The role of vitamin D signalling in the interaction between mesenchymal mammary tumour cells and macrophages

The transition of epithelial-like tumour cells to those exhibiting mesenchymal characteristics (Epithelial-to-mesenchymal Transition; EMT) is an integral process in breast cancer metastasis. EMT can be promoted by Transforming growth factor-beta (TGF-β) which can be found at high levels in the tumour stroma. Tumour-associated macrophages (TAMs) can also induce EMT in breast cancer cells, which is one way that they promote breast cancer metastasis. Vitamin D signalling has been implicated in EMT suppression and plays a role in modulating macrophage differentiation and stimulating their anti-inflammatory functions. This project had two major aims. First, we aimed to create and verify a unique fluorescent reporter gene construct designed to evaluate the dynamics of EMT in real-time and at the single-cell level. While some components of this reporter system were successfully validated, work to complete the final reporter construct is ongoing. The second and main aspect of this project focused on exploring the ability of 1,25-dihydroxyvitamin D3 (1,25D3) to modulate the interaction between mesenchymal mammary tumour cells and TAMs. Unexpectedly, in short-term treatment (48 hours) studies of 4T1 murine mammary tumour cells, we observed that 1,25D3 and TGF-β signalling work together to increase expression of the mesenchymal markers, Snai1, Fn1, and Col1a1. 1,25D3 and TGF-β also synergistically activate transcription of the gene encoding the 1,25D3-catabolizing enzyme, Cyp24a1. The ability of 1,25D3 and TGF-β to enhance expression of these genes was diminished in a long-term treatment (14 days) of 4T1 cells, and this effect was accompanied by a decrease in cell proliferation. 1,25D3 may also cooperate with cytokines produced by normal macrophages and macrophages considered to be TAM-like. Conditioned media experiments revealed that in the presence of factors from normal macrophages, 1,25D3 enhanced expression of Fn1, and in the presence of factors from TAM-like macrophages, 1,25D3 enhanced expression of Fn1 and Cyp24a1. Rather than mitigating the interaction as hypothesized, 1,25D3 may exacerbate the tumour-promoting effects of the EMT-TAM relationship. Also, signalling pathways involved in the EMT-TAM relationship may synergize with 1,25D3 to upregulate Cyp24a1 expression. These findings are important for understanding the potential of vitamin D compounds to be used in the treatment of breast cancer.

A disappearing act performed by magnesium: the nucleotide exchange mechanism of Ran GTPase by Quantum mechanics/Molecular mechanics studies

The Ran GTPase protein is a guanine nucleotide-binding protein (GNBP) with an acknowledged profile in cancer onset, progression and metastases. The complex mechanism adopted by GNBPs in exchanging GDP for GTP is an intriguing process and crucial for Ran viability. The successful completion of the process is a fundamental aspect of propagating downstream signalling events. QM/MM molecular dynamics simulations were employed in this study to provide a deeper mechanistic understanding of the initiation of nucleotide exchange in Ran. Results indicate significant disruption of the metal-binding site upon interaction with RCC1 (the Ran guanine nucleotide exchange factor), overall culminating in the prominent shift of the divalent magnesium ion. The observed ion drifting is reasoned to occur as a consequence of the complex formation between Ran and RCC1 and is postulated to be a critical factor in the exchange process adopted by Ran. This is the first report to observe and detail such intricate dynamics for a protein in Ras superfamily.

Using Vibrational Infrared Biomolecular Spectroscopy to Detect Heat-Induced Changes of Molecular Structure in Relation to Nutrient Availability of Prairie Whole Oat Grains on a Molecular Basis

Background To our knowledge, there is little study on the interaction between nutrient availability and molecular structure changes induced by different processing methods in dairy cattle. The objective of this study was to investigate the effect of heat processing methods on interaction between nutrient availability and molecular structure in terms of functional groups that are related to protein and starch inherent structure of oat grains with two continued years and three replication of each year. Method The oat grains were kept as raw (control) or heated in an air-draft oven (dry roasting: DO) at 120 °C for 60 min and under microwave irradiation (MIO) for 6 min. The molecular structure features were revealed by vibrational infrared molecular spectroscopy. Results The results showed that rumen degradability of dry matter, protein and starch was significantly lower (P <0.05) for MIO compared to control and DO treatments. A higher protein α-helix to β-sheet and a lower amide I to starch area ratio were observed for MIO compared to DO and/or raw treatment. A negative correlation (−0.99, P < 0.01) was observed between α-helix or amide I to starch area ratio and dry matter. A positive correlation (0.99, P < 0.01) was found between protein β-sheet and crude protein. Conclusion The results reveal that oat grains are more sensitive to microwave irradiation than dry heating in terms of protein and starch molecular profile and nutrient availability in ruminants.

Structural characterization of interaction of K11 and K63 mixed-linkage polyubiquitin chains with the tUIMs of epsin1

Ubiquitylation or covalent attachment of ubiquitin (Ub) to a variety of substrate proteins in cells is a versatile post-translational modification involved in the regulation of numerous cellular processes. The distinct messages that polyubiquitylation encodes are attributed to the multitude of conformations possible through attachment of ubiquitin monomers within a polyubiquitin chain via a specific lysine residue. Thus the hypothesis is that linkage defines polyubiquitin conformation which in turn determines specific recognition by cellular receptors. Ubiquitylation of membrane surface receptor proteins plays a very important role in regulating receptor-mediated endocytosis as well as endosomal sorting for lysosomal degradation. Epsin1 is an endocytic adaptor protein with three tandem UIMs (Ubiquitin Interacting Motifs) which are responsible for the highly specific interaction between epsin and ubiquitylated receptors. Epsin1 is also an oncogenic protein and its expression is upregulated in some types of cancer. Recently it has been shown that novel K11 and K63 mixed-linkage polyubiquitin chains serve as internalization signal for MHC I (Major Histocompatibility Complex I) molecule through their association with the tUIMs of epsin1. However the molecular mode of action and structural details of the interaction between polyubiquitin chains on receptors and tUIMs of epsin1 is yet to be determined. This information is crucial for the development of anticancer therapeutics targeting epsin1. The molecular basis for the linkage-specific recognition of K11 and K63 mixed-linkage polyubiquitin chains by the tandem UIMs of the endocytic adaptor protein epsin1 is investigated using a combination of NMR methods.

Novas estratégias para entender o sistema de influxo de antibióticos e a resistência antimicrobiana a nível molecular

As fluoroquinolonas são antibióticos que têm um largo espectro de ação contra bactérias, especialmente Gram-negativas. O seu mecanismo de ação assenta na inibição de enzimas responsáveis pela replicação do DNA. Porém, devido ao seu uso indevido, o surgimento de resistência bacteriana a estes antibióticos tem-se tornado um grave problema de saúde pública. Uma vez que os seus alvos de ação se situam no meio intracelular, a redução da permeabilidade da membrana externa de bactérias Gram-negativas constitui um dos mecanismos de resistência mais conhecidos. Esta redução é associada à baixa expressão ou mutações em porinas necessárias para permitir o seu transporte, mais concretamente, da OmpF. Estudos prévios demonstraram que a coordenação de fluoroquinolonas com iões metálicos divalentes e 1,10-fenantrolina (genericamente designados metaloantibióticos) são potenciais candidatos como alternativa às fluoroquinolonas convencionais. Estes metaloantibióticos exibem um efeito antimicrobiano comparável ou superior à fluoroquinolona na forma livre, mas parecem ter uma via de translocação diferente, independente de porinas. Estas diferenças no mecanismo de captura podem ser fundamentais para contornar a resistência bacteriana. De forma a compreender o papel dos lípidos no mecanismo de entrada dos metaloantibióticos, estudou-se a interação e localização dos metaloantibióticos da Ciprofloxacina (2ª geração), da Levofloxacina (3ª geração) e Moxifloxacina (4ª geração) com um modelo de membranas de Escherichia coli desprovido de porinas. Estes estudos foram realizados através de técnicas de espectroscopia de fluorescência, por medições em modo estacionário e resolvida no tempo. Os coeficientes de partição determinados demonstraram uma interação mais elevada dos metaloantibióticos relativamente às respetivas fluoroquinolonas na forma livre, um facto que está diretamente relacionado com as espécies existentes em solução a pH fisiológico. Os estudos de localização mostraram que estes metaloantibióticos devem estar inseridos na membrana bacteriana, confirmando a sua entrada independente de porinas. Este mecanismo de entrada, pela via hidrofóbica, é potenciado por interações eletrostáticas entre as espécies catiónicas de metaloantibiótico que existem a pH 7,4 e os grupos carregados negativamente dos fosfolípidos da membrana. Desta forma, os resultados obtidos neste estudo sugerem que a via de entrada dos metaloantibióticos e das respetivas fluoroquinolonas deve ser diferente. Os metaloantibióticos são candidatos adequados para a realização de mais testes laboratoriais e uma alternativa promissora para substituir as fluoroquinolonas convencionais, uma vez que parecem ultrapassar um dos principais mecanismos de resistência bacteriana a esta classe de antibióticos.

Advances in gastropod immunity from the study of the interaction between the snail Biomphalaria glabrata and its parasites: A review of research progress over the last decade

This review summarizes the research progress made over the past decade in the field of gastropod immunity resulting from investigations of the interaction between the snail Biomphalaria glabrata and its trematode parasites. A combination of integrated approaches, including cellular, genetic and comparative molecular and proteomic approaches have revealed novel molecular components involved in mediating Biomphalaria immune responses that provide insights into the nature of host-parasite compatibility and the mechanisms involved in parasite recognition and killing. The current overview emphasizes that the interaction between B. glabrata and its trematode parasites involves a complex molecular crosstalk between numerous antigens, immune receptors, effectors and anti-effector systems that are highly diverse structurally and extremely variable in expression between and within host and parasite populations. Ultimately, integration of these molecular signals will determine the outcome of a specific interaction between a B. glabrata individual and its interacting trematodes. Understanding these complex molecular interactions and identifying key factors that may be targeted to impairment of schistosome development in the snail host is crucial to generating new alternative schistosomiasis control strategies.

A influência da incorporação de ácido α-eleosteárico, purificado e isolado do óleo de tungue, na dinâmica molecular de lipossomos

Este trabalho descreve o isolamento e purificação do ácido α-eleosteárico (α-ESA) a partir do óleo de tungue e sua caracterização por espectroscopia de infravermelho com transformada de Fourier (FTIR), cromatografia gasosa acoplada com espectrometria de massas (GC-MS) e espectroscopia de ressonância magnética nuclear (RMN) de 1H e 13C. O α-ESA apresenta atividades biológicas (antitumorais, anti-inflamatórias e antioxidantes), tornando-se importante compreender sua interação com membranas lipídicas. Assim, este trabalho também descreve resultados referentes ao efeito da incorporação de α-ESA na dinâmica molecular de lipossomos compostos por fosfatidilcolina. O sistema lipossomal puro e contendo α-ESA foi caracterizado através do uso de espectroscopia de UV-visível, FTIR, RMN e calorimetria de varredura diferencial (DSC). Como resultados da purificação do α-ESA, obtivemos uma pureza de 95,9% utilizando acetona como solvente de recristalização em detrimento dos 92,2% em solução etanólica. Na incorporação em lipossomos, observou-se uma maior interação do α-ESA com a parte polar, de interface e os primeiros metilenos da região apolar da fosfatidilcolina. Além disso, α-ESA apresentou um efeito de redução da fluidez de lipossomos. Os resultados contribuem para a geração de conhecimento para o desenvolvimento de novos sistemas farmacológicos.

Low molecular weight compounds from mushrooms as potential Bcl-2 inhibitors: docking and virtual screening studies

Mushrooms have the ability to promote apoptosis in tumor cell lines, but the mechanism of action is not quite well understood. Inhibition of the interaction between Bcl-2 and pro-apoptotic proteins could be an important step that leads to apoptosis. Therefore, the discovery of compounds with the ability to inhibit Bcl-2 is an ongoing research topic in drug discovery. In this study, we started by analyzing Bcl-2 experimental structures that are currently available in Protein Data Bank database. After analysis of the more relevant Bcl-2 structures, 4 were finally selected. An analysis of the best docking methodology was then performed using a cross-docking and re-docking approach while testing 2 docking softwares: AutoDock 4 and AutoDock Vina. Autodock4 provided the best docking results and was selected to perform a virtual screening study applied to a dataset of 40 Low Molecular Weight (LMW) compounds present in mushrooms, using the selected Bcl-2 structures as target. Results suggest that steroid are the more promising family, among the analyzed compounds, and may have the ability to interact with Bcl-2 and this way promoting tumor apoptosis. The steroids that presented lowest estimated binding energy (ΔG) were: Ganodermanondiol, Cerevisterol, Ganoderic Acid X and Lucidenic Lactone; with estimated ΔG values between -8,45 and -8,23 Kcal/mol. A detailed analysis of the docked conformation of these 4 top ranked LMW compounds was also performed and illustrates a plausible interaction between the 4 top raked steroids and Bcl-2, thus substantiating the accuracy of the predicted docked poses. Therefore, tumoral apoptosis promoted by mushroom might be related to Bcl-2 inhibition mediated by steroid family of compounds.

Host-guest system of 4-nerolidylcatechol in 2-hydroxypropyl-beta-cyclodextrin: preparation, characterization and molecular modeling

The interaction of 4-nerolidylcatechol (4-NRC), a potent antioxidant agent, and 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was investigated by the solubility method using Fourier transform infrared (FTIR) methods in addition to UV-Vis, (1)H-nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. The inclusion complexes were prepared using grinding, kneading and freeze-drying methods. According to phase solubility studies in water a B(S)-type diagram was found, displaying a stoichiometry complexation of 2:1 (drug:host) and stability constant of 6494 +/- A 837 M(-1). Stoichiometry was established by the UV spectrophotometer using Job's plot method and, also confirmed by molecular modeling. Data from (1)H-NMR, and FTIR, experiments also provided formation evidence of an inclusion complex between 4-NRC and HP-beta-CD. 4-NRC complexation indeed led to higher drug solubility and stability which could probably be useful to improve its biological properties and make it available to oral administration and topical formulations.

mRNA-Seq and microarray development for the Grooved carpet shell clam, Ruditapes decussatus: a functional approach to unravel host -parasite interaction

Background The Grooved Carpet shell clam Ruditapes decussatus is the autochthonous European clam and the most appreciated from a gastronomic and economic point of view. The production is in decline due to several factors such as Perkinsiosis and habitat invasion and competition by the introduced exotic species, the manila clam Ruditapes philippinarum. After we sequenced R. decussatus transcriptome we have designed an oligo microarray capable of contributing to provide some clues on molecular response of the clam to Perkinsiosis. Results A database consisting of 41,119 unique transcripts was constructed, of which 12,479 (30.3%) were annotated by similarity. An oligo-DNA microarray platform was then designed and applied to profile gene expression in R. decussatus heavily infected by Perkinsus olseni. Functional annotation of differentially expressed genes between those two conditionswas performed by gene set enrichment analysis. As expected, microarrays unveil genes related with stress/infectious agents such as hydrolases, proteases and others. The extensive role of innate immune system was also analyzed and effect of parasitosis upon expression of important molecules such as lectins reviewed. Conclusions This study represents a first attempt to characterize Ruditapes decussatus transcriptome, an important marine resource for the European aquaculture. The trancriptome sequencing and consequent annotation will increase the available tools and resources for this specie, introducing the possibility of high throughput experiments such as microarrays analysis. In this specific case microarray approach was used to unveil some important aspects of host-parasite interaction between the Carpet shell clam and Perkinsus, two non-model species, highlighting some genes associated with this interaction. Ample information was obtained to identify biological processes significantly enriched among differentially expressed genes in Perkinsus infected versus non-infected gills. An overview on the genes related with the immune system on R. decussatus transcriptome is also reported.

RGS14 (414)-mediated prevention of an episodic memory loss: a study of molecular mechanism

A large proportion of human populations suffer memory impairments either caused by normal aging or afflicted by diverse neurological and neurodegenerative diseases. Memory enhancers and other drugs tested so far against memory loss have failed to produce therapeutic efficacy in clinical trials and thus, there is a need to find remedy for this mental disorder. In search for cure of memory loss, our laboratory discovered a robust memory enhancer called RGS14(414). A treatment in brain with its gene produces an enduring effect on memory that lasts for lifetime of rats. Therefore, current thesis work was designed to investigate whether RGS14(414) treatment can prevent memory loss and furthermore, explore through biological processes responsible for RGS-mediated memory enhancement. We found that RGS14(414) gene treatment prevented episodic memory loss in rodent models of normal aging and Alzheimer´s disease. A memory loss was observed in normal rats at 18 months of age; however, when they were treated with RGS14(414) gene at 3 months of age, they abrogated this deficit and their memory remained intact till the age of 22 months. In addition to normal aging rats, effect of memory enhancer treatment in mice model of Alzheimer´s disease (AD-mice) produced a similar effect. AD-mice subjected to treatment with RGS14(414) gene at the age of 2 months, a period when memory was intact, showed not only a prevention in memory loss observed at 4 months of age but also they were able to maintain normal memory after 6 months of the treatment. We posit that long-lasting effect on memory enhancement and prevention of memory loss mediated through RGS14(414) might be due to a permanent structural change caused by a surge in neuronal connections and enhanced neuronal remodeling, key processes for long-term memory formation. A neuronal arborization analysis of both pyramidal and non-pyramidal neurons in brain of RGS14(414)-treated rats exhibited robust rise in neurites outgrowth of both kind of cells, and an increment in number of branching from the apical dendrite of pyramidal neurons, reaching to almost three times of the control animals. To further understand of underlying mechanism by which RGS14(414) induces neuronal arborization, we investigated into neurotrophic factors. We observed that RGS14 treatment induces a selective increase in BDNF. Role of BDNF in neuronal arborization, as well as its implication in learning and memory processes is well described. In addition, our results showing a dynamic expression pattern of BDNF during ORM processing that overlapped with memory consolidation further support the idea of the implication of this neurotrophin in formation of long-term memory in RGS-animals. On the other hand, in studies of expression profiling of RGS-treated animals, we have demonstrated that 14-3-3ζ protein displays a coherent relationship to RGS-mediated ORM enhancement. Recent studies have demonstrated that the interaction of receptor for activated protein kinase 1 (RACK1) with 14-3-3ζ is essential for its nuclear translocation, where RACK1-14-3-3ζ complex binds at promotor IV region of BDNF and promotes an increase in BDNF gene transcription. These observations suggest that 14-3-3ζ might regulate the elevated level of BDNF seen in RGS14(414) gene treated animals. Therefore, it seems that RGS-mediated surge in 14-3-3ζ causes elevated BDNF synthesis needed for neuronal arborization and enhanced ORM. The prevention of memory loss might be mediated through a restoration in BDNF and 14-3-3ζ protein levels, which are significantly decreased in aging and Alzheimer’s disease. Additionally, our results demonstrate that RGS14(414) treatment could be a viable strategy against episodic memory loss.

Prospection for genes expressed during wheat - Magnaporthe oryzae interaction.

2013

Structural and functional interaction between polyamine related molecules and biological membranes

Changes induced by PA on nucleic acid (NA) conformation and synthesis is proven to be a major reason for PA essentiality (1-3). However, PA interactions with other polyanions, for instance polyanionic membrane lipid bilayers and glyosaminoglycans have received less attention (3-4). The functional importance of these interactions still is an obscure but interesting area of cell and molecular biology, especially in mammalian cells for which specific PA transport systems are not fully characterized (5). In mammals, activity and turnover of the polyamine (PA) synthesis key enzyme is controlled by a set of proteins: Antizymes (OAZ1-3) and antizyme inhibitors (AZIN1 and 2). It is demonstrated that AOZ modulate polyamine uptake (6), and that PA transport to mitochondria is linked to the respiratory chain state and modulates mitochondrial permeability transition (7). Antizyme expression variants have been located in mitochondria, being proposed as a proapoptotic factor (7-8). AZIN 2 is only expressed in a reduced set of tissues that includes mast cells, where it is associated to mast cell granules membrane (9). This fact, together to the abnormalities observed in bone marrow derived mast cell granules when they are differentiated under restricted PA synthesis conditions (10 and unpublished results), point out to important roles of PA and their related proteins in structure and function of mast cell granules. We will also present novel biophysical results on tripartite interactions of PA that remark the interest of the characterization of PA interactions with lipid bilayers for biomedicine and biotechnology. Thus, the information reported in this paper integrates previously reported information with our still unpublished results, all indicating that PA and their related proteins also are important factors for structure and dynamics of biological membranes and their associated functions essential in human physiology; for instance, solute interchange with the environment (uptake and secretion), oxidative metabolism and apoptosis. The importance of these involved processes for human homeostasis claim for further research efforts. 1. Ruiz-Chica J, Medina MA, Sánchez-Jiménez F and Ramírez FJ (2001) Fourier Transform Raman study of the structural specificities on the interaction between DNA and biogenic polyamines. Biophysical J. 80:443-454. 2. Lightfoot HL, Hall J (2014) Endogenous polyamine function--the RNA perspective. Nucleic Acids Res. 42:11275-11290. 3. Igarashi K, Kashiwagi K (2010) Modulation of cellular function by polyamines. Int J Biochem Cell Biol. 42:39-51. 4. Finger S, Schwieger C, Arouri A, Kerth A, Blume A (2014) Interaction of linear polyamines with negatively charged phospholipids: the effect of polyamine charge distance. Biol Chem. 395:769-778. 5. Poulin R, Casero RA, Soulet D. (2012) Recent advances in the molecular biology of metazoan polyamine transport. Amino Acids. 42:711-723. 6. Kahana C (2009) Regulation of cellular polyamine levels and cellular proliferation by antizyme and antizyme inhibitor. Essays Biochem. 4:47-61. 7. Agostinelli E, Marques MP, Calheiros R, Gil FP, Tempera G, Viceconte N, Battaglia V, Grancara S, Toninello A (2010) Polyamines: fundamental characters in chemistry and biology. Amino Acids 38:393-403. 8. Liu GY, Liao YF, Hsu PC, Chang WH, Hsieh MC, Lin CY, Hour TC, Kao MC, Tsay GJ, Hung HC (2006) Antizyme, a natural ornithine decarboxylase inhibitor, induces apoptosis of haematopoietic cells through mitochondrial membrane depolarization and caspases' cascade. Apoptosis 11:1773-1788. 9. Kanerva K, Lappalainen J, Mäkitie LT, Virolainen S, Kovanen PT, Andersson LC (2009). Expression of antizyme inhibitor 2 in mast cells and role of polyamines as selective regulators of serotonin secretion. PLoS One 31:e6858. 10. García-Faroldi G, Rodríguez CE, Urdiales JL, Pérez-Pomares JM, Dávila JC, Pejler G, Sánchez-Jiménez F, Fajardo I (2010) Polyamines are present in mast cell secretory granules and are important for granule homeostasis. PLoS One 30:e15071.

Caractérisation des structures de septines hautement organisées chez la drosophile et leur interaction avec le cytosquelette d’actine

Les septines sont des GTPases conservées dérégulées dans le cancer et les maladies neurodégénératives. Elles servent de protéines d’échafaudage et forment une barrière de diffusion à la membrane plasmique et au corps central lors de la cytokinèse. Elles interagissent avec l’actine et s’organisent en complexes qui polymérisent et forment des structures hautement organisées (anneaux et filaments). Leur dynamique d’assemblage et leur rôle dans la cellule restent à être élucidés. La Drosophile est un modèle simple pour l’étude des septines puisqu’on n’y retrouve que 5 gènes (sep1, sep2, sep4, sep5, peanut) comparativement aux 13 gènes chez l’humain. À l’aide d’un anticorps contre Pnut, nous avons identifié des structures tubulaires dans 30% des cellules S2 de Drosophile. Mon projet a comme but de caractériser ces tubes en élucidant leurs constituants, leur comportement et leurs propriétés pour mieux clarifier le mécanisme par lequel les septines forment des structures hautement organisées et interagissent avec le cytosquelette d’actine. Par immunofluorescence, j’ai pu démontrer que ces tubes sont cytoplasmiques, en mitose ou interphase, ce qui suggère qu’ils ne sont pas régulés par le cycle cellulaire. Pour investiguer la composition et les propriétés dynamiques de ces tubes, j’ai généré une lignée cellulaire exprimant Sep2-GFP qui se localise aux tubes et des ARNi contre les cinq septines. Trois septines sont importantes pour la formation de ces tubes et anneaux notamment Sep1, Sep2 et Pnut. La déplétion de Sep1 cause la dispersion du signal GFP en flocons, tandis que la déplétion de Sep2 ou de Pnut mène à la dispersion du signal GFP uniformément dans la cellule. Des expériences de FRAP sur la lignée Sep2-GFP révèlent un signal de retour très lent, ce qui indique que ces structures sont très stables. J’ai aussi démontré une relation entre l’actine et les septines. Le traitement avec la Latrunculin A (un inhibiteur de la polymérisation de l’actine) ou la Jasplakinolide (un stabilisateur des filaments d’actine) mène à la dépolymérisation rapide (< 30 min) des tubes en anneaux flottants dans le cytoplasme, même si ces tubes ne sont pas reconnus suite à un marquage de la F-actine. L’Actin05C-mCherry se localise aux tubes, tandis que le mutant déficient de la polymérisation, Actin05C-R62D-mCherry perd cette localisation. On observe aussi que la déplétion de la Cofiline et de l’AIP1 (ce qui déstabilise l’actine) mène au même phénotype que le traitement avec la Latrunculine A ou la Jasplakinolide. Alors on peut conclure qu’un cytosquelette d’actine dynamique est nécessaire pour la formation et le maintien des tubes de septines. Les futures études auront comme but de mieux comprendre l’organisation des septines en structures hautement organisées et leur relation avec l’actine. Ceci sera utile pour l’élaboration du réseau d’interactions des septines qui pourra servir à expliquer leur dérégulation dans le cancer et les maladies neurodégénératives.

A THEORETICAL STUDY OF INTERACTION OF NANOPARTICLES WITH BIOMOLECULE

Many types of materials at nanoscale are currently being used in everyday life. The production and use of such products based on engineered nanomaterials have raised concerns of the possible risks and hazards associated with these nanomaterials. In order to evaluate and gain a better understanding of their effects on living organisms, we have performed first-principles quantum mechanical calculations and molecular dynamics simulations. Specifically, we will investigate the interaction of nanomaterials including semiconducting quantum dots and metallic nanoparticles with various biological molecules, such as dopamine, DNA nucleobases and lipid membranes. Firstly, interactions of semiconducting CdSe/CdS quantum dots (QDs) with the dopamine and the DNA nucleobase molecules are investigated using similar quantum mechanical approach to the one used for the metallic nanoparticles. A variety of interaction sites are explored. Our results show that small-sized Cd4Se4 and Cd4S4 QDs interact strongly with the DNA nucleobase if a DNA nucleobase has the amide or hydroxyl chemical group. These results indicate that these QDs are suitable for detecting subcellular structures, as also reported by experiments. The next two chapters describe a preparation required for the simulation of nanoparticles interacting with membranes leading to accurate structure models for the membranes. We develop a method for the molecular crystalline structure prediction of 1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1,2-Dimyristoyl-sn-glycero-3-phosphorylethanolamine (DMPE) and cyclic di-amino acid peptide using first-principles methods. Since an accurate determination of the structure of an organic crystal is usually an extremely difficult task due to availability of the large number of its conformers, we propose a new computational scheme by applying knowledge of symmetry, structural chemistry and chemical bonding to reduce the sampling size of the conformation space. The interaction of metal nanoparticles with cell membranes is finally carried out by molecular dynamics simulations, and the results are reported in the last chapter. A new force field is developed which accurately describes the interaction forces between the clusters representing small-sized metal nanoparticles and the lipid bilayer molecules. The permeation of nanoparticles into the cell membrane is analyzed together with the RMSD values of the membrane modeled by a lipid bilayer. The simulation results suggest that the AgNPs could cause the same amount of deformation as the AuNPs for the dysfunction of the membrane.