Palmitate induces insulin resistance and a pro-atherogenic phenotype in monocytes

The present study investigated the effect of the two most abundant FFA in plasma – palmitate and oleate – on insulin sensitivity and vascular function (monocyte phenotype and adhesion to endothelium) using in vitro cell culture models and Wistar rats. Palmitate at 300µM for 6h induced insulin resistance in THP-1 monocytes and L6 monocytes. The ceramide synthesis pathway partly accounted for the palmitate-induced insulin resistance in THP-1 monocytes but not for L6 myotubes. Oleate treatment did not induce insulin resistance in either cell type and co-incubation with oleate protected cells from palmitate-induced insulin resistance. Palmitate at 300µN for 24h significantly increased cell surface CD11b and CD36 expression in U937 monocytes. The increase in CD11b and CD36 expression was effectively inhibited by Fumonisin B1, an inhibitor of ceramide synthesis. Oleate treatment did not show any effect on CD11b and CD36 expression and co-incubation with oleate antagonised the effect of palmitate on CD11b and CD36 expression in U937 monocytes. The increase in CD11b expression did not affect U937 monocyte adhesion to ICAM-1. Treating Wistar rats with palmitate for 6h caused a transient delay in glucose disposal and an increase in adhesion of U937 monocytes to the aortic endothelium, particularly at bifurcations. In conclusion, the present study demonstrates that the saturated free fatty acid palmitate induces insulin resistance and a pro-atherogenic phenotype for monocytes, whereas the unsaturated free fatty acid oleate does not. In vivo studies also confirmed that palmitate induces insulin resistance and an increase in monocyte adhesion to aorta.

NT2 derived neuronal and astrocytic network signalling

A major focus of stem cell research is the generation of neurons that may then be implanted to treat neurodegenerative diseases. However, a picture is emerging where astrocytes are partners to neurons in sustaining and modulating brain function. We therefore investigated the functional properties of NT2 derived astrocytes and neurons using electrophysiological and calcium imaging approaches. NT2 neurons (NT2Ns) expressed sodium dependent action potentials, as well as responses to depolarisation and the neurotransmitter glutamate. NT2Ns exhibited spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling. Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, NT2 astrocytes (NT2As) exhibited morphology and functional properties consistent with this glial cell type. NT2As responded to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. NT2As also generated propagating calcium waves that were gap junction and purinergic signalling dependent. Our results show that NT2 derived astrocytes exhibit appropriate functionality and that NT2N networks interact with NT2A networks in co-culture. These findings underline the utility of such cultures to investigate human brain cell type signalling under controlled conditions. Furthermore, since stem cell derived neuron function and survival is of great importance therapeutically, our findings suggest that the presence of complementary astrocytes may be valuable in supporting stem cell derived neuronal networks. Indeed, this also supports the intriguing possibility of selective therapeutic replacement of astrocytes in diseases where these cells are either lost or lose functionality.

Physiological effects of oxidized phospholipids and their cellular signaling mechanisms in inflammation

Oxidized phospholipids, such as the products of the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine by nonenzymatic radical attack, are known to be formed in a number of inflammatory diseases. Interest in the bioactivity and signaling functions of these compounds has increased enormously, with many studies using cultured immortalized and primary cells, tissues, and animals to understand their roles in disease pathology. Initially, oxidized phospholipids were viewed largely as culprits, in line with observations that they have proinflammatory effects, enhancing inflammatory cytokine production, cell adhesion and migration, proliferation, apoptosis, and necrosis, especially in vascular endothelial cells, macrophages, and smooth muscle cells. However, evidence has emerged that these compounds also have protective effects in some situations and cell types; a notable example is their ability to interfere with signaling by certain Toll-like receptors (TLRs) induced by microbial products that normally leads to inflammation. They also have protective effects via the stimulation of small GTPases and induce up-regulation of antioxidant enzymes and cytoskeletal rearrangements that improve endothelial barrier function. Oxidized phospholipids interact with several cellular receptors, including scavenger receptors, platelet-activating factor receptors, peroxisome proliferator-activated receptors, and TLRs. The various and sometimes contradictory effects that have been observed for oxidized phospholipids depend on their concentration, their specific structure, and the cell type investigated. Nevertheless, the underlying molecular mechanisms by which oxidized phospholipids exert their effects in various pathologies are similar. Although our understanding of the actions and mechanisms of these mediators has advanced substantially, many questions do remain about their precise interactions with components of cell signaling pathways.

Regulation of signal transduction by calcitonin gene-related peptide receptors

Calcitonin gene-related peptide (CGRP) plays a pivotal role in migraine, activating its cognate receptor to initiate intracellular signalling. This atypical receptor comprises a distinct assembly, made up of a G protein-coupled receptor (GPCR), a single transmembrane protein, and an additional protein that is required for Ga(s) coupling. By altering the expression of individual receptor components, it might be possible to adjust cellular sensitivity to CGRP. In recognition of the increasing clinical significance of CGRP receptors, it is timely to review the signalling pathways that might be controlled by this receptor, how the activity of the receptor itself is regulated, and our current understanding of the molecular mechanisms involved in these processes. Like many GPCRs, the CGRP receptor appears to be promiscuous, potentially coupling to several G proteins and intracellular pathways. Their precise composition is likely to be cell type-dependent, and much work is needed to ascertain their physiological significance.

Influence of Egr-1 in cardiac tissue-derived mesenchymal stem cells in response to glucose variations

Mesenchymal stem cells (MSCs) represent a promising cell population for cell therapy and regenerative medicine applications. However, how variations in glucose are perceived by MSC pool is still unclear. Since, glucose metabolism is cell type and tissue dependent, this must be considered when MSCs are derived from alternative sources such as the heart. The zinc finger transcription factor Egr-1 is an important early response gene, likely to play a key role in the glucose-induced response. Our aim was to investigate how short-term changes in in vitro glucose concentrations affect multipotent cardiac tissue-derived MSCs (cMSCs) in a mouse model of Egr-1 KO (Egr-1-/-). Results showed that loss of Egr-1 does not significantly influence cMSC proliferation. In contrast, responses to glucose variations were observed in wt but not in Egr-1 -/- cMSCs by clonogenic assay. Phenotype analysis by RT-PCR showed that cMSCs Egr-1-/- lost the ability to regulate the glucose transporters GLUT-1 and GLUT-4 and, as expected, the Egr-1 target genes VEGF, TGFβ-1, and p300. Acetylated protein levels of H3 histone were impaired in Egr-1-/- compared to wt cMSCs. We propose that Egr-1 acts as immediate glucose biological sensor in cMSCs after a short period of stimuli, likely inducing epigenetic modifications. © 2014 Daniela Bastianelli et al.

Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence

The optical redox ratio as a measure of cellular metabolism is determined by an altered ratio between endogenous fluorophores NADH and flavin adenine dinucleotide (FAD). Although reported for other cancer sites, differences in optical redox ratio between cancerous and normal urothelial cells have not previously been reported. Here, we report a method for the detection of cellular metabolic states using flow cytometry based on autofluorescence, and a statistically significant increase in the redox ratio of bladder cancer cells compared to healthy controls. Urinary bladder cancer and normal healthy urothelial cell lines were cultured and redox overview was assessed using flow cytometry. Further localisation of fluorescence in the same cells was carried out using confocal microscopy. Multiple experiments show correlation between cell type and redox ratio, clearly differentiating between healthy cells and cancer cells. Based on our preliminary results, therefore, we believe that this data contributes to current understanding of bladder tissue fluorescence and can inform the design of endoscopic probes. This approach also has significant potential as a diagnostic tool for discrimination of cancer cells among shed urothelial cells in voided urine, and could lay the groundwork for an automated system for population screening for bladder cancer.

The development of functional astrocyte-neuron networks derived from stem cells

There is currently great scientific and medical interest in the potential of tissue grown from stem cells. These cells present opportunities for generating model systems for drug screening and toxicological testing which would be expected to be more relevant to human outcomes than animal based tissue preparations. Newly realised astrocytic roles in the brain have fundamental implications within the context of stem cell derived neuronal networks. If the aim of stem cell neuroscience is to generate functional neuronal networks that behave as networks do in the brain, then it becomes clear that we must include and understand all the cellular components that comprise that network, and which are important to support synaptic integrity and cell to cell signalling. We have shown that stem cell derived neurons exhibit spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling (1). Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, astrocytes exhibit morphology and functional properties consistent with this glial cell type. Astrocytes also respond to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. Astroctyes also generate propagating calcium waves that are gap junction and purinergic signalling dependent. Our results show that stem cell derived astrocytes exhibit appropriate functionality and that stem cell neuronal networks interact with astrocytic networks in co-culture. Using mixed cultures of stem cell derived neurons and astrocytes, we have also shown both cell types also modulate their glucose uptake, glycogen turnover and lactate production in response to glutamate as well as increased neuronal activity (2). This finding is consistent with their neuron-astrocyte metabolic coupling thus demonstrating a tractable human model, which will facilitate the study of the metabolic coupling between neurons and astrocytes and its relationship with CNS functional issues ranging from plasticity to neurodegeneration. Indeed, cultures treated with oligomers of amyloid beta 1-42 (Aβ1-42) also display a clear hypometabolism, particularly with regard to utilization of substrates such as glucose (3). Both co-cultures of neurons and astrocytes and purified cultures of astrocytes showed a significant decrease in glucose uptake after treatment with 2 and 0.2 μmol/L Aβ at all time points investigated (p <0.01). In addition, a significant increase in the glycogen content of cells was also measured. Mixed neuron and astrocyte co-cultures as well as pure astrocyte cultures showed an initial decrease in glycogen levels at 6 hours compared with control at 0.2 μmol/L and 2 μmol/L P <0.01. These changes were accompanied by changes in NAD+/NADH (P<0.05), ATP (P<0.05), and glutathione levels (P<0.05), suggesting a disruption in the energy-redox axis within these cultures. The high energy demands associated with neuronal functions such as memory formation and protection from oxidative stress put these cells at particular risk from Aβ-induced hypometabolism. As numerous cell types interact in the brain it is important that any in vitro model developed reflects this arrangement. Our findings indicate that stem cell derived neuron and astrocyte networks can communicate, and so have the potential to interact in a tripartite manner as is seen in vivo. This study therefore lays the foundation for further development of stem cell derived neurons and astrocytes into therapeutic cell replacement and human toxicology/disease models. More recently our data provides evidence for a detrimental effect of Aβ on carbohydrate metabolism in both neurons and astrocytes. As a purely in vitro system, human stem cell models can be readily manipulated and maintained in culture for a period of months without the use of animals. In our laboratory cultures can be maintained in culture for up to 12 months months thus providing the opportunity to study the consequences of these changes over extended periods of time relevant to aspects of the disease progression time frame in vivo. In addition, their human origin provides a more realistic in vitro model as well as informing other human in vitro models such as patient-derived iPSC.

Rôle des protéoglycanes à héparane sulfate dans le transfert de gène des cellules CHO et HEK293

La possibilité de programmer une cellule dans le but de produire une protéine d’intérêt est apparue au début des années 1970 avec l’essor du génie génétique. Environ dix années plus tard, l’insuline issue de la plateforme de production microbienne Escherichia coli, fut la première protéine recombinante (r-protéine) humaine commercialisée. Les défis associés à la production de r-protéines plus complexes et glycosylées ont amené l’industrie biopharmaceutique à développer des systèmes d’expression en cellules de mammifères. Ces derniers permettent d’obtenir des protéines humaines correctement repliées et de ce fait, biologiquement actives. Afin de transférer le gène d’intérêt dans les cellules de mammifères, le polyéthylènimine (PEI) est certainement un des vecteurs synthétiques le plus utilisé en raison de son efficacité, mais aussi sa simplicité d’élaboration, son faible coût et sa stabilité en solution qui facilite son utilisation. Il est donc largement employé dans le contexte de la production de r-protéines à grande échelle et fait l’objet d’intenses recherches dans le domaine de la thérapie génique non virale. Le PEI est capable de condenser efficacement l’ADN plasmidique (vecteur d’expression contenant le gène d’intérêt) pour former des complexes de petites tailles appelés polyplexes. Ces derniers doivent contourner plusieurs étapes limitantes afin de délivrer le gène d’intérêt au noyau de la cellule hôte. Dans les conditions optimales du transfert de gène par le PEI, les polyplexes arborent une charge positive nette interagissant de manière électrostatique avec les protéoglycanes à héparane sulfate (HSPG) qui décorent la surface cellulaire. On observe deux familles d’HSPG exprimés en abondance à la surface des cellules de mammifères : les syndécanes (4 membres, SDC1-4) et les glypicanes (6 membres, GPC1-6). Si l’implication des HSPG dans l’attachement cellulaire des polyplexes est aujourd’hui largement acceptée, leur rôle individuel vis-à-vis de cet attachement et des étapes subséquentes du transfert de gène reste à confirmer. Après avoir optimisées les conditions de transfection des cellules de mammifères CHO et HEK293 dans le but de produire des r-protéines secrétées, nous avons entrepris des cinétiques de capture, d’internalisation des polyplexes et aussi d’expression du transgène afin de mieux comprendre le processus de transfert de gène. Nous avons pu observer des différences au niveau de ces paramètres de transfection dépendamment du système d’expression et des caractéristiques structurelles du PEI utilisé. Ces résultats présentés sous forme d’articles scientifiques constituent une base solide de l’enchaînement dans le temps des évènements essentiels à une transfection efficace des cellules CHO et HEK293 par le PEI. Chaque type cellulaire possède un profil d’expression des HSPG qui lui est propre, ces derniers étant plus ou moins permissifs au transfert de gène. En effet, une étude menée dans notre laboratoire montre que les SDC1 et SDC2 ont des rôles opposés vis-à-vis du transfert de gène. Alors que tous deux sont capables de lier les polyplexes, l’expression de SDC1 permet leur internalisation contrairement à l’expression de SDC2 qui l’inhibe. De plus, lorsque le SDC1 est exprimé à la surface des cellules HEK293, l’efficacité de transfection est augmentée de douze pourcents. En utilisant la capacité de SDC1 à induire l’internalisation des polyplexes, nous avons étudié le trafic intracellulaire des complexes SDC1 / polyplexes dans les cellules HEK293. De plus, nos observations suggèrent une nouvelle voie par laquelle les polyplexes pourraient atteindre efficacement le noyau cellulaire. Dans le contexte du transfert de gène, les HSPG sont essentiellement étudiés dans leur globalité. S’il est vrai que le rôle des syndécanes dans ce contexte est le sujet de quelques études, celui des glypicanes est inexploré. Grâce à une série de traitements chimiques et enzymatiques visant une approche « perte de fonction », l’importance de la sulfatation comme modification post-traductionnelle, l’effet des chaînes d’héparanes sulfates mais aussi des glypicanes sur l’attachement, l’internalisation des polyplexes, et l’expression du transgène ont été étudiés dans les cellules CHO et HEK293. L’ensemble de nos observations indique clairement que le rôle des HSPG dans le transfert de gène devrait être investigué individuellement plutôt que collectivement. En effet, le rôle spécifique de chaque membre des HSPG sur la capture des polyplexes et leur permissivité à l’expression génique demeure encore inconnu. En exprimant de manière transitoire chaque membre des syndécanes et glypicanes à la surface des cellules CHO, nous avons déterminé leur effet inhibiteur ou activateur sur la capture des polyplexes sans pouvoir conclure quant à l’effet de cette surexpression sur l’efficacité de transfection. Par contre, lorsqu’ils sont présents dans le milieu de culture, le domaine extracellulaire des HSPG réduit l’efficacité de transfection des cellules CHO sans induire la dissociation des polyplexes. Curieusement, lorsque chaque HSPG est exprimé de manière stable dans les cellules CHO, seulement une légère modulation de l’expression du transgène a pu être observée. Ces travaux ont contribué à la compréhension des mécanismes d'action du vecteur polycationique polyéthylènimine et à préciser le rôle des protéoglycanes à héparane sulfate dans le transfert de gène des cellules CHO et HEK293.

Functions of the SLC36 transporter Pathetic in growth control of Drosophila sensory neurons

Thesis (Ph.D.)--University of Washington, 2016-08

Mesenchymal stromal cells (MSCs) and colorectal cancer - a troublesome twosome for the anti-tumour immune response?

The tumour microenvironment (TME) is an important factor in determining the growth and metastasis of colorectal cancer, and can aid tumours by both establishing an immunosuppressive milieu, allowing the tumour avoid immune clearance, and by hampering the efficacy of various therapeutic regimens. The tumour microenvironment is composed of many cell types including tumour, stromal, endothelial and immune cell populations. It is widely accepted that cells present in the TME acquire distinct functional phenotypes that promote tumorigenesis. One such cell type is the mesenchymal stromal cell (MSC). Evidence suggests that MSCs exert effects in the colorectal tumour microenvironment including the promotion of angiogenesis, invasion and metastasis. MSCs immunomodulatory capacity may represent another largely unexplored central feature of MSCs tumour promoting capacity. There is considerable evidence to suggest that MSCs and their secreted factors can influence the innate and adaptive immune responses. MSC-immune cell interactions can skew the proliferation and functional activity of T-cells, dendritic cells, natural killer cells and macrophages, which could favour tumour growth and enable tumours to evade immune cell clearance. A better understanding of the interactions between the malignant cancer cell and stromal components of the TME is key to the development of more specific and efficacious therapies for colorectal cancer. Here, we review and explore MSC- mediated mechanisms of suppressing anti-tumour immune responses in the colon tumour microenvironment. Elucidation of the precise mechanism of immunomodulation exerted by tumour-educated MSCs is critical to inhibiting immunosuppression and immune evasion established by the TME, thus providing an opportunity for targeted and efficacious immunotherapy for colorectal cancer growth and metastasis.

Concussion: Examining the Effect of Neuronal Oxidative Stress on the Pathophysiology of Brain and Blood Brain Barrier Cells

Neuronal stretching during concussion alters glucose transport and reduces neuronal viability, also affecting other cells in the brain and the Blood Brain Barrier (BBB). Our hypothesis is that oxidative stress (OS) generated in neurons during concussions contributes to this outcome. To validate this, we investigated: (1) whether OS independently causes alterations in brain and BBB cells, namely human neuron-like, neuroblastoma cells (NCs), astrocyte cells (ACs) and brain microvascular endothelial cells (ECs), and (2) whether OS originated in NCs (as in concussion) is responsible for causing the subsequent alterations observed in ACs and ECs. We used H2O2 treatment to mimic OS, validated by examining the resulting reactive oxygen species, and evaluated alterations in cell morphology, expression and localization of the glucose transporter GLUT1, and the overall cell viability. Our results showed that OS, either directly affecting each cell type or originally affecting NCs, caused changes in several morphological parameters (surface area, Feret diameter, circularity, inter-cellular distance), slightly varied GLUT1 expression and lowered the overall cell viability of all NCs, ACs, and ECs. Therefore, we can conclude that oxidative stress, which is known to be generated during concussion, caused alterations in NCs, ACs, and ECs whether independently originated in each cell or when originated in the NCs and could further propagate the ACs and ECs.

A study of computational methods to analyze gene expression data

The recent advent of new technologies has led to huge amounts of genomic data. With these data come new opportunities to understand biological cellular processes underlying hidden regulation mechanisms and to identify disease related biomarkers for informative diagnostics. However, extracting biological insights from the immense amounts of genomic data is a challenging task. Therefore, effective and efficient computational techniques are needed to analyze and interpret genomic data. In this thesis, novel computational methods are proposed to address such challenges: a Bayesian mixture model, an extended Bayesian mixture model, and an Eigen-brain approach. The Bayesian mixture framework involves integration of the Bayesian network and the Gaussian mixture model. Based on the proposed framework and its conjunction with K-means clustering and principal component analysis (PCA), biological insights are derived such as context specific/dependent relationships and nested structures within microarray where biological replicates are encapsulated. The Bayesian mixture framework is then extended to explore posterior distributions of network space by incorporating a Markov chain Monte Carlo (MCMC) model. The extended Bayesian mixture model summarizes the sampled network structures by extracting biologically meaningful features. Finally, an Eigen-brain approach is proposed to analyze in situ hybridization data for the identification of the cell-type specific genes, which can be useful for informative blood diagnostics. Computational results with region-based clustering reveals the critical evidence for the consistency with brain anatomical structure.

Are iron oxide nanoparticles safe? Current knowledge and future perspectives

Due to their unique physicochemical properties, including superparamagnetism, iron oxide nanoparticles (ION) have a number of interesting applications, especially in the biomedical field, that make them one of the most fascinating nanomaterials. They are used as contrast agents for magnetic resonance imaging, in targeted drug delivery, and for induced hyperthermia cancer treatments. Together with these valuable uses, concerns regarding the onset of unexpected adverse health effects following exposure have been also raised. Nevertheless, despite the numerous ION purposes being explored, currently available information on their potential toxicity is still scarce and controversial data have been reported. Although ION have traditionally been considered as biocompatible - mainly on the basis of viability tests results - influence of nanoparticle surface coating, size, or dose, and of other experimental factors such as treatment time or cell type, has been demonstrated to be important for ION in vitro toxicity manifestation. In vivo studies have shown distribution of ION to different tissues and organs, including brain after passing the blood-brain barrier; nevertheless results from acute toxicity, genotoxicity, immunotoxicity, neurotoxicity and reproductive toxicity investigations in different animal models do not provide a clear overview on ION safety yet, and epidemiological studies are almost inexistent. Much work has still to be done to fully understand how these nanomaterials interact with cellular systems and what, if any, potential adverse health consequences can derive from ION exposure.

Rôle des protéoglycanes à héparane sulfate dans le transfert de gène des cellules CHO et HEK293

La possibilité de programmer une cellule dans le but de produire une protéine d’intérêt est apparue au début des années 1970 avec l’essor du génie génétique. Environ dix années plus tard, l’insuline issue de la plateforme de production microbienne Escherichia coli, fut la première protéine recombinante (r-protéine) humaine commercialisée. Les défis associés à la production de r-protéines plus complexes et glycosylées ont amené l’industrie biopharmaceutique à développer des systèmes d’expression en cellules de mammifères. Ces derniers permettent d’obtenir des protéines humaines correctement repliées et de ce fait, biologiquement actives. Afin de transférer le gène d’intérêt dans les cellules de mammifères, le polyéthylènimine (PEI) est certainement un des vecteurs synthétiques le plus utilisé en raison de son efficacité, mais aussi sa simplicité d’élaboration, son faible coût et sa stabilité en solution qui facilite son utilisation. Il est donc largement employé dans le contexte de la production de r-protéines à grande échelle et fait l’objet d’intenses recherches dans le domaine de la thérapie génique non virale. Le PEI est capable de condenser efficacement l’ADN plasmidique (vecteur d’expression contenant le gène d’intérêt) pour former des complexes de petites tailles appelés polyplexes. Ces derniers doivent contourner plusieurs étapes limitantes afin de délivrer le gène d’intérêt au noyau de la cellule hôte. Dans les conditions optimales du transfert de gène par le PEI, les polyplexes arborent une charge positive nette interagissant de manière électrostatique avec les protéoglycanes à héparane sulfate (HSPG) qui décorent la surface cellulaire. On observe deux familles d’HSPG exprimés en abondance à la surface des cellules de mammifères : les syndécanes (4 membres, SDC1-4) et les glypicanes (6 membres, GPC1-6). Si l’implication des HSPG dans l’attachement cellulaire des polyplexes est aujourd’hui largement acceptée, leur rôle individuel vis-à-vis de cet attachement et des étapes subséquentes du transfert de gène reste à confirmer. Après avoir optimisées les conditions de transfection des cellules de mammifères CHO et HEK293 dans le but de produire des r-protéines secrétées, nous avons entrepris des cinétiques de capture, d’internalisation des polyplexes et aussi d’expression du transgène afin de mieux comprendre le processus de transfert de gène. Nous avons pu observer des différences au niveau de ces paramètres de transfection dépendamment du système d’expression et des caractéristiques structurelles du PEI utilisé. Ces résultats présentés sous forme d’articles scientifiques constituent une base solide de l’enchaînement dans le temps des évènements essentiels à une transfection efficace des cellules CHO et HEK293 par le PEI. Chaque type cellulaire possède un profil d’expression des HSPG qui lui est propre, ces derniers étant plus ou moins permissifs au transfert de gène. En effet, une étude menée dans notre laboratoire montre que les SDC1 et SDC2 ont des rôles opposés vis-à-vis du transfert de gène. Alors que tous deux sont capables de lier les polyplexes, l’expression de SDC1 permet leur internalisation contrairement à l’expression de SDC2 qui l’inhibe. De plus, lorsque le SDC1 est exprimé à la surface des cellules HEK293, l’efficacité de transfection est augmentée de douze pourcents. En utilisant la capacité de SDC1 à induire l’internalisation des polyplexes, nous avons étudié le trafic intracellulaire des complexes SDC1 / polyplexes dans les cellules HEK293. De plus, nos observations suggèrent une nouvelle voie par laquelle les polyplexes pourraient atteindre efficacement le noyau cellulaire. Dans le contexte du transfert de gène, les HSPG sont essentiellement étudiés dans leur globalité. S’il est vrai que le rôle des syndécanes dans ce contexte est le sujet de quelques études, celui des glypicanes est inexploré. Grâce à une série de traitements chimiques et enzymatiques visant une approche « perte de fonction », l’importance de la sulfatation comme modification post-traductionnelle, l’effet des chaînes d’héparanes sulfates mais aussi des glypicanes sur l’attachement, l’internalisation des polyplexes, et l’expression du transgène ont été étudiés dans les cellules CHO et HEK293. L’ensemble de nos observations indique clairement que le rôle des HSPG dans le transfert de gène devrait être investigué individuellement plutôt que collectivement. En effet, le rôle spécifique de chaque membre des HSPG sur la capture des polyplexes et leur permissivité à l’expression génique demeure encore inconnu. En exprimant de manière transitoire chaque membre des syndécanes et glypicanes à la surface des cellules CHO, nous avons déterminé leur effet inhibiteur ou activateur sur la capture des polyplexes sans pouvoir conclure quant à l’effet de cette surexpression sur l’efficacité de transfection. Par contre, lorsqu’ils sont présents dans le milieu de culture, le domaine extracellulaire des HSPG réduit l’efficacité de transfection des cellules CHO sans induire la dissociation des polyplexes. Curieusement, lorsque chaque HSPG est exprimé de manière stable dans les cellules CHO, seulement une légère modulation de l’expression du transgène a pu être observée. Ces travaux ont contribué à la compréhension des mécanismes d'action du vecteur polycationique polyéthylènimine et à préciser le rôle des protéoglycanes à héparane sulfate dans le transfert de gène des cellules CHO et HEK293.

Evaluation of the genotoxicity of cellulose nanofibers

Background: Agricultural products and by products provide the primary materials for a variety of technological applications in diverse industrial sectors. Agro-industrial wastes, such as cotton and curaua fibers, are used to prepare nanofibers for use in thermoplastic films, where they are combined with polymeric matrices, and in biomedical applications such as tissue engineering, amongst other applications. The development of products containing nanofibers offers a promising alternative for the use of agricultural products, adding value to the chains of production. However, the emergence of new nanotechnological products demands that their risks to human health and the environment be evaluated. This has resulted in the creation of the new area of nanotoxicology, which addresses the toxicological aspects of these materials.Purpose and methods: Contributing to these developments, the present work involved a genotoxicological study of different nanofibers, employing chromosomal aberration and comet assays, as well as cytogenetic and molecular analyses, to obtain preliminary information concerning nanofiber safety. The methodology consisted of exposure of Allium cepa roots, and animal cell cultures (lymphocytes and fibroblasts), to different types of nanofibers. Negative controls, without nanofibers present in the medium, were used for comparison.Results: The nanofibers induced different responses according to the cell type used. In plant cells, the most genotoxic nanofibers were those derived from green, white, and brown cotton, and curaua, while genotoxicity in animal cells was observed using nanofibers from brown cotton and curaua. An important finding was that ruby cotton nanofibers did not cause any significant DNA breaks in the cell types employed.Conclusion: This work demonstrates the feasibility of determining the genotoxic potential of nanofibers derived from plant cellulose to obtain information vital both for the future usage of these materials in agribusiness and for an understanding of their environmental impacts.