Les bactériophages sont des virus qui injectent leur génome dans une bactérie après fixation à des récepteurs sur la surface de celle-ci, puis effectuent un cycle de multiplication de leur ADN, la synthèse des protéines de structure, l’encapsidation du génome viral et la lyse de la bactérie. Les phages virulents réalisent uniquement des cycles lytiques alors que les phages tempérés peuvent également intégrer leur génome dans celui de la bactérie, donnant ainsi naissance à une bactérie dite lysogène. Les phages peuvent parfois être maintenus dans la bactérie sans effectuer un cycle lytique ni s’intégrer, dans un état encore peu compris, connu sous le nom de pseudolysogénie. Pseudomonas aeruginosa est une espèce bactérienne présente dans l’environnement et associée à de nombreux hôtes, végétaux et animaux. Elle est responsable de graves infections nosocomiales et on observe de plus en plus souvent des souches multirésistantes aux antibiotiques, ayant une grande capacité à former des biofilms, et en conséquence très difficiles à éradiquer. Il faut donc absolument trouver des approches thérapeutiques nouvelles telle que la phagothérapie. De nombreuses données cliniques obtenues dans les pays de l’est de l’Europe et en Russie attestent de l’efficacité et de l’innocuité de la phagothérapie, mais il reste des incertitudes en particulier concernant la nature et la fréquence des résistances naturelles. Notre projet vise à évaluer le potentiel thérapeutique des phages et à mieux comprendre la dynamique de leur interaction avec leur hôte. Nous avons étudié les mécanismes de résistance mis en place par la souche de P. aeruginosa, PAO1, à quatre bactériophages virulents appartenant à des genres différents: deux podovirus, Ab05 (ФKMV-like) et Ab09 (LIT1-like), et deux myovirus, Ab27 (PB1-like) et Ab17 (KPP10-like), tous isolés par notre laboratoire. Des infections simples ou multiples de PAO1 ont été réalisées, et une collection de variants résistants aux phages a été isolée et étudiée. La fréquence des bactéries résistantes était de 10⁻⁵ pour l'infection par un phage seul et 10⁻⁶ pour les infections par des combinaisons de deux ou quatre phages. Le phénotype et la mobilité des variants résistants étaient fréquemment affectés.Le génome de 27 variants a été entièrement séquencé par la technologie Illumina, et la comparaison avec le génome de la souche PAO1 a permis l'identification de mutations ponctuelles ou de petites indels. Quatre variants supplémentaires ont été caractérisés par une approche «gène candidat». Des mutations affectant 14 gènes différents et 1 région régulatrice ont été observées. Les gènes mutés codent pour des protéines impliquées dans la biosynthèse des pili de type IV (T4P) et des lipopolysacharides (LPS), très fréquemment utilisés comme récepteurs par les phages. Des mutations de la synthèse des alginates ont été également observées. La moitié des variants possède des mutations de variation de phase qui se sont révélées être instables. Par contre, les gènes impliqués dans la biosynthèse du T4P montrent des délétions stables. Nous avons aussi observé que la pseudolysogénie est une conséquence fréquente de l'infection par ces phages virulents et que la sélection de mutants (très souvent des mutants doubles) est favorisée par la production continue de phages par les pseudolysogènes. La présence d'ADN de phage libre a été observée en liaison avec l'exclusion de surinfection. Pour conclure, si les phages sélectionnent des bactéries résistantes possédant des altérations dans les gènes impliqués dans la biogenèse ou la régulation des déterminants de la virulence, celle-ci sera probablement modifiée, d'une manière bénéfique ou préjudiciable, ce qui reste à étudier. L'utilisation du cocktail par rapport à l’infection simple, ne réduit pas de manière significative la fréquence de la résistance aux phages et en outre, nous montrons que la pseudolysogénie est un acteur majeur de la sélection de mutations. / Bacteriophages are obligate parasites of bacteria that can be defined as virulent or temperate according to their lifestyle: virulent phages perform a lytic cycle by injecting their genome in the bacterial cell and immediately multiply. Temperate phages, instead, can either perform a lytic, or a lysogenic cycle by integrating their genome into the bacterial chromosome and persisting in a dormant state until the lytic cycle is resumed. The viral genome can also be maintained in the bacterial cell in an episomal form for an undetermined period of time in a stage known as pseudolysogeny. P. aeruginosa, a bacterium commonly found in the environment and in association with many hosts including plants and animals, is responsible for severe nosocomial infections. A proportion of clinical strains are multidrug-resistant, possessing a high ability to form biofilms which are very difficult to eradicate with conventional treatments. It is therefore essential to find new therapeutic approaches, such as phage therapy. Numerous clinical data obtained in Eastern Europe and Russia attest the effectiveness and safety of phage therapy. However, there remain uncertainties related to their therapeutic use and particularly the high frequency of natural resistance. Our project aimed to better understand the dynamic of phage/bacteria interactions by studying the resistance mechanisms acting in the reference strain P. aeruginosa PAO1, against virulent phages. Infections were performed by combining phages belonging to four different genera: Ab05, a ФKMV-like podovirus, Ab09, a LIT1-like podovirus, Ab27, a PB1-like myovirus and Ab17, a KPP10-like myovirus, all isolated in our laboratory. Single or multiple infections of P. aeruginosa PAO1 were performed, and a collection of phage-resistant variants was isolated and analysed. The frequency of phage-resistant variants selection was 10⁻⁵ for single phage infection, and 10⁻⁶ for infections with cocktails of two or four phages. The phenotype and mobility of the variants was often affected, as compared to the parental strain. The genome of 27 variants was entirely sequenced by Illumina technology in order to identify mutations responsible for the resistance. Other variants were analysed by a candidate gene approach. We identified point mutations or small indels: in total, 27 independent mutations affected 14 genes and 1 regulatory region. The affected genes encode proteins involved in biosynthesis of type IV pili (T4P) and lipopolysaccharide (LPS), frequently used as receptors by the phages. Other mutations were observed in genes necessary for alginate production. Of interest, we found that half of the variants with mutations in genes involved in LPS biosynthesis possessed unstable phase variation mutations, responsible for translation frameshift. In contrast, genes involved in pilus type IV biogenesis were mainly subjected to deletions. Surprisingly, the presence of free phage DNA was found in association with exclusion of superinfection in half of the variants and no chromosomal mutation could be found in three of them. Thus, we showed that pseudolysogeny is a frequent outcome of infection by virulent phages of P. aeruginosa. Moreover, double mutants were selected at high frequency and this could presumably due to evolutionary pressure exerted by re-activation of lytic cycle in some cells of the pseudolysogen population. In conclusion, if phage predation selects for variants with alterations in genes involved in biogenesis or regulation of virulence determinants such as LPS or alginate, the resulting phage-resistant variants could potentially exhibit altered levels of virulence in a beneficial or detrimental way. The use of cocktail does not lower significantly the frequency of phage-resistance and in addition we show that pseudolysogeny is a major actor in the selection of mutations.

P. aeruginosa fait partie des bactéries classées comme multirésistantes. Ce bacille est un agent pathogène opportuniste susceptible d’être présent dans les réseaux d’eau. Les contaminations sont souvent localisées au niveau des points d’usage et sont à l’origine de risques sanitaires et économiques pour les établissements de santé et les industries. Bien que différents procédés de traitements soient couramment appliqués certaines contaminations persistent sous la forme de biofilm et altèrent la qualité de l’EDCH, tout en devenant un potentiel réservoir de dissémination. L’absence de traitements efficaces et l’impact négatif des biocides sur l’environnement sont en faveur du développement de nouvelles alternatives. Les bactériophages sont exclusivement des virus de bactéries. Ces prédateurs naturels sont omniprésents dans l'environnement, ce qui nous permet de disposer d'une grande diversité et ont l’avantage des’auto-répliquer en présence de leur hôte. Dans ce contexte, cette étude évalue le potentiel de ces virus en tant qu'agents de biocontrôle pour éliminer les biofilms de P. aeruginosa. Neuf souches de P. aeruginosa, incluant la souche référence PAO1 et des souches environnementales, ont été utilisées pour étudier l'activité de neuf phages appartenant à la famille des Caudovirales. Un screening a été réalisé permettant par la méthode des spots test de sélectionner les phages les plus efficaces et les souches sensibles. Les bactéries ont ensuite été cultivées dans un milieu mineral minimum et l'efficacité des phages a été étudiée sur une culture exponentielle. Le suivi de la densité optique a permis de mettre évidence trois profils d’activités différents. Sur la base de ces résultats, deux phages et deux souches ont été conservés pour réaliser des tests sur des biofilms de 24 h implantés à la surface de coupons en INOX, représentatif des surfaces industrielles ou thermales. L’efficacité du traitement par les phages durant 14 h a été évaluée par qPCR viable. Une réduction maximale de 1,7 équivalent Log UFC.cm2 /coupon a été obtenu selon le couple étudié. Les résultats mettent également en avant une répartition des phages en faveur des cellules planctoniques, contrôlant ainsi efficacement la dissémination du biofilm dans l’environnement. Cette étude met en évidence une action des phages qui est dépendante de la souche de P. aeruginosa ainsi que de l'état physiologiques des cellules (planctoniques ou sessiles) ce qui rend difficile l’élimination d’un biofilm, même jeune. Dans le but d’améliorer l’infection de ces structures il pourrait être envisagé d'associer l'activité de plusieurs phages dans un cocktail ou de les combiner à d'autres molécules d’intérêts. / P. aeruginosa is one of the bacteria classified as multiresistant. This bacillus is an opportunistic pathogen that may be present in water networks. Contaminations are often located at the point of use and are at the origin of health and economic issues for health facilities and industries. Although different treatment processes are commonly applied, certain contaminations persist in the form of biofilm, alter the quality of EDCH and represent a reservoir of dissemination. The lack of effective treatments and the negative impact of biocides on the environment favor the development of new alternatives. Bacteriophages are exclusively bacterial viruses. These natural predators are ubiquitous in the environment, which allows us to have a great diversity, and have the advantage of self replicationin the presence of their host. In this context, this work explores the potential of these viruses as biocontrol agents to eliminate P. aeruginosa biofilms in addition to existing solutions. Nine strains of P. aeruginosa, including PAO1 reference strain and environmental strains were used to study the activity of nine phages belonging to the family Caudovirales. A screening was carried out allowing by the spottest method to select the most effective phages and sensitives trains. A screening was carried out allowing by the spot test method to select the most effective phages and sensitive strains. Bacteria were then cultivated in a mineral minimum medium and the efficiency of the phages was studied on an exponential culture phase. Monitoring of optical density has enabled to highlight three different activity profiles. On the basis of these results, two phages and two strains were kept for testing on 24-hour biofilms implanted on the surface of stainless steel coupons, representative of industrial or thermal surfaces. The efficacy of phage treatment for 14 h was evaluated by viable qPCR. A maximum reduction of 1.7 log equivalent UFC.cm2 / coupon was obtained according to the couple studied. The results also highlight a phage distribution in favor of planktonic cells, effectively controlling the release of biofilm into the environment. This study demonstrates a phage action that is dependent on the P. aeruginosa strain as well as the physiological state of the cells (planktonic or sessile). The complexity of the biofilms’ structure makes it difficult to eliminate them, even when young. In order to improve the infection of these structures it could be considered to associate the activity of several phages in a cocktail or to combine them with other molecules of interest.

<p>Antimicrobial resistant bacteria pose a serious threat to global public health. With the development of antibioticresistance far outpacing the discovery of new antibiotics, there is a need to  develop  alternative  strategies  to  control  bacterial  infections without increasing  antibiotic resistance. This study focused on developing bacteriophage therapy as a non-antibiotic means to control colibacillosis in poultry. Avian colibacillosis causes significant mortality and economic loss to poultry industries around the world. The etiological agent is Avian Pathogenic<em> E.  coli</em> (APEC), with serotypes are O78, O1, O2 and O5 most often associated with infections. Here, seven bacteriophages (AHP, MP1, MP2, AKA, MKA, AHC and MIA) were isolated from human and poultry wastewater samples to target these four APEC serotypes.</p> <p>The host-spectrum analysis of these phages revealed that all seven phages lysed at least two different APEC strains, with four phages lysing four or more distinct APEC serotypes. Taken together, the isolated phages covered 90% (9/10) of O-serotyped APEC strains targeted in our study. When co-cultured with the targeted APEC strain, bacterial concentrations in phage treated APEC cultures (average OD₆₀₀= 0.09) were significantly (P < 0.05) lower than those of untreated cultures  (average  OD₆₀₀=  1.22)  after  4  hr  incubation.  However,  exposure  of  the  phages  to simulated gastric fluid (pH 2.2–2.5) reduced viability of three of the seven phages by 2.35 –4.01 log PFU/mL after 90 min and to undetectable levels after 5 min for other four phages. In contrast, phage viability was not impacted by simulated intestinal fluid (SIF) with no reductions in phage concentrations after exposure to SIF for 3 hr. All seven bacteriophages were encapsulated in sodium  alginate  microcapsules  with  encapsulation  efficiencies  between  94.4%  to  98.9%.  In contrast to unprotected phages, viability of encapsulated phages was reduced by only0.74 –1.21  log PFU/mL when exposed to SGF for 90 min. Incubating the encapsulated phages sequentially in SGF (1 hr) and SIF (3 hr) indicated that 85% -90% of phages were released from the microcapsules after1 hr incubation in SIF with the maximum release of phages from the microcapsules occurring after 3 hr of incubation in SIF. To assess the in vivoefficacy of the phage treatment, broiler chicks were challenged with APEC and treated with a mixture of unprotected and encapsulated phages. Concentrations of the APEC in the ceca of phage treated birds (2.79 log CFU/g) were significantly lower (P < 0.05) than those of untreated birds (6.18 log CFU/g) by 4 d post-challenge. Additionally, in  most  cases,  APEC  was  not  recovered  from  the  lungs  of  phage  treated  birds  whereas concentrations of APEC in lungs of untreated birds was 4.81 log CFU/g. Hence, these results indicate that phage treatmenteffectively controlled APEC colonization and replication in the ceca and lungs of APEC-challenged chickens and provide further evidence of the viability of phage-based treatments as a non-antibiotic means of controlling bacterial infections in chickens.</p>

Bacterial contamination of food is a global concern. Methods of treating bacterial contamination are limited. Bacteriophages, bacterial viruses, offer a promising solution. However, bacteriophages may have limited application for foods that undergo sterilization processing, are inhospitable to organisms, or must be maintained in a dry state. This thesis focused on methods to expand the application of bacteriophages. First, bacteriophages were subjected to generalized stresses of desiccation, heat, and acidity, under laboratory conditions to propagate new populations with improved stress resistance. However, testing of these stress-resistant populations under real-world conditions failed to produce results comparable to generalized laboratory conditions. Success in the application of selected bacteriophages may require high situational specificity during selection, including in terms of food matrix and stress mechanics. The focus of our research shifted from the modification of bacteriophage populations themselves to the development of food-safe protective matrices. Designed matrices encapsulate bacteriophage for integration with modern food production and even the food products themselves. A pullulan-trehalose sugar powder was developed for the protection of a model bacteriophage from pasteurization. Microparticles were engineered such that the majority of the particle would be composed of trehalose as a stabilizer and polysaccharide pullulan was designed to accumulate at the particle surface to slow dissolution. This structure resulted in a bacteriophage powder that remained intact and protective over short-term high-temperature pasteurization, whereas unprotected bacteriophage experienced significant loss in titer. Leucine-lactose and leucine-lactose-maltodextrin microparticles were engineered for the inclusion of bacteriophage in powdered infant formula. The bacteriophage powder was designed as a dormant protection system that could activate upon reconstitution. The excipient system was formulated to not significantly affect the pH, composition, and dissolution of commercial infant formula. The bacteriophage powder was also engineered to match the shelf life and secondary shelf life of infant formula. Altogether, this thesis demonstrates that bacteriophage application in different foods can be expanded through particle engineering. / Thesis / Doctor of Philosophy (PhD) / Bacterial contamination of food can lead to widespread outbreaks and subsequent preventable deaths. Our best tool against bacteria, antibiotics, cannot be widely applied to food for risk to the natural human biome and creation of resistant bacteria. Bacteriophages, viruses that infect bacteria, are a naturally occurring bactericide that offer an alternative solution. This thesis focuses on improving the application of bacteriophages in food. First, bacteriophages are selected for resistance to common food processing stresses, such as heat, drying, and acidity, to prepare future generations that are stress-resistant. Second, a protective sugar powder was designed that could be used to add bacteriophages to milk before pasteurization. Post-pasteurization, the sugar would dissolve and release bacteriophage into the milk to deal with any post-processing contamination. Lastly, an infant-safe bacteriophage powder was developed that could be intermixed with powdered infant formula in an effort to reduce infant death due to the ingestion of bacteria.

Experiments show that MgSO4 salt has a non-monotonic effect as a function of MgSO4 concentration on the ejection of DNA from bacteriophage lambda. There is a concentration, N0, at which the minimum amount of DNA is ejected. At lower or higher concentrations, more DNA is ejected. We propose that this non-monotonic behavior is due to the overcharging of DNA at high concentration of Mg⁺² counterions. As the Mg⁺² concentration increases from zero, the net charge of ejected DNA changes its sign from negative to positive. N0 corresponds to the concentration at which DNA is neutral. Our theory fits experimental data well. The DNA-DNA electrostatic attraction is found to be -0.004 kBT/nucleotide. Simulations of DNA-DNA interaction of a hexagonal DNA bundle support our theory. They also show the non-monotonic DNA-DNA interaction and reentrant behavior of DNA condensation by divalent counterions. Three problems in understanding the capsid assembly for a retrovirus are studied: First, the way in which the viral membrane affects the structure of in vivo assembled HIV-1 capsid is studied. We show that conical and cylindrical capsids have similar energy at high surface tension of the viral membrane, which leads to the various shapes of HIV-1 capsids. Secondly, the problem of RNA genome packaging inside spherical viruses is studied using RNA condensation theory. For weak adsorption strength of capsid protein, most RNA genomes are located at the center of the capsid. For strong adsorption strength, RNA genomes peak near the capsid surface and the amount of RNA packaged is proportional to the capsid area instead its volume. Theory fits experimental data reasonably well. Thirdly, the condensation of RNA molecules by nucleocapsid (NC) protein is studied. The interaction between RNA molecules and NC proteins is important for the reverse transcription of viral RNA which relates to the viral infectivity. For strong adsorption strength of the NC protein, there is a screening effect by RNA molecules around a single NC protein.

Les infections nosocomiales sont causées par des germes opportunistes souvent résistants aux antibiotiques et persistants sur les surfaces, représentant une source constante de risque d’infection en milieu hospitalier. Dans ce contexte, l’isolement et la caractérisation de bactériophages s’attaquant spécifiquement aux bactéries nosocomiales telles que Staphylococcus aureus résistant (SARM), Enterococcus résistant (ERV), Pseudomonas aeruginosa et Acinetobacter baumanii, pourraient fournir une alternative bactéricide naturelle contre la transmission de ces infections. Des phages isolés des eaux usées, ont été sélectionnés selon leur capacité d’amplification, leur profil génomique et leur potentiel lytique envers différentes souches bactériennes cliniques. Les meilleurs ont été caractérisés en détail pour s’assurer de leur spécificité, sécurité, stabilité et efficacité préalablement à leur utilisation in vivo. Sept phages contre SARM et trois contre Acinetobacter baumanii ont été caractérisés. Quatre phages SARM s’avèrent être de bons candidats potentiels et pourraient être testés en milieu hospitalier comme agents désinfectants dans le but de lutter contre les infections nosocomiales. / Nosocomial infections are directly related to opportunistic germs, which are often resistant to antibiotics and persistent on surfaces, representing a high infectious risk in hospitals. In this context, the isolation and characterization of bacteriophages specifically targeting nosocomial bacteria such as resistant Staphylococcus aureus (MRSA), resistant Enterococcus (VRE), Pseudomonas aeruginosa and Acinetobacter baumanii, could provide a natural bactericidal alternative against the transmission of these infections. Phages, isolated from waste water, were selected according to their capacity of amplification, their genomic profile and lytic potential towards various bacterial clinical strains. The best ones were characterized in detail to primarily ensure their specificity, safety, stability and effectiveness, before studying their in vivo usage. Seven phages against MRSA and three against Acinetobacter baumanii were characterized. Four MRSA phages proved to be good potential candidates and could be tested in hospitals as disinfectant agents with the aim of fighting nosocomial infections.

Résumé : La compréhension des contraintes qui régissent l'assemblage des machineries supramoléculaires – qu'elles soient solubles ou bien ancrées dans les membranes biologiques – est un enjeu scientifique majeur.Le système de de sécrétion type VI (T6SS) est un organelle bactérien récemment mis en évidence qui a pour particularité de posséder une origine évolutive commune avec le bactériophage T4. En raison de cette origine évolutive commune, certaines sous unités du T6SS et du bactériophage T4 présentent des structures comparables. Cependant, un grand nombre des sous unités du T6SS reste à caractériser. Parmi celles-ci, les protéines SciB et SciC sont retrouvées dans tous les systèmes de sécrétion de type VI suggérant que ces deux protéines participent à la formation du "core-complexe": le complexe minimal requis pour le fonctionnement du T6SS. / The recently identified type VI secretion system has been demonstrated to be involved in most of these processes. The T6SS is a highly complex macromolecular machine that allows Gram-negative bacteria to deliver effector proteins to both prokaryotic and eukaryotic cells in a contact-dependent manner. The T6SS promotes therefore antibacterial competition, virulence towards eukaryotes or even both. The T6SS is composed of a minimal set of 13 subunits, which are currently believed to form the core apparatus. They assemble two distinct sub-complexes: one is a cytosolic contractile structure related to the tail of contractile bacteriophages, whereas the other spans the whole cell envelope. Therefore, the T6SS is generally depicted as an inverted phage tail anchored to the cell envelope through its membrane-associated complex. Contractile tails are currently thought to assemble from four structural elements: the baseplate, the internal tube, the contractile sheath and the tail terminator. The aim of my Ph.D. work was to further characterize the assembly and function of the T6SS phage tail-like complex in enteroaggregative E. coli. In this thesis document, I provide evidence that the internal tube assembles from Hcp hexamers stacked in a head-to-tail manner and that this internal cylinder is used as a template during sheath assembly. I also characterized a sub-complex of three proteins (TssEFG) that forms the baseplate of the T6SS and controls the polymerization of the tube and sheath. Finally, I recently showed that the T6SS functions like a nano-crossbow to kill target cells as the contraction of the T6SS results in prey cell death during interbacterial competition.

Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2017-08-30T13:20:01Z No. of bitstreams: 2 Deisi_Tapparo2017.pdf: 1334800 bytes, checksum: e1f7fae75650df8c87e6f134d2dad8cf (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-08-30T13:20:01Z (GMT). No. of bitstreams: 2 Deisi_Tapparo2017.pdf: 1334800 bytes, checksum: e1f7fae75650df8c87e6f134d2dad8cf (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The swine breeding stands out on worldwide due to its constant growth. However, environmental problems have increased as the activity gains recognition, consequently, planning and management of waste produced have been required in the systems. Animal carcasses disposal inside or outside the animal rearing farms is under concern and object of discussion about good practices to do it considering biosecurity aspects. One alternative is the swine manure and swine carcass co-digestion in order to improve biogas production. At the same time, it is known that animal carcass has some pathogenic microorganisms of zoonotic importance. In this context, this research aimed at studying swine carcass co-digestion and swine manure as well as its implications on biogas yield and digestate sanitation. This study was carried out under mesophilic temperature (37 ºC) and triplicate tests. Biogas volume was measured using eudiometer tubes, according to VDI 4630. The trials used the carcass sample digestion in a separate way and loading rates of carcass/manure were 3, 7.5 and 15kg.m-3manure, which represented 1, 2.5 and 5 times of mortality/manure production rates on typical swine farms (Mortality rate of 7% .year-1 for matrices). The inactivation trials were carried out in separated. Then it was evaluated the inactivation of the following microorganisms models (E. coli, Salmonella enterica serovar Senftenberg, PCV2, and bacteriophage MS2 and PhiX-174). Four inactivation strategies were carried out at two temperatures (24 ºC and 37 ºC) and two swine carcass/swine manure ratios were also studied (3kgcarcass.m-3manure and 15 kgcarcass.m-3manure). The swine carcass presented biochemical methane potential (BMP) of 1076 ± 48 LNbiogas.kgSVadic-1, and its co-digestion with swine manure increases biogas production potential when compared to manure monodigestion. The model microorganisms such as E. coli, S. Senftenberg and PCV2 (37°C) were completely inactivated until 30 days, while PCV2 (24°C), MS2 and PhiX-174 were more resistant. The temperature of co-digestion process influences the time required for microorganisms’ inactivation. Inactivation results suggest that, during co-digestion at 37°C, there is a greater pathogen reduction when compared to the same process at 24° C. It is recommended to use a pre-treatment process to biodigestor without heating system and under mesophilic temperature to ensure biosafety. / A suinocultura se destaca no cenário mundial devido ao seu constante crescimento. Todavia, os problemas ambientais aumentam à medida que a atividade ganha reconhecimento, por isso, são necessários planejamento e gestão dos resíduos produzidos nos sistemas. Um dos desafios da atividade é a destinação de animais mortos, logo, são necessárias alternativas que aliem proteção do meio ambiente e saúde pública bem como opções de baixo custo. Uma das alternativas é realizar a codigestão de carcaça suína com dejeto suíno para melhorar a produção de biogás. Ao mesmo tempo, é de conhecimento que a carcaça animal possui micro-organismos patogênicos de importância zoonótica. Nesse contexto, o objetivo desta pesquisa foi estudar a codigestão de dejeto suíno e carcaça suína, suas implicações sobre a produção de biogás e sanitização do digestato. Os estudos de digestão foram conduzidos em batelada, sob condições mesofílicas (37ºC) e em triplicata. Foram utilizados tubos eudiômetros para mensurar o biogás produzido, conforme norma VDI 4630. Os experimentos envolveram a digestão de amostras de carcaça em separado e das relações de mistura entre carcaça e dejeto nas seguintes proporções: 3; 7,5 e 15 kgcarcaça.m-3dejeto, as quais representaram 1; 2,5 e 5 vezes a mortalidade encontrada em granjas comerciais (mortalidade de 7%.ano-1 para matrizes). Os experimentos de inativação de patógenos foram conduzidos em separado. Avaliou-se a inativação de micro-organismos modelos (E. coli, Salmonella entérica serovar Senftenberg, PCV2, MS2 e PhiX-174). Quatro estratégias de inativação foram estudadas: duas temperaturas (24ºC e 37ºC) e duas relações de carcaça suína/dejeto suíno (3kg.m-3 e 15kg.m-3). Os resultados mostraram que a carcaça suína tem um potencial de 1076 ± 48 LNbiogás.kgSVadic-1, e a sua codigestão com dejeto suíno aumenta o potencial de produção de biogás comparando com a monodigestão do dejeto. Os micro-organismos modelo E. coli, S. Senftenberg e PCV2 (37 ºC) foram totalmente inativados em 30 dias, enquanto o PCV2 (24 ºC), MS2 e PhiX -174 foram mais resistentes. A temperatura do processo de codigestão influencia no tempo necessário para a inativação dos micro-organismos. Os resultados de inativação sugerem que, durante a codigestão a 37 ºC, ocorre maior redução de patógenos quando comparada ao mesmo processo a 24 ºC. Recomenda-se a utilização de processo de pré-tratamento para biodigestores sem sistema de aquecimento e em temperaturas mesofílicas, a fim de garantir a biossegurança.

Traditional topics such as physics, chemistry and mathematics have immensely changed the world in the twentieth century, but the twenty-first century seems to be that of soft condensed matter physics, which has already shown its tremendous possibilities to influence the everyday human life through its technological manifestations such as biotechnology and soft nano technology. Unlike the traditional topics, soft condensed matter physics has an interdisciplinary nature. It studies systems that usually come under chemistry or biology, using the methods of physics and mathematics and hence, transcends the frontiers between the subjects. Soft matter may be classified into three main classes; colloidal dispersions, polymers and polymer melts and liquid crystals. Study of single polymer chains is a fascinating topic that provides insights to understand many processes occurring in biological systems. Here, we present analytical studies of a few such processes, involving single polymer chains. In fact, there are a number of biological processes, which involve the dynamics of a single polymer chain. Due to the importance of Brownian motion at the mesoscopic level, soft matter systems are always studied using the analytical as well as computational methods of statistical mechanics. The statistical mechanics of polymers has been developed into a fascinating topic due to the contributions from the theory of random walks and path integrals. The dynamical behavior of many-particle systems has been described traditionally by the so-called rate theories. Here, we use these classical approaches to study a few biological processes that involve single polymer chains. The kind of processes that we have investigated may be categorized into two, namely the processes that leads to conformational changes in a chain molecule and the processes involving spatial translocation of a polymer. In the first category, we have considered the dynamics of semiflexible polymer loops. Loop formation of chain molecules has a key role in biological processes like DNA replication, gene regulation and protein folding. Hence, the dynamics of a polymer closing to form a loop as well as opening of the loop are topics of considerable theoretical/experimental interest. For closing, results are available in the completely flexible limit. Wilemski and Fixman, (J. Chem. Phys.60, 878 (1974)) have studied the closing and opening reactions in a single flexible polymer chain and using their approach Doi found the closing time to vary with the length of the chain as L2 . Szabo, Schul-ten, and Schulten, (J. Chem. Phys. 72, 4350 (1980)) have used mean first passage time approach and they find that the closure time vary as L3/2. Both approaches have been compared with simulations (Pastor et.al, J. Chem. Phys., 105, 3878 (1996), Srinivas et. al,116,7276 (2002)). In the case of semiflexible chains, studies are fewer in comparison. However, real polymers such as DNA, RNA or proteins are not flexible and therefore, it is important to incorporate the intrinsic stiffness of the chain into account. In the worm-like chain model, the chain is described as a continuous, inextensible and differentiable space curve represented by the position vector r(s), where s is the arc length. Inextensibility of the chain requires that the tangent vector, u(s) = ∂r(s)/∂s, at any point on the curve must have unit magnitude, i.e, |u(s)| = 1. But incorporating this constraint has been a difficult problem in dealing with semiflexible polymers. Yamakawa and Stockmayer (J. Chem. Phys., 57, 2843 (1972)) and Shimada and Yamakawa (Macromolecules, 17, 689 (1984)) have calculated ring closure probabilities for worm-like chains and helical worm-like chains. Cherayil and Dua (J. Chem. Phys., 116, 399 (2002)) have calculated closure time for a semiflexible chain using the approximate model for semiflexible chains by Har-nau, Winkler and Reineker (J. Chem. Phys., 101, 8119 (1994)) and find that the closure time ~ Lν where ν is in the range 2.2 to 2.4. Physically, one expects that the closing time should decrease exponentially with length in the very short chain limit and then increase with length for longer chains. Hence, the closing time has a minimum at an intermediate length. The reason for this behavior is that, for short chains, the bending energy contributes significantly to the activation energy for the process. The activation energy ~ const./L and hence, the closing time τ ~ exp(const./L). For longer chains, the free energy barrier for closing is due to the configurational entropy and hence, τ obeys a power law. Recently, Jun et. al (Europhys. Lett., 64, 420 (2003)) have followed an approximate one dimensional Kramers approach to reproduce this behavior and obtained the minimum at a length Lmin = 3.4lp, where lp is the persistence length of the chain. Monte Carlo simulations by Chen et.al (Europhys. Lett., 65, 407 (2004)) lead Lmin = 2.85lp. We investigate (K. P. Santo and K. L. Sebastian, Phys. Rev. E, 73, 031923, (2006)) in detail the problem of loop opening for semiflexible polymers. The inextensibility constraint is incorporated rigorously by setting u(s) to be a unit vector in the angular direction (θ, φ) and the conformations of the polymer are then represented by Brownian motion over a unit sphere in the tangent vector space. We use the worm-like chain model, which takes into account the bending rigidity of the polymer. The bending energy can then be given in terms of the angle coordinates θ and φ. For the dynamics, we make use of a semiclassical approach, which is based on expanding the bending energy about a minimum energy path. For the sake of simplicity, we take the great circle on the unit sphere to be the minimum energy configuration of the loop and expand the bending energy up to second order in terms of fluctuations about this configuration. We find that, this is a very good approximation in the large stiffness limit, as this approach leads to a minimum energy value, which is very close to the exact calculations. The loop is unstable, unless the ends are bound to each other with a potential. Once the two ends have been brought together, they can separate from each other in any of the three directions in space. Considering the ring to be in the XY plane with the ends meeting in the Y-axis, we find that the separation in the X and Z directions are unstable as motion in these directions lead to decrease in bending energy. But the motion in the other direction, that is, the Y direction leads to increase in energy and is stable. Therefore, we choose the potential to be of Morse type in the X-direction and stable harmonic ones in the other two directions. With this, the potential energy surface for opening can be found and the rate of opening can be calculated using classical Transition State Theory. The effects of friction on the rate can also be incorporated using the standard coupling to a bath of harmonic oscillators . We find that for short chains, the rate is strongly length dependent and is well-described by the equation Aexp(B/x)/xν, with A and B constants, x = L/lp, L the length of the chain, lp the persistence length and ν ~ 1.2. However, for long chains, the rate is found to obey a power law. But in view of the fact that our approximations, while sensible for short semiflexible chains, are not expected to be valid for long flexible chains and therefore, this result is not expected to be correct. We also present results for the seemingly more biologically important reverse process, the closing of a semiflexible polymer, thus presenting a rather complete theory of dynamics of semiflexible polymer loops. In this work, we give a detailed multidimensional analysis of the closing dynamics of semiflexible chains by making use of the approximation scheme developed in the previous study of loop opening. We use the formalism of Wilemski and Fixman for the diffusion-controlled intra-chain reactions of polymers and their "closure" approximation for an arbitrary sink function. In this procedure, the closing time is expressed in terms of a sink-sink correlation function. We calculate this sink-sink correlation function through a normal mode analysis on the chain. The closing times, τclose for different lengths of the chain are then obtained. We find that τclose(L) ~ L4.5W(L), where W(L) was found to be described by B'exp(A'/L) with A' and B' constants. τclose(L) is found to have a minimum at Lmin = 2.4lp, which is to be compared with the values obtained through a one dimensional analysis (Europhys. Lett., 64, 420 (2003)) and simulations (Europhys. Lett., 65, 407 (2004)). We thus present a multidimensional analysis that give results that are physically expected. There does not seem to be any previous analysis which leads to these results shown through one-dimensional studies and simulations. In the category of translocation problems, we consider DNA packaging in viruses. DNA Packaging into the viral capsid is an essential step in any kind of viral infection. The mechanism of packaging in bacteriophage φ — 29 has recently been studied (Simpson et. al, Nature (London), 408, 745 (2000)). The study revealed the structure of the molecular motor that packages the DNA. In another experimental study, Smith et. Al (Nature (London), 408, 745 (2001)) have investigated the effect of applied external force on the packaging. Motivated by this study, we suggest (K. P. Santo and K. L. Sebastian, Phys. Rev. E, 65, 052902 (2002)) a simple model to explain the kinetics of packaging of DNA the external force, which tries to prevent it. The model suggests a Butler-Volmer kind of dependence of the rate of packaging on the pulling force. We find that our model explains the experimental data very well. Another very interesting situation that arises in biological contexts is the translocation of a polymer across a membrane through a pore. The uptake of DNA into the cell nucleus and the translocation of cytosolic protein into endoplasmic reticulum are examples. There have been two main classes of polymer translocation problems; translocation in presence of a field or driven by a molecular motor and the translocation assisted by the adsorption of molecules onto the chain in the region into which it is translocated. While the first class of problems is reasonably well understood, for the second class of problems a complete understanding still does not exist in the literature. The existing understanding of this kind of polymer translocation is mainly due to Simon, Peskin and Oster (Proc. Natl. Accad. Sci. USA, 89, 3770 (1992)), who describe the translocation as kind of biased Brownian motion, which is known as the Brownian Ratchet. But Brownian Ratchet is an idealization and can only be realized in certain limits and therefore, it does not account for the detailed dynamics of polymer and the binding particles. We present a simple statistical description of the problem. We find that in the regime where number of binding particles are larger than the number of adsorption sites on the chain, the translocation proceeds as if it is driven by a constant force and hence, seems to be governed by a mechanism similar to the kink mechanism (K. L. Sebastian and Alok. K. R. Paul, Phys. Rev. E, 62, 927 (2000), K. L. Sebastian, 61, 3245 (2000)) that has been suggested in the case translocation in presence of an external field. In the other regime, where the number of binding particles are less than the number of binding sites on the chain, the translocation was found to be predominantly diffusive.

Les infections nosocomiales sont causées par des germes opportunistes souvent résistants aux antibiotiques et persistants sur les surfaces, représentant une source constante de risque d’infection en milieu hospitalier. Dans ce contexte, l’isolement et la caractérisation de bactériophages s’attaquant spécifiquement aux bactéries nosocomiales telles que Staphylococcus aureus résistant (SARM), Enterococcus résistant (ERV), Pseudomonas aeruginosa et Acinetobacter baumanii, pourraient fournir une alternative bactéricide naturelle contre la transmission de ces infections. Des phages isolés des eaux usées, ont été sélectionnés selon leur capacité d’amplification, leur profil génomique et leur potentiel lytique envers différentes souches bactériennes cliniques. Les meilleurs ont été caractérisés en détail pour s’assurer de leur spécificité, sécurité, stabilité et efficacité préalablement à leur utilisation in vivo. Sept phages contre SARM et trois contre Acinetobacter baumanii ont été caractérisés. Quatre phages SARM s’avèrent être de bons candidats potentiels et pourraient être testés en milieu hospitalier comme agents désinfectants dans le but de lutter contre les infections nosocomiales. / Nosocomial infections are directly related to opportunistic germs, which are often resistant to antibiotics and persistent on surfaces, representing a high infectious risk in hospitals. In this context, the isolation and characterization of bacteriophages specifically targeting nosocomial bacteria such as resistant Staphylococcus aureus (MRSA), resistant Enterococcus (VRE), Pseudomonas aeruginosa and Acinetobacter baumanii, could provide a natural bactericidal alternative against the transmission of these infections. Phages, isolated from waste water, were selected according to their capacity of amplification, their genomic profile and lytic potential towards various bacterial clinical strains. The best ones were characterized in detail to primarily ensure their specificity, safety, stability and effectiveness, before studying their in vivo usage. Seven phages against MRSA and three against Acinetobacter baumanii were characterized. Four MRSA phages proved to be good potential candidates and could be tested in hospitals as disinfectant agents with the aim of fighting nosocomial infections.