The current environment for drug discovery and disease treatment relies heavily on genomic analysis, structural biology and chemical biology techniques. With the enormous advances in genomic analysis and structural biology, the use of and desire for targeted therapies has increased. However, as more genomic data for cancer disease state pathology becomes available we must ask increasingly difficult questions and even produce new technologies, such as activity-based probes, to answer these questions. In particular, targeted kinase inhibitors for the treatment of cancer has become a mainstay for drug development for both industry and academia, but it is evident that the genomic data is not always indicative of protein expression. Additionally, protein expression alone does not completely characterize functional activity. Therefore, in order to more accurately validate drug targets and predict drug efficacy, we must not only identify possible targets but also determine their activity in vivo. The goal of this work was to develop a probe for Protein Kinase A that would act by alkylating a conserved cysteine in the substrate-binding pocket of the enzyme. We hypothesized that by targeting the substrate-binding pocket we could effectively utilize the natural substrate selectivity filters as well as take into account multiple endogenous regulatory mechanisms. We produced probes utilizing portions of the pseudosubstrate inhibitor PKI that demonstrate the ability to label the catalytic subunit of Protein Kinase A in an activity-dependent manner, thus making it an important first step in a new class of activity-based probes for the kinome.

Calcium (Ca2+)/calmodulin-dependent kinase 2 (CaMK-II) is one member of a family of Ca2+/calmodulin-dependent protein kinases that responds to intracellular Ca2+ signals (Hudmon, A. and H. Schulman (2002)). CaMK-II is a multifunctional regulator of transcription, cell cycle progression, cell motility and neuronal development. (Wang, C., et al. (2008), Easley, C. A. IV, et al. (2008), Osterhoff, M., et al. (2003), Faison, M. O., et al. (2002)). Recently, CaMK-II has been shown to be important in the early development of vertebrates. In developing zebrafish, disruption of CaMK-II expression has been shown to induce phenotypes similar to those documented in several human diseases. The identification of the tissue-specific binding partners and substrates of CaMK-II which are responsible for specific developmental fates remains a key step in understanding this important protein kinase family. In this thesis research, specific “substrate-trapping” mutants of CaMK-II were designed, introduced into a variety of rodent and human cell lines in culture and used in conjunction with tandem mass spectrometry to identify binding partners, such as β-actin, tropomodulin-3 and Fli-I as well as novel, putative substrates, such as the tumor suppressor protein 53 (p53). This approach was subsequently applied to zebrafish embryos where an overlapping subset of CaMK-II binding proteins to those found in mammalian cell culture were identified. This project represents one of the first studies to identify binding proteins in zebrafish embryos using epitope tagging and mass spectrometry. This research has also established a technical framework for the use of mass spectrometry to characterize the developmental proteome of whole zebrafish embryos or specific zebrafish tissues at any developmental time point.

Different methods were developed for the preparation of bulk ZnO substrates. Remarkable improvement on the surface, optical and crystalline quality of the bulk ZnO substrate was achieved. ZnO substrates with an atomically flat surface exhibiting terrace-like features were used as a substrate for GaN grown by MBE. High-resolution x-ray diffraction and low temperature PL results show that similar high quality GaN layers can be achieved on both annealed O-face and Zn-face ZnO substrates. The prospect of the device applications of GaN epitaxy on ZnO, including AlGaN/GaN MODFET structure on ZnO and GAN/ZnO based p-n junction were discussed.

Filamentous aeroterestrial green algae genus Klebsormidium occurs in a very wide range of freshwater and terrestrial habitats. Recent results of molecular investigations led to the finding that the diversity within this genus is far greater than expected on the basis of the morphological features, and that the traditional phenotypic species concept is insufficient. I tried to differentiate phylogenetic lineages within the genus Klebsormidium by thein different biogeographical distribution and environmental preferences. Since no study dealing with the biogeographic pattern of aeroterrestrial algae was so far undertaken, another aim of this work was to test validity of the protist ubiquity model in aeroterrestrial habitats. I studied this issues based on the chloroplast rbcL molecular marker. Based on the obtained data I found that the geographic definition of particular Klebsormidium lineages turns out to be unusable because of the cosmopolitan occurrence of almost all genotypes. However, the data obtained from the substrate specificity study shows that clear ecological preferences exist within the genus Klebsormidium and could be simply used to define different lineages within the genus.

An ever-increasing demand for wider bandwidths in communication, radar, and imaging systems has emerged. In order to facilitate this growing demand, progressive research into millimeter-wave technologies has become vital in achieving next generation networks such as 5G. Being cost effective and easy to manufacture, Substrate Integrated Waveguide (SIW) circuits have been demonstrated as a viable candidate for high-frequency applications due to their low-loss, high quality-factor, and high power-handling capabilities.Research on beam-forming networks, specifically the Butler matrix, has demonstrated powerful beam-steering capabilities through the use of passive component networks. Through these clever configurations, a cost effective and robust option is available for us to use. In order to further millimeter-wave research in this area, this thesis presents a modified configuration of the Butler Matrix in SIW that is physically reconfigurable; by separating the Butler matrix from the antenna array at a pre-selected point, the array can be easily interchanged with other 1-Dimensional,and 2-Dimensional slot antenna arrays. Although this system does not fall under the rigorous definitions of Reconfigurable Antennas, it should be noted that the interchangeability of 1 and 2 dimensional arrays is not typically expressed in Butler matrix configurations. Design and simulations are carried out in CST Microwave Studio to inspect individual components as well as system characteristics. Circuit prototypes are then manufactured and tested in an anechoic chamber to validate simulation results and the design approach. / Graduate / 2020-07-17

The Gangliosidoses, to include GM1 gangliosidosis and Sandhoff disease are a class of incurable lysosomal storage disorders characterized by an abnormal accumulation of gangliosides leading to progressive neurodegeneration and eventually death. GM1 gangliosidosis is caused by a genetic defect in the lysosomal-specific acid β-galactosidase, which results in the massive accumulation of ganglioside GM1 primarily in the central nervous system (CNS). Sandhoff disease (SD) results from a defect in the β-subunit of β- Hexosaminidase A and leads to the accumulation of ganglioside GM2 and its asialo derivative (GA2). As there are no effective therapies for these glycosphingolipid (GSL) storage disorders, I studied substrate reduction therapy (SRT), stem cell therapy, and adeno-associated viral (AAV) gene therapy in neonatal mice as early intervention therapies and were effective in reducing CNS GSL storage. In addition, AAV gene therapy was also evaluated in the adult GM1 gangliosidosis mice. Furthermore, analysis of the brain lipids in mice, cats, and humans with Sandhoff disease revealed that the SD cat model is intermediate between the SD mouse and the SD patient with respect to GM2 and GA2 accumulation. These findings are the first to compare the different therapies and provide valuable information for the translation of mouse studies to clinical trials in patients. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Student Number : 0002482E - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Proteins in the glutathione transferase family share a common fold. The close packing of secondary structures in the thioredoxin fold in domain 1 forms a compact hydrophobic core. This fold has a bababba topology and most proteins/domains with this fold have a topologically conserved isoleucine residue at the N-terminus of a-helix 3. Class Alpha glutathione transferases are one of 12 classes within the glutathione transferase family. To investigate the role of the conserved isoleucine residue in the structure, function and stability of glutathione transferases, homodimeric human glutathione transferase A1-1 (hGST A1-1) was used as a representative of the GST family. Ile71 was replaced with valine and the properties of I71V hGST A1-1 were compared with those of wildtype hGST A1-1. The spectral properties monitored using far-UV CD and tryptophan fluorescence indicated little change in secondary or tertiary structure confirming the absence of any gross structural changes in hGST A1-1 due to the incorporation of the mutation. Both wildtype and mutant dimeric proteins were determined to have a monomeric molecular mass of 26 kDa. The specific activity of I71V hGST A1-1 (130 mmol/min/mg) was three times that of wildtype hGST A1-1 (48 mmol/min/mg). I71V hGST A1-1 showed increased kinetic parameters compared to wildtype with a 10-fold increase in kcat/Km for CDNB. The increase in Km of I71V hGST A1-1 suggests the mutation had a negative effect on substrate binding. The DDG for transition state stabilisation was –5.82 kJ/mol which suggest the I71V mutation helps stabilise the transition state of the SNAR reaction involving the conjugation of reduced glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB). A 2-fold increase in the IC50 value for I71V hGST A1-1 (11.3 mM) compared to wildtype (5.4 mM) suggests that the most noticeable change due to the mutation occurs at the H-site of the active site. Conformational stability studies were performed to determine the contribution of Ile71 to protein stability. The non-superimposability of I71V hGST A1-1 unfolding curves and the decreased m-value suggest the formation of an intermediate state. The conformational stability of I71V hGST A1-1 (16.5 kcal/mol) was reduced when compared to that of the wildtype (23 kcal/mol). ITC was used to dissect the binding energetics of Shexylglutathione to wildtype and I71V hGSTA1-1. The ligand binds 5-fold more tightly to wildtype hGST A1-1 (0.07 mM) than I71V hGST A1-1 (0.37 mM). The I71V mutant displays a larger negative DCp than wildtype hGST A1-1 (DDCp = -0.41 kJ/mol/K). This indicates that a larger solvent-exposed hydrophobic surface area is buried for I71V hGST A1-1 than for wildtype hGST A1-1 upon the binding of S-hexylglutathione. Overall the results suggest that Ile71 conservation is for the stability of the protein as well as playing a pivotal indirect role in catalysis and substrate binding.

Thesis advisor: Thomas N. Seyfried / Gangliosides are glycosphingolipids (GSLs) containing sialic acids that play numerous roles in neuronal maturation, apoptotic signaling, angiogenesis, and cell surface receptor activity. The GM1 and GM2 gangliosidoses are a series of autosomal recessive lysosomal storage disorders (LSDs) characterized by an inability to degrade these lipid molecules. GM1 gangliosidosis is caused by a mutation in the lysosomal hydrolase β-galactosidase, resulting in neuronal storage of ganglioside GM1 and asialo GA1. Tay-Sachs (TS) and Sandhoff Disease (SD) are GM2 gangliosidoses caused by mutations in either the α or β subunits, respectively, of the heterodimeric protein β- hexosaminidase A, resulting in the storage of ganglioside GM2 and asialo GA2. The accumulation of excess ganglioside in the central nervous system leads to abnormal intracellular vacuoles, neuronal loss, demyelination, ataxia, dementia, and premature death. In my studies, I have shown that accumulation of GM1 ganglioside may not coincide with secondary storage of cholesterol, by providing evidence that cholesterol-binding fluorescent molecule filipin reacted to GM1 ganglioside in the absence of cholesterol. In an effort to combat the early-onset gangliosidoses, I have explored the effects of combining Neural Stem Cells (NSCs) with Substrate Reduction Therapy (SRT) in juvenile Sandhoff mice. The analysis showed that SRT was more effective than NSCs in reducing stored GM2 and GA2 in young mice, and no synergy was observed. In adult GM1 gangliosidosis, Tay- Sachs, and Sandhoff mice, Adeno-Associated Viral (AAV) vector gene therapy was used to restore therapeutic levels of wild-type enzyme to the CNS. AAV therapy corrected ganglioside storage and ameliorated myelin-associated lipid loss in all tissues assayed, increasing motor performance and life in effected animals. Lastly, AAV therapy was also successful in a feline model of Sandhoff disease. These results in juvenile and adult model systems point the way towards multiple effective clinical therapies in the near future. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Os crustáceos estão entre os invertebrados marinhos mais diversos e tendem a se associar com outros organismos a fim de encontrar abrigo, oferta de alimento e um lugar seguro para reprodução. Aspectos reprodutivos como fecundidade, tamanho dos embriões e energia investida na reprodução são os principais parâmetros para a compreensão da história natural de uma espécie e/ou população. Assim, esse trabalho foi desenvolvido no sentido de investigar tais parâmetros e a influência do tipo de substrato ocupado. A espécie utilizada como modelo de estudo foi o caranguejo porcelanídeo Pachycheles monilifer coletado em dois substratos biológicos diferentes, no briozoário Schizoporella errata no píer do Itaguá e no poliqueto Phragmatopoma caudata na praia Grande, ambas as paias localizadas em Ubatuba, litoral norte de SP. Somente os embriões em estágio inicial de desenvolvimento foram utilizados para os cálculos de fecundidade e investimento reprodutivo (RO). Os embriões foram contados e medidos sob esteromicroscópio. Para obtenção do RO foram utilizados os pesos secos das fêmeas ovígeras e das massas de embriões, previamente secos em estufa. Além dos aspectos reprodutivos, análises de distância genética também foram realizadas pelo sequenciamento de genes mitocôndrias (16S e COI) de exemplares de P. monilifer. A estimativa da porcentagem de variação genética intra e interespecífica, a possibilidade de fluxo gênico entre indivíduos de diferentes praias do estado de São Paulo foram verificadas. A partir desses dados confirmou-se a validade da espécie, assim como o fluxo gênico contínuo entre indivíduos dessas duas praias. A baixa divergência genética permite concluir que as diferenças reprodutivas encontradas são, portanto um reflexo das condições proporcionadas pelo ambiente. Os animais simbiontes do briozoário, assim como as fêmeas ovígeras, são maiores que os associados ao poliqueta e também possuem maior fecundidade e investimento reprodutivo, porém não foram encontradas diferenças em relação ao tamanho dos embriões. No substrato do briozoário o estresse reprodutivo parece ser menor que no substrato do poliqueto, já que aquele substrato proporciona um ambiente fisicamente mais estável para os organismos simbiontes. No substrato do poliqueto, além do alto hidrodinamismo local, o espaço disponível na colônia parece ser mais restrito e pode limitar o tamanho de seus habitantes. Os animais simbiontes do briozoário parecem não serem influenciados por fatores externos à colônia, entretanto o mesmo não foi observado nos simbiontes do poliqueto. Tais diferenças reprodutivas refletem o tipo de substrato ocupado, evidenciando a plasticidade fenotípica da espécie para uma melhor adaptação local. / The crustaceans are the most diverse of the marine invertebrates and many of them tend to associate with other organisms where they can find shelter, food supply and a safe place to reproduce. Reproductive traits as fecundity, egg size and reproductive output (RO) are the most important aspects to understand the species life history. This study aimed to investigate the reproductive traits in relation to the substrate that the organisms live. The studied species used as model was the porcellanid crab Pachycheles monilifer that was collected in two different substrates, one of them is the bryozoan Schizoporella errata at the Itaguá beach and the other is the polychaete Phragmatopoma caudata at Grande beach, both are located in Ubatuba, northern coast of São Paulo. Only the initial stage of eggs was used to estimate the crab fecundity and RO. The eggs were counted and measured in a stereomicroscope. To obtain the RO the female and the egg mass dry weight, which were previously dried in an oven, were used. Besides the reproductive aspects, the genetic distance between the organisms was valued with partial sequences of the mitochondrial genes 16S and COI. The estimate of the intra and interspecific genetic variation and the possibility of gene flow between the individuals from the beaches in SP were analyzed too. Therefore, with all these analyses it was possible to confirm that the species is valid and that there is gene flow among the populations. The low genetic divergence allowed us to conclude that the different reproductive traits found among the populations were due to the different substrates that the organisms inhabit. The bryozoan symbionts are larger than the polychaete symbionts as well as the females fecundity and the RO, but there is no difference in egg size. In the bryozoan substrate it seems like the physical stress is smaller than in polychaete and it promotes a more stable habitat for the crabs reproduce. In the polychaete substrate, beyond the high hydrodynamic location, the available space in the colony appears to be narrower, which could limit the size of its inhabitants. The bryozoan symbionts do not seem to be influenced by the external factors of the colony, though the same was not observed in the polychaete colony. Such differences in reproduction reflect the kind of substrate inhabited, highlighting the phenotypic plasticity of the species to a local better adaptation.

Este experimento avaliou, em casa de vegetação, o desenvolvimento de mudas de cafeeiro produzidas em tubetes, sob o efeito de malhas utilizadas para sombreamento. Os tratamentos utilizados foram a malha negra 50%, conhecida comercialmente como "sombrite" ou "tela preta", considerada padrão para o desenvolvimento destas mudas, e malhas termo-refletoras com quatro diferentes percentagens de atenuação da radiação solar, sendo as malhas 40%, 50%, 60% e 70%. O delineamento experimental adotado foi em blocos casualizados contendo os 5 tratamentos, com 4 repetições, totalizando-se assim 20 parcelas. As parcelas foram representadas por 20 bancadas, cada qual contendo fechamento superior com um tipo de malha para sombreamento, e também fechamento nas laterais, atenuando a radiação solar lateral. Cada parcela foi composta por 175 mudas, sendo 161 para bordadura, e 14 para avaliações. As irrigações, realizadas diariamente, foram implementadas com a utilização de uma barra motorizada semi-automatizada que se deslocava longitudinalmente na casa de vegetação em ambos os sentidos. A lâmina desejada era ajustada de acordo com a velocidade de deslocamento da barra que era regulada por meio de um inversor de freqüência, responsável por este controle. Para monitorar o microclima, foi instalado interiormente a cada parcela um psicrômetro não ventilado e de tempos em tempos foram realizadas medidas de radiação solar global, fotossinteticamente ativa e o saldo de radiação (SR), estabelecendo-se relações de transmissividade global (TRG) e transmissividade fotossinteticamente ativa (TRFA). As variáveis fisiológicas utilizadas para avaliar o desenvolvimento das mudas foram área foliar, altura das plantas, diâmetro do caule, e, ao final do experimento, foram realizadas análises de matéria seca da parte aérea, do sistema radicular e matéria seca total. A avaliação dos dados psicrométricos mostrou que a temperatura do ar pouco variou nas parcelas, assim como a umidade relativa do ar. Com relação a TRG e TRFA, verificou-se que malhas com menor atenuação da radiação solar apresentaram em geral maior transmissividade, conforme esperado. Para a variável saldo de radiação, a presença de interferências ambientais inviabilizou a maior parte dos dados, verificando-se em apenas parte deles uma tendência de maior SR nas malhas 40% e 50%. A avaliação das variáveis fisiológicas mostrou que a área foliar não apresentou diferença significativa entre os tratamentos. Já para altura da planta e diâmetro do caule, verificou-se que as mudas produzidas sob as malhas termo-refletoras 40% e 50% foram superiores àquelas produzidas sob malha negra 50% e malhas termo-refletoras 60% e 70%, respectivamente. Nas avaliações de matéria seca, verificou-se que as mudas produzidas sob as malhas termo-refletoras 40% e 50%, e malha negra 50% acumularam mais matéria seca comparativamente àquelas produzidas sob malhas termo-refletoras 60% e 70%. Assim, nas condições em que desenvolveu-se o experimento, concluiu-se que as malhas termo-refletoras 40% e 50% e negra 50% apresentaram melhores resultados que as malhas termo-refletoras 60% e 70%. / This experiment evaluated, in greenhouse conditions, the development of coffee seedlings produced in plastic tubes, under the effect of screens used for shading. The treatments were black screen 50%, known commercially as "sombrite" or as "black screen", considered standard for the development of these seedlings, and thermo-reflective screens with four different percentages of attenuation of the solar radiation, being meshes 40%, 50%, 60%, and 70%. The adopted experimental delineation was randomized blocks with 5 treatments and 4 replicates, making up 20 parcels. The parcels were represented by 20 benches; each one with superior closing system with one type of screen, and also side closing system, attenuating the side solar radiation. Each parcel was composed by 175 seedlings, from which 161 were used as a border, and 14 used for the evaluations. The irrigation, daily carried out, was implemented with the use of a semi-automated motorized bar that dislocated longitudinally along the greenhouse in both directions. The desired water amount was set in accordance to the bar displacement speed that was established by means of a frequency inverter, responsible for this control. To monitor the microclimate, one psicrometer was installed inside each parcel and, from time to time, global solar radiation, photosynthetically active radiation, and net radiation (SR) measures were taken, establishing, this way, relations of global transmissivity (TRG) and photosynthetically active transmissivity (TRFA). The physiological variables used to evaluate the development of the seedlings were leaf area, seedlings height, and stalk diameter. Analyses on the dry matter of aerial part and root system and total dry matter were carried out at the end of the experiment. The evaluation of the psicrometers data showed that the temperature of air gently varied in the parcels, as well as the air relative humidity. Relating to TRG and to TRFA, it was verified that screens with less attenuation of the solar radiation presented higher transmissivity in general, as expected. For the variable net radiation, the presence of environment interference made most data impracticable, verifying a partial trend of higher SR in the screens 40% and 50%. The evaluation of the physiological variables showed that the leaf area did not present significant difference among the treatments. Yet, it was verified that, for seedlings height and stalk diameter, the seedlings produced under thermo-reflective screens 40% and 50% were superior to those produced under black screen 50% and thermo-reflective screen 60% and 70%. It was also verified that, for the evaluations of dry matter, the seedlings produced under thermo-reflective screens 40% and 50%, and black screen 50% comparatively accumulated more dry matter than those produced under thermo-reflective screens 60% and 70%. Thus, under the conditions where the experiment was carried out, it can be concluded that the thermo-reflective screens 40% and 50% and black screen 50% presented better results than the thermo-reflective screens 60% and 70%.