the active compounds block the metabolic functions or intracellular signaling pathways of the fungus. Some molecules can induce dramatic morphological changes of a fungus, the so-called phenotypes, that are observable in microscopy and can be associated with the (known or unknown) mode of action of the molecule. Each molecule must be tested at various concentrations since the phenotype is exhibited only above a certain dose. To date, the analysis of microscopy images is done manually. Therefore, it represents a significant cost which can be drastically reduced by computer analysis. Within this framework, this PhD thesis aims at discovering the relationships “Family of Molecules <=> Mode of Action <=> Phenotype” for new molecules. In order to characterize the different phenotypes of Botrytis cinerea, we developed an automatic analysis of the microscopy images. The first steps rely on image processing and extraction of morphometric features. Then, a method of automatic classification of the phenotypes including a rejection class for unknown phenotypes was developed. It proposes a general strategy in a supervised context based on three main steps: learning a model independently for each class, learning one threshold per model based on the interactions between the classes, and a prediction procedure based on the responses of models with respect to their threshold. An "expert system" able to take into account all the decisions at the different concentrations of a molecule has been developed to propose a hypothesis of the Mode of Action of the molecule. Besides the conclusion on the mechanism of action, this procedure allows to obtain an analysis of the tested molecule, in particular by providing indications on its degree of effectiveness. We have also developed an alternative classification approach based on optimal transport whose strength lies in its ability to take into account the geometry of the sample distribution. We proposed to transform the data so that they follow a simple model (in practice a Gaussian model), the complexity of the data then being "hidden" in the transport transformation. Note that this approach also offers an original way to estimate the probability density function underlying a population sample. Finally, we studied the growth of the fungus over time in order to understand or even predict the appearance of a phenotype. For each phenotype, different morphometric features are estimated from temporal sequences in microscopy. This is done by analyzing the evolution of these features as a function of the tested molecule, its concentration, and the incubation time. Then, we designed growth models calibrated from these ground-truth data. The models are discrete-time stochastic processes using discrete and continuous probability laws to control the triggering of the different events (growth, creation of a branch, etc.) and their magnitude. We then simulated the growth of fungi in contexts corresponding to different phenotypes. This work has provided a better understanding of the growth of Botrytis cinerea in the presence of an antifungal molecule, i.e., for a given mode of action

Mestrado em Biologia Molecular e Celular
The neuronal cytoskeleton is an interconnected network of filamentous polymers, having in its constitution three major components: actin, microtubules and intermediate filaments. Up to the discovery of axon actin rings, the neuronal actin cytoskeleton has gained relevance. Still, the molecular details of the regulation of the actin cytoskeleton in neurons are largely unknown. Helping in the actin cytoskeleton regulation and maintenance, there is adducin. Adducin is organized in heterotetamers of heterodimers which comprises α/β and α/γ subunits. In the nervous system, the depletion of α subunit results in an almost complete absence of functional adducin. Given this, α-adducin KO mice arose as relevant models to study the role of this protein in actin cytoskeleton. Results from our group showed that α-adducin KO mice develop progressive axon enlargement and degeneration. As defects in axonal transport have been related to axon enlargement, we determined the importance of adducin in the axonal cytoskeleton and, more specifically, in axonal transport. Although no differences were found in the retrograde transport of CTB in the optic nerve, the lack of adducin resulted in a decreased speed of axonal transport of mitochondria and lysosomes. Several neurodegenerative disorders have been associated with axonal transport deficits and, consequently, with alterations in MT-based transport. Although no differences were found in the levels of acetylated and de-tyrosinated tubulin, the levels of tyrosinated tubulin were significantly decreased in α-adducin KO brains, suggesting a less dynamic status of the MT cytoskeleton in the absence of adducin. Besides differential PTMs of tubulin the decreased axonal transport speed may result from the decreased levels of dynein and kinesin in α-adducin KO mice. Lastly, we hypothesized that adducin might be involved in the organization and/or plasticity of the AIS that requires actin dynamics. In α-adducin KO animals, although the AIS forms normally, neurons do not have the ability to relocate it in response to chronic depolarization. Still, the specific role of actin and its associated proteins, like adducin, in this process remains unclear. In sum, with this Thesis we contributed to understand the relevance of the actin in cytoskeleton, more specifically, of the actin-binding protein adducin in neuron biology.
O citoesqueleto neuronal é maioritariamente constituído por três componentes: actina, microtúbulos e filamentos intermédios. Com a descoberta dos anéis de actina presentes no axónio, o citoesqueleto neuronal de actina tem vindo a ganhar bastante relevância. Contudo, os mecanismos moleculares envolvidos na regulação da actina no citoesqueleto continuam por esclarecer. Nesta tese focámo-nos no estudo da importância da aducina, uma proteína de ligação à actina, na regulação do citoesqueleto neuronal. A aducina é uma proteína constituída por heterotetrameros de heterodímeros das subunidades α/β e α/γ, sendo que no sistema nervoso, a depleção da subunidade α resulta numa completa ausência da proteína no seu estado funcional. Assim, murganhos KO para α-aducina demonstraram ser um modelo animal relevante para o estudo do papel desta proteína no citoesqueleto de actina. Resultados do nosso grupo demonstraram que murganhos KO para α-aducina desenvolvem uma degeneração progressiva e um aumento do calibre do axónio. Uma vez que defeitos no transporte axonal têm vindo a ser relacionados com o alargamento axonal, tornou-se importante determinar o papel da aducina no citoesqueleto axonal, mais especificamente no transporte ao longo do axónio. Apesar de não terem sido encontradas diferenças no transporte da toxina da cólera no nervo óptico, a ausência da aducina resultou num decréscimo significativo na velocidade de transporte axonal de mitocôndrias e lisossomas. Diversos distúrbios neurodegenerativos têm sido associados com deficiências no transporte axonal consequentes de alterações na maquinaria de transporte incluindo microtúbulos e proteínas relacionadas. Apesar de não terem sido encontradas diferenças nos níveis de acetilação e de-tirosinação da tubulina em amostras de cérebro α-aducina KO, os níveis de tirosinação da tubulina estão significativamente diminuídos quando comparados com aqueles encontrados em amostras WT, sugerindo uma menor dinâmica dos microtúbulos na ausência de aducina. Além das modificações da tubulina, a diminuição da velocidade de transporte axonal poderá resultar também do decréscimo dos níveis de ambos os motores moleculares dineina e cinesina nos murganhos α-aducina KO. Por fim, sugere-se que a aducina poderá também estar envolvida na organização e/ou plasticidade do segmento inicial do axónio dada a sua ligação ao citoesqueleto de actina, importante para a sua função e organização. Nos murganhos KO para α-aducina, foi verificado que apesar da formação do segmento inicial ser normal, as células não têm a capacidade para o relocalizar após uma depolarização crónica. Porém, o papel específico da actina e das suas proteínas associadas, tal como a aducina, neste processo deverá ser investigado com maior detalhe. Sumariamente, com esta tese, foi possível contribuir para uma melhor compreensão da relevância da actina, mais especificamente, da proteína de ligação à actina aducina, na biologia de um neurónio.

Nous avons compare l'absorption et le largage de deux herbicides phloeme-mobile (glyphosate, amitrole) et d'un fongicide non phloeme-mobile (iprodione) par des tissus foliaires de feve (vicia faba l. ) depourvus de leur epiderme inferieur. Le modele "disque foliaire" qui permet une visualisation des evenements localises au niveau du phloeme semble mieux adapte a l'etude de la systemie liberienne que les modeles "parenchymes" frequemment utilises. Les autoradiographies montrent que le glyphosate et l'amitrole sont localises preferentiellement dans les nervures, alors que le marquage du a l'iprodione concerne principalement les parenchymes

An investigation was conducted to in vivo study the interaction between sulphate and molybdate during transport across the epithelium of the renal tubule and ileum in the sheep. In the kidney experiments, plasma concentration of sulphate and molybdate were progressively elevated by constant intravenous infusion. Kinetic analysis of the data indicated the presence of a common carrier system (C1) upon which a fully competitive inhibition between the two anions occurs for tubular reabsorption against the prevailing anion concentration in plasma. This has been characterized as active transport and the proximal tubule is postulated as a probable site for its occurrence. Further observation and analysis revealed the presence of a sulphate-activated system for molybdate tubular secretion. This has been characterized as facilitated diffusion and the distal tubule is postulated as a most probable site for its occurrence. There was no evidence to suggest a tubular secretion of sulphate. In the ileal experiments, a continuous perfusion technique was used to progressively increase the concentration of sulphate and molybdate in the lumen of the lower ileum. Kinetic study of the absorption data revealed the existence of two dissimilar machanisms for the transport of each anion across the ileal mucosa. One mechanism has shown the characteristics of a common carrier system (C1) where sulphate and molybdate exhibit a fully competitive inhibition for their transport across the Heal epithelium. This has been classified as active transport. The close similarity between kinetic parameters of C1 of the ileum and that of the renal tubule suggests that this is probably the same carrier system in both sites. The ileal absorption of sulphate by another mechanism has been observed to follow the anion concentration gradient; thus appeared to be diffusion. The sulphate absorption rates by this mechanism have been demonstrated to disobey Fick's law and to decrease with the progressive increase of molybdate concentration in the ileal lumen. Hence it has been declared facilitated diffusion mediated by a carrier system (Ds) It is also observed that Ds contributes to the majority of sulphate absorption from the ovine ileum up to 77%. The other route for molybdate ileal absorption (C2) has been found to be a sulphate-dependent system which is essentially activated by sulphate and proportionally accelerated with the progressive increase of sulphate concentration in the ileal lumen. It has shown the characteristics of active transport and contributed to about 78% in the total ileal absorption of molybdate. A mobile carrier model for the relationship between Ds and 02 is proposed. In this model, once Ds has attained a sulphate equilibrium sulphate is suggested to recycle to the mucosal membrane to be handled by C2. There a driving force or energy source may be found that enables the system to secrete sulphate back into the luminal pool in exchange for molybdate from that pool as countertransport. Unloaded carriers at the mucosal surface cannot bind molybdate but can bind sulphate only.

Fung, Chun Kit.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 121-135).
Abstracts in English and Chinese.
Declaration --- p.i
Acknowledgement --- p.ii
Abbreviations --- p.iii
Abstract in English --- p.v
Abstract in Chinese --- p.vii
Table of Contents --- p.ix
List of Figures --- p.xii
Chapter Chapter 1 - --- Introduction
Chapter 1.1 --- Genus Cordyceps --- p.1
Chapter 1.2 --- Cordyceps militaris --- p.4
Chapter 1.3 --- Biological Functions and Chemical Constituents of Cordyceps militaris --- p.9
Chapter 1.4 --- "Human Bronchial Epithelial Cell Line, 16HBE14o-" --- p.13
Chapter 1.5 --- Ion Transport in Human Bronchial Epithelial Cells --- p.16
Chapter 1.6 --- Objectives of the Experiments --- p.20
Chapter Chapter 2 - --- Materials and Methods
Chapter 2.1 --- Solutions and Chemicals --- p.21
Chapter 2.2 --- Preparation of Hot Water Cordyceps militaris Extract --- p.22
Chapter 2.3 --- Culture of Cells --- p.23
Chapter 2.4 --- Short-Circuit Current (lsc) Measurement --- p.24
Chapter 2.5 --- Short-Circuit Current (lsc) Measurement in Nystatin-Permeabilized Monolayer --- p.29
Chapter 2.6 --- Measurements of [Ca2+]i --- p.31
Chapter 2.7 --- Measurement of PKA Activity --- p.36
Chapter 2.8 --- Statistical Analysis --- p.37
Chapter Chapter 3 - --- Results
Chapter 3.1 --- Regulation of Ion Transport in 16HBE14o- Cells by CM Water Extract --- p.38
Chapter 3.1.1 --- Dose-Dependent Relationship of CM Water Extract --- p.39
Chapter 3.1.2 --- "Involvement of CI"" Transport in CM-induced lsc Response" --- p.42
Chapter 3.1.3 --- Involvement of K+ channels in CM-induced lsc Response --- p.47
Chapter 3.1.4 --- Involvement of Adenylate Cyclase/cAMP/Protein Kinase A Pathway in CM-induced lsc Response --- p.52
Chapter 3.1.5 --- Involvement of Ca2+-Dependent Pathway in CM-induced lsc Response --- p.57
Chapter 3.1.6 --- "Effect of CM Extract on Apical CI"" Current and Basolateral K+ Current in Nystatin-Permeabilized Epithelia" --- p.61
Chapter 3.1.7 --- Effect of CM Extract on PKA Activity --- p.67
Chapter 3.2 --- Regulation of Ion Transport in 16HBE14o- Cells by Cordycepin --- p.70
Chapter 3.2.1 --- Dose-Dependent Relationship of Cordycepin --- p.70
Chapter 3.2.2 --- "Involvement of CI"" Transport in Cordycepin-induced lsc Response" --- p.73
Chapter 3.2.3 --- Involvement of K+ channels in Cordycepin-induced lsc Response --- p.79
Chapter 3.2.4 --- Involvement of Adenylate Cyclase/cAMP/Protein Kinase A Pathway in Cordycepin-induced lsc Response --- p.84
Chapter 3.2.5 --- Involvement of Ca2+-Dependent Pathway in Cordycepin-induced lsc Response --- p.89
Chapter 3.2.6 --- Effect of Cordycepin on Intracellular Ca2+ Concentrations --- p.93
Chapter 3.2.7 --- "Effect of Cordycepin on Apical CI"" Current and Basolateral K+ Current in Nystatin-Permeabilized Epithelia" --- p.98
Chapter 3.2.8 --- Effect of Cordycepin on PKA Activity --- p.104
Chapter Chapter 4 - --- Discussion
Chapter 4.1 --- Regulation of Ion Transport in 16HBE14o- Cells by CM Extract --- p.107
Chapter 4.2 --- Intracellular Signaling Mechanisms behind CM-induced lsc Responses --- p.110
Chapter 4.3 --- Regulation of Ion Transport in 16HBE14o- Cells by Cordycepin --- p.111
Chapter 4.4 --- Intracellular Signaling Mechanisms behind Cordycepin-induced lsc Responses --- p.114
Chapter Chapter 5 - --- Conclusion
Chapter 5.1 --- Summary --- p.117
Chapter 5.2 --- Future Directions --- p.120
Chapter Chapter 6 - --- References --- p.121
Chapter Chapter 7 - --- Publications --- p.136

Vacuolar-type H+-pumping pyrophosphatases (V-H+-PPases) are primary active transporters that utilise the energy from PPi (inorganic pyrophosphate) hydrolysis to translocate H+ ions across a biological membrane. These proteins have been well characterised in plant cells where they function in parallel with the ubiquitous vacuolar-type H+-pumping ATPase (V-H+-ATPase) to generate proton gradients across the plant vacuole membrane. V-H+-PPases have also been identified on the plasma membrane and intracellular organelle membranes of several protozoan parasites responsible for causing severe disease in humans. The identification of V-H+-PPases in these disease-causing agents, and the absence of this class of proton pump in human cells, has exciting implications for its potential as an antiparasitic drug target. The P. falciparum genome encodes two genes, PfVP1 and PfVP2, that encode putative K+-sensitive (type I) and K+-independent (type II) V-H+-PPases respectively. Furthermore, V-H+-PPase activity in P. falciparum has been demonstrated in the asexual blood-stage parasite, both in terms of PPi-induced acidification of subcellular parasite compartments and PPi hydrolysis activity in parasite membrane preparations. V-H+-PPase activity in the blood-stage parasite was further characterised in this study. PPi-induced acidification of the parasite digestive vacuole (DV; a large lysosome-like organelle within the parasite cytosol) was sensitive to Ca2+, which is known to inhibit V-H+-PPase activity, but was insensitive to the V-H+-PPase type-specific inhibitor aminomethylenediphosphonate (AMDP). PPi hydrolysis in membrane-permeabilised, intact cells was also sensitive to Ca2+. This study also confirmed previous observations of K+ sensitivity of both H+ translocation and PPi hydrolysis, consistent with the presence of a K+ sensitive, type I V-H+-PPase in the intraerythrocytic parasite. This study specifically investigated the role of the putative type II V-H+-PPase, PfVP2, using PfVP2 knockout (KO) parasites. Although PfVP2 was not active on the DV membrane, it extruded H+ ions out of the parasite cytosol in the D10 strain of P. falciparum (only apparent when the parasite V-H+-ATPase was inhibited with the V-H+-ATPase inhibitor concanamycin A). PfVP2 KO parasites also displayed a small but significant decrease in PPi hydrolysis compared to wild type (WT) parasites in the D10 strain, consistent with PfVP2 hydrolysing PPi to Pi (inorganic phosphate). Despite these results supporting the presence of a functional PfVP2 protein in the intraerythrocytic-stage parasite, PfVP2 gene disruption was not associated with a fitness cost or increased sensitivity to the antiplasmodial agents concanamycin A, chloroquine and artemisinin in both the 3D7 and D10 strains. The subcellular localisation of PfVP2 was also investigated in this study. PfVP2, tagged with the green fluorescent protein (GFP) localised to punctate structures within the intraerythrocytic parasite cytosol. Disruption of PfVP2-GFP by brefeldin A (a compound known to interfere with the secretory pathway) and partial co-localisation of PfVP2-GFP with the P. falciparum Golgi Reassembly and Stacking Protein or PfGRASP, are consistent with a Golgi localisation of PfVP2.

The chondrocyte is the only cell type in articular cartilage, and its role is to maintain cartilage integrity by synthesizing and releasing macromolecules into the extracellular matrix (ECM) or breaking down its damaged constituents (Stockwell, 1991). The two major constituents of the ECM are type II collagen and aggrecans (aggregating proteoglycans). Proteoglycans have a high negative charge which attracts cations and increases the osmolarity, while also lowering the pH of the interstitial fluid. The fibrillar collagen matrix constrains ECM swelling that results from the Donnan osmotic pressure produced by proteoglycans (Wilkins et al., 2000). Activities of daily living produce fluctuating mechanical loads on the tissue which also alter the mechano-electro-chemical environment of chondrocytes embedded in the ECM. These conditions affect the physiology and function of chondrocytes directly (Wilkins et al., 2000; Guilak et al., 1995; Guilak et al., 1999). Relatively few studies of in situ chondrocyte mechanics have been reported in the biomechanics literature, in contrast to the more numerous experimental studies of the mechanobiological response of live cartilage explants to various culture and loading conditions. Analyses of chondrocyte mechanics can shed significant insights in the interpretation of experimental mechanobiological responses. Predictions from carefully formulated biomechanics models may also generate hypotheses about the mechanisms that transduce signals to chondrocytes via mechanical, electrical and chemical pathways. Therefore, computational tools that can model the response of cells, embedded within a charged hydrated ECM, to various loading conditions may serve a valuable role in mechanobiological studies. Computational modeling has become a necessary tool to study biomechanics with complex geometries and mechanisms (De et al., 2010). Usually, theoretical and computational models of cell physiology and biophysics are formulated in 1D, deriving solutions by solving ordinary differential equations, such as cell volume regulation (Tosteson and Hoffman, 1960), pH regulation (Boron and De Weer, 1976), and Ca2+ regulation (Schuster et al., 2002). Cell modeling software, such as The Virtual Cell (vcell.org Moraru et al. (2008)), analyze stationary cell shapes and isolated cells. To model the cell-ECM system while accounting for ECM deformation, the fibrillar nature of the ECM, interstitial fluid flow, solute transport, and electrical potential arising from Donnan or streaming effects, we adopt the multiphasic theory framework (Ateshian, 2007). This framework serves as the foundation of multiphasic analyses in the open source finite element software FEBio (Maas et al., 2012; Ateshian et al., 2013), which was developed specifically for biomechanics and biophysics, and offers a suitable environment to solve complex models of cell-ECM interactions in 3D. In the studies proposed here, we will extend the functionality of FEBio to further investigate the cell-ECM system. These extensions and studies are summarized in the following chapters: Chapter 1: This introductory chapter provides the general background and specific aims of this dissertation. Chapter 2: Cell-ECM interactions depend significantly on the ECM response to external loading conditions. For fibrillar soft tissues such as articular cartilage, it has been shown that modeling the ECM using a continuous fiber distribution produces much better agreement with experimental measurements of its response to loading. However, evaluating the stress and elasticity tensors for such distributions is computationally very expensive in a finite element analysis. In this aim we develop a new numerical integration scheme to calculate these tensors more efficiently than standard techniques, only accounting for fibers that are in tension. Chapter 3: Cell-ECM interactions also depend significantly on accurate modeling of selective transport across the cell membrane. However, the thickness of this membrane is typically three orders of magnitude smaller than the cell size, which poses significant numerical challenges when modeling the membrane using the finite element method, such as element locking. To date, no existing finite element software offers a multiphasic membrane element. In this aim, we formulate and implement a new membrane element in FEBio, which can accommodate fluid and solute transport within the biphasic and multiphasic framework, to model passive and selective transport across the cell membrane. Chapter 4: This aim extends Aim 2 to incorporate reactions across multiphasic membrane elements in FEBio, to model the conformational reactions of cell membrane transporters, such as carrier-mediated transporters and membrane pumps. This implementation is verified against standard models for the regulation of cell volume, pH, and Ca2+. Chapter 5: This final chapter provides a summary of the advances contributed in this dissertation, along with suggestions for future aims related to the topics covered here. With the completion of these aims, we have extended the modeling capabilities for cell physiology and mechanobiology to more complex multicellular systems embedded within their ECM, while subjected to a range of varying mechanical, electrical or chemical loading conditions.

A Dissertation Submitted to the Department of Physiology, University of the Witwatersrand,Johannesburg for the Degree of Master ot Science
Extensive disruption of muscle fibres has been shown to occur after short term eccentric exercise where high mechanical forces are generated. This study tested whether downhill running acts as a stimulus for inducing eccentric damage, and results in greater muscle damage and deterioration in muscular performance than an equal workload of uphill running. The study aimed at determining whether an adaptation or training effect takes place such that the muscle is more resistant to the damaging effects of a repeated bout of the same exercise. In. addition, the study aimed at determining whether the lower muscle volumes and forces of muscular contractions in females compared to males, makes females less susceptible to the damaging effects of eccentric contraction.(Abbreviation abstract)
Andrew Chakane 2019

The maintenance of a low intracellular [Na⁺] ([Na⁺]i) is a crucial aspect of cellular physiology. In mammalian cells this is achieved through the extrusion of Na⁺ via the well-characterised Na⁺/K⁺-ATPase. Approximately 12 hr after invasion of the human erythrocyte by the malaria parasite there is a profound increase in the permeability of the erythrocyte membrane to a wide range of solutes, including Na⁺. Na⁺ enters the infected erythrocyte via parasite-induced 'New Permeability Pathways' and there is, as a result, an increase in [Na⁺] in the erythrocyte compartment, with [Na⁺]i eventually reaching levels similar to those in the extra erythrocytic plasma (~130 mM). The parasite itself maintains a low [Na⁺]i. The resulting large inwardly-directed electrochemical Na⁺ gradient across the parasite plasma membrane energises the accumulation within the parasite of at least one essential nutrient (inorganic phosphate). The aim of this thesis was to characterise the mechanisms involved in Na⁺ regulation in the mature asexual 'trophozoite' stage of the human malaria parasite Plasmodium falciparum. The Na⁺-sensitive, fluorescent dye Sodium-binding BenzoFuran Isophthalate (SBFI) was used to measure [Na⁺]i in parasites functionally isolated from their host cells by saponin-permeabilisation of the host erythrocyte membrane. Under physiologically relevant conditions the resting [Na⁺]i in isolated trophozoites was estimated to be ~11 mM. Maintenance of [Na⁺]i was sensitive to the P-type ATPase inhibitor orthovanadate, consistent with Na⁺ extrusion being via a P-type Na⁺-ATPase, similar to the ENA (exitus natru; exit of sodium)-type ATPases that operate in some other protozoa, fungi and lower plants. ENA ATPases have been predicted to antiport H⁺ and the data obtained here are consistent with this being true of the P. falciparum Na⁺ extrusion system. The P. falciparum genome encodes a number of putative P-type ATPases; one of these, PfATP4, was found to share significant sequence similarities to ENA ATPases of other protozoa. A recent study showed that mutations in PfATP4 confer resistance to a newly-described class of antimalarials, the spiroindolones. The effect of the spiroindolones on ion regulation was therefore investigated. Several spiroindolones were shown to cause a profound disruption of [Na⁺]i regulation. In parasites with mutant PfATP4 there was both an impairment of Na⁺ regulation and a decrease in the spiroindolone-sensitivity of Na⁺ regulation. These results are consistent with PfATP4 being a Na⁺-ATPase and the target of the spiroindolones. The physiological role of another putative Na⁺ transporter, the Na⁺/H⁺-exchanger PfNHE was also investigated, as previous studies on its contribution to regulation of [Na⁺]i and intracellular pH (pHi) have been controversial. On the basis of a bioinformatics analysis it was predicted that the protein functions as an amiloride-insensitive, plasma membrane Na⁺-extruder, like its closely related plant homologues. However physiological studies revealed no significant role for such an NHE in either pHi or [Na⁺]i regulation in the P. falciparum trophozoite. This study constitutes a significant advance in our understanding of fundamental aspects of the cell physiology of the intraerythrocytic parasite, as well as shedding light on the mode of action of what promises to be an important new class of antimalarials, the spiroindolones.

42 h.; ils.; grafs.; tabls. Contiene Referencia Bibliográfica
La alantoína es un metabolito derivado de la degradación de las purinas, que se acumula en Arabidopsis thaliana en condiciones de estrés abiótico. Numerosos autores han relacionado este hecho con la tolerancia al estrés. Puesto que se conoce que diferentes tipos de estrés abiótico conducen al aumento de los niveles de especies activas del oxígeno (EAO), situación conocida como estrés oxidativo; se propuso que la acumulación de alantoína podría estar relacionada con la tolerancia a dicho estrés. En este trabajo mutantes alantoinasa knockout (aln-1) que acumulan alantoína en forma constitutiva, presentaron signos de mayor tolerancia al estrés oxidativo inducido por H2O2; sin embargo, no presentaron diferencias respecto al genotipo silvestre al ser tratados con alta intensidad lumínica, metilviológeno o aminotriazol. También se ha propuesto que el transporte a larga distancia de alantoína, mediante el transportador Ureido Permeasa 5 (AtUPS5), podría estar implicado en la tolerancia al estrés. Por ello, se estudió la relación de la translocación de alantoína con la regulación de las EAO endógenas, en líneas WT y ups5. Luego de 3 horas de tratamiento con alantoína exógena, las plantas ups5 mostraron una disminución en sus niveles de O2·- foliares. Sin embargo, luego de 24 horas de tratamiento, ambos genotipos presentaron un aumento en los niveles de O2·- y H2O2, lo que sugiere la implicancia de otros transportadores en la translocación de alantoína y modulación de las EAO en parte aérea. Se discuten las posibles relaciones de este hecho, con las respuestas de tolerancia al estrés abiótico de plantas que acumulan alantoína o tratadas con alantoína exógena descriptas.

Indiana University-Purdue University Indianapolis (IUPUI)
Approximately 2.8% of the world population is currently infected with hepatitis C virus (HCV). Neutralizing antibodies (nAbs) are often generated in chronic hepatitis C patients yet fail to control the infection. In the first two chapters of this study, we focused on two alternative routes of HCV transmission, which may contribute to HCV’s immune evasion and establishment of chronic infection. HCV was transmitted via a cell-cell contact-mediated (CCCM) route and in the form of exosomes. Formation of HCV infection foci resulted from CCCM HCV transfer and was cell density-dependent. Moreover, CCCM HCV transfer occurred rapidly, involved all four known HCV receptors and intact actin cytoskeleton, and led to productive HCV infection. Furthermore, live cell imaging revealed the temporal and spatial details of the transfer process. Lastly, HCV from HCV-infected hepatocytes and patient plasma occurred in both exosome-free and exosome-associated forms and the exosome-associated HCV remained infectious, even though HCV infection did not significantly alter exosome secretion. In the third chapter, we characterized HCV interaction with astrocytes, one of the putative HCV target cells in the brain. HCV infection causes the central nervous system (CNS) abnormalities in more than 50% of chronically infected subjects but the underlying mechanisms are largely unknown. We showed that primary human astrocytes (PHA) were very inefficiently infected by HCV, either in the free virus form or through cell-cell contact. PHA expressed all known HCV receptors but failed to support HCV entry. HCV IRES-mediated translation was functional in PHA and further enhanced by miR122 expression. Nevertheless, PHA did not support HCV replication regardless of miR122 expression. To our great surprise, HCV exposure induced robust IL-18 expression in PHA and exhibited direct neurotoxicity. In summary, we showed that CCCM HCV transfer and exosome-mediated HCV infection constituted important routes for HCV infection and dissemination and that astrocytes did not support productive HCV infection and replication, but HCV interactions with astrocytes and neurons alone might be sufficient to cause CNS dysfunction. These findings provide new insights into HCV infection of hepatocytes and astrocytes and shall aid in the development of new and effective strategies for preventing and treating HCV infection.