As reações aldólicas vêm ganhando destaque com os recentes avanços na área de biocatálise. Uma alternativa para o uso de aldolases, que não atuam sobre uma grande variedade de substratos e possuem custos elevados, é o emprego de enzimas de outras classes que possam exercer como função promíscua a atividade aldolase. Neste trabalho lipases e celulases foram capazes de catalisar a reação aldólica entre o 4-nitrobenzaldeído e a cicloexanona. A lipase de Rhizopus niveus (RNL) catalisou a reação aldólica em solventes orgânicos na presença de água e também em tampões aquosos. As reações condicionais influenciaram o rendimento (0-99%) e a enantiosseletividade do produto aldólico anti (6-55% eeanti). O produto aldólico com enantiosseletividade foi observado mesmo com a enzima inativa e em condições desnaturantes. Portanto, as reações aldólicas procederam por catálise proteica inespecífica com enantiosseletividades moderadas e não por atividade promíscua. Contudo, este estudo identificou um novo catalisador verde para reações aldólicas, pois foram obtidos produtos com bons rendimentos, ee e excesso diastereoisomérico (ed), demonstrando novas possibilidades para lipases, especialmente para a RNL que é pouco estudada na literatura. A partir destes resultados obtidos, uma nova abordagem baseada em sistemas reacionais de miniemulsão foi elaborada. Superando limitações para este sistema fluido não convencional como o emprego de reagente sólido (4-nitrobenzaldeído), substratos relativamente polares, solução tampão e o desenvolvimento de um procedimento de isolamento do produto. Desta maneira, foram descritos neste trabalho métodos de produção de sistemas reacionais de miniemulsão estáveis e aplicáveis, como relatado para reações aldólicas pela RNL. Empregando planejamento experimental minimizou-se a quantidade de catalisador (de 20 para 6 mg mL-1) em comparação com as reações realizadas em mistura de solvente orgânico e água. Mesmo com menor quantidade de enzima, o rendimento aumentou de 25% para valores de até 65% para 48 h de reação e o eeanti de 10% foi maximizado para valores acima de 30%, valor não considerado satisfatório, entretanto bem superior ao observado inicialmente. Com a interpretação estatística dos dados obtidos foi possível apresentar compreensões satisfatórias para as variações observadas de ee, ed, rendimento e tamanho médio de diâmetro das gotículas de miniemulsão, possibilitando uma compreensão mais completa deste sistema e permitindo uma melhor racionalização deste meio reacional que ainda não foi amplamente estudado e divulgado na literatura para promover reações biocatalíticas. Este estudo demonstrou a potencialidade deste método para diferentes tipos de reações orgânicas. / The aldol reactions have received attention with the recent advances in biocatalysis. In this sense, studies showed that an alternative for the use of aldolases, which do not act on a great variety of substrates and have high costs, is the use of enzymes of other classes that can present the promiscuous aldolase activity. In this work lipases and cellulases were able to catalyze the aldol reaction between 4-nitrobenzaldehyde and cyclohexanone. Rhizopus niveus lipase (RNL) catalyzed the aldol reaction in organic solvents with water or aqueous buffers. The reactional conditions affected the yield (0-99%) and the enantioselectivity of the anti-aldol product (6-55% eeanti). The aldol product with enantioselectivity was observed even with the inactive enzyme and under denaturing conditions. Therefore, the aldol reaction proceeded by non-specific protein catalysis with moderate enantioselectivity and not by promiscuous activity. However, this study identified a new green catalyst for aldol reactions, since products with good yields, ee and diastereoisomeric excess (ed) were obtained, and new possibilities for lipases, especially for RNL that is understudied. From these results, a new approach based on the miniemulsion reactional system was developed. Overcoming limitations to this unconventional fluid system such as the use of a solid reagent (4-nitrobenzaldehyde), relatively polar substrates, buffer solutions and the development of a product isolation method. In this way, we have described in this work methods for the production of stable and applicable miniemulsion reaction systems for aldol reactions by RNL. Using experimental design, the amount of catalyst (from 20 to 6 mg mL -1) was minimized in comparison with the reactions performed in organic solvent and water. Even with a lower amount of enzyme, the yield increased from 25% to 65% for 48 hours of reaction and the eeanti of 10% was maximized to values above 30%, a value not considered satisfactory, however higher than the enantioselectivity initially observed. The statistics applied to the obtained data presented satisfactory interpretations for the observed ee, ed, yield and average diameter size of the miniemulsion droplets, allowing a more complete understanding of the miniemulsion system and allowing a better rationalization of this reactional medium that is understudied to promote biocatalytic reactions. This study demonstrated the potential of this methodology for different types of organic reactions.

As reações aldólicas vêm ganhando destaque com os recentes avanços na área de biocatálise. Uma alternativa para o uso de aldolases, que não atuam sobre uma grande variedade de substratos e possuem custos elevados, é o emprego de enzimas de outras classes que possam exercer como função promíscua a atividade aldolase. Neste trabalho lipases e celulases foram capazes de catalisar a reação aldólica entre o 4-nitrobenzaldeído e a cicloexanona. A lipase de Rhizopus niveus (RNL) catalisou a reação aldólica em solventes orgânicos na presença de água e também em tampões aquosos. As reações condicionais influenciaram o rendimento (0-99%) e a enantiosseletividade do produto aldólico anti (6-55% eeanti). O produto aldólico com enantiosseletividade foi observado mesmo com a enzima inativa e em condições desnaturantes. Portanto, as reações aldólicas procederam por catálise proteica inespecífica com enantiosseletividades moderadas e não por atividade promíscua. Contudo, este estudo identificou um novo catalisador verde para reações aldólicas, pois foram obtidos produtos com bons rendimentos, ee e excesso diastereoisomérico (ed), demonstrando novas possibilidades para lipases, especialmente para a RNL que é pouco estudada na literatura. A partir destes resultados obtidos, uma nova abordagem baseada em sistemas reacionais de miniemulsão foi elaborada. Superando limitações para este sistema fluido não convencional como o emprego de reagente sólido (4-nitrobenzaldeído), substratos relativamente polares, solução tampão e o desenvolvimento de um procedimento de isolamento do produto. Desta maneira, foram descritos neste trabalho métodos de produção de sistemas reacionais de miniemulsão estáveis e aplicáveis, como relatado para reações aldólicas pela RNL. Empregando planejamento experimental minimizou-se a quantidade de catalisador (de 20 para 6 mg mL-1) em comparação com as reações realizadas em mistura de solvente orgânico e água. Mesmo com menor quantidade de enzima, o rendimento aumentou de 25% para valores de até 65% para 48 h de reação e o eeanti de 10% foi maximizado para valores acima de 30%, valor não considerado satisfatório, entretanto bem superior ao observado inicialmente. Com a interpretação estatística dos dados obtidos foi possível apresentar compreensões satisfatórias para as variações observadas de ee, ed, rendimento e tamanho médio de diâmetro das gotículas de miniemulsão, possibilitando uma compreensão mais completa deste sistema e permitindo uma melhor racionalização deste meio reacional que ainda não foi amplamente estudado e divulgado na literatura para promover reações biocatalíticas. Este estudo demonstrou a potencialidade deste método para diferentes tipos de reações orgânicas. / The aldol reactions have received attention with the recent advances in biocatalysis. In this sense, studies showed that an alternative for the use of aldolases, which do not act on a great variety of substrates and have high costs, is the use of enzymes of other classes that can present the promiscuous aldolase activity. In this work lipases and cellulases were able to catalyze the aldol reaction between 4-nitrobenzaldehyde and cyclohexanone. Rhizopus niveus lipase (RNL) catalyzed the aldol reaction in organic solvents with water or aqueous buffers. The reactional conditions affected the yield (0-99%) and the enantioselectivity of the anti-aldol product (6-55% eeanti). The aldol product with enantioselectivity was observed even with the inactive enzyme and under denaturing conditions. Therefore, the aldol reaction proceeded by non-specific protein catalysis with moderate enantioselectivity and not by promiscuous activity. However, this study identified a new green catalyst for aldol reactions, since products with good yields, ee and diastereoisomeric excess (ed) were obtained, and new possibilities for lipases, especially for RNL that is understudied. From these results, a new approach based on the miniemulsion reactional system was developed. Overcoming limitations to this unconventional fluid system such as the use of a solid reagent (4-nitrobenzaldehyde), relatively polar substrates, buffer solutions and the development of a product isolation method. In this way, we have described in this work methods for the production of stable and applicable miniemulsion reaction systems for aldol reactions by RNL. Using experimental design, the amount of catalyst (from 20 to 6 mg mL -1) was minimized in comparison with the reactions performed in organic solvent and water. Even with a lower amount of enzyme, the yield increased from 25% to 65% for 48 hours of reaction and the eeanti of 10% was maximized to values above 30%, a value not considered satisfactory, however higher than the enantioselectivity initially observed. The statistics applied to the obtained data presented satisfactory interpretations for the observed ee, ed, yield and average diameter size of the miniemulsion droplets, allowing a more complete understanding of the miniemulsion system and allowing a better rationalization of this reactional medium that is understudied to promote biocatalytic reactions. This study demonstrated the potential of this methodology for different types of organic reactions.

A satisfying sexual relationship is an important aspect of general marital satisfaction. Considering that most people will marry, understanding the factors influencing marital sexual satisfaction becomes important in understanding what makes happy, satisfied relationships and individuals in our society. This study builds on previous research, which has indicated that there are a number of "risky factors" associated with having numerous premarital sexual partners, by demonstrating that there are risks associated with the future marital relationship. Using exchange theory concepts, this study empirically examines the relationship between premarital sexual promiscuity and marital sexual satisfaction. The sample included 313 married males and females between the ages of 18-40 using data from the NHSLS survey, a United States national random sample. The dichotomous dependent variable combined measures of emotional and physical sexual satisfaction. Premarital sexual promiscuity was measured continuously. Implications for clinicians are discussed. Of males, 87.7% reported being extremely satisfied with their marital sexual relationship and 12.3% reported being moderately satisfied. Of females, 84.6% of reported extreme satisfaction and 15.4% reported moderate satisfaction with their marital sexual relationship. The range of partners including the marital partner was 1-191 for men and 1-66 for women. The mean number of partners was 11.35 (SD 19.77) for men and 4.25 (SD 6.3) for women. Results indicate that for every additional premarital sexual partner an individual has, not including the marital sexual partner, the likelihood that they will say their current marital sexual relationship is extremely satisfying versus only being moderately satisfying goes down 3.9%. Additionally, when running models separately for males and females, the male model was more significant at 5.3%. The female model approached significance. Control variables for the models were not significant except that males who were not White, Black, or Hispanic, were 8.9% less likely than White males to report being extremely satisfied with their marital sexual relationship as compared to only being moderately satisfied. This model does not account for individuals who had sex with their spouse before marriage and the results should not be interpreted to include such.

In this thesis paper, I will recount the creative and technical processes of making my graduate thesis film, KOOL. I will describe how the story was developed and written, then analyze the phases of pre-production, principle photography, and post production. I will then evaluate my leadership skills, and strengths and weaknesses as a film director.

In a lipase investigation, Candida antarctica lipase B (CALB) are explored for enzyme catalytic promiscuity. Enzyme catalytic promiscuity is shown by enzymes catalyzing alternative catalytic transformations proceeding via different transition state structures than normal. CALB normally performs hydrolysis reactions by activating and coordinating carboxylic acid/ester substrates in an oxyanion hole prior to nucleophilic attack from an active-site serine resulting in acyl enzyme formation. The idea of utilizing the carbonyl activation oxyanion hole in the active-site of CALB to catalyze promiscuous reactions arose by combining catalytic and structural knowledge about the enzyme with chemical imagination. We choose to explore conjugate addition and direct epoxidation activities in CALB by combining molecular modeling and kinetic experiments. By quantum-chemical calculations, the investigated promiscuous reactions were shown to proceed via ordered reaction mechanisms that differ from the native ping pong bi bi reaction mechanism. The investigated promiscuous activities were shown to take place in the enzyme active-site by various kinetic experiments, but despite this, no enantioselectivity was displayed. The reason for this is unknown, but can be a result of a too voluminous active-site or the lack of covalent coordination of the substrates during enzyme-catalysis (Paper I-IV). Combining enzyme structural knowledge with chemical imagination may provide numerous novel enzyme activities to be discovered. In an ω-transaminase investigation, two (S)-selective ω-transaminases from Arthrobacter citreus (Ac-ωTA) and Chromobacterium violaceum (Cv-ωTA) are explored aiming to improve their catalytic properties. Structural knowledge of these enzymes was provided by homology modeling. A homology structure of Ac-ωTA was successfully applied for rational design resulting in enzyme variants with improved enantioselectivity. Additionally, a single-point mutation reversed the enantiopreference of the enzyme from (S) to (R), which was further shown to be substrate dependent (Paper V). A homology structure of Cv-ωTA guided the creation of an enzyme variant showing reduced isopropyl amine inhibition. / QC20100609

The African continent has been hit the hardest by the HIV/AIDS epidemic which has seen more women becoming infected than men (Hunter, 2003; UNAIDS, 2004). This has been attributed to the permissible nature of the African culture which is lenient on male promiscuity (Foreman, 1999; Colvin 2000; Leclerc-Madlala 2001; Dube 2003). African women are not only vulnerable to infection but are also vulnerable to negative disclosure experiences when they disclose their status to sexual partners. This double impact of culture has not been addressed by past researches (UNAIDS 2004). The present research thus attempts to fill that gap. It explores the interplay between culture and HIV transmission as well as the interplay between culture and disclosure experiences. The study’s setting is Magunje Township, a rural village in Mashonaland Central province in Zimbabwe. Taking into consideration the limitations of the present analysis, data gathered indicates that African women are vulnerable to both HIV infection and negative disclosure experiences. Their vulnerability also hampers prevention and treatment efforts as it makes them decide to keep their status a secret from sexual partners for fear of losing the financial income tied to sexual partners. The present researcher concludes with some suggestions for policy makers and programme implementers, highlighting the importance of focusing on HIV/AIDS status disclosure as a prevention and treatment method in the absence of a cure for HIV.

Protein kinases are important regulatory enzymes in signal transduction and in cell regulation. Understanding inhibition mechanisms of kinases is important for the further development of new therapies for cancer and inflammatory diseases. I have developed a statistical approach based on the Mantel test to find the relationship between the shapes of ATP binding sites and their affinities for inhibitors. My shape-based dendrogram shows clustering of the kinases based on similarity in shape. I investigate the pocket in terms of conservation of surrounding amino acids and atoms in order to identify the key determinants of ligand binding. I find that the most conserved regions are the main chain atoms in the hinge region and I show that the tetrahydropyran ring of staurosporine causes induced-fit of the glycine rich loop. I apply multiple linear regression to select distances measured between the distinctive parts of residues which correlate with the binding constants. This method allows me to understand the importance of the size of the gatekeeper residue and the closure between the first glycine of the GXGXXG motif and the aspartate of the DFG loop, which act together to promote tight binding to staurosporine. I also find that the greater the number of hydrogen bonds made by the kinase around the methylamine group of staurosporine, the tighter the binding to staurosporine. The website I have developed allows a better understanding of cross reactivity and may be useful for narrowing down the options for a synthetic strategy to design kinase inhibitors.

The aim of this thesis is to investigate aspects of molecular evolution and enzyme engineering using the experimental evolution of Tobacco Etch Virus cysteine protease (TEV) as a model. I map key features of the local fitness landscape and characterise how they affect details of enzyme evolution. In order to investigate the evolution of core active site machinery, I mutated the nucleophile of TEV to serine. The differing chemical properties of oxygen and sulphur force the enzyme into a fitness valley with a >104-fold activity reduction. Nevertheless, directed evolution was able to recover function, resulting in an enzyme able to utilise either nucleophile. High-throughput screening and sequencing revealed how the array of possible beneficial mutations changes as the enzyme evolves. Potential adaptive mutations are abundant at each step along the evolutionary trajectory, enriched around the active site periphery. It is currently unclear how seemingly neutral mutations affect further adaptive evolution. I used high-throughput directed evolution to accumulate neutral variation in large, evolving enzyme populations and deep sequencing to reconstruct the complex evolutionary dynamics within the lineages. Specifically I was able to observe the emergence of robust enzymes with improved mutation tolerance whose descendants overtake later populations. Lastly, I investigate how evolvability towards new substrate specificities changed along these neutral lineages, dissecting the different determinants of immediate and long-term evolvability. Results demonstrate the utility of evolutionary understanding to protease engineering. Together, these experiments forward our understanding of the molecular details of both fundamental evolution and enzyme engineering.

Extra-pair paternity, resulting from sexual promiscuity, is frequently detected in socially monogamous passerines. Previous studies on extra-pair paternity in birds have identified several traits correlated with increased fertilization success of males. However, the effect of experimental manipulation of ornament expression on male fertilization success has only seldom been evaluated. The aim of this thesis is to reveal the potential link between the size of a trait suposedly playing a role in female mate choice decision and male fertilization success in collared flycatchers (Ficedula albicollis), using experimental manipulation of male forehead white patch size. In addition, mating success, mating speed of the manipulated and control individuals and size of a brood (a proxy for female reproductive investments) were evaluated. Results indicate that forehead patch size manipulation did not affect male social pairing success (mating speed), his social mate reproductive investments (brood size) or male extra-pair and within-pair fertilization success. Keywords: extra-pair paternity, sexual selection, reproductive success, collared flycatcher

Within a lifetime of an individual, there are changes in the organism leading to an an impairment of phenotype, survival and reproduction. The expression of phenotypic traits often increases with age. Higher expression of these traits in the young age can lead to decreased probability of survival till the next season, influencing reproductive success of an individual. That is why organisms are challenged with trade-offs, when they have to allocate their limited resources either into the reproduction in form of expression of extravagant sex ornaments or into the life span. If the ornament evolved as a handicap, its exaggerated size could reduce viability of low quality individuals. Therefore, the ornament becomes a predictor of quality of its bearer and its size indicates the ability to survive till the next season. Both sexual ornamentation and inter-individual variation in life span may result in differences in reproductive success (fitness) and targeted by sexual or natural selection. Variability in reproductive success, mainly in monogamous species, can be increased also by sexual promiscuity and extra-pair mating and extra-pair copulations. This strategy can increase the opportunity for selection of traits that play role in choosing of extra-pair mate. In this work, I focused on two potential...