Emerging rise in microbial drug resistance and the slow-growing characteristic of some intracellular pathogens such as MAP (Mycobacterium avium subspecies paratuberculosis) strongly urges the need for an effective approach for unconventional drug susceptibility testing. We designed a molecular-based PCR method for the evaluation of rifabutin (RFB) and rifampicin (RIF) resistance based on probable determinant regions within the rpoB gene of MAP, including the 81 bp variable site located between nucleotides 1363 and 1443. The minimum inhibitory concentration (MIC) for RIF was also determined against 10 MAP isolates in attempt to seek correlation with rpoB sequences. We determined that MAP strain 18 had an MIC ≥ 30 ug/ml and ≥ 5 ug/ml for RIF and RFB respectively, and a significant rpoB mutation C1367T, compared to an MIC of ≤ 1.0 ug/ml for both drugs in the wild type MAP. The 30-fold increase in the MIC was a direct result of the rpoB mutation C1367T, which caused an amino acid change Thr456 to Ile456 in the drug's binding site; the beta subunit of RNA polymerase. Our in vitro induced mutation in MAP strain UCF5 resulted in the generation of a new resistant strain (UCF5-RIF16r) that possessed T1442C rpoB mutation and an MIC ≥ 30 ug/ml and ≥ 10 ug/ml for RIF and RFB respectively. The T1442C mutation resulted in a Leu481 to Pro481 amino acid change, consequently altering the beta subunit sequence. Sequencing the entire 3.5 kb rpoB in strains 18 and UCF5-RIF16r revealed no additional expressed nucleotide mutation. Of the 10 MAP strains analyzed, an additional one strain (UCF4) exhibited a slight increase in the MIC against RIF and RFB compared to the wild-type. Nucleotide sequencing of the rpoB gene revealed an A2284C mutation in strain UCF4 that occurred further downstream of the expected probable rpoB region and resulted in an amino acid alteration Asn762 to His762. The location of this mutation outside the binding site and its correlation with the minor increase in MIC suggests a possible secondary interaction between the drug and the beta subunit. We have provided three dimensional images through the utilization of PyMol Molecular-based Graphics to display a clear comparison of the mutations observed in the beta subunit for MAP strains 18, UCF5-RIF16r, and UCF4. We propose that these alterations may have caused a less stable interaction between RIF and the beta subunit, resulting in the observed increased in MIC. Furthermore, the change in amino acid sequence did not affect the viability for our RIF resistant strains. The data clearly illustrates that clinical and in vitro-induced MAP mutants with rpoB mutations result in resistance to RIF and RFB. Consequently, unconventional drug susceptibility testing such as our molecular approach will be beneficial for evaluation of antibiotic effectiveness. This molecular approach may also serve as a model for other drugs used for treatment of MAP infections.

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The following study was conducted to investigate the antibacterial and feed additive potential of medicinal plants. Ethanol extracts of different medicinal plants including Curcuma longa (Turmeric), Zingiber officinale (Ginger), Piper nigrum (Black Pepper), Cinnamomum cassia (Cinnamon), Thymus vulgaris (Thyme), Laurus nobilis (Bay leaf), and Syzgium aromaticum (Clove) were tested using the disc diffusion method for their antimicrobial activity against the common poultry pathogens E. coli, S. typhimurium, E. faecium, and E. faecalis. Cinnamon extract (CE), at 130 mg/disk, exhibited antibacterial activity against E. coli, S. typhimurium, and E. faecalis. Thyme extract (TE), at 30 mg/disk, exhibited antibacterial activity against E. coli, E. faecium, and E. faecalis while the remaining medicinal plants extracts showed no activity. The minimum inhibitory concentration (MIC) of the cinnamon and thyme ranged from 31.25 to 250 mg/ml by the dilution method. From this in vitro antibacterial study, cinnamon and thyme were selected for a 21-d feeding trial in broilers to study their influence on feed consumption, body weight gain, and feed conversion. There were 6 dietary treatments groups: 1) negative control (NC) containing no plant extracts or antibiotic, 2) positive control (PC) containing BMD (bacitracin) at 50g/ton of feed, 3) Diet 1 plus low level of cinnamon extract (LCE) at 290 gm/100 kg of feed, 4) Diet 1 plus high level of cinnamon extract (HCE) at 580 gm/ 100 kg of feed, 5) Diet 1 plus low level of thyme extract (LTE) at 290 gm/100kg of feed, and 6) Diet 6 plus high level of thyme extract (HTE) at 580 gm/100 kg of feed. No significant changes in body weight gain were observed with the cinnamon extracts compared to the NC or PC at 7, 14, or 21 d. The HTE reduced body weight gain compare to the NC and PC at 7, 14, and 21 d (P < 0.02). No difference in feed efficiency was observed with any of the treatments except LCE which reduced feed efficiency compared to other treatments. No difference in feed consumption was found among any of the treatments. These results suggest that cinnamon and thyme have antibacterial activity in vitro, and thyme has an activity that reduces body weight. Since cinnamon caused no significant change in body weight gain compared to positive or negative controls, it warrants further study as a substitute for antibiotics in the diet. / Master of Science

No / Nanotechnology can offer new opportunities in the fight against infection. The aim of current work was to investigate an eco-friendly method for synthesis of silver nanoparticles (AgNP) which have the ability to load lipophilic compounds onto their surface. Pharmaceutically acceptable hydrophilic lipid (Gelucire® 50/13) has been used as a reducing agent for in situ reduction of silver nitrate so as to obtain silver nanoparticles. Propolis is used as model molecule for loading onto surface of AgNP owing to its well reported broad range of pharmacological activities including anti-inflammatory, antioxidant and antimicrobial activity. Propolis loaded silver nanoparticles (PLSN) were prepared and characterized for silver content, surface plasmon resonance, particle size, XRD, FTIR, TEM, antibacterial activity and burn wound healing in wistar rats. Propolis constituents were successfully loaded onto surface of AgNP using the proposed conceptual method. The formation of PLSN having size 24.3 ± 2.5 nm was confirmed using surface plasmon resonance, FTIR, XRD and TEM. The combination of propolis with AgNP significantly reduced minimum inhibitory concentration of AgNP alone when tested against Staphylococcus aureus. PLSN gel showed comparable burn wound healing in wistar rats when tested against marketed silver sulfadiazine gel. The use of Gelucire® as solubilizing agent for lipophillic drugs was effectively utilized for loading lipophillic constituents of propolis onto the AgNP. This potentially provides an effective method for the green synthesis of AgNP which can be used to load lipophillic molecules onto their surface whenever such combination is required.

Les anticorps sont des molécules de reconnaissance du non-soi permettant de distinguer spécifiquement des marqueurs antigéniques appelés épitopes. Deux types d’anticorps ont été découverts jusqu’à présents : les anticorps « classiques » et les anticorps de camélidés, également appelés nanobody. Cette thèse porte sur des études structurales de fragments d’anticorps d’intérêt thérapeutique et biotechnologique. Au cours d’un premier volet, j’ai résolu la structure par cristallographie aux rayons X du fragment d’un anticorps d’intérêt thérapeutique, MIC12, à une résolution de 1,5 Å. Dans le but de résoudre la structure des complexes de MIC12 avec des protéines homologues du CMH de classe I, j’ai obtenu des cristaux diffractant à une résolution allant jusqu’à 7 Å. En parallèle, des analyses par diffusion de rayons X aux petits angles (SAXS) combinées à des prédictions d’interaction par docking ont été conduites afin d’obtenir une première description de la région globale d’interaction de MIC12 sur l’une de ses cibles. Concernant le second volet, 4 nanobody ont été obtenus contre la protéine périplasmique PorM (pPorM) du système de sécrétion de type 9 de Porphyromonas gingivalis. J’ai résolu la structure par cristallographie du nanobody nb02, à une résolution de 1,5 Å. Leur utilisation comme chaperonne de cristallisation m’a permis de résoudre la structure de la partie N-terminale de pPorM. J’ai également mené une étude par SAXS de la protéine pPorM entière. L’ensemble des résultats que j’ai obtenu par cristallographie et par SAXS, combinés aux résolutions des structures des autres domaines de pPorM, ont permis de proposer un modèle structural de la protéine pPorM entière. / Antibodies are non-self recognition molecules wich help to specifically distinguish antigenic markers called epitopes. Two types of antibody were discovered so far: “classic” antibodies and camelid antibodies, also called nanobodies. This PhD deals with structural studies of antibodies fragments of therapeutic and biotechnological interest. During the first part, I solved the structure of the therapeutic antibody targeting homologous proteins of the MHC class I, MIC12, using X-ray crystallography at a resolution of 1.5 Å. In order to solve the structure of the complexes of MIC12 with its MIC antigens, I obtained crystals of the complexes diffracting at a resolution of up to 7 Å. In parallel, analyses by Small Angles X-ray Scattering (SAXS) combined with in silico docking predictions were led to obtain a first description of the global binding region of MIC12 on one of its targets. Concerning the second part, 4 nanobodies were obtained against the periplasmic protein PorM (pPorM) of the secretion system type 9 from Porphyromonas gingivalis. I solve the structure of the nanobody nb02, at a resolution of 1.5 Å. Their use as chaperones of crystallization helped me to solve the structure of the N-terminal part of pPorM. I also conducted a study by SAXS of the whole pPorM protein. All these results, obtained by crystallography and SAXS studies, combined with the solving of the structures of the other domains of pPorM, made it possible to propose a structural model of the entire pPorM protein.

L’accès aux données de base, que sont les sections efficaces, constitue le principal goulot d’étranglement aux performances dans la résolution des équations du transport neutronique par méthode Monte Carlo (MC). Ces sections efficaces caractérisent les probabilités de collisions des neutrons avec les nucléides qui composent le matériau traversé. Elles sont propres à chaque nucléide et dépendent de l’énergie du neutron incident et de la température du matériau. Les codes de référence en MC chargent ces données en mémoire à l’ensemble des températures intervenant dans le système et utilisent un algorithme de recherche binaire dans les tables stockant les sections. Sur les architectures many-coeurs (typiquement Intel MIC), ces méthodes sont dramatiquement inefficaces du fait des accès aléatoires à la mémoire qui ne permettent pas de profiter des différents niveaux de cache mémoire et du manque de vectorisation de ces algorithmes.Tout le travail de la thèse a consisté, dans une première partie, à trouver des alternatives à cet algorithme de base en proposant le meilleur compromis performances/occupation mémoire qui tire parti des spécificités du MIC (multithreading et vectorisation). Dans un deuxième temps, nous sommes partis sur une approche radicalement opposée, approche dans laquelle les données ne sont pas stockées en mémoire, mais calculées à la volée. Toute une série d’optimisations de l’algorithme, des structures de données, vectorisation, déroulement de boucles et influence de la précision de représentation des données, ont permis d’obtenir des gains considérables par rapport à l’implémentation initiale.En fin de compte, une comparaison a été effectué entre les deux approches (données en mémoire et données calculées à la volée) pour finalement proposer le meilleur compromis en termes de performance/occupation mémoire. Au-delà de l'application ciblée (le transport MC), le travail réalisé est également une étude qui peut se généraliser sur la façon de transformer un problème initialement limité par la latence mémoire (« memory latency bound ») en un problème qui sature le processeur (« CPU-bound ») et permet de tirer parti des architectures many-coeurs. / Monte Carlo (MC) neutron transport simulations are widely used in the nuclear community to perform reference calculations with minimal approximations. The conventional MC method has a slow convergence according to the law of large numbers, which makes simulations computationally expensive. Cross section computation has been identified as the major performance bottleneck for MC neutron code. Typically, cross section data are precalculated and stored into memory before simulations for each nuclide, thus during the simulation, only table lookups are required to retrieve data from memory and the compute cost is trivial. We implemented and optimized a large collection of lookup algorithms in order to accelerate this data retrieving process. Results show that significant speedup can be achieved over the conventional binary search on both CPU and MIC in unit tests other than real case simulations. Using vectorization instructions has been proved effective on many-core architecture due to its 512-bit vector units; on CPU this improvement is limited by a smaller register size. Further optimization like memory reduction turns out to be very important since it largely improves computing performance. As can be imagined, all proposals of energy lookup are totally memory-bound where computing units does little things but only waiting for data. In another word, computing capability of modern architectures are largely wasted. Another major issue of energy lookup is that the memory requirement is huge: cross section data in one temperature for up to 400 nuclides involved in a real case simulation requires nearly 1 GB memory space, which makes simulations with several thousand temperatures infeasible to carry out with current computer systems.In order to solve the problem relevant to energy lookup, we begin to investigate another on-the-fly cross section proposal called reconstruction. The basic idea behind the reconstruction, is to do the Doppler broadening (performing a convolution integral) computation of cross sections on-the-fly, each time a cross section is needed, with a formulation close to standard neutron cross section libraries, and based on the same amount of data. The reconstruction converts the problem from memory-bound to compute-bound: only several variables for each resonance are required instead of the conventional pointwise table covering the entire resolved resonance region. Though memory space is largely reduced, this method is really time-consuming. After a series of optimizations, results show that the reconstruction kernel benefits well from vectorization and can achieve 1806 GFLOPS (single precision) on a Knights Landing 7250, which represents 67% of its effective peak performance. Even if optimization efforts on reconstruction significantly improve the FLOP usage, this on-the-fly calculation is still slower than the conventional lookup method. Under this situation, we begin to port the code on GPGPU to exploit potential higher performance as well as higher FLOP usage. On the other hand, another evaluation has been planned to compare lookup and reconstruction in terms of power consumption: with the help of hardware and software energy measurement support, we expect to find a compromising solution between performance and energy consumption in order to face the "power wall" challenge along with hardware evolution.

Hudens normalflora består av lågpatogena bakterier där stafylokockerna är de vanligaste förekommande bakterierna. Studier visar att antimikrobiella medel kan förändra hudbakteriepopulationer och att dessa förändringar kan leda till kritiska konsekvenser för hudens försvar. Propylenglykol är en substans som klassas som antimikrobiell och bakteriedödande. Propylenglykol har ett brett användningsområde och används ofta som hjälpmedel i en mängd olika läkemedel. Den finns bland annat i kosmetika såsom hudprodukter där den fungerar som fuktbindande och som konserveringsmedel. Vanliga koncentrationer av propylenglykol som fuktighetsbevarande ämne i topikaler är cirka 15 % och i kosmetika finns propylenglykol i koncentrationerna &lt;0,1 % - &gt;50 %. Det saknas i nuläget forskning om propylenglykols effekt på hudens bakterieflora. Syftet med studien var att med olika koncentrationer av propylenglykol bestämma MIC (minsta hämmande koncentration) och MBC (minsta baktericida koncentration) på vanliga stafylokocker (S. epidermidis, S. aureus, S. hominis och S. capitis) som ingår i hudens normalflora. För att bestämma MIC och MBC användes buljongspädningsmetoden där propylenglykol späddes ut i olika koncentrationer med buljong i en mikrotiterplatta. Sedan tillsattes valda testbakterier och OD600 mättes i 24 timmar. Resultatet visade att MIC och MBC för propylenglykol var 12,5 % respektive 25 % på vanliga stafylokocker som finns på huden. Vid koncentrationer av propylenglykol på 12,5 % hämmades synlig bakterietillväxt av S. epidermidis, S. aureus, S. hominis och S. capitis och vid 25 % uppstod en baktericid effekt på bakterierna. Mer forskning behövs dock för att få reda på hur hudens bakterier påverkas av propylenglykol och konsekvenserna av det. / The normal flora of the human skin is consisting of low pathogen bacteria, where the staphylococci are the most common bacteria. Studies show that antimicrobial substances can alter populations of skin bacteria and that these alterations can lead to critical consequences for the resistance of the skin. Propylene glycol is a substance that is classified as antimicrobial and bactericidal and the substance has a wide area of use and is frequently used as a supportive substance in various pharmaceuticals. Propylene glycol can be found in cosmetics and skincare products where it functions as moisture-binding and preservative. Normal concentrations of propylene glycol as moisture-binding substance in topicals is approximately 15 % and in cosmetics the concentration of propylene glycol is &lt;0,1 % - &gt;50 %. As of today, there are few scientific studies regarding the effects of propylene glycol to the bacterial flora of the human skin. The purpose of this study was to determine MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) for normal staphylococci (S. epidermidis, S. aureus, S. hominis och S. capitis) included in the normal flora of the skin using various concentrations of propylene glycol. To be able to determine MIC and MBC the broth dilution method was used, where propylene glycol was diluted in various concentrations with broth in a microtiter plate. Hereafter, selected test bacteria were added and OD600 was measured during 24 hours. The results implicated that MIC and MBC for propylene glycol were 12,5 % and 25 % for common staphylococci located on the skin. At concentrations of propylene glycol of 12,5 %, visible bacterial growth of S. epidermidis, S. aureus, S. hominis and S. capitis was inhibited and at 25 % a bactericidal effect occurred on the bacteria. It shall be noted that further research is needed to find out how the skin's bacteria are affected by propylene glycol and its consequences.

This thesis focuses on solution antimicrobial effectiveness for copolyoxetanes with quaternary ammonium and PEG-like side chains. Ring opening copolymerization of 3-((4-bromobutoxy)methyl)-3-methyloxetane (BBOx) and 3-((2-(2-methoxyethoxy) ethoxy) methyl)-3-methyloxetane (ME2Ox) yielded random copolymers with 14-100 (m) mole% BBOx designated P[(BBOx-m)(ME2Ox)]. Reaction of P[(BBOx-m)(ME2Ox)] with dodecyl dimethylamine gave the corresponding quaternary P[(C12-m)(ME2Ox)] polycation salts, designated C12-m. Mole ratios and molecular weights were obtained from 1H-NMR and end group analysis. Differential scanning calorimetry (DSC) studies showed Tg’s between 69 and -34 °C. Minimum inhibitory concentrations (MIC) against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa showed MIC decreasing with increasing C12 mole% reaching a minimum between C12-43 and C12-60. C12-43 had the lowest MIC for all strains. At 5× MIC (challenge:108 cfu/ml), C12 43 kills ≥ 99% of the tested strains within 1 hr. C12-m copolyoxetane cytotoxicity toward human red blood cells, HFF (Human Foreskin Fibroblast) and HDF (Human Dermal Fibroblast) was low, indicating good prospects for biocompatibility. Cx-m copolyoxetane antimicrobial efficacy, hemolytic activity and cytotoxicity were further explored by changing quaternary alkyl chain length. Copolyoxetanes are represented as Cx-50, where 50 is the mole percent quaternary repeat units and ‘x’ is quaternary alkyl chain length (2 to 16 carbons). Reaction of P[(BBOx-m)(ME2Ox)] with a series of tertiary amines yielded the desired quaternary ammonium segment. DSC studies showed Tg’s between -40 °C and -60 °C and melting endotherms for C14-50 and C16-50. A systematic dependence of alkyl chain length on MIC was found with C8-50 being the most effective antimicrobial. Kill kinetics for C8-50 (5× MIC, challenge: 108 cfu/ml) effected >99% kill in 1 hour for S. aureus (7 log reduction). C8-50 efficacy on biomass and cell viability of P. aeruginosa biofilms was investigated. Crystal violet (CV) staining assays demonstrate that C8-50 had no effect on adhesion of already established P. aeruginosa biofilms, but reduced biofilm formation by killing cells prior to attachment. For anti-adhesion assays, noticeable reduction in biofilm mass occurred at concentrations greater than 2× MIC. Viability studies show a substantial log reduction of 2.1 at MIC. The low cytotoxicity of Cx-m copolyoxetanes coupled with low MICs and favorable biofilm results indicate good prospects for therapeutic applications.