La société Nanobactérie développe un traitement thermique innovant contre le cancer qui repose sur l‘utilisation de nanoparticules d‘oxyde de fer d‘origine bactérienne, appelées magnétosomes. Celles-ci sont injectées directement dans la tumeur puis activées par le champ magnétique alternatif. Cette activation crée une augmentation locale de la température provoquant la destruction de la tumeur, sans affecter les tissus sains environnants. Afin d‘éviter les problèmes de toxicité liés à la présence d‘endotoxines bactériennes à la surface des magnétosomes, un processus de purification est utilisé. Il permet l‘élimination de toute la membrane organique immunogène et de garder ainsi le minéral responsable de l‘activité thermique. Cependant, l‘élimination de cette membrane entraîne l‘agrégation des magnétosomes. La première étape de ce travail de thèse a donc consisté à stabiliser les magnétosomes purifiés, et l'‘identification du meilleur revêtement a été évaluée. Dans une deuxième partie, une nouvelle modalité de chauffage a été mise au point pour augmenter l‘efficacité de l‘hyperthermie magnétique dans la destruction de cellules cancéreuses. / The Nanobactérie company develops a novel strategy of cancer treatment using iron oxide nanoparticles of bacterial origin, called magnetosomes. These nanoparticles are injected directly into the tumor and then activated by an alternating magnetic field. Activated nanoparticles trigger a highly localized rise of temperature, inducing the destruction of the tumor without any adverse effects on adjacent healthy tissues. To avoid the problems of toxicity caused by the presence of bacterial endotoxin which present on the surface of magnetosomes extracted from bacteria, a process of purification is realized to eliminate all the immunogenic organic membrane and keep only the mineral responsible for the thermal activity. However, since elimination of this membrane causes the aggregation of the magnetosomes which become unstable in aqueous solution, the first part of this work consisted in stabilizing the purified magnetosomes by a modification of their surface. The identification of the best coating was then evaluated. Moreover, in the second part of this work, a new heating modality was assessed to increase the efficiency of the magnetic hyperthermia in the destruction of cancer cells.

During the past few years, gold nanoparticles (AuNPs) have received considerable attention in many fields due to their optical properties, photothermal effect and biocompatibility. AuNPs, particularly AuNCs and AuNRs, exhibit great potential in diagnostics and imaging. In the present study, AuNCs were used to selectively image and quantify intracellular antioxidants. It was reported by Chen et al. that the strong fluorescence of AuNCs is quenched by highly reactive oxygen species (hROS). Most of applications depend on fluorescence quenching, however, for our project we designed turn-on fluorescent sensors using AuNCs that sense antioxidants. In the presence of antioxidants, AuNCs fluorescence switch on, while in the absence of antioxidants their fluorescence immediately turn off due to hROS effect. AuNRs were also used for cellular imaging in which AuNRs were conjugated to Cy3-labelled molecular beacon (MB) DNA. Next, the previous complex was loaded in two different strains of magnetotactic bacteria (MTB). MTB were used as a targeted delivery vehicle in which magnetosomes direct the movement of bacteria. The DNA sequence was specific to a certain sequence in mitochondria. The exposure of MTB to an alternating magnetic field (AMF) leads to the increase of temperature inside the bacteria, which destruct the cell wall, and hence, bacterial payload is released. When MD-DNA hybrid with the target sequence, AuNR and Cy3 separate from each other, the fluorescence of the Cy3 is restored.

Iron is a common cation in biomineral sand; it is present for example in magnetite produced by magnetotactic bacteria and in iron sulfides produced by sulfate reducing microorganisms. The work presented in this thesis focused on two types of microorganisms capable of forming iron biominerals. In the first project I have studied the effect of O2 on the respiratory physiology and the formation of magnetosomes by Magnetospirillum magneticum AMB-1. In the second project I have studied the relationship between olivine and the activity of dissimilatory sulfate reducing (DSR) microorganisms. For the first project, I grew cells of AMB-1 in cultures with various concentrations of O2 and monitored growth and the formation of magnetic mineral particles (MMP). Results have shown that AMB-1 cells grew better at 100-225 uMO2(aq) than at lower [O2], yet the formation of MMP was repressed at ~45 uM O2(aq) and strongly inhibited at >100 uM O2(aq).These results have helped better understand the dissimilarity between the optimal growth conditions of magnetotactic bacteria and the conditions needed for the formation of MMPs. My results have also shown that the reaction between H2S produced by DSRs and olivine is abiotic, not catalyzed and exergonic. The pH did not vary significantly during this reaction and pH variation (in the 5-9 range) did not significantly influence this chemical reaction. Bicarbonate inhibited the reaction between H2S and olivine, but not the chemical equilibrium. Phosphate, a weak iron chelator, influenced the equilibrium of the reaction and it is assumed to help increase the rate of olivine weathering in the presence of DSRs. The activity of DSRs was positively influenced by the presence and abundance of olivine. Based on my results I propose that olivine help DSR obtain energy more efficiently, but does not represent a source of energy or nutrients for the cells. These results helped better understand the formation of iron biominerals and signatures of this activity.

<p>Magnetotactic bacteria (MTB) are ubiquitous, multi-phylogenetic bacteria that actively synthesize chains of magnetic, membrane bound; single domain magnetite (Fe₃O₄) or greigite (Fe₃S₄) crystals, termed magnetosomes in order to better navigate to their preferred chemical environment using the Earth’s magnetic field. Discovered in 1963, the field is now focused on understanding magnetosome chain formation and associated processes through genetic studies as well as analytical techniques such as Transmission Electron Microscopy (TEM) and Scanning Transmission X-ray Microscopy – X-ray Magnetic Circular Dichroism (STXM-XMCD).</p> <p>This thesis performed studies on <em>Candidatus Magnetovibrio blakemorei</em> strain MV-1 using STXM at the C 1s, O 1s, Ca 2p and Fe 2p edges. STXM-XMCD was used to determine the magnetism of individual magnetosomes and quantitatively determine magnetic properties such as the magnetic moment of individual chains. A sub-population of MV-1 cells was identified as having anomalous magnetic orientations of magnetosome sub-chains when separated spatial gaps. The frequency of this event and the underlying implications to magnetosome formation are discussed.</p> / Master of Science (MSc)

Les bactéries magnétotactiques (MTB) appartiennent à différents phyla procaryotes et ont la capacité de synthétiser des magnetosomes (cristaux de magnétite entourés par une membrane). Durant la thèse, nous avons procédé à l’analyse génomique de 2 bactéries magnétotactiques: Magnetospira sp. QH-2 et Magnetococcus MO-1. La synthénie et la correlation génique des gènes impliqués dans la formation des magnétosomes montrent que l'insertion de cet îlot chez QH-2 a eu lieu après la divergence entre les Magnetospirillum sp et Magnetospira sp. L'analyse comparative a mis en évidence trois groupes distincts de MTB : Groupe I, comprenant les souches Magnetospirillum spp. et Magnetospira; Groupe II avec MO-1 et M. marinus MC-1 et le Groupe III, avec D. magneticus RS-1. QH-2 montre aussi une évolution adaptative distincte par comparaison aux souches marines ou d'eau douce. L'analyse comparative des réseaux métaboliques révèle une très grande similitude intra-Groupe et une importante variabilité inter-Groupe. Cela est probablement dû aux enzymes impliqués dans les voies métaboliques anoxiques, qui représentent ainsi la contrainte à une distribution taxonomique large des MTB. Ces enzymes permettent ainsi de prédire le phénotype métabolique nécessaire à la production des magnétosomes. Différentes analyses (des protéines ribosomales au genome entier) indiquent une composition taxonomique chimérique des gènes de MO-1 et MC-1, et peut représenter une nouvelle lignée taxonomique chez les Protéobactéries. J’ai aussi participé à l'analyse des génomes de deux bactéries piezophiles, d’une bactérie photosynthétique pourpre et l’analyse phylogénomique des tyrosine-Kinases bactériennes. / Magnetotactic bacteria (MTB) are a diverse group of aquatic prokaryotes, which synthesize membrane-Enclosed magnetic crystals known as magnetosomes. In this thesis, the genome sequences of two marine MTB strains, Magnetospira sp. QH-2 and magneto-Ovoid strain MO-1 were analyzed. The magnetosome gene cluster synteny and mam gene correlation indicate that the insertion of the magnetosome island into QH-2 chromosome occurred after divergence between freshwater and marine magnetospirilla. Comparative genomic analysis revealed three distinct groups of sequenced MTB strains: Group I with Magnetospirillum spp. strains and Magnetospira strain, Group II with MO-1 strain and M. marinus MC-1, and Group III including Desulfovibrio magneticus RS-1. In addition, it shows an adaptive evolution of two marine MTB strains to marine sediments in comparison with closely related freshwater species. Moreover, comparative metabolic network analysis reveals high level of intra-Group similarity and inter-Group variety in MTB. With anoxic network enzymes, potential “MTB” strains are predicted, and are consistent with recently isolated MTB strains. It suggested that the anoxic metabolic network might be one restricted constraint for MTB distribution in bacterial lineages. Interestingly, analyses from ribosomal proteins to the whole MTB genome strongly support a taxonomic chimeric nature of MO-1 and MC-1 genes, and may represent a novel Proteobacteria lineage. Additionally, I also participate to genome analyses of piezophilic Desulfovibrio and Phaeospirillum molischianum strains as well as genome-Wide analysis of bacterial tyrosine kinases.

Les bactéries magnétotactiques représentent un groupe d’une grande diversité écologique et phylogénétique. Elles sont capables de biominéraliser des nanocristaux de magnétite [un oxyde de fer (Fe(II)Fe(III)2O4)] ou de greigite [un sulfure de fer (Fe(II)Fe(III)2S4)] dans leurs magnétosomes, organites alignés en chaînes permettant la navigation le long des lignes de champ magnétique terrestre. Jusqu'à récemment, seules des souches produisant de la magnétite étaient disponibles en culture pure, conduisant à des études sur les mécanismes de biominéralisation de cet oxyde de fer. En 2011, une nouvelle bactérie capable de former de la magnétite et de la greigite, Desulfamplus magnetovallimortis souche BW-1, a été cultivée avec succès en laboratoire. Dans cette thèse, nous proposons d'utiliser une approche intégrée et multidisciplinaire pour comprendre les mécanismes de biominéralisation de la greigite en utilisant comme modèle d’étude la souche BW-1. Nous avons donc cherché à déterminer les conditions environnementales et biologiques favorisant la formation de la magnétite et de la greigite. Ces travaux ont également conduit à la caractérisation physiologique et phylogénétique de BW-1. Puis, l’utilisation d’approches globales et ciblées de transcriptomique ont permis d'évaluer le taux d'expression des gènes impliqués dans la formation des magnétosomes (magnétite vs. greigite) dans diverses conditions de croissance. Une approche de protéomique a permis d’apporter des informations supplémentaires à cette étude. Ces résultats ont permis de progresser dans la compréhension fondamentale de la biominéralisation in vivo, en particulier pour des bactéries formant de la greigite. / Magnetotactic bacteria represent a phylogenetically and ecologically diverse group of prokaryotes able to biomineralize magnetic nanocrystals composed of magnetite [an iron oxide (Fe(II)Fe(III)2O4)] or greigite [an iron sulfide (Fe(II)Fe(III)2S4)] in their magnetosomes, a prokaryotic organelle whose cytoplasmic alignement in chain allows the cell to navigate along the Earth’s magnetic field lines. Until recently, only magnetite-producing strains were available in pure culture. Thus, only the magnetite biomineralization has been studied. In 2011, a new bacterium able to form both magnetite and greigite, Desulfamplus magnetovallimortis strain BW-1, was isolated from Death Valley, California and cultivated in pure culture. In this work, we propose to use an integrated and multidisciplinary approach to understand the mechanisms involved in greigite biomineralization in BW-1 strain. First, we determined the environmental and biological conditions in which magnetite and greigite are formed. This first part of my thesis also contributed to the physiologic and phylogenetic characterization of this bacterium. Secondly, we used global and targeted transcriptomic approaches to evaluate the transcription levels of genes putatively involved in magnetosomes formation (magnetite vs. greigite) under various growth conditions. A proteomic approach provided additional informations to this study.Results obtained during my thesis contribute to the understanding of in vivo biomineralization, particularly for greigite production in magnetotactic bacteria.

Cette thèse étudie quelques situations dans lesquelles un système actif, composé de particules auto-propulsées, est soumis à des contraintes extérieures. Dans un premier chapitre, nous étudions le comportement d'une assemblée de bactéries magnétotactiques —capables de s'aligner sur un champ magnétique extérieur— forcées au travers d'une constriction en forme de sablier. Nous caractérisons les propriétés dynamiques de ce système, à l'échelle individuelle mais également à celle de l'embouteillage formé et du jet émergeant. En particulier, nous montrons dans les zones concentrées en bactéries des couplages reliant densité en bactéries, vitesse de nage et forçage magnétique beaucoup plus complexes que ce qui avait été considéré jusqu'à maintenant dans les modèles théoriques.Le deuxième chapitre aborde un nouveau système actif constitué de disques de camphres posés à la surface de l'eau. Dans une première étape, nous avons étudié en détails les propriétés de nage individuelle de ces objets qui brisent spontanément la symétrie du système pour se mettre en mouvement. En particulier, nous montrons que les données recueillies peuvent être rationnalisées à l'aide d'une approche théorique très simple de ce problème couplé d'hydrodynamique et de transport de tensio-actif. Dans un troisième chapitre, nous avons abordé la dynamique d'une assemblée de ces nageurs interfaciaux interagissant via les champs hydrodynamiques et chimiques qu'ils génèrent. À concentration intermédiaire en nageurs, un régime de nage intermittente caractérisé par des bouffées pseudo-périodiques d'activité des nageurs apparaît. En utilisant des outils et concepts issus du domaine de la turbulence nous montrons que de façon remarquable, ce système très simple exhibe des comportements canoniques de la turbulence tels que prédits par Kolmogorov (1941), ouvrant ainsi des perspectives concrètes sur des analogies très riches entre turbulence et systèmes actifs / This work address different situations where active matter, made out of self-propelled particles, is submitted to external constraints.In a first part, we consider the response of magnetotactic bacteria –capable of swim alignment along magnetic field lines- directed through an hourglass shape geometry. We characterize the dynamic properties of the system, both at the individual bacteria scale and at the scale of the jammed region or of the induced outgoing jet. We show that in high density regions, couplings between the bacteria interactions, swim velocity and magnetic forcing take a much more complex form than had been considered so far in theoretical models.In a second chapter, we are addressing a new active system made out of camphor disks lying at the air-water interface. First of all, we study in details the individual swim properties of such particles, which spontaneously break the system symmetry to start moving. In particular, we show that all experimental data can be rationalized within the framework of a very simple model of this complex problem where hydrodynamic flows and surfactant transports are coupled through Marangoni stress.In a last chapter, we addressed the collective dynamics of an assembly of such interfacial swimmers that interact through the flow and chemical fields they generate. At intermediate swimmers concentrations, an intermittent swim regime appears characterized by pseudo-periodic activity bursts. Using tools and concepts from the turbulence domain, we show that, remarkably, this simple system exhibits dynamical properties matching the ones of canonical turbulence as predicted by Kolmogorov in the 40s. This demonstration opens up rich perspectives in the historical domain of turbulence together with in the emerging one of active matter

Submitted by Biblioteca de Pós-Graduação em Geoquímica BGQ (bgq@ndc.uff.br) on 2017-07-03T17:14:01Z No. of bitstreams: 1 Dissertação Rodrigo Sobrinho.pdf: 3591957 bytes, checksum: 2927de76042f83d833c2294256be9c4a (MD5) / Made available in DSpace on 2017-07-03T17:14:01Z (GMT). No. of bitstreams: 1 Dissertação Rodrigo Sobrinho.pdf: 3591957 bytes, checksum: 2927de76042f83d833c2294256be9c4a (MD5) / Fundação de Amparo a Pesquisa do Rio de Janeiro / Universidade Federal Fluminense. Instituto de Química. Programa de Pós-Graduação em Geoquímica, Niterói, RJ / Candidatus Magnetoglobus Multicelularis são organizações multi celulares, classificada como pertencentes ao grupo de sulfato redutoras e que possuem em seu citoplasma cristais de greigita, que dá a eles a característica magnetotática. São encontrados em ambientes aquáticos na região de tra nsição oxica-anóxica onde há uma estratificação química vertical. Entretanto, po uco conhecimento se tem sobre as características ambientais que favorecem seu cresci mento e sua sobrevivência, o que impede seu cultivo em laboratório e a compreens ão sobre sua influência nos ciclos biogeoquímicos dos ambientes onde são encont rados. O presente trabalho tem, neste sentido, o objetivo de caracterizar as c ondições geoquímicas que exercem influência sobre a população de Candidatus Magnetoglobus Multicelularis através de uma avaliação das variáveis mais relevan tes para seu metabolismo. Foram realizadas, para isso, duas coletas nos meses de março e junho de 2007 em três pontos da Lagoa de Araruama, quantificando a d istribuição vertical da população de Ca . M. multicellularis em extratos de sedimento e as variáveis abióticas nos mesmos extratos. Os resultados mostra ram que o ferro exerce influência sobre o crescimento populacional na maio ria dos pontos. Entretanto, no ponto da Praia da baleia, onde foram encontrados 89 % de todos os indivíduos quantificados, o ferro encontra-se em concentrações maiores e outros fatores exercem maior influência sobre o crescimento popula cional, como a disponibilidade de enxofre e nitrogênio. O trabalho conclui que Ca . M. multicellularis são encontrados em maior abundância em sedimentos que f avorecem a formação de sulfetos de ferro e a população concentra-se em ext ratos onde as condições ambientais favorecem a formação e a manutenção dos cristais encontrados no citoplasma ou onde há maior disponibilidade de nutr ientes, de acordo com a necessidade metabólica das células. / andidatus Magnetoglobus Multicelularis consist of multicellul ar aggregate that are classified as sulfate reductors, with grei gite crystals in their cytoplasm, which give them magnetotactic characteristics. They are f ound in aquatic environments, on the oxi-anoxi transition zone, where a vertical che mical stratification. However, little is known about the environmental characteristics that favor their survival and growth. This fact prevents their controlled breed and cultu re in a laboratorial environment and also impairs the understanding of their influence o n biogeochemical cycles of the environments where they are found. Therefore, the o bjective of the present work is to characterize the environmental conditions that favo r the Ca . M. multicellularis survival through an in situ evaluation of the variables that are more importan t to the cellular metabolism. In order to obtain this data, two sampl e collections were conducted in three spots of the Araruama lagoon, in the months m arch and in june of 2007 quantifying the vertical distribution of the Ca . M. multicellularis population in sediment extracts and their abiotic variables. The results s howed that the quantified iron acts as a limiting factor in most of the collection spot s. However, in Praia da Baleia, where 89% of the total quantified individuals were found, a higher concentration of iron was also found and it does not act as a limiting factor . At the latter collection spot, other factors such as sulfur and nitrogen availability re gulate the population growth. As a conclusion, this work demonstrates that Ca . M. multicellularis are found in more abundance in sediments that enable iron sulfide for mations. Moreover, they use their magnetotactic characteristics to adapt themselves t o environmental variations that occur frequently in the referred sediment region an d they try to find a region where the environmental conditions may allow the formatio n and maintenance of the crystals found in the cytoplasm or where there are more nutrients available.

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