Fibronectin is an important protein that is able to bind to other fibronectin molecules and to cell surface receptors. In doing so, the interactions fibronectin can perform is important for the processes of cell migration and tissue formation. Understanding the properties of fibronectin and fibril assembly is useful for areas such as wound healing, where fibronectin molecules are assembled to protect the tissue and to perform other tasks. Because of these reasons, it is important to understand how fibronectin is assembled and how its properties affect the fibril assembly, which in return affects the way the cell matrix operates. Previously published papers illustrate that the properties of fibronectin affect the mechanotransduction process, the cell conversion of mechanical stimulus to chemical, and this leads to various changes of the fibril assembly. However, the question that now comes to focus is what variables affect the fibril assembly? The two main variables that come into question is the substrate stiffness (ksub) (pN/nm) and the actin velocity (Vu) (nm/s). In order to test this hypothesis, several fibril assembly simulations were performed via MATLAB based upon the Weinberg-Mair-Lemmon Fibronectin Model. These simulations were performed by varying the parameters of substrate stiffness and actin velocity as well as fibril size, which affect the various measurements of the fibronectin, such as stretched length, relaxed length, etc. Through these various experiments, it was determined that the actin velocity and fibril size had the greatest impacts in affecting the fibronectin’s properties and its assembly.

L’utilisation de revêtements et de couches minces déposés sur substrats est très recherchée dans de nombreuses applications. Les objectifs de ces revêtements et dépôts sont multiples (améliorer la durabilité des structures, leur résistance à l’usure et à la fatigue, etc.). D'autre part, les matériaux à gradient sont également développés en vue de répondre à de nouvelles exigences fonctionnelles, comme de meilleures tenues en température, en usure, en corrosion. Pour toutes ces applications, la caractérisation de ces revêtements et de ces matériaux à gradients, afin d’en déterminer leurs propriétés (épaisseur, constantes élastiques, adhérence, contraintes résiduelles, …etc), est déterminante pour le contrôle santé des pièces et pour leur fonctionnement optimal au cours de leur utilisation. Pour caractériser ces structures, nous avons choisi d’exploiter la dispersion des ondes de surface sur une large gamme de fréquences (10 à 60 MHz). Afin d’exciter ces ondes, des capteurs MEMS de type IDT-SAW ont été réalisés à différentes fréquences couvrant la totalité de la gamme fréquentielle considérée. L’excitation quasi-harmonique a été privilégiée dans le but d’obtenir des mesures précisesde vitesses de phase. Nous avons montré les potentialités de cette approche en caractérisant premièrement des structures à couche mince allant jusqu’à 500 nm et deuxièmement des structures amorphes à gradient de contraintes. / The use of coatings and thin layers deposited on substrates is highly sought in many applications. The objectives of these coatings and deposits are multiple (improve the durability of structures, their wear resistance and fatigue, etc.). On the other hand, gradient materials are being developed to meet new functional requirements, such as a better resistance to temperature, wear and corrosion. For all of these applications, the characterization of these coatings and gradient materials, in order to determine their properties (thickness, elastic constants, adherence, residual stresses, etc…), is decisive for the health control of pieces and for their optimum operation during their use. To characterize these structures, wechose to exploit the dispersion of surface acoustic waves over a wide frequency range (10 to 60 MHz).To excite these waves, SAW-IDT MEMS sensors have been carried out at different frequencies covering the entire frequency range we considered. The quasi-harmonic excitation was preferred to obtain accurate measures of phase velocities. We showed the potential of this approach by characterizing, first, thin layers structures (500 nm) and second, amorphous structures with a stressesgradient.

Cette thèse est centrée sur l’analyse du rôle de l’adhésion cellule-substrat et des facteurs de détectionde Quorum sur la migration amibienne de Dictyostelium . Des outils pour automatiser les enregistrementsde vidéoomicroscopie et l’analyse d’image ont été développés afin de travailler avec de trèsgrands échantillons de cellules et de quantifier la migration cellulaire. Un dispositif microfluidique dedétachement cellulaire sous flux hydrodynamique combiné une platine motorisée a permis une étude statistique de l’adhésion mais aussi de la dynamique de détachement. L’analyse de la migration de Dictyostelium en milieu non nutritif met en évidence le rôle de la densité sur la différentiation des celluleset leur capacité de migration. Nous observons la présence d’une vitesse maximale de migrationaprès 6h de carence. Nous montrons que l’adhésion sur verre est deux fois plus faible en milieu carenc´equ’en milieu nutritif. Les exp´eriences de migration en milieu nutritif ont révélé la présence d’un facteur de détection de densité sécrété par les cellules et régulant leur migration aléatoire. Le coefficient de diffusion, la persistance du mouvement et la morphologie des cellules varient en fonction de la concentrationde ce facteur. Ce facteur ne modifie pas l’adhésion cellulaire mais uniquement la dynamique de d´etachement. Enfin, le protocole d’analyse développé nous a permis de faire une étude comparative de la migration en faisant varier d’autres paramètres tel que la surface ou la composition chimique du milieu expérimental. Ce travail se conclue en exposant le possible rôle de l’adhésion sur la migrationchez Dictyostelium en milieu nutritif. / This thesis focuses on the analysis of the role of adhesion between substrate and cell and factors of Quorum sensing on the migration of Dictyostelium amoeba. Tools to automate the recordings of videomicroscopy and image analysis have been developed to work with very large samples of cells and toquantify cell migration. A microfluidic device for cell detachment in hydrodynamic flow combined witha motorized stage has allowed a statistical study of adhesion but also the dynamics of detachment. The analysis of the migration of Dictyostelium in non nutritive medium highlights the role of density on celldifferentiation and migration capacity. We observe the presence of a maximum speed of migration after6 hours of starvation. We show that the adhesion to glass is twice as low in deprivation buffer as inthe nutrient medium. The experiences of migration in growth medium revealed the presence of a factorof detection of density secreted by the cells and regulating their random migration. The diffusion coefficient, the persistence of the movement and morphology of cells vary depending on the concentrationof this factor. This factor does not affect cell adhesion but only the dynamics of detachment. Finally, the testing protocol developed allowed us to make a comparative study of migration by varying otherparameters such as surface or the chemical composition of experimental medium. This work concludesby outlining the possible role of adhesion to the migration of Dictyostelium in nutrient medium.

The photosynthetic water oxidation reaction is catalyzed by an inorganic Mn4OxCaClyHCO3-z cluster at the heart of the oxygen evolving complex (OEC) in photosystem II. In the absence of an atomic resolution crystal structure, the precise molecular organization of the OEC remains unresolved. Accordingly, the role of the protein and inorganic cofactors of PSII (Ca2+, HCO3- and Cl-) in the mechanism of O2-evolution await clarification. In this study, rapid 18O-isotope exchange measurements were applied to monitor the substrate-water binding kinetics as a function of the intermediate S-states of the catalytic site (i.e. S3, S2 and S1) in Triton X-100 solubilized membrane preparations that are enriched in photosystem II activity and are routinely used to evaluate cofactor requirements. Consistent with the previous determinations of the 18O exchange behavior in thylakoids, the initial 18O exchange measurements of native PSII membranes at m/e = 34 (which is sensitive to the 16O18O product) show that the ‘fast’ and ‘slowly’ exchanging substrate-waters are bound to the catalytic site in the S3 state, immediately prior to O2 release. Although the slowly exchanging water is bound throughout the entire S-state cycle, the kinetics of the fast exchanging water remains too fast in the S2, S1 [and S0] states to be resolved using the current instrumentation, and left open the possibility that the second substrate-water only binds to the active site after the formation of the S3 state. Presented is the first direct evidence to show that fast exchanging water is already bound to the OEC in the S2 state. Rapid 18O-isotope exchange measurements for Ex-depleted PSII (depleted of the 17- and 23-kDa extrinsic proteins) in the S2 state reveals a resolvable fast kinetic component of 34k2 = 120 ± 14 s-1. The slowing down of the fast phase kinetics is discussed in terms of increased water permeation and the effect on the local dielectric following removal of the extrinsic subunits. In addition, the first direct evidence to show the involvement of calcium in substrate-water binding is also presented. Strontium replacement of the OEC Ca2+-site reveals a factor of ~3-4 increase in the 18O exchange of the slowly exchanging water across the S3, S2 and S1 states while the kinetics of the fast exchanging water remain unchanged. Finally, a re-investigation of the proposed role for bicarbonate as an oxidizable electron donor to photosystem II was unable to discern any 18O enrichment of the photosynthetically evolved O2 in the presence of 18O-bicarbonate. A working model for O2-evolution in terms of these results is presented.

Fasciola cause considerable monetary loss in the agriculture industry, while parasitism of humans is an emerging disease. Fasciola cathepsin L-like proteases are believed to aid parasite invasion and survival through a range of functions including feeding, immune evasion and modulation, tissue migration, egg production and excystment. As such these proteases are considered good targets for chemotherapies and vaccine development. Fasciola cathepsins are evolutionarily divided into clades that reflect function and life stage of expression. Analysis of F. gigantica genomic DNA and mRNA identified novel cathepsin L-like sequences which are incorporated into a phylogenetic analysis of the complete Fasciola cathepsin L-like protease family. Analysis of mRNA transcripts isolated in this study also points to trans-splicing occurring amongst cathepsin transcripts, the first time this has been identified in Fasciola species. S2 subsite specificity is important in determining substrate interactions with cathepsin L-like proteases. Previous work has shown that amino acid substitutions at this site can dramatically influence substrate specificity. A number of substitutions, specifically those that have been observed, or predicted to occur during the evolution of Fasciola cathepsins L-like proteases, were introduced into the S2 subsite of FhCatL5 at aa69 to determine their influence. The introduction of L69C and L69S substitutions resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants amongst fluke. The L69F variant showed an increase in the ability to cleave substrates with P2 proline, indicating F69 variants expressed by fluke are also likely to have this ability, similar to that shown with L69Y and FhCatL2. The introduction of a L69W substitution leads to increased cleavage of substrates with P2 proline, along with a decrease in cleavage of substrates with P2 phenylalanine. FgCatL1G transcripts were isolated from F. gigantica metacercariae. This contrasts with FhCatL5 and FhCatL2 which have been isolated in adult F. hepatica. These cathepsins differ at aa69, possessing tryptophan, leucine and tyrosine respectively. The processing and substrate specificities of each recombinant enzyme was analysed and compared. While FhCatL5 and FhCatL2 process in vitro in a manner similar to that reported for FhCatL1, FgCatL1G requires different processing conditions, including neutral pH. Combined with FgCatL1G possessing increased stability at acidic pH, this reflects the different environment into which FgCatL1G is expressed by immature compared to the adult flukes. The substrate specificity of FgCatL1G also differed from previously reported cathepsins, with a preference for P2 proline and low activity against substrates with P2 phenylalanine. This is the first time recombinant expression and purification of a cathepsin L-like protease specific to the immature life stages of Fasciola has been undertaken and had enzyme specificity analysed. This work has expanded knowledge of the repertoire of cathepsin proteases expressed at various life-stages of the liver fluke. Vaccination and/or drug inhibition studies may in the future be targeted towards cathepsins that are expressed in either the adult or immature stage, or perhaps both in a multi-targeted approach. The knowledge gained in this study may allow such targets to be chosen.

<p>RNase P is an essential ribonuclease responsible for removal of the 5’ leader of tRNA precursors. Bacterial RNase P consists of an RNA subunit and a small basic protein. The catalytic activity is associated with the RNA subunit, i.e. bacterial RNase P RNA is a ribozyme. The protein subunit is, however, essential for activity in vivo. RNase P RNA, as well as the holoenzyme, requires the presence of divalent metal ions for activity. The aim of this thesis was to increase our understanding of the catalytic mechanism of RNase P RNA mediated cleavage. The importance of the nucleotides close to the cleavage site and the roles of divalent metal ions in RNase P RNA-catalyzed reaction were investigated. Escherichia coli RNase P RNA (M1 RNA) was used as a model system.</p><p>It was shown that different metal ions have differential effects on cleavage site recognition. Cleavage activity was rescued by mixing metal ions that do not promote cleavage activity by themselves. This suggests that efficient and correct cleavage is the result of metal ion cooperativity in the RNase P RNA-mediated cleavage reaction. The results suggested that one of the metal ions involved in this cooperativity is positioned in the vicinity of a well-known interaction between RNase P RNA and its substrate. Based on my studies on how different metal ions bind to RNA and influence its activity we raise the interesting possibility that the activity of biocatalysts that depend on RNA for activity are up- or downregulated depending on the intracellular concentrations of the bulk biological metal ions Mg²⁺ and Ca²⁺.</p><p>The nucleotides upstream of the cleavage site in the substrate were found to influence the cleavage efficiency. This was not exclusively due to intermolecular base pairing within the substrate but also dependent on the identities of the nucleotides at position –2 and –1. The strength of the base pair at position –1/+73 was demonstrated to affect cleavage efficiency. These observations are in keeping with previous suggestion that the nucleotides close to the cleavage site are important for RNase P cleavage. We conclude that the residue at -1 is a positive determinant for cleavage by RNase P. Hence, my studies extend our understanding of the RNase P cleavage site recognition process.</p>

<p>Extensive use of the sulphonamide drugs against the bacterium <i>Neisseria meningitidis</i> has resulted in drug resistance development. Sulphonamide resistance in <i>N. meningitidis</i> is caused by alterations in the chromosomal <i>folP</i> gene, coding for DHPS (dihydropteroate synthase). One type of resistant DHPS has high sequence divergence compared to DHPS from susceptible strains. This divergent DHPS has a duplication of two amino acids, crucial for resistance, and an altered amino acid in position 68, important for both resistance and substrate binding. When introduced into a susceptible DHPS, these two alterations did not incur resistance and resulted in abnormal substrate binding properties. This indicated that the divergent DHPS was not directly developed by mutations, but rather had been acquired by horizontal transfer of <i>folP</i> from another species.</p><p>Commensal <i>Neisseria</i> species are implied as the origin of the horizontally transferred resistance. Sulphonamide-resistant commensal <i>Neisseria</i> isolates were detected in throat swabs from healthy individuals not exposed to these drugs; however, transformation of resistance from these commensals to <i>N. meningitidis</i> was restricted in the laboratory. A comparison of the genomic region surrounding <i>folP</i> revealed differences in gene organisation and in the DNA uptake sequence between <i>N. meningitidis</i> and distantly related commensals. These differences are likely to restrict transformation between distantly related <i>Neisseria</i> species.</p><p>DHPS participates in the folate biosynthesis pathway. The enzyme preceding DHPS in the pathway, HPPK (hydroxymethyl-dihydropterin pyrophosphokinase), from <i>N. meningitidis</i> was characterised and a method for studying substrate channelling from HPPK to DHPS was developed. The information gained could be exploited in the search for new antibiotics.</p><p>In conclusion, well-adapted sulphonamide-resistant strains of <i>N. meningitidis</i> and commensal <i>Neisseria</i> are established in the bacterial population and resistance can be horizontally spread by natural transformation. This may explain the abundance of sulphonamide-resistant <i>N. meningitidis</i>, although these drugs are no longer used against this bacterium.</p>

This dissertation investigates the influence of surface heterogeneities on colloid deposition. First, deposition of colloidal particles on a nanofiltration membrane during cross flow membrane filtration was studied under different operating pressures and solution chemistries. An atomic force microscope (AFM) was then used to observe the deposit morphology formed on the membrane. At the initial stages of fouling, more particles preferentially accumulate near the peaks than in the valleys of the rough nanofiltration membrane surface. This study demonstrates that it is difficult to isolate, correlate and assess the effects that physical (roughness) heterogeneity and chemical heterogeneity has on colloid deposition based on experiments involving surfaces where the physical and chemical heterogeneities are uncorrelated or randomly distributed. In the second phase of the study, the deposition of model colloidal particles onto patterned charge-heterogeneous surfaces was studied both experimentally and theoretically. Controlled charge heterogeneity was created experimentally employing self assembled monolayers of alkanethiols patterned onto gold substrates using a soft lithographic technique. Model colloidal particles and fluorescent nanoparticles were sequentially deposited onto the patterned substrate under no flow (quiescent) conditions, and the deposited structures and the micro-patterns were imaged in situ using a combination of phase contrast and fluorescence microscopy. This study indicates that particles tend to preferentially deposit at the edges of the chemically favourable stripes. The theoretical investigation involved the formulation of a mathematical model based on Random Sequential Adsorption (RSA). This study showed that a simple binary probability distribution assumed in the model is able to predict the experimental deposit morphology adequately, particularly the periodicity of the underlying patterns on the substrate. Furthermore, the effect of charge heterogeneity on the electrostatic double layer interaction between a particle and a charge heterogeneous planar surface was studied numerically employing a 3D finite element model. In this system, significant lateral forces at close separation distances were observed, and found to be appreciably higher when the particle is near the edge of a heterogeneous region of the substrate. From the above studies, it can be concluded that by altering/controlling the chemical heterogeneity of the substrate, it is possible to achieve significant control on the resulting deposit morphology.

Mobile communication has become an essential part of our daily life. We love the flexibility of wireless cell phones and even accept their lower quality of service when compared to wired links. Similarly, we are looking forward to the day that we can continue watching our favorite TV programs while travelling anywhere and everywhere. Mobility, flexibility, and portability are the themes of the next generation communication. Motivated and fascinated by such technology breakthroughs, this effort is geared towards enhancing the quality of wireless services and bringing mobile satellite reception one step closer to the market. Meanwhile, phased array antennas are vital components for RADAR applications where the antenna is required to have certain scan capabilities. One of the main concerns in that perspective is how to avoid the potential of scan blindness in the required scan range. Targeting to achieve wide-band wide-scan angle phased arrays free from any scan blindness our efforts is also geared. Conventionally, the key to lower the profile of the antenna is to use planar structures. In that perspective microstrip patch antennas have drawn the attention of antenna engineers since the 1970s due to their attractive features of being low profile, compact size, light weight, and amenable to low-cost PCB fabrication processes. However, patch elements are basically resonating at a single frequency, typically have <2% bandwidth, which is a major deficit that impedes their usage in relatively wide-band applications. There are various approaches to enhance the patch antennas bandwidth including suspended substrates, multi-stack patches, and metalized cavities backing these patches. Metalized cavity-backed patch structures have been demonstrated to give the best performance, however, they are very expensive to manufacture. In this dissertation, we develop an alternative low-cost bandwidth enhancement topology. The proposed topology is based on substrate-integrated waveguides. The great potential of the proposed structure lies in being amenable to the conventional PCB fabrication. Moreover, substrate-integrated cavity-backed structures facilitate the design of sophisticated arrays that are very expensive to develop using the conventional metalized cavity-backed topology, which includes the common broadside arrays used in fixed-beam applications and the scanned phased arrays used in RADAR applications.

Section 303(d) of the Clean Water Act (CWA) requires states to assess and list all streams that do not meet water quality criteria for their designated use classes. In Tennessee, the Tennessee Department of Environment and Conservation (TDEC) uses macroinvertebrate surveys to assess the condition of streams designated for “fish and aquatic life” and the progress of targeted waterbodies toward meeting established standards for sediment. As of yet, no substrate metric has been established to monitor water quality or to document progress toward water quality improvement with respect to fish and aquatic life in Tennessee. A substrate metric that could be efficiently measured and would represent the needs of aquatic species would be valuable for monitoring streams with known sediment impairment to detect water quality improvement. The objectives of this study were to (1) investigate the relationships between riffle substrates and benthic macroinvertebrate data, provided by TDEC; (2) assess the potential use of substrate metrics as a monitoring tool for benthic habitat status; and (3) examine variation in riffle substrates over time in the Ridge and Valley Ecoregion of Tennessee. Bed and interstitial sediment were characterized at sites corresponding with TDEC macroinvertebrate sampling stations. Bed sediment characteristics were significantly correlated with benthic macroinvertebrate data; however, interstitial fines yielded no significant correlations with benthic macroinvertebrate data. Substrate metrics did not differ significantly between varying levels of impairment; however, they did differ significantly when all impaired sites were combined into a single impairment group. The lack of significant differences between varying classes of reach impairment suggests that substrate metrics may not be able to distinguish impairment at the level necessary for monitoring impairment. However, substrate metrics may be of potential use in monitoring sites where impairment is less ambiguous. To investigate change in riffle substrate over time, three sites were monitored over the course of a year. Preliminary observations showed little change in riffle substrate during the study period, suggesting that seasonal restrictions on substrate surveys are unneccessary.