Ultraviolet (UV) light reactors or banks are often arranged in series in order to meet microbial inactivation credit requirements. It has been assumed that UV doses given by each reactor in series are mathematically additive, though work done to substantiate the hypothesis has been inconsistent. Based on previously developed theory of reactor additivity and the reactor additivity factor (RAF), three types of UV reactors are modelled using computational fluid dynamics and their RAFs are computed. It is noted that the assumption of perfect mixing may not be valid depending on the distance between reactors in series. It is discussed that the original formulation of the RAF is inadequate when dealing with wastewater. It is shown unexpectedly that even with perfect mixing performance, worse than additivity would be achieved. A new performance factor (PF) is introduced and the implications of this are further discussed in the context of UV reactor validation.

Ultraviolet (UV) light reactors or banks are often arranged in series in order to meet microbial inactivation credit requirements. It has been assumed that UV doses given by each reactor in series are mathematically additive, though work done to substantiate the hypothesis has been inconsistent. Based on previously developed theory of reactor additivity and the reactor additivity factor (RAF), three types of UV reactors are modelled using computational fluid dynamics and their RAFs are computed. It is noted that the assumption of perfect mixing may not be valid depending on the distance between reactors in series. It is discussed that the original formulation of the RAF is inadequate when dealing with wastewater. It is shown unexpectedly that even with perfect mixing performance, worse than additivity would be achieved. A new performance factor (PF) is introduced and the implications of this are further discussed in the context of UV reactor validation.

CK2 is a ubiquitous serine/threonine protein kinase implicated in numerous cellular processes as well as in tumorigenesis. CK2 is composed of two catalytic (αα, αα’, α’α’) subunits and two regulatory (ββ) subunits that assemble to form the active CK2 holoenzyme. CK2 has been shown to phosphorylate, interact with, and regulate other proteins, including other protein kinases. CK2 substrates can be initially bound by the CK2β regulatory subunit, which acts as a docking site to facilitate phosphorylation and mediate CK2 substrate specificity. In a screen to identify novel CK2β interacting proteins, I identified three novel CK2β interactors, including the mitogen- and stress-activated kinase 2 (MSK2), which I pursued for further characterization. MSK2, and the closely related isoform MSK1, are nuclear kinases that are activated following mitogen stimulation or cellular stress, including UV radiation, by the ERK1/2 and p38-MAPK signaling cascades, respectively. However, factors that differentially regulate MSK1 and MSK2 have not been well characterized. In my thesis, I demonstrate that CK2, which contributes to NF-κB activation following UV radiation in a p38-dependent manner, physically interacts with MSK2 but not MSK1 and that CK2 inhibition specifically impairs UV-induced MSK2 kinase activation. A putative site of CK2 phosphorylation was mapped to MSK2 residue serine-324 and when substituted to alanine (S324A) also compromised MSK2 activity. RNA interference-mediated depletion of MSK2 in human MDA-MB-231 cells, but not MSK1 depletion, resulted in impaired UV-induced phosphorylation of NF-κB p65 at serine-276 in vivo, which was restored by the ectopic expression of MSK2 but not by MSK2-S324A. Furthermore, UV-induced p65 transactivation capacity was dependent on MSK2, MSK2 residue S324, and p65-S276. These results suggest that MSK1 and MSK2 are differentially regulated by CK2 during the UV response and that MSK2 is the major protein kinase responsible for the UV-induced phosphorylation of p65 at S276 that positively regulates NF-κB activity in MDA-MB-231 cells.

This thesis is an investigation of the vacuum uv spectra of the phosphorus, phosphoryl, and thiophosphoryl trichlorofluorides in the region 1250 to 3000A. Assignments for absorption bands are made utilizing results from photoelectron spectra and ab initio calculations, oscillator strengths for absorption bands, and CNDO/2 molecular orbital calculations. Results from CNDO/2 calculations are compared with theoretical calculations, and experimental data are discussed with regard to the bonding in the compounds.

Master of Science / Department of Chemistry / Christopher T. Culbertson / Chemists have been trying to relate the structure and composition of different cereal proteins to their physical properties to better inform their product use for more than 250 years now. Among these cereals, wheat is considered the most important due to its unique ability to form viscoelastic dough and retain gas during fermentation, the latter being important for bread making. This property is due to the endosperm part of wheat that contains proteins mostly gliadins and glutens. It is known that the composition and relative ratio of these proteins is determined by both the growing environment and genetics. Manipulation of the genetics allows one for control of only about 50% of the end use quality of the wheat and the rest is controlled by environment. Currently, the bread making quality of wheat is determined by baking test loaves of bread. This process is time consuming and wasteful. The main goal of this project was to create fingerprints of gliadin proteins for different wheat cultivars as a function of environmental conditions. This would then allow wheat kernels to be analyzed and assessed right after harvest to determine their appropriateness for making the various wheat products. Researchers have tried to create a catalogue of information for individual wheat cultivars by ‘fingerprinting’ the gliadins proteins in wheat using various analytical techniques including capillary electrophoresis (CE). CE offers advantages like high separation efficiency, and faster analysis. Further miniaturization of CE on microfluidic devices has enhanced the speed and efficiency of separation. Furthermore, it is possible to integrate multiple chemical analysis processes like sample preparation, separation and detection in a single microfluidics device. Microfluidic uses micron sized separation channels defined in a glass, quartz or polymer. This dissertation is focused on fabricating multilayer microfluidic devices from Poly(dimethylsiloxane) (PDMS) and using these devices to electrophoretically separate wheat gliadin proteins followed by detection using UV absorption in less than 5 min. PDMS is cheap, easy to fabricate and is optically transparent above ~230nm. Initial results of the UV absorbance detector developed for this device are presented.

Nanomaterials such as gold nanowires and gold nanoparticles were self-assembled with several peptides derived from betaamyloid peptide. The peptides propensity to form fibrilar structures was exploited. The products obtained by aggregation of the peptides with the nano materials were studied using HPLC, UV-vis spectroscopy, TEM and optical light microscopy.

Concerns about increased stratospheric ozone depletion increasing ambient levels of ultraviolet-B radiation (UV-B), and the fact that some ecosystems are naturally exposed to high levels, has resulted in an increased interest in the effects of UV-B on plant communities. Despite this, there has been a paucity of studies into its effects on plant competition. Artificial plant communities consisting of Lolium perenne and Lotus corniculatus and a sub-montane community consisting of Agrostis tenuis, Festuca ovina and Galium saxatile (also including different nitrogen levels) were created using the response surface approach. The long-term effects of UV-B were also studied on a natural sub-Arctic community in Abisko, Sweden. In addition, all plant samples were analysed by Fourier-Transform Infrared Spectroscopy (FT-IR) to obtain a ‘metabolic fingerprint’ which was used to detect chemical differences to the whole biochemical complement of the sample. The results showed that enhanced UV-B altered the competitive interaction of Lolium perenne and Lotus corniculatus in favour of Lolium perenne although ambient levels of UV-B did not elicit an effect in the sub-montane community. Only one dwarf shrub species in the sub-Arctic experiment, Vaccinium myrtillus, was negatively affected by UV-B. In most cases, elevated UV-B elicited a change in the metabolic fingerprint in the samples and in some cases an alteration in competitive stress altered the metabolome. This suggests that FT-IR can be used as a screening tool to detect for both abiotic stress and competitive biochemical alterations. In addition, this thesis proposes that the facilitative effect between the grass-legume mixture of Lolium perenne and Lotus corniculatus is not related to nitrogen fixation in the early stages of competition which has traditionally been believed.

Objetivos: O objetivo deste estudo foi avaliar a influência do Diabetes Mellitus do tipo 2 na farmacocinética da glimepirida em ratos Wistar e descrever o perfil através de modelo farmacocinética populacional (popPK). Metodologia: Os experimentos com animais foram aprovados pelo CEUA/UFRGS (protocolo #27892). O diabetes foi induzido com administração intraperitoneal de 100 mg/kg de nicotinamida, 15 minutos antes da administração intravenosa de 65 mg/kg de STZ. Os animais com nível de glicemia > 250 mg/dL foram considerados diabéticos. A glimepirida foi administrada na dose de 5 mg/kg via i.v. nos animais sadios (n = 11) e diabéticos (n = 9) e quantificada por CLAE-UV. A ligação às proteínas plasmáticas foi determinada por método de ultracentrifugação (Centrifree®). A análise farmacocinética não compartimental (software Phoenix®) foi realizada, assim como a modelagem farmacocinética populacional (software Monolix ®). Resultados e Discussão: A metodologia analítica para quantificação da glimepirida em plasma foi desenvolvida e validada, seguindo os critérios do FDA, apresentou sensibilidade, exatidão e precisão. O modelo de indução da diabetes produziu glicemia > 250 mg/dL. A ligação às proteínas plasmáticas não foi afetada pela doença (LPPSaudáveis = 99,3 ± 0,09%, LPPDiabéticos = 99,13 ± 0,075%, p > 0,05). O modelo farmacocinético populacional estrutural de 2 compartimentos com eliminação de primeira-ordem com covariável categórica (diabetes), foi usado para descrever os perfis plasmáticos de concentração-tempo da glimepirida após administração intravenosa na dose de 5 mg/kg a ratos saudáveis e diabéticos. O CL e a ASC0-inf dos animais diabéticos foram estatisticamente diferentes dos animais saudáveis, CLpop Saudáveis= 0,066 L/h para CLpop diabéticos = 0,024 L/h e ASCpop saudáveis = 19,24 μg/mL.h para ASCpop diabéticos = 59,64 μg.h/mL, indicando que a eliminação foi alterada nos animais diabéticos induzidos STZ. Conclusões: A modela gempossibilitou identificação do parâmetro que atribuiu variabilidade entre os grupos. Desta forma, a variabilidade interindividual foi quantificada e incluída no modelo. O modelo popPK final, nos permitiu elucidar os fatores que afetam a farmacocinética da glimepirida e prever mudanças na exposição em uma população específica. / Objective: The aim of this study was to evaluate the influence of diabetes mellitus type 2 on the pharmacokinetics of glimepiride in rats and describe the profile in population pharmacokinetic model (popPK). Methods: The experiments with animals were approved by CEUA/UFRGS (protocol number). The diabetes was induced by intraperitoneal administration of NA (100 mg/kg) dissolved in saline 15 min before an intravenous administration of 65 mg/kg STZ in citrate buffer (pH 4.5) to overnight fasted rats. Animals with blood glucose level> 250 mg/dL were considered diabetic. After administered of glimepiride at a dose of 5 mg/kg i.v. bolus in healthy (n = 11) and diabetic animals (n = 9). Method HPLC-UV developed and validated quantified plasma concentrations. The plasma protein binding was determined by method ultracentrifugation (Centrifree®). Noncompartmental analysis of pharmacokinetic in Phoenix® software was performed, as well as the model pharmacokinetic population using Monolix®. Performed by Student's t-test for SigmaStat® software. Results and Discussion: The HPLC-UV method for quantification of glimepiride in plasma was developed and validated following requirements by FDA showing sensitivity, accuracy and precision. Induced diabetes model produced glucose> 250 mg / dL. The plasma protein binding was not affect by the disease (LPPSaudáveis = 99.3 ± 0.09%, LPPDiabéticos = 99.13 ± 0.075%, p> 0.05). The model pharmacokinetic population 2 compartments with eliminating first-order with categorical covariates diabetic was used to describe the plasma profile concentration-time glimepiride. The CL and AUC 0-inf of diabetic animals were significantly different. In healthy animals was Clpop Healthy = 0.066 L/h for diabetics Clpop = 0.024 L/h and healthy ASCpop = 19.24 g/mL.h ASCpop for diabetics = 59.64 g/mL.h, indicating that elimination was decreased in induced diabetic rats STZ. Conclusions: popPk enabled identification of the parameter assigned variability between the groups. Thus, the inter subject variability was measured and included in the model. The PBPK final model, allowed us to elucidate the factors that affect the pharmacokinetics of glimepiride and predict changes in exposure in a specific population.

Photocatalytic treatment has the potential to become a cost effective method of organic contaminant removal from water. Photocatalytic materials are semiconductors that enhance chemical reactions such as the breakdown of organic molecules in the presence of light. One of the most studied photocatalysts for water purification is titanium dioxide (TiO2). Variations in the composition of photocatalysts can affect the outcome of the experiments, the detection of the change in behaviour of the photocatalyst is of significant scientific interest. It requires minimisation of the impact of all other factors affecting the photocatalytic process, such as temperature, light intensity, wavelength and uniformity. Repeatability of the experiments is also affected by these factors. If their impact is not considered and addressed the outcome of multiple seemingly identical experiments with a single sample of the photocatalyst will produce different results. Light is one of the most important factors in a photocatalytic process. The undoped TiO2 has a sharp drop in its light absorbtion characteristics between UV and visible spectral regions. It is theregion of the spectrum where most efficient UV LEDs radiate. As the characteristics of the light produced by LEDs are temperature dependent, heat management is important in achieving light with stable characteristics and prolonged lifetime of the LEDs. One of the contributions of this thesis is a novel method of not only stabilisation of the LED radiation parameters, namely optical output power and wavelength, but also the independent control of these parameters. The importance of LED calibration is also a significant contribution as commercial LEDs have dierent radiation parameters between devices. Possibility of independent control of optical power and wavelength of the LEDs has allowed to demonstrate the importance of radiant flux (total spectral power) over the peak spectral flux (power of a single wavelength component) for TiO2 activation, which is another significant contribution of this work. Uniformity of the produced light is another factor that needs to be addressed when a light source for the photocatalytic experimentation is designed. Non-uniform light distribution in a photocatalytic reactor will result in bright spot formation that will affect the overall performance of the photocatalytic sample. This together with the temperature control of the photocatalyst and the water sample are key issues that need to be addressed for achieving ecient and repeatable experimentation outcomes. Photocatalytic reactors developed from simulation to the working prototypes and tested during the work described in this thesis address the problem of light distribution uniformity. They have been designed to remove as many sources of uncertainty usually present in photocatalytic reactors as possible, such as for example temperature stability of the liquid sample, dierent sizes or of the photocatalytic samples and same volume of the liquid sample. As such, these novel reactors together with LED light sources provide a contribution of having a potential of becoming a photocatalytic experimentation standard for achieving the repeatable and comparable results.

Protein-nucleic acid interactions play essential roles in all living cells in various cellular functions. The study of these interactions can reveal important structural and functional information. UV cross-linking of nucleic acids to proteins in combination with mass spectrometry is a powerful technique to identify proteins, peptides and the amino acids involved in intermolecular interactions within nucleic acid-protein complexes. However, the mass spectrometric identification of cross-linked nucleic acid-protein heteroconjugates in complex mixtures and MS/MS characterisation of the specific sites of cross-linking is a challenging task. In this investigation, novel tools and methods have been developed for the investigation of DNA-protein interactions using UV cross-linking and mass spectrometry. These tools were developed towards their application for the characterisation of the complex between the eukaryotic DNA repair protein Tyrosyl-DNA phosphodiesterase 1 (Tdp1) and its DNA substrates. DNA-Tdp1 UV cross-linking was optimised using purified recombinant human Tdp1 and radioactively labelled DNA oligonucleotides containing UV photoactivatable 4- thio-thymidine or 5-iodouracil. Tdp1-DNA heteroconjugates were detected by SDS PAGE and Phosphorimaging. In order to analyse the DNA-Tdp1 heteroconjugates by mass spectrometry, they must first be enriched and hydrolysed by a protease and a nuclease. Here, a novel sample preparation protocol was developed for the enrichment of Tdp1 oligonucleotide-peptide heteroconjugates. Detection and analysis of oligonucleotide-peptide heteroconjugates using mass spectrometry is a challenging task. As a tool to optimise the various parameters involved, a synthetic DNA oligonucleotide-peptide heteroconjugate was constructed using click chemistry. RP-HPLC/ESI-FT-ICR-MS on a Bruker 12T SolariX in conjunction with CID fragmentation was used to unambiguously identify the site of the cross-link. Lastly, a novel 18O labeling approach was introduced to facilitate the identification of DNA-protein cross-links. This approach was shown to be suitable for the labeling of heteroconjugate species by testing it with the click heteroconjugate.