Structure-Guided Design and Biophysical Characterization of Novel Anti-HIV Reagents

Despite over 30 years of effort, an HIV-1 vaccine that elicits protective antibodies still does not exist. Recent clinical studies have identified that during natural infection about 20% of the population is capable of mounting a potent and protective antibody response. Closer inspection of these individuals reveal that a subset of these antibodies, recently termed potent VRC01-like (PVL), derive exclusively from a single human germline heavy chain gene. Induced clonal expansion of the B cell encoding this gene is the first step through which PVL antibodies may be elicited. Unfortunately, naturally occurring HIV gp120s fail to bind to this germline, and as a result cannot be used as the initial prime for a vaccine regimen. We have determined the crystal structure of an important germline antibody that is a promising target for vaccine design efforts, and have set out to engineer a more likely candidate using computationally-guided rational design.

In addition to prevention efforts on the side of vaccine design, recently characterized broadly neutralizing anti-HIV antibodies have excellent potential for use in gene therapy and passive immunotherapy. The separation distance between functional Fabs on an antibody is important due to the sparse distribution of envelop spikes on HIV compared to other viruses. We set out to build and characterize novel antibody architectures by incorporating structured linkers into the hinge region of an anti-HIV antibody b12. The goal was to observe whether these linkers increased the arm-span of the IgG dimer. When incorporated, flexible Gly4Ser repeats did not result in detectable extensions of the IgG antigen binding domains, by contrast to linkers including more rigid domains such as β2-microglobulin, Zn-α2-glycoprotein, and tetratricopeptide repeats (TPRs). This study adds an additional set of linkers with varying lengths and rigidities to the available linker repertoire, which may be useful for the modification and construction of antibodies and other fusion proteins.

New optoelectronic devices using GaAs-GaAlAs epitaxy

Three subjects related to epitaxial GaAs-GaAlAs optoelectronic devices are discussed in this thesis. They are:

1. Embedded Epitaxy

This is a technique of selective multilayer growth of GaAs- Ga_1-xAl_xAs single crystal structures through stripe openings in masking layers on GaAs substrates. This technique results in prismatic layers of GaAs and Ga_1-xAl_xAs "embedded" in each other and leads to controllable uniform structures terminated by crystal faces. The dependence of the growth habit on the orientation of the stripe openings has been studied. Room temperature embedded double heterostructure lasers have been fabricated using this technique. Threshold current densities as low as 1.5 KA/cm² have been achieved.

2. Barrier Controlled PNPN Laser Diode

It is found that the I-V characteristics of a PNPN device can be controlled by using potential barriers in the base regions. Based on this principle, GaAs-GaAlAs heterostructure PNPN laser diodes have been fabricated. GaAlAs potential barriers in the bases control not only the electrical but also the optical properties of the device. PNPN lasers with low threshold currents and high breakover voltage have been achieved. Numerical calculations of this barrier controlled structure are presented in the ranges where the total current is below the holding point and near the lasing threshold.

3. Injection Lasers on Semi-Insulating Substrates

GaAs-GaAlAs heterostructure lasers fabricated on semi-insulating substrates have been studied. Two different laser structures achieved are: (1) Crowding effect lasers, (2) Lateral injection lasers. Experimental results and the working principles underlying the operation of these lasers are presented. The gain induced guiding mechanism is used to explain the lasers' far field radiation patterns. It is found that Zn diffusion in Ga_1-xAl_xAs depends on the Al content x, and that GaAs can be used as the diffusion mask for Zn diffusion in Ga_1-xAl_xAs. Lasers having very low threshold currents and operating in a stable single mode have been achieved. Because these lasers are fabricated on semi-insulating substrates, it is possible to integrate them with other electronic devices on the same substrate. An integrated device, which consists of a crowding effect laser and a Gunn oscillator on a common semi-insulating GaAs substrate, has been achieved.

Heavy metal pollution in sediments at shipbreaking area of Bangladesh

The research was carried out to assess the trace metal concentration in sediments of ship breaking area in Bangladesh. The study areas were separated into Ship breaking Zone and Reference Site for comparative analysis. Metals like Iron ( Fe) was found at 11932 to 41361.71µg.g-1 in the affected site and 3393.37 µg.g-1 in the control site. Manganese (Mn) varied from 2.32 to 8.25 µg.g-1 in the affected site where as it was recorded as 1.8 µg.g-1 in the control area. Chromium(Cr), Nickel (Ni), Zinc(Zn) and Lead (Pb) were also varied from 22.89 to 86.72 µg.g-1; 23.12 to 48.6;83.78 to 142.85 and 36.78 to 147.83 µg.g-1 respectively in the affected site whereas these were recorded as 19; 3.98; 22.22 and 8.82 µg.g-1 in the control site. Copper (Cu); Cadmium (Cd) and Mercury (Hg) concentration were varied from 21.05 to 39.85; 0.57 to 0.94 and 0.05 to 0.11 µg.g-1 in the affected site and 33.0; 0.115 and 0.01 µg.g-1 in the control site. It may conclude that heavy metal pollution in sediments at ship breaking area of Bangladesh is at alarming stage.

Enhanced Thermoelectric Performance at the Superionic Phase Transitions of Mixed Ion-Electron Conducting Materials

The quality of a thermoelectric material is judged by the size of its temperature de- pendent thermoeletric-figure-of-merit (zT ). Superionic materials, particularly Zn₄Sb₃ and Cu₂Se, are of current interest for the high zT and low thermal conductivity of their disordered, superionic phase. In this work it is reported that the super-ionic materials Ag₂Se, Cu₂Se and Cu_1.97Ag_0.03Se show enhanced zT in their ordered, normal ion-conducting phases. The zT of Ag₂Se is increased by 30% in its ordered phase as compared to its disordered phase, as measured just below and above its first order phase transition. The zT ’s of Cu₂Se and Cu_1.97Ag_0.03Se both increase by more than 100% over a 30 K temperatures range just below their super-ionic phase transitions. The peak zT of Cu₂Se is 0.7 at 406 K and of Cu_1.97Ag_0.03Se is 1.0 at 400 K. In all three materials these enhancements are due to anomalous increases in their Seebeck coefficients, beyond that predicted by carrier concentration measurements and band structure modeling. As the Seebeck coefficient is the entropy transported per carrier, this suggests that there is an additional quantity of entropy co-transported with charge carriers. Such co-transport has been previously observed via co-transport of vibrational entropy in bipolaron conductors and spin-state entropy in Na_xCo₂O₄. The correlation of the temperature profile of the increases in each material with the nature of their phase transitions indicates that the entropy is associated with the thermodynamcis of ion-ordering. This suggests a new mechanism by which high thermoelectric performance may be understood and engineered.

Caracterização físico-química e avaliação dos níveis de elementos-traço em sedimentos de manguezais das baías da Ilha Grande e de Sepetiba, RJ, por metodologias de digestão ácida assistidas por micro-ondas

O presente trabalho teve como objetivo principal comparar dois métodos de extração de elementos-traço em sedimentos que são assistidos por radiação micro-ondas. Foram coletadas amostras de sedimentos superficiais em um manguezal localizado na baía da Ilha Grande e em três manguezais localizados na baía de Sepetiba, RJ. As amostras de sedimentos coletadas foram secas, destorroadas e peneiradas. Sub-amostras foram submetidas a análises de caracterização física e química. Os métodos de extração de elementos-traço comparados foram o EPA-3051A e o método água régia assistido por micro-ondas. Os elementos-traço analisados foram: Cd, Cr, Cu, Ni, Pb e Zn. As concentrações dos elementos-traço nos extratos produzidos foram determinadas por FAAS (Espectrometria de absorção atômica com chama). Foram encontradas nos sedimentos dos manguezais estudados concentrações acima dos níveis estabelecidos pela Resolução CONAMA 344/2004 para os elementos-traço: Cd, Ni, Pb e Zn. Os resultados mostraram que as capacidades de extração de elementos-traço dos métodos EPA-3051A e água régia assistido por micro-ondas variam em função das características físico-químicas dos sedimentos. A análise estatística pela técnica de regressão linear simples mostrou que as duas metodologias são estatisticamente similares apenas para o Cr e os valores dos coeficientes de determinação (R2) obtidos obedeceram à seqüência: Cu > Pb > Zn > Ni > Cr > Cd. Na análise pela técnica estatística de regressão linear múltipla ocorreram melhorias estatisticamente significativas nos coeficientes de determinação quando foram incluídas as seguintes variáveis independentes: potencial redox e fósforo total para o Cd, argila para o Cr, areia para o Ni e fósforo total para o Pb e para o Zn

Electron transfer in chemically and genetically modified myoglobins

The temperature dependences of the reduction potentials (E^o') of wildtype human myoglobin (Mb) and three site-directed mutants have been measured by using thin-layer spectroelectrochemistry. Residue Val68, which is in van der Waals contact with the heme in Mb, has been replaced by Glu, Asp, and Asn. At pH 7.0, reduction of the heme iron (III) in the former two proteins is accompanied by uptake of a proton by the protein. The changes in E^o', and the standard entropy (ΔS^o') and enthalpy (ΔH^o') of reduction in the mutant proteins were determined relative to values for wild-type; the change in E^o' at 25°C was about -200 millivolts for the Glu and Asp mutants, and about -80 millivolts for the Asn mutant. Reduction of Fe(III) to Fe(II) in the Glu and Asp mutants is accompanied by uptake of a proton. These studies demonstrate that Mb can tolerate substitution of a buried hydrophobic group by potentially charged and polar residues, and that such amino acid replacements can lead to substantial changes in the redox thermodynamics of the protein.

Through analysis of the temperature dependence and shapes of NMR dispersion signals, it is determined that a water molecule is bound to the sixth coordination site of the ferric heme in the Val68Asp and in the Val68Asn recombinant proteins while the carboxyl group of the sidechain of Glu68 occupies this position in Val68Glu. The relative rhombic distortions in the ESR spectra of these mutant proteins combined with H₂¹⁷O and spin interconversion experiments performed on them confirm the conclusions of the NMRD study.

The rates of intramolecular electron transfer (ET) of (NH₃)₅Ru-His48 (Val68Asp, His81GIn, Cys110AIa)Mb and (NH₃)₅Ru-His48 (Val68GIu,His81GIn,Cys110Ala)Mb were measured to be .85(3)s^-1 and .30(2)s^-1, respectively. This data supports the hypothesis that entropy of 111 reduction and reorganization energy of ET are inversely related. The rates of forward and reverse ET for (NH₃)₅ Ru-His48 (Val68GIu, His81 GIn, Cys110AIa)ZnMb -7.2(5)•10⁴s^-1and 1.4(2)•10⁵s^-1, respectively- demonstrate that the placement of a highly polar residue nearby does not significantly change the reorganization energy of the photoactive Zn porphyrin.

The distal histidine imidazoles of (NH₃)₄isnRu-His48 SWMb and (NH₃)₅Ru-His48 SWMb were cyanated with BrCN. The intramolecular ET rates of these BrCN-modified Mb derivatives are 5.5(6)s^-1 and 3.2(5)s^-1, respectively. These respective rates are 20 and 10 times faster than those of their noncyanated counterparts after the differences in ET rate from driving force are scaled according to the Marcus equation. This increase in ET rate of the cyanated Mb derivatives is attributed to lower reorganization energy since the cyanated Mb heme is pentacoordinate in both oxidation states; whereas, the native Mb heme loses a water molecule upon reduction so that it changes from six to five coordinate. The reorganization energy from Fe-OH₂ dissociation is estimated to be .2eV. This conclusion is used to reconcile data from previous experiments in our lab. ET in photoactive porphyrin-substituted myoglobins proceed faster than predicted by Marcus Theory when it is assumed that the only difference in ET parameters between photoactive porphyrins and native heme systems is driving force. However, the data can be consistently fit to Marcus Theory if one corrects for the smaller reorganization in the photoactive porphyrin systems since they do not undergo a coordination change upon ET.

Finally, the intramolecular ET rate of (NH₃)₄isnRu-His48 SWMb was measured to be 3.0(4)s^-1. This rate is within experimental error of that for (NH₃)₄pyrRu-His48 SWMb even though the former has 80mV more driving force. One likely possibility for this observation is that the tetraamminepyridineruthenium group undergoes less reorganization upon ET than the tetraammineisonicotinamideruthenium group. Moreover, analysis of the (NH₃)₄isnRu-His48 SWMb experimental system gives a likely explanation of why ET was not observed previously in (NH₃)₄isnRu-Cytochrome C.

Light-matter Interactions in Semiconductors and Metals: From Nitride Optoelectronics to Quantum Plasmonics

This thesis puts forth a theory-directed approach coupled with spectroscopy aimed at the discovery and understanding of light-matter interactions in semiconductors and metals.

The first part of the thesis presents the discovery and development of Zn-IV nitride materials.The commercial prominence in the optoelectronics industry of tunable semiconductor alloy materials based on nitride semiconductor devices, specifically InGaN, motivates the search for earth-abundant alternatives for use in efficient, high-quality optoelectronic devices. II-IV-N2 compounds, which are closely related to the wurtzite-structured III-N semiconductors, have similar electronic and optical properties to InGaN namely direct band gaps, high quantum efficiencies and large optical absorption coefficients. The choice of different group II and group IV elements provides chemical diversity that can be exploited to tune the structural and electronic properties through the series of alloys. The first theoretical and experimental investigation of the ZnSnxGe1−xN2 series as a replacement for III-nitrides is discussed here.

The second half of the thesis shows ab−initio calculations for surface plasmons and plasmonic hot carrier dynamics. Surface plasmons, electromagnetic modes confined to the surface of a conductor-dielectric interface, have sparked renewed interest because of their quantum nature and their broad range of applications. The decay of surface plasmons is usually a detriment in the field of plasmonics, but the possibility to capture the energy normally lost to heat would open new opportunities in photon sensors, energy conversion devices and switching. A theoretical understanding of plasmon-driven hot carrier generation and relaxation dynamics in the ultrafast regime is presented here. Additionally calculations for plasmon-mediated upconversion as well as an energy-dependent transport model for these non-equilibrium carriers are shown.

Finally, this thesis gives an outlook on the potential of non-equilibrium phenomena in metals and semiconductors for future light-based technologies.

Mass measurements of some proton-rich nuclei

The Q values and 0^o cross sections of (He³, n) reactions forming seven proton-rich nuclei have been measured with accuracies varying from 6 to 18 keV. The Q values (in keV) are: Si²⁶ (85), S³⁰ (-573), Ar³⁴ (-759), Ti⁴² (-2865), Cr⁴⁸ (5550), Ni⁵⁶ (4513) and Zn⁶⁰ (818). At least one excited state was found for all but Ti⁴². The first four nuclei complete isotopic spin triplets; the results obtained agree well with charge-symmetry predictions. The last three, all multiples of the α particle, are important in the α and e-process theories of nucleo-synthesis in stars. The energy available for β decay of these three was found by magnetic spectrometer measurements of the (He³, p) Q values of reactions leading to V⁴⁸, Co⁵⁶, and Cu⁶⁰. Many excited states were seen: V⁴⁸ (3), Co⁵⁶ (15), Cu⁶⁰ (23). The first two states of S³⁰ are probably 0⁺ and 2⁺ from (He³, n) angular distribution measurements. Two NaI γ-ray measurements are described: the decay of Ar³⁴ (measured ^Ƭ1/2 = 1.2 ± 0.3s) and the prompt γ-ray spectrum from Fe⁵⁴(He³, nγ)Ni⁵⁶. Possible collective structure in Ni⁵⁶ and Ca⁴⁰, both doubly magic, is discussed.

The (He³, n) neutron energy and yield measurements utilized neutron-induced nuclear reactions in a silicon semiconductor detector. Cross sections for the most important detection processes, Si²⁸ (n, α) Mg²⁵ and Si²⁸ (n, p) Al²⁸, are presented for reactions leading to the first four states of both residual nuclei for neutron energies from 7.3 to 16.4 MeV. Resolution and pulse-height anomalies associated with recoil Mg²⁵ and Al²⁸ ions are discussed. The 0^o cross section for Be⁹ (α, n) C¹², used to provide calibration neutrons, has been measured with a stilbene spectrometer for n_o (5.0 ≤ E_α ≤ 12 MeV), n₁ (4.3 ≤ E_α ≤ 12.0 MeV) and n₂ (6.0 ≤ E_α ≤ 10.1 MeV). Resonances seen in the n_o yield may correspond to nine new levels in C¹³.

Mutagênese ambiental: estabelecimento de valores de referência para manguezais da Baía de Ilha Grande

Estudos ambientais têm demonstrado que substâncias geradas por processos antropogênicos podem causar efeitos prejudiciais interferindo no equilíbrio natural do ecossistema. Manguezais exercem funções essenciais nos ciclos biológicos e constituem Área de Proteção Permanente no Brasil. Infelizmente, eles estão sendo degradados acima do seu limite de suporte, levando a uma redução das áreas remanescentes no mundo. Este trabalho apresenta os resultados de mutagenicidade e genotoxicidade observados em quatro amostragens (PI, V, O e PII) entre 2009 e 2010, relacionados com metais e hidrocarbonetos policíclicos aromáticos (HPA) em sedimento de mangue para a caracterização dos valores de referência. Os testes de genotoxicidade foram feitos a partir de hemócitos do caranguejo Goniopsis cruentata, coletados em um ecossistema potencialmente não poluído do Brasil, localizado no sul do Rio de Janeiro (Parati/RJ), chamado de "Saco do Mamanguá". Coleta, armazenamento e manipulação dos sedimentos e material biológico de cinco pontos de amostragem (M1- M5) foram processados de acordo com normas norte-americanas reconhecidas. A identificação das substâncias químicas foi realizada com extratos de sedimentos e utilizada no bioensaio Salmonella/microssoma (Kado). A avaliação de potenciais danos genotóxicos estabelecidos foi realizada através do Teste de Micronúcleo, que apresentou valores significativos na amostra V. Resultados negativos foram observados para as cepas de Salmonella typhimurium TA97, TA98, TA100 e TA102, tanto na ausência quanto na presença de fração de metabolização exógena de mamíferos (S9 mix 4%) em todas as análises. A quantificação por cromatografia gasosa com detecção por espectrometria de massas dos 16 HPA prioritários em termos de conservação ambiental apresentou valores baixos nas duas primeiras amostragens (PI e V) e nulos nas coletas seguintes (O e PII), nos mesmos pontos. De acordo com os valores utilizados nos Estados Unidos e Canadá como referência, os detectados por nós não são considerados como toxicantes ambientais positivos, com exceção do Benzo(a)pireno, que em M1V apresentou valores um pouco acima do limite a partir do qual já podem ser observados pequenos efeitos na biota. A análise dos metais (Cd, Cr, Cu, Fe, Mn, Ni, Pb e Zn) por Espectrometria de Absorção Atômica inicialmente realizada com a água intersticial foi melhor interpretada a partir da matriz sedimento. Este estudo contribuirá com a implementação de indicadores para valores de referência em mangue.