The retinoblastoma-susceptibility gene product binds directly to the human TATA-binding protein-associated factor TAFII250.

RB, the protein product of the retinoblastoma tumor-suppressor gene, regulates the activity of specific transcription factors. This regulation appears to be mediated either directly through interactions with specific transcription factors or through an alternative mechanism. Here we report that stimulation of Sp1-mediated transcription by RB is partially abrogated at the nonpermissive temperature in ts13 cells. These cells contain a temperature-sensitive mutation in the TATA-binding protein-associated factor TAFII250, first identified as the cell cycle regulatory protein CCG1. The stimulation of Sp1-mediated transcription by RB in ts13 cells at the nonpermissive temperature could be restored by the introduction of wild-type human TAFII250. Furthermore, we demonstrate that RB binds directly to hTAFII250 in vitro and in vivo. These results suggest that RB can confer transcriptional regulation and possibly cell cycle control and tumor suppression through an interaction with TFIID, in particular with TAFII250.

p53-dependent growth arrest of REF52 cells containing newly amplified DNA.

The rat cell line REF52 is not permissive for gene amplification. Simian virus 40 tumor (T) antigen converts these cells to a permissive state, as do dominant negative mutants of p53, suggesting that the effect of T antigen is due mainly to its ability to bind to p53. To manipulate permissivity, we introduced a temperature-sensitive mutant of T antigen (tsA58) into REF52 cells and selected for resistance to N-(phosphonacetyl)-L-aspartate (PALA). Most freshly isolated PALA-resistant colonies, each of approximately 200 cells, selected at a permissive temperature, arrested when shifted to a nonpermissive temperature. Growth arrest was stable, with no evidence of apoptosis, as long as T antigen was absent but was reversed when T antigen was restored. In contrast, PALA-resistant clones grown to approximately 10(7) cells at a permissive temperature did not arrest when shifted to a nonpermissive temperature. All PALA-resistant clones examined had amplified carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) genes, present in structures consistent with a mechanism involving bridge-breakage-fusion (BBF) cycles. We propose that p53-mediated growth arrest operates only early during the complex process of gene amplification, when newly formed PALA-resistant cells contain broken DNA, generated in BBF cycles. During propagation under permissive conditions, the broken DNA ends are healed, and, even though the p53-mediated pathway is still intact at a nonpermissive temperature and the cells contain amplified DNA, they are not arrested in the absence of broken DNA. The data support the hypothesis that BBF cycles are an important mechanism of amplification and that the broken DNA generated in each cycle is a key signal that regulates permissivity for gene amplification.

Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast.

The CDC47 gene was isolated by complementation of a cdc47 temperature-sensitive mutant in Saccharomyces cerevisiae and was shown to encode a predicted polypeptide, Cdc47, of 845 aa. Cdc47 belongs to the Cdc46/Mcm family of proteins, previously shown to be essential for initiation of DNA replication. Using indirect immunofluorescence microscopy and subcellular fractionation techniques, we show that Cdc47 undergoes cell cycle-regulated changes in its subcellular localization. At mitosis, Cdc47 enters the nucleus, where it remains until soon after the initiation of DNA replication, when it is rapidly exported back into the cytoplasm. Cdc47 protein levels do not vary with the cell cycle, but expression of CDC47 and nascent synthesis of Cdc47 occur late in the cell cycle, coinciding with mitosis. Together, these results show that Cdc47 is not only imported into the nucleus at the end of mitosis but is also exported back into the cytoplasm at the beginning of S phase. The observation that Cdc47 is exported from the nucleus at the beginning of S phase has important implications for how initiation of DNA replication is controlled.

Library of medium-resolution Fiber Optic Echelle spectra of F, G, K, and M field dwarfs to giant stars

We present a library of Penn State Fiber Optic Echelle (FOE) observations of a sample of field stars with spectral types F to M and luminosity classes V to I. The spectral coverage is from 3800 to 10000 Å with a nominal resolving power of 12,000. These spectra include many of the spectral lines most widely used as optical and near-infrared indicators of chromospheric activity such as the Balmer lines (Hα to H epsilon), Ca II H & K, the Mg I b triplet, Na I D_1, D_2, He I D_3, and Ca II IRT lines. There are also a large number of photospheric lines, which can also be affected by chromospheric activity, and temperature-sensitive photospheric features such as TiO bands. The spectra have been compiled with the goal of providing a set of standards observed at medium resolution. We have extensively used such data for the study of active chromosphere stars by applying a spectral subtraction technique. However, the data set presented here can also be utilized in a wide variety of ways ranging from radial velocity templates to study of variable stars and stellar population synthesis. This library can also be used for spectral classification purposes and determination of atmospheric parameters (T_eff, log g, [Fe/H]). A digital version of all the fully reduced spectra is available via ftp and the World Wide Web (WWW) in FITS format.

Degradação enzimática de clorofenol em microrreator.

O microrreator faz parte de conjunto de dispositivos de uma nova e promissora tecnologia, que podem ser chamados de micro fabricados, atuante em campos como a da química, biológica, farmacêutica, engenharia química e biotecnologia. Trata-se de um dispositivo que possibilita reação química, tais como os reatores convencionais, mas com dimensões menores, com canais na escala micrométrica. A tecnologia de miniaturização de dispositivos para reações químicas vem se expandindo promovendo uma importante evolução, com microssistemas que abrange dispositivos mais eficazes, com configuração e geometrias específicas e menor consumo de energia, onde reações com elevadas taxas de transporte podem ser usadas para muitas finalidades diferentes, tais como, reações rápidas, mistura, reações sensíveis à temperatura, temperatura de homogeneização, ou até mesmo precipitação de nano partículas. Devido sua escala ser extremamente reduzida em relação à escala macro, oferecem um sistema que permite uma investigação do processo em um curto espaço de tempo, sendo muito útil para o rastreio de substratos, enzimas, condições de reação, bem como a determinação de parâmetros cinéticos. O presente trabalho teve por objetivo estudar a biodegradação enzimática de 2,4,6-Triclorofenol, com a utilização das enzimas Lacase e Soybean Peroxidase em microrreator da Syrris com volume de 250 ?l, que permite o estudo de cinéticas muito rápidas. Para as análises de degradação utilizou-se duas enzimas, a Lacase em concentrações de 0,05; 0,1 e 0,2 mg/ml; e a Soybean Peroxidase em concentrações de 0,0005; 0,001 e 0,002 mg/ml com a adição de Peróxido de Hidrogênio. Através dos ensaios realizados obteve-se dados experimentais da reação enzimática, possibilitando a verificação da taxa inicial de reação e sua cinética. Posteriormente, realizou-se as análises em simulação utilizando os dados experimentais, que através de um sistema de EDOs estimando inicialmente as constantes cinéticas k1, k2 e k3 usando a ferramenta ESTIMA, onde apresentaram duas respostas, uma resposta típica de mínimos quadrados, e a outra resposta que a velocidade inicial, que foi melhor representada pelos parâmetros obtidos. O método empregado na degradação do substrato, o microrreator mostrou-se eficiente, permitindo a detecção de baixo consumo de substrato para a determinação da taxa inicial, em curto tempo de residência. Perante os ensaios realizados com Lacase e Soybean Peroxidase, o microrreator é também um equipamento eficaz na repetitividade e na reprodutibilidade dos dados obtidos em diferentes concentrações.

Filmes de óxido de zinco e nitreto de zinco depositados por magnetron sputtering com diferentes pressões de argônio, oxigênio e nitrogênio.

O óxido de zinco é um material semicondutor que apresenta alta transparência óptica no espectro visível, alta energia de ligação de éxcitons e piezoeletricidade. Por suas propriedades, ele é utilizado na área de sensores, eletrodos transparentes e dispositivos optoeletrônicos. No entanto, sua utilização ainda é limitada pela dificuldade de obtenção de condutividade tipo p, cujo principal dopante é o nitrogênio, devido à assimetria de dopagem ocasionada por defeitos intrínsecos do material, dopagem em valências diferentes das esperadas e formação de níveis de aceitadores profundos na banda proibida. A aplicação em dispositivos piezoelétricos também exige alta resistividade e ótimas propriedades cristalinas. Muitos processos de deposição estabelecidos hoje ainda utilizam altas temperaturas, o que impede sua deposição sobre superfícies ou substratos sensíveis a altas temperaturas. O objetivo deste trabalho é desenvolver técnicas de deposição de filmes de ZnO, principalmente em baixas temperaturas ( 100°C), pelo método de magnetron sputtering de rádio frequência, para avaliar a influência dos gases de processo nas características estruturais, estequiométricas, elétricas e ópticas dos filmes. Para isso, foram obtidos filmes utilizando pressão total de argônio, e pressões parciais de argônio e oxigênio e argônio e nitrogênio, utilizando alvo cerâmico de óxido de zinco ou alvo metálico de zinco. Para alvo de ZnO, filmes com condutividade tipo n foram obtidos em ambiente de argônio, em condições que geraram deficiências de oxigênio. Filmes altamente resistivos foram obtidos com a utilização de pressão parcial de oxigênio no gás de processo, em condições que resultaram em filmes estequiométricos, inclusive com condutividade tipo p. Condutividade tipo p mais alta foi observada, apenas por ponta quente, para uma amostra obtida em argônio logo após a utilização de nitrogênio na câmara de processo, que provavelmente sofreu influência da dopagem não intencional do cobre, que foi identificado como um contaminante do processo devido à estrutura da câmara. Para alvo de Zn, observou-se a formação de nitreto de zinco, que demonstrou alta capacidade de oxidação em ambiente atmosférico, e portanto, transforma-se naturalmente ao longo do tempo ou por processos de oxidação térmica em ZnO dopado com nitrogênio. Filmes de ZnO produzidos a partir de nitreto de zinco foram os únicos dos testados que apresentaram fotoluminescência característica do ZnO, mesmo para processos onde não houve aquecimento intencional.

On the initiation of boudinage and folding instabilities in layered elasto-visco-plastic solids - Insights from natural microstructures and numerical simulations

The present understanding of the initiation of boudinage and folding structures is based on viscosity contrasts and stress exponents, considering an intrinsically unstable state of the layer. The criterion of localization is believed to be prescribed by geometry-material interactions, which are often encountered in natural structures. An alternative localization phenomenon has been established for ductile materials, in which instability emerges for critical material parameters and loading rates from homogeneous conditions. In this thesis, conditions are sought under which this type of instability prevails and whether localization in geological materials necessarily requires a trigger by geometric imperfections. The relevance of critical deformation conditions, material parameters and the spatial configuration of instabilities are discussed in a geological context. In order to analyze boudinage geometries, a numerical eigenmode analysis is introduced. This method allows determining natural frequencies and wavelengths of a structure and inducing perturbations on these frequencies. In the subsequent coupled thermo-mechanical simulations, using a grain size evolution and end-member flow laws, localization emerges when material softening through grain size sensitive viscous creep sets in. Pinch-and-swell structures evolve along slip lines through a positive feedback between the matrix response and material bifurcations inside the layer, independent from the mesh-discretization length scale. Since boudinage and folding are considered to express the same general instability, both structures should arise independently of the sign of the loading conditions and for identical material parameters. To this end, the link between material to energy instabilities is approached by means of bifurcation analyses of the field equations and finite element simulations of the coupled system of equations. Boudinage and folding structures develop at the same critical energy threshold, where dissipative work by temperature-sensitive creep overcomes the diffusive capacity of the layer. This finding provides basis for a unified theory for strain localization in layered ductile materials. The numerical simulations are compared to natural pinch-and-swell microstructures, tracing the adaption of grain sizes, textures and creep mechanisms in calcite veins. The switch from dislocation to diffusion creep relates to strain-rate weakening, which is induced by dissipated heat from grain size reduction, and marks the onset of continuous necking. The time-dependent sequence uncovers multiple steady states at different time intervals. Microstructurally and mechanically stable conditions are finally expressed in the pinch-and-swell end members. The major outcome of this study is that boudinage and folding can be described as the same coupled energy-mechanical bifurcation, or as one critical energy attractor. This finding allows the derivation of critical deformation conditions and fundamental material parameters directly from localized structures in the field.

Molecular characterization of three gonad cell lines

To facilitate the study of the regulation and downstream interactions of genes involved in gonad development it is important to have a suitable cell culture model. We therefore aimed to characterize molecularly three different mouse gonad cell lines. TM3 and TM4 cells were originally isolated from prepubertal mouse gonads and were tentatively identified as being of Leydig cell and Sertoli cell origin, respectively, based upon their morphology and hormonal responses. The third line is a conditionally immortalized cell line, derived from 10.5-11.5 days post-coitum (dpc) male gonads of transgenic embryos carrying a temperature-sensitive SV40 large T-antigen. We studied by reverse transcription-polymerase chain reaction (RT-PCR) the expression profiles of a number of genes known to be important for early gonad development. Moreover, we assessed these cell lines for their capacity to induce Sox9 transcription upon expression of Sry, a key molecular event occurring during sex determination. We found that all three cell lines were unable to upregulate Sox9 expression upon transfection of Sry-expression constructs, even though these cells express many of the studied embryonic gonad genes. These observations point to a requirement for SRY cofactors for direct or indirect upregulation of Sox9 expression during testis determination. Copyright © 2003 S. Karger AG, Basel

Domain and nucleotide dependence of the interaction between Saccharomyces cerevisiae translation elongation factors 3 and 1A

Eukaryotic translation elongation factor 3 (eEF3) is a fungal-specific ATPase proposed to catalyze the release of deacylated-tRNA from the ribosomal E-site. In addition, it has been shown to interact with the aminoacyl-tRNA binding GTPase elongation factor 1A (eEF1A), perhaps linking the E and A sites. Domain mapping demonstrates that amino acids 775-980 contain the eEF1A binding sites. Domain III of eEF1A, which is also involved in actin-related functions, is the site of eEF3 binding. The binding of eEF3 to eEF1A is enhanced by ADP, indicating the interaction is favored post-ATP hydrolysis but is not dependent on the eEF1A-bound nucleotide. A temperature-sensitive P915L mutant in the eEF1A binding site of eEF3 has reduced ATPase activity and affinity for eEF1A. These results support the model that upon ATP hydrolysis, eEF3 interacts with eEF1A to help catalyze the delivery of aminoacyl-tRNA at the A-site of the ribosome. The dynamics of when eEF3 interacts with eEF1A may be part of the signal for transition of the post to pre-translocational ribosomal state in yeast.

In situ diagnostics and prognostics of solder fatigue in IGBT modules for electric vehicle drives

This paper proposes an in situ diagnostic and prognostic (D&P) technology to monitor the health condition of insulated gate bipolar transistors (IGBTs) used in EVs with a focus on the IGBTs' solder layer fatigue. IGBTs' thermal impedance and the junction temperature can be used as health indicators for through-life condition monitoring (CM) where the terminal characteristics are measured and the devices' internal temperature-sensitive parameters are employed as temperature sensors to estimate the junction temperature. An auxiliary power supply unit, which can be converted from the battery's 12-V dc supply, provides power to the in situ test circuits and CM data can be stored in the on-board data-logger for further offline analysis. The proposed method is experimentally validated on the developed test circuitry and also compared with finite-element thermoelectrical simulation. The test results from thermal cycling are also compared with acoustic microscope and thermal images. The developed circuitry is proved to be effective to detect solder fatigue while each IGBT in the converter can be examined sequentially during red-light stopping or services. The D&P circuitry can utilize existing on-board hardware and be embedded in the IGBT's gate drive unit.

Climate variability in the Aegean Sea (marine record M2, Athos basin) during the past 1500 years

We provide new evidence on sea surface temperature (SST) variations and paleoceanographic/paleoenvironmental changes over the past 1500 years for the north Aegean Sea (NE Mediterranean). The reconstructions are based on multiproxy analyses, obtained from the high resolution (decadal to multi-decadal) marine record M2 retrieved from the Athos basin. Reconstructed SSTs show an increase from ca. 850 to 950 AD and from ca. 1100 to 1300 AD. A cooling phase of almost 1.5 °C is observed from ca. 1600 AD to 1700 AD. This seems to have been the starting point of a continuous SST warming trend until the end of the reconstructed period, interrupted by two prominent cooling events at 1832 ± 15 AD and 1995 ± 1 AD. Application of an adaptive Kernel smoothing suggests that the current warming in the reconstructed SSTs of the north Aegean might be unprecedented in the context of the past 1500 years. Internal variability in atmospheric/oceanic circulations systems as well as external forcing as solar radiation and volcanic activity could have affected temperature variations in the north Aegean Sea over the past 1500 years. The marked temperature drop of approximately ~2 °C at 1832 ± 15 yr AD could be related to the 1809 ?D 'unknown' and the 1815 AD Tambora volcanic eruptions. Paleoenvironmental proxy-indices of the M2 record show enhanced riverine/continental inputs in the northern Aegean after ca. 1450 AD. The paleoclimatic evidence derived from the M2 record is combined with a socio-environmental study of the history of the north Aegean region. We show that the cultivation of temperature-sensitive crops, i.e. walnut, vine and olive, co-occurred with stable and warmer temperatures, while its end coincided with a significant episode of cooler temperatures. Periods of agricultural growth in Macedonia coincide with periods of warmer and more stable SSTs, but further exploration is required in order to identify the causal links behind the observed phenomena. The Black Death likely caused major changes in agricultural activity in the north Aegean region, as reflected in the pollen data from land sites of Macedonia and the M2 proxy-reconstructions. Finally, we conclude that the early modern peaks in mountain vegetation in the Rhodope and Macedonia highlands, visible also in the M2 record, were very likely climate-driven.

LGM, planktic foraminifera (EPILOG version)

[1] We used planktic foraminiferal assemblages in 70 sediment cores from the tropical and subtropical South Atlantic Ocean (10°N-37°S) to estimate annual mean sea surface temperatures (SSTs) and seasonality for the Last Glacial Maximum with a modified version of the Imbrie-Kipp transfer function method (IKTF) that takes into account the abundance of rare but temperature sensitive species. In contrast to CLIMAP Project Members [1981], the reconstructed SSTs indicate cooler glacial SSTs in the entire tropical/subtropical South Atlantic with strongest cooling in the upwelling region off Namibia (7-10°C) and smallest cooling (1-2°C) in the western subtropical gyre. In the western Atlantic, our data support recent temperature estimates from other proxies. In the upwelling regions in the eastern Atlantic, our data conflict with SST reconstructions from alkenones, which may be due to an environmental preference of the alkenone-producing algae or to an underestimation of foraminiferal SSTs due to anomalous high abundances of N. pachyderma (sinistral).

Structural and Biochemical Dissection of the Trehalose Biosynthetic Complex in Pathogenic Fungi

Trehalose is a non-reducing disaccharide essential for pathogenic fungal survival and virulence. The biosynthesis of trehalose requires the trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. More importantly, the trehalose biosynthetic pathway is absent in mammals, conferring this pathway as an ideal target for antifungal drug design. However, lack of germane biochemical and structural information hinders antifungal drug design against these targets.

In this dissertation, macromolecular X-ray crystallography and biochemical assays were employed to understand the structures and functions of proteins involved in the trehalose biosynthetic pathway. I report here the first eukaryotic Tps1 structures from Candida albicans (C. albicans) and Aspergillus fumigatus (A. fumigatus) with substrates or substrate analogs. These structures reveal the key residues involved in substrate binding and catalysis. Subsequent enzymatic assays and cellular assays highlight the significance of these key Tps1 residues in enzyme function and fungal stress response. The Tps1 structure captured in its transition-state with a non-hydrolysable inhibitor demonstrates that Tps1 adopts an “internal return like” mechanism for catalysis. Furthermore, disruption of the trehalose biosynthetic complex formation through abolishing Tps1 dimerization reveals that complex formation has regulatory function in addition to trehalose production, providing additional targets for antifungal drug intervention.

I also present here the structure of the Tps2 N-terminal domain (Tps2NTD) from C. albicans, which may be involved in the proper formation of the trehalose biosynthetic complex. Deletion of the Tps2NTD results in a temperature sensitive phenotype. Further, I describe in this dissertation the structures of the Tps2 phosphatase domain (Tps2PD) from C. albicans, A. fumigatus and Cryptococcus neoformans (C. neoformans) in multiple conformational states. The structures of the C. albicans Tps2PD -BeF3-trehalose complex and C. neoformans Tps2PD(D24N)-T6P complex reveal extensive interactions between both glucose moieties of the trehalose involving all eight hydroxyl groups and multiple residues of both the cap and core domains of Tps2PD. These structures also reveal that steric hindrance is a key underlying factor for the exquisite substrate specificity of Tps2PD. In addition, the structures of Tps2PD in the open conformation provide direct visualization of the conformational changes of this domain that are effected by substrate binding and product release.

Last, I present the structure of the C. albicans trehalose synthase regulatory protein (Tps3) pseudo-phosphatase domain (Tps3PPD) structure. Tps3PPD adopts a haloacid dehydrogenase superfamily (HADSF) phosphatase fold with a core Rossmann-fold domain and a α/β fold cap domain. Despite lack of phosphatase activity, the cleft between the Tps3PPD core domain and cap domain presents a binding pocket for a yet uncharacterized ligand. Identification of this ligand could reveal the cellular function of Tps3 and any interconnection of the trehalose biosynthetic pathway with other cellular metabolic pathways.

Combined, these structures together with significant biochemical analyses advance our understanding of the proteins responsible for trehalose biosynthesis. These structures are ready to be exploited to rationally design or optimize inhibitors of the trehalose biosynthetic pathway enzymes. Hence, the work described in this thesis has laid the groundwork for the design of Tps1 and Tps2 specific inhibitors, which ultimately could lead to novel therapeutics to treat fungal infections.

La perception du temps et sa modulation par la température chez les guêpes parasitoïdes

Les modèles d'optimalité postulent que les animaux en quête de ressources utilisent le taux de gain de valeur adaptative pour optimiser plusieurs comportements tels que la répartition du temps lors de l’exploitation d‘un agrégat et l'investissement en progénitures. Bien que la durée de plusieurs comportements doit être régulée, peu d’évidences de la perception du temps sont actuellement disponibles pour les insectes et aucune pour les guêpes parasitoïdes, et ce malgré leur importance en tant que modèles écologiques. De plus, puisque les guêpes parasitoïdes sont poïkilothermes, cette capacité pourrait être affectée par la température. Nous avons supposé que les guêpes parasitoïdes auraient la capacité de percevoir le temps, à la fois de façon prospective (mesure du temps écoulé) et rétrospective (durée d'un événement passé), afin d'optimiser les décisions liées à l'exploitation d’agrégats d’hôtes et à la reproduction. Nous avons également émis l'hypothèse que la température aurait une incidence sur la perception du temps des guêpes parasitoïdes. Pour la mesure prospective du temps, nous avons utilisé la capacité d’apprentissage associatif de Microplitis croceipes (Hymenoptera: Braconidae). Les guêpes ont été entraînées à associer une odeur à la durée d'un intervalle entre des hôtes. Après leur entraînement, elles ont été testées dans un tunnel de vol avec un choix d’odeurs. Les guêpes ont choisi majoritairement l'odeur associée à l'intervalle de temps auquel elles étaient testées. Nous avons également investigué le rôle de la dépense énergétique sur la mesure du temps. Suite à une restriction de mouvement des guêpes pendant l'intervalle de temps entre les hôtes, elles choisissaient aléatoirement dans le tunnel de vol. L'absence de dépense énergétique les aurait rendues incapables de mesurer le temps. La dépense d'énergie est donc un substitut essentiel pour mesurer le temps. Pour la mesure rétrospective du temps, nous avons utilisé le processus d'évaluation de l'hôte de Trichogramma euproctidis (Hymenoptera: Trichogrammatidae). Certains trichogrammes utilisent la durée du transit initial sur l'œuf hôte afin d’en évaluer la taille et d’ajuster le nombre d’œufs à y pondre. Nous avons augmenté artificiellement la durée de transit initiale de T. euproctidis en suspendant l'œuf hôte pour le faire paraître plus gros qu'un œuf de taille similaire. Une augmentation de la durée de transit initiale a augmenté la taille de la ponte. Ceci démontre la capacité de T. euproctidis de mesurer la durée du transit initial, et donc d’une mesure du temps rétrospective. Pour déterminer si la température modifie la mesure du temps dans les espèces poïkilothermes, nous avons utilisé le comportement d’exploitation d’agrégats d’hôtes de T. euproctidis. Les modèles d’optimalités prédisent que les guêpes devraient rester plus longtemps et quitter à un faible taux de gain de valeur adaptative suite à un déplacement de longue durée plutôt que pour un déplacement de courte durée. Nous avons testé l'impact d'un déplacement de 24 h à différentes températures sur l'exploitation d’agrégats d’hôtes. Un déplacement à température chaude augmente le temps de résidence dans l’agrégat et diminue le taux de gain de valeur adaptative au moment de quitter ; ces comportements sont associés à un trajet de longue durée. L'inverse a été observé lors d’un déplacement à une température froide. Les températures chaude et froide ont modulé la mesure du temps en accélérant ou ralentissant l'horloge biologique, faisant paraître le déplacement respectivement plus long ou plus court qu’il ne l’était réellement. Ces résultats démontrent clairement que les guêpes parasitoïdes ont la capacité de mesurer le temps, autant rétrospectivement que prospectivement. Des preuves directes de leur capacité sont maintenant disponibles pour au moins deux espèces de guêpes parasitoïdes, une composante essentielle des modèles d'optimalité. Le rôle de la dépense énergétique dans la mesure du temps a aussi été démontré. Nos résultats fournissent également la preuve de l'impact de la température sur la perception du temps chez les insectes. L'utilisation de la dépense énergétique en tant que proxy pour mesurer le temps pourrait expliquer une partie de sa thermosensibilité, puisque les guêpes parasitoïdes sont poïkilothermes. Cette mesure du temps sensible à la température pourrait affecter des stratégies de lutte biologique. Sur le terrain, au début de la journée, la température de l'air sera similaire à la température de l'air autour des plantes infestées par des parasites, alors qu'elle sera plus chaude pendant la journée. En lutte biologique augmentative, les guêpes parasitoïdes libérées resteraient plus longtemps dans les agrégats d’hôtes que celles relâchées en début de journée.

Microcantilever sensors for biochemical detection and analysis

Biochemical agents, including bacteria and toxins, are potentially dangerous and responsible for a wide variety of diseases. Reliable detection and characterization of small samples is necessary in order to reduce and eliminate their harmful consequences. Microcantilever sensors offer a potential alternative to the state of the art due to their small size, fast response time, and the ability to operate in air and liquid environments. At present, there are several technology limitations that inhibit application of microcantilever to biochemical detection and analysis, including difficulties in conducting temperature-sensitive experiments, material inadequacy resulting in insufficient cell capture, and poor selectivity of multiple analytes. This work aims to address several of these issues by introducing microcantilevers having integrated thermal functionality and by introducing nanocrystalline diamond as new material for microcantilevers. Microcantilevers are designed, fabricated, characterized, and used for capture and detection of cells and bacteria. The first microcantilever type described in this work is a silicon cantilever having highly uniform in-plane temperature distribution. The goal is to have 100 μm square uniformly heated area that can be used for thermal characterization of films as well as to conduct chemical reactions with small amounts of material. Fabricated cantilevers can reach above 300C while maintaining temperature uniformity of 2−4%. This is an improvement of over one order of magnitude over currently available cantilevers. The second microcantilever type is a doped single crystal silicon cantilever having a thin coating of ultrananocrystalline diamond (UNCD). The primary application of such a device is in biological testing, where diamond acts as a stable, electrically isolated reaction surface while silicon layer provides controlled heating with minimum variations in temperature. This work shows that composite cantilevers of this kind are an effective platform for temperature-sensitive biological experiments, such as heat lysing and polymerase chain reaction. The rapid heat-transfer of Si-UNCD cantilever compromised the membrane of NIH 3T3 fibroblast and lysed the cell nucleus within 30 seconds. Bacteria cells, Listeria monocytogenes V7, were shown to be captured with biotinylated heat-shock protein on UNCD surface and 90% of all viable cells exhibit membrane porosity due to high heat in 15 seconds. Lastly, a sensor made solely from UNCD diamond is fabricated with the intention of being used to detect the presence of biological species by means of an integrated piezoresistor or through frequency change monitoring. Since UNCD diamond has not been previously used in piezoresistive applications, temperature-denpendent piezoresistive coefficients and gage factors are determined first. The doped UNCD exhibits a significant piezoresistive effect with gauge factor of 7.53±0.32 and a piezoresistive coefficient of 8.12×10^−12 Pa^−1 at room temperature. The piezoresistive properties of UNCD are constant over the temperature range of 25−200C. 300 μm long cantilevers have the highest sensitivity of 0.186 m-Ohm/Ohm per μm of cantilever end deflection, which is approximately half that of similarly sized silicon cantilevers. UNCD cantilever arrays were fabricated consisting of four sixteen-cantilever arrays of length 20–90 μm in addition to an eight-cantilever array of length 120 μm. Laser doppler vibrometry (LDV) measured the cantilever resonant frequency, which ranged as 218 kHz−5.14 MHz in air and 73 kHz−3.68 MHz in water. The quality factor of the cantilever was 47−151 in air and 18−45 in water. The ability to measure frequencies of the cantilever arrays opens the possibility for detection of individual bacteria by monitoring frequency shift after cell capture.