This PhD thesis focuses on improving the limit of detection (LOD) of Raman biosensors by using surface enhanced Raman scattering (SERS) and/or hollow core photonic crystal fibers (HC-PCF), in conjunction with statistical methods. Raman spectroscopy is a multivariate phenomenon that requires statistical analysis to identify the relationship between recorded spectra and the property of interest. The objective of this research is to improve the performance of Raman biosensors using SERS techniques and/or HC-PCF, by applying partial least squares (PLS) regression and principal component analysis (PCA). I began my research using Raman spectroscopy, PLS analysis and two different validation methods to monitor heparin, an important blood anti-coagulant, in serum at clinical levels. I achieved lower LOD of heparin in serum using the Test Set Validation (TSV) method. The PLS analysis allowed me to distinguish between weak Raman signals of heparin in serum and background noise. I then focused on using SERS to further improve the LOD of analytes, and accomplished simultaneous detection of GLU-GABA in serum at clinical levels using the SERS and PLS models. This work demonstrated the applicability of using SERS in conjunction with PLS to measure properties of samples in blood serum. I also used SERS with HC-PCF configuration to detect leukemia cells, one of the most recurrent types of pediatric cancers. This was achieved by applying PLS regression and PCA techniques. Improving LOD was the next objective, and I was able to achieve this by improving the PLS model to decrease errors and remove outliers or unnecessary variables. The results of the final optimized models were evaluated by comparing them with the results of previous models of Heparin and Leukemia cell detection in previous sections. Finally, as a clinical application of Raman biosensors, I applied the enhanced Raman technique to detect polycystic ovary syndrome (PCOS) disease, and to determine the role of chemerin in this disease. I used SERS in conjunction with PCA to differentiate between PCOS and non-PCOS patients. I also confirmed the role of chemerin in PCOS disease, measured the level of chemerin, a chemoattractant protein, in PCOS and non-PCOS patients using PLS, and further improved LOD with the PLS regression model, as proposed in previous section.

In recent decades the interest of wheat with non-standard discoloration of caryopsis has increased, because of its positive impacts on health. The pigments anthocyanins, which are included in the protective layer of grain, have antioxidant effects. A group of genotypes from Agrotest fyto Ltd. Kroměříž was chosen which consisted of eighteen samples of wheat with non-standard discoloration of caryopsis. Fourteen samples were blue (aleuron), tree yellow (endosperm) and one sample had a red (pericarp). By these samples PCR-based analyses were performed to detect the Waxy gene zero allele, the Glu-A3 glutenin locus and the hardness of grain by puroindoline genes. These parameters are significant indicators of wheat quality. By all genotypes the presence of allele Pina-D1a/b was detected and by a few the allele Pinb-D1a/c/d. These detected alleles, which are included in the analyzed wheat, should be sorted to the hardness wheats because they are suitable for baking purposes. The identified material (V1 104 15), a so-called waxy genotype, was positive in the presence of all three zero alleles in the Waxy gene. Similar lines were used in Asia for the preparation of pastas. At the locus Glu-A3, the Glu-A3d allele was detected in all tested genotypes. Along with this, Glu-A3f was founded in some genotypes. These alleles have a positive effect on dough quality, especially on ductility. The results will be used for further breeding work.

This research was conducted to discover how the U.S. building construction and forest products sectors could benefit from the development of tall, cross-laminated (CLT) and mass timber buildings. Barriers that may restrict such development were also investigated. The primary benefits were discovered to be eco-performance and job creation. Code restrictions and material performance misconceptions were found to be the largest obstacles. Case studies of Treet, Tamedia, and the WIDC were conducted to demonstrate the benefits of tall wood buildings and the various paths around potential barriers. Opportunities for tall wood buildings in the U.S. are also discussed. This research discovered that a tall wood movement is gathering momentum in the U.S. To fully realize this potential, accurate information regarding the use of wood and the performance capacities of mass timber systems needs to be disseminated. Co-operation between academia and industry will also be necessary.

Les microtubules (MT) sont des structures dynamiques contrôlant divers aspects essentiels de l'architecture<br />cellulaire. En particulier, les bouts plus des MT (bouts +) sont capables d'accumuler des protéines spécifiques jouant un rôle dans le contrôle de la dynamique microtubulaire et dans l'interaction des MT avec le cortex cellulaire. Ces interactions sont notamment décisives pour le positionnement correct du fuseau mitotique. La description des mécanismes permettant l'accumulation de protéines aux bouts + est donc importante pour la compréhension des fonctions microtubulaires.<br />La tyrosine C-terminale de la tubuline α est cruciale pour l'interaction de protéines à domaine CAP-Gly avec les bouts +. En effet, CLIP-170 et son homologue de S.cerevisiae Bik1p se lient moins bien aux bouts + des MT dépourvus de tyrosine C-terminale (MT Glu).<br />Dans ce travail de thèse, nous avons étudié les perturbations associées au déficit de liaison de Bik1p aux bouts + des MT Glu dans S. cerevisiae.<br />Les modèles actuels proposent que Bik1p est amenée aux bouts + par son association avec la kinésine Kip2p. La dynéine (Dyn1p) est alors recrutée par Bik1p aux bouts + pour être ciblée vers le cortex. Nous montrons<br />que, dans des levures n'exprimant que de la tubuline détyrosinée (souche tub1-Glu), Kip2p et Dyn1p sont<br />correctement associées aux bouts +, malgré le déficit de liaison de Bik1p. Nous proposons que, dans les cellules<br />sauvages, le complexe Kip2p/Bik1p transporte Dyn1p le long des MT vers les bouts +. Kip2, Bik1p et Dyn1p<br />s'associent alors aux bouts + de façon indépendante.<br />De plus, nous montrons que des formes constitutionnellement actives de la petite protéine G Rho1p favorisent l'association de Bik1p avec les bouts +. Ces données seront importantes pour comprendre le rôle des Rho GTPases dans la régulation des MT, notamment dans la migration cellulaire.<br />L'ensemble de ce travail suggère de nouveaux modèles pour la formation et la fonction des complexes protéiques associés aux bouts +.<br />Finalement, nous avons recherché de manière systématique les mutations qui, associées avec la mutation tub1-Glu, sont létales chez la levure (létalité synthétique). Ce crible a identifié des composants participant à la formation de la membrane et de la paroi cellulaire. Ces gènes pourraient être impliqués, au niveau cortical, dans la mise en place des interactions des microtubules avec le cortex, et montrent l'importance de la tyrosine C-terminale de la tubuline α dans cette fonction.

The primary purpose of this study was to investigate the combination of experimental and computational methods in the search for reproducible colloidal surface-enhanced Raman scattering of pharmaceutical compounds. In the search for optimal experimental conditions for colloidal surface-enhance Raman scattering, the amphipathic β-blocker propranolol was used as the target molecule. Fractional factorial designs of experiments were performed and a multiobjective evolutionary algorithm was used to find acceptable solutions, from the results, that were Pareto ranked. The multiobjective evolutionary algorithm suggested solutions outside of the fractional factorial design and the experiments were then performed in the laboratory. The results observed from the suggested solutions agreed with the solutions that were found on the Pareto front. One of the experimental conditions observed on the Pareto front was then used to determine the practical limit of detection of propranolol. The experimental conditions that were chosen for the limit of detection took into account reproducibility and enhancement, the two most important parameters for analytical detection using surface-enhanced Raman scattering. The principal conclusion to this study was that the combination of computational and experimental methods can reduce the need for experiments by > 96% and then selecting solutions from the Pareto front improved limit of detection by a factor of 24.5 when it was compared to the previously reported limit of detection for propranolol. Using the same experimental conditions that were used for the limit of detection, these experiments were extended to plasma spiked with propranolol in order to test detection of this pharmaceutical in biofluids. Concentrations of propranolol were prepared using plasma as the solvent and measured for detection using colloidal surface-enhanced Raman scattering. Detection was determined as <130 ng/mL, within physiological concentrations, previously achieved using separation techniques. The second part of this thesis also involved a combination of experimental and computational methods. Raman optical activity was utilized to investigate secondary structure of amino acids and diamino acid peptides in combination with density functional theory calculations. Amino acids are important biological molecules that have vital functions in the biological system. They have been recognized as neurotransmitters and implicated in neurodegenerative diseases. Raman and Raman optical activity experimental results were compared to determine site-specific acetylation, marker bands for constitutional isomers and identification of functional groups that interact with the solvent. The experimental spectra were then compared to those from the density functional theory calculations. The results indicated that; constitutional isomers cannot be distinguished from the Raman spectra but can be distinguished from the Raman optical activity spectra, site-specific acetylation can be identified from the Raman spectra, however, Raman optical activity provides more structural information in relation to acetylation. When the results were compared to the density functional theory calculations for the diamino acid peptides the results agreed reasonably well, however, agreement was not as good for the monoamino acids because diamino acid peptides support fewer conformations due to the peptide bond whereas monoamino acids can adopt a far greater number of conformations. Combined computational and experimental techniques have developed the ability to detect and characterize biomedical compounds, a significant move in the advancement of Raman spectroscopies.

Joints are critical parts of timber structures, transmitting static and dynamic forces between structural members. The ultimate behavior of e.g. a building depends strongly on the structural configuration and the capacity of its joints. The complete collapse of a building or other less extensive accidents that may occur usually start as a local failure inside or in the vicinity of a joint. Such serious failures have recently occurred in the Nordic countries. Especially the collapses of two large glued-laminated timber (glulam) structures clearly indicate the need of an improved joint design. The trend toward larger and more complex structures even further increases the importance of a safer design of the joints. One aim of this partly experimentally and partly numerically based work has been to investigate if the short term capacity of steel-timber dowel joints loaded parallel to the grain is affected by an initial drying exposure. The experimental results showed that the load-bearing capacity of the joints is indeed reduced by such moisture changes. Moisture induced stresses was mentioned to be the explanation. The key point is that the climates chosen in the present work (20°C / 65% RH and 20°C / 20% RH) are equivalent to service class 1 according to EC5 (Eurocode 5 2004). Thus, EC5 predicts no decrease in load-bearing capacity, in relation to the standard climate used during testing. A decrease in load-bearing capacity in the range of 5-20%, which was found in the present work, is of course not negligible and, therefore, there could be a need to introduce the effect of drying in design codes. Because similar results were also observed for a double-tapered glulam beam, further work should consider timber structures in general. Two numerical methods in order to predict the capacity of multiple steel-timber dowel joints loaded parallel to the grain were tested in the thesis. For the first method, where fracture mechanics (LEFM) concepts were implemented, a good correlation with the experimental results was seen. Also for the second method, where the capacity for a single dowel-type joint as given in EC5 was used as a failure criterion, a good correlation to traditional EC5 calculations of multiple dowel-type joints was seen. One advantage of using numerical methods in design is that the capacity of the joint can be calculated also for cases when the dowels are placed in more complex patterns. From both a structural and an architectural point of view this can be very important. In addition, such numerical methods are effective tools for the structural engineer when considering complicated loading situations in joints, i.e. eccentric loading giving moments in the joint.

One-third of the world’s population is latently infected with Mycobacterium tuberculosis, which causes over 2 million deaths every year. The current live attenuated vaccine, Bacille Calmette-Guerin (BCG), protects against miliary tuberculosis in children, but fails to consistently protect against pulmonary tuberculosis in adults. The global resurgence of tuberculosis, together with the HIV pandemic and emerging multi-drug resistance, has heightened the need for an effective vaccine. Completion of the M. tuberculosis genome sequence paved way for identification of many new candidate antigens for protective vaccine against tuberculosis. This includes the discovery of two multigene families of proteins PE and PPE which constitute 10% of the coding capacity of the M. tuberculosis genome. Members of the PE and PPE protein families are characterized by highly conserved N-terminal domains and the C-terminus, however, exhibit considerable variation in the number of residues as well as in the sequence. Till date, little is known about the functional role of the proteins of PPE or PE family in the biology of M.tuberculosis. Some of the PE_PGRS proteins have been found to be associated with the cell wall and influence interactions with other cells. PE and PPE family of proteins are of potential interest from the point of view of immune response, since they show antigenic variation which may play a role in immune evasion. Very little is known about the immunogenecity of these two classes of proteins and only few proteins have been shown to be potent B or T cell antigens, like Rv3873, Mtb39 and Rv0915c. Two proteins from PE_PGRS subfamily, Rv1759c and Rv3367 are expressed during infection and show antibody response in humans and rabbits, respectively. Rv1196 and Rv0915c from PPE family have been shown to be good T cell antigens. Another study has shown that the PE domain of PE_PGRS protein Rv1818c upon immunization into mice induces good cell mediated immune response in mice, whereas the PGRS domain is responsible for good humoral response. In humans there is increasing evidence to suggest that CD8+ T cells are elicited in response to infection with mycobacteria. CD8+ CTL may play an important role through several mechanisms. They produce potent anti-bacterial cytokines such as IFN-γ and TNF-α in response to antigenic stimulation and IFN-γ is critical for immunity to TB. Thus, identification of antigens and peptides that induce T cell responses could be useful for designing new vaccines to protect against TB. Relatively few epitopes in mycobacterial antigens have so far been identified for human CD8 T cells. In this regard, release of genome sequences of M. tuberculosis has provided an opportunity to identify proteins with vaccine potential that could give immune protection in individuals with different HLA backgrounds. Objectives and scope of the present work 1. Prediction of putative T cell antigens in PE and PPE family of proteins of Mycobacterium tuberculosis through immuno-informatics approach 2. Evaluation of immune response to three of the PE and PPE proteins in mouse model. 3. Evaluation of immune response against chosen PE and PPE proteins of Mycobacterium tuberculosis with Human Peripheral Blood Mononuclear Cells (PBMCs) from PPD positive healthy donors and TB patients. 4. Immune response to multi-epitope DNA vaccine construct for Mycobacterium tuberculosis. Prediction of MHC class I peptides from PE and PPE proteins. In an effort to identify potential T cell antigens from PE and PPE family of proteins, we have carried out a systematic in silico analysis of the 167 different PE and PPE proteins. Employing immuno-informatics approach, a set of HLA class I binding peptides have been identified from these proteins. Further, their binding abilities have been ascertained using independent methods such as molecular modeling and structural analysis methods. The nonameric sequences from PE and PPE families of proteins were predicted to contain high percentage of binding peptides to human class I HLA, whereas PE_PGRS proteins show relatively low level of binding. This difference is seen in spite of PE and PE_PGRS being Sub-families of the same family, PE. Seventy-one high- as well as low-affinity peptides from both PE and PPE proteins have been analyzed for structural compatibility with crystal structures of HLA in terms of intermolecular energies and were found to correlate well with the corresponding affinities predicted by the BIMAS algorithm. Most of the peptides binding to HLA are specific with very few promiscuous binders. Identification of T cell epitopes from three of the PE/PPE proteins using DNA immunization This work describes the evaluation of immune responses to three of the PE and PPE proteins in mouse model. Three of PE and PPE proteins, coded by Rv1818c, Rv3812 and Rv3018c genes were chosen based on immuno-informatics approach. They were cloned, expressed in prokaryotic and mammalian expression vectors and recombinant protein expressing stable cell lines were made. T lymphocytes from DNA immunized mice recognize synthetic peptides from chosen proteins in vitro, indicating that these peptides are being processed and presented by MHC molecules to T cells. By MHC stabilization assay, 5 of the synthetic peptides were found to stabilize the MHC class I molecules on the cell surface for more than 6 hrs, validating the computational prediction. Recognition of T cell epitopes derived from PE/PPE proteins by human PBMCs This work describes the evaluation of immune response against three of PE and PPE proteins of Mycobacterium tuberculosis with Human Peripheral Blood Mononuclear Cells (PBMCs) from PPD positive Healthy donors and TB patients. Proliferation response of PBMCs from ten PPD positive healthy donors as well as from ten TB patients, indicated that the peptides from PE and PPE proteins of Mtb can sensitize naive T cells and induce peptide specific IFN-γ and also the T cell response to the chosen peptides was both HLA class I restricted and CD8 mediated. After the peptide specific expansion, significant percentage of CD8+ T cells were shown to secrete IFN-γ and stained positive for perforin. Antigen specific CD8+ T cells were found to have cytolytic potential in addition to their cytokine function. Immune response to a multiepitope DNA vaccine in mouse model Minigene poly-epitope vaccine constructs coding for nine peptides derived from identified T cell antigens of PE and PPE proteins and three of the experimentally mapped epitopes from M tuberculosis was designed and constructed. The minigene was used to immunize mice and the immune response was tested. The DNA primed splenocytes recognized the full length poly-epitope protein as well as the individual peptides. T cell response to epitopes was enhanced by mere presence in multi-epitope construct compared to full length antigens. Human PBMCs derived from both PPD+ve and TB patients also recognized the peptides in vitro. It is thus obvious that a large cocktail of proteins are required to achieve reasonable population coverage. Besides, this work suggests the feasibility of designing haplotype specific subunit vaccine, which can be given to individuals with known HLA haplotype. The haplotype specific vaccines can be combined to target a population where the distribution of HLA alleles is known. This work also indicates that use of single or limited number of genes in a DNA vaccine may not be suitable to cover a given population.

Systematic studies of basal nonsense suppression, orthogonality of tRNAPyl variants, and cross recognition between codons and tRNA anticodons are reported. E. coli displays detectable basal amber and opal suppression but shows a negligible ochre suppression. Although detectable, basal amber suppression is fully inhibited when a pyrrolysyl-tRNA synthetase (PylRS)-tRNAPyl_CUA pair is genetically encoded. trnaPyl_CUA is aminoacylated by an E. coli aminoacyl-tRNA synthetase at a low level, however, this misaminoacylation is fully inhibited when both PylRS and its substrate are present. Besides that it is fully orthogonal in E. coli and can be coupled with PylRS to genetically incorporate a NAA at an ochre codon, tRNAPyl_UUA is not able to recognize an UAG codon to induce amber suppression. This observation is in direct conflict with the wobble base pair hypothesis and enables using an evolved M. jannaschii tyrosyl-tRNA synthetase-tRNAPyl_UUA pair and the wild type or evolved PylRS-tRNAPyl_UUA pair to genetically incorporate two different NAAs at amber and ochre codons. tRNAPyl_UCA is charged by E. coli tryptophanyl-tRNA synthetase, thus not orthogonal in E. coli. Mutagenic studies of trnaPyl_UCA led to the discovery of its G73U form which shows a higher orthogonality. Mutating trnaPyl_CUA to trnaPyl_UCCU not only leads to the loss of the relative orthogonality of tRNAPyl in E. coli but also abolishes its aminoacylation by PylRS.