Cytokinesis and Midzone Microtubule Organization in Caenorhabditis elegans Require the Kinesin-like Protein ZEN-4

Members of the MKLP1 subfamily of kinesin motor proteins localize to the equatorial region of the spindle midzone and are capable of bundling antiparallel microtubules in vitro. Despite these intriguing characteristics, it is unclear what role these kinesins play in dividing cells, particularly within the context of a developing embryo. Here, we report the identification of a null allele of zen-4, an MKLP1 homologue in the nematode Caenorhabditis elegans, and demonstrate that ZEN-4 is essential for cytokinesis. Embryos deprived of ZEN-4 form multinucleate single-celled embryos as they continue to cycle through mitosis but fail to complete cell division. Initiation of the cytokinetic furrow occurs at the normal time and place, but furrow propagation halts prematurely. Time-lapse recordings and microtubule staining reveal that the cytokinesis defect is preceded by the dissociation of the midzone microtubules. We show that ZEN-4 protein localizes to the spindle midzone during anaphase and persists at the midbody region throughout cytokinesis. We propose that ZEN-4 directly cross-links the midzone microtubules and suggest that these microtubules are required for the completion of cytokinesis.

“Coarse” stability and bifurcation analysis using time-steppers: A reaction-diffusion example

Evolutionary, pattern forming partial differential equations (PDEs) are often derived as limiting descriptions of microscopic, kinetic theory-based models of molecular processes (e.g., reaction and diffusion). The PDE dynamic behavior can be probed through direct simulation (time integration) or, more systematically, through stability/bifurcation calculations; time-stepper-based approaches, like the Recursive Projection Method [Shroff, G. M. & Keller, H. B. (1993) SIAM J. Numer. Anal. 30, 1099–1120] provide an attractive framework for the latter. We demonstrate an adaptation of this approach that allows for a direct, effective (“coarse”) bifurcation analysis of microscopic, kinetic-based models; this is illustrated through a comparative study of the FitzHugh-Nagumo PDE and of a corresponding Lattice–Boltzmann model.

A new Eocene archaeocete (Mammalia, Cetacea) from India and the time of origin of whales

Himalayacetus subathuensis is a new pakicetid archaeocete from the Subathu Formation of northern India. The type dentary has a small mandibular canal indicating a lack of auditory specializations seen in more advanced cetaceans, and it has Pakicetus-like molar teeth suggesting that it fed on fish. Himalayacetus is significant because it is the oldest archaeocete known and because it was found in marine strata associated with a marine fauna. Himalayacetus extends the fossil record of whales about 3.5 million years back in geological time, to the middle part of the early Eocene [≈53.5 million years ago (Ma)]. Oxygen in the tooth-enamel phosphate has an isotopic composition intermediate between values reported for freshwater and marine archaeocetes, indicating that Himalayacetus probably spent some time in both environments. When the temporal range of Archaeoceti is calibrated radiometrically, comparison of likelihoods constrains the time of origin of Archaeoceti and hence Cetacea to about 54–55 Ma (beginning of the Eocene), whereas their divergence from extant Artiodactyla may have been as early as 64–65 Ma (beginning of the Cenozoic).

Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin

A finely tuned Ca2+ signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca2+ channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca2+ permeation properties suggest an important function in Ca2+ uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca2+-dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.

Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution in real time

The molecular reaction mechanism of the GTPase-activating protein (GAP)-catalyzed GTP hydrolysis by Ras was investigated by time resolved Fourier transform infrared (FTIR) difference spectroscopy using caged GTP (P3-1-(2-nitro)phenylethyl guanosine 5′-O-triphosphate) as photolabile trigger. This approach provides the complete GTPase reaction pathway with time resolution of milliseconds at the atomic level. Up to now, one structural model of the GAP⋅Ras⋅GDP⋅AlFx transition state analog is known, which represents a “snap shot” along the reaction-pathway. As now revealed, binding of GAP to Ras⋅GTP shifts negative charge from the γ to β phosphate. Such a shift was already identified by FTIR in GTP because of Ras binding and is now shown to be enhanced by GAP binding. Because the charge distribution of the GAP⋅Ras⋅GTP complex thus resembles a more dissociative-like transition state and is more like that in GDP, the activation free energy is reduced. An intermediate is observed on the reaction pathway that appears when the bond between β and γ phosphate is cleaved. In the intermediate, the released Pi is strongly bound to the protein and surprisingly shows bands typical of those seen for phosphorylated enzyme intermediates. All these results provide a mechanistic picture that is different from the intrinsic GTPase reaction of Ras. FTIR analysis reveals the release of Pi from the protein complex as the rate-limiting step for the GAP-catalyzed reaction. The approach presented allows the study not only of single proteins but of protein–protein interactions without intrinsic chromophores, in the non-crystalline state, in real time at the atomic level.

Insulin-like growth factor II mediates epidermal growth factor-induced mitogenesis in cervical cancer cells.

There is increasing evidence that activation of the insulin-like growth factor I (IGF-I) receptor plays a major role in the control of cellular proliferation of many cell types. We studied the mitogenic effects of IGF-I, IGF-II, and epidermal growth factor (EGF) on growth-arrested HT-3 cells, a human cervical cancer cell line. All three growth factors promoted dose-dependent increases in cell proliferation. In untransformed cells, EGF usually requires stimulation by a "progression" factor such as IGF-I, IGF-II, or insulin (in supraphysiologic concentrations) in order to exert a mitogenic effect. Accordingly, we investigated whether an autocrine pathway involving IGF-I or IGF-II participated in the EGF-induced mitogenesis of HT-3 cells. With the RNase protection assay, IGF-I mRNA was not detected. However, IGF-II mRNA increased in a time-dependent manner following EGF stimulation. The EGF-induced mitogenesis was abrogated in a dose-dependent manner by IGF-binding protein 5 (IGFBP-5), which binds to IGF-II and neutralizes it. An antisense oligonucleotide to IGF-II also inhibited the proliferative response to EGF. In addition, prolonged, but not short-term, stimulation with EGF resulted in autophosphorylation of the IGF-I receptor, and coincubations with both EGF and IGFBP-5 attenuated this effect. These data demonstrate that autocrine secretion of IGF-II in HT-3 cervical cancer cells can participate in EGF-induced mitogenesis and suggest that autocrine signals involving the IGF-I receptor occur "downstream" of competence growth factor receptors such as the EGF receptor.

Stopped-flow NMR spectroscopy: real-time unfolding studies of 6-19F-tryptophan-labeled Escherichia coli dihydrofolate reductase.

Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues that have been replaced with 6-19F-tryptophan. The 19F NMR assignments are known in the native, unliganded form and the unfolded form. We have used these assignments with stopped-flow 19F NMR spectroscopy to investigate the behavior of specific regions of the protein in real time during urea-induced unfolding. The NMR data show that within 1.5 sec most of the intensities of the native 19F resonances of the protein are lost but only a fraction (approximately 20%) of the intensities of the unfolded resonances appears. We postulate that the early disappearance of the native resonances indicates that most of the protein rapidly forms an intermediate in which the side chains have considerable mobility. Stopped-flow far-UV circular dichroism measurements indicate that this intermediate retains native-like secondary structure. Eighty percent of the intensities of the NMR resonances assigned to the individual tryptophans in the unfolded state appear with similar rate constants (k approximately 0.14 sec-1), consistent with the major phase of unfolding observed by stopped-flow circular dichroism (representing 80% of total amplitude). These data imply that after formation of the intermediate, which appears to represent an expanded structural form, all regions of the protein unfold at the same rate. Stopped-flow measurements of the fluorescence and circular dichroism changes associated with the urea-induced unfolding show a fast phase (half-time of about 1 sec) representing 20% of the total amplitude in addition to the slow phase mentioned above. The NMR data show that approximately 20% of the total intensity for each of the unfolded tryptophan resonances is present at 1.5 sec, indicating that these two phases may represent the complete unfolding of the two different populations of the native protein.

Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences.

Early Alzheimer disease-like histopathology increases in frequency with age in mice transgenic for beta-APP751.

beta-Amyloid deposition and neurofibrillary tangle formation are two histopathological features of Alzheimer disease. We have previously reported that beta-amyloid immunoreactive deposits form in the brains of transgenic mice programmed for neuronal expression of the 751-amino acid isoform of human beta-amyloid precursor protein (beta-APP751) and now describe that these animals also display Alz50 intraneuronal immunoreactivity similar to that seen in early Alzheimer disease. This suggests that abnormal beta-APP expression and/or beta-amyloid deposition promotes pathogenic alterations in tau protein. The frequency of both beta-amyloid deposition and Alz50-positive neurons was twice as prevalent in brains from old (22 months) as compared to young (2-3 months) beta-APP751 transgenic mice. This increase in histopathology with age in beta-APP751 transgenic mice parallels the time-dependent progression seen in the human disease.

Determination of absorption curves, dissolution profiles and establishment of in vitro-in vivo correlation by in silico methods using GastroPlusTM and DDDPlusTM

The use of computer programs to predict drug absorption in humans and to simulate dissolution profiles has become a valuable tool in the pharmaceutical area. The objective of this study was to use in silico methods through software GastroPlusTM and DDDPlusTM to simulate drug absorption curves and dissolution profiles, and to establish in vitro-in vivo correlations (IVIVCs). The work presented herein is divided into five chapters and includes the drugs ketoprofen, pyrimethamine, metronidazole, fluconazole, carvedilol and doxazosin. In Chapter 1, simulated plasma curves for ketoprofen matrix tablets are presented and IVIVC was established. The use of simulated intrinsic dissolution tests for pyrimethamine and metronidazole as a tool for biopharmaceutics classification is detailed in Chapter 2. In Chapter 3, simulation of plasma curves for fluconazole capsules with different dissolution profiles is demonstrated as a tool for biowaiver. IVIVC studies were also conducted for carvedilol immediate-release tablets from dissolution profiles in Chapter 4. Chapter 5 covers the application of simulated dissolution tests for development of doxazosin extended-release formulations. Simulation of plasma curves and IVIVC using the software GastroPlusTM as well as intrinsic dissolution tests and dissolution profiles using the software DDDPlusTM proved to be a tool of wide application in predicting biopharmaceutical characteristics of drugs and formulations, allowing the reduction of time and costs of experimental laboratory work.

Análise de acetona em ar exalado: desenvolvimento de método eletroanalítico e algoritmo para processamento de sinais

Propõe-se método novo e completo para análise de acetona em ar exalado envolvendo coleta com pré-concentração em água, derivatização química e determinação eletroquímica assistida por novo algoritmo de processamento de sinais. Na literatura recente a acetona expirada vem sendo avaliada como biomarcador para monitoramento não invasivo de quadros clínicos como diabetes e insuficiência cardíaca, daí a importância da proposta. Entre as aminas que reagem com acetona para formar iminas eletroativas, estudadas por polarografia em meados do século passado, a glicina apresentou melhor conjunto de características para a definição do método de determinação por voltametria de onda quadrada sem a necessidade de remoção de oxigênio (25 Hz, amplitude de 20 mV, incremento de 5 mV, eletrodo de gota de mercúrio). O meio reacional, composto de glicina (2 mol·L-1) em meio NaOH (1 mol·L-1), serviu também de eletrólito e o pico de redução da imina em -1,57 V vs. Ag|AgCl constituiu o sinal analítico. Para tratamento dos sinais, foi desenvolvido e avaliado um algoritmo inovador baseado em interpolação de linha base por ajuste de curvas de Bézier e ajuste de gaussiana ao pico. Essa combinação permitiu reconhecimento e quantificação de picos relativamente baixos e largos sobre linha com curvatura acentuada e ruído, situação em que métodos convencionais falham e curvas do tipo spline se mostraram menos apropriadas. A implementação do algoritmo (disponível em http://github.com/batistagl/chemapps) foi realizada utilizando programa open source de álgebra matricial integrado diretamente com software de controle do potenciostato. Para demonstrar a generalidade da extensão dos recursos nativos do equipamento mediante integração com programação externa em linguagem Octave (open source), implementou-se a técnica da cronocoulometria tridimensional, com visualização de resultados já tratados em projeções de malha de perspectiva 3D sob qualquer ângulo. A determinação eletroquímica de acetona em fase aquosa, assistida pelo algoritmo baseado em curvas de Bézier, é rápida e automática, tem limite de detecção de 3,5·10-6 mol·L-1 (0,2 mg·L-1) e faixa linear que atende aos requisitos da análise em ar exalado. O acetaldeído, comumente presente em ar exalado, em especial, após consumo de bebidas alcoólicas, dá origem a pico voltamétrico em -1,40 V, contornando interferência que prejudica vários outros métodos publicados na literatura e abrindo possibilidade de determinação simultânea. Resultados obtidos com amostras reais são concordantes com os obtidos por método espectrofotométrico, em uso rotineiro desde o seu aperfeiçoamento na dissertação de mestrado do autor desta tese. Em relação à dissertação, também se otimizou a geometria do dispositivo de coleta, de modo a concentrar a acetona num volume menor de água gelada e prover maior conforto ao paciente. O método completo apresentado, englobando o dispositivo de amostragem aperfeiçoado e o novo e efetivo algoritmo para tratamento automático de sinais voltamétricos, está pronto para ser aplicado. Evolução para um analisador portátil depende de melhorias no limite de detecção e facilidade de obtenção eletrodos sólidos (impressos) com filme de mercúrio, vez que eletrodos de bismuto ou diamante dopado com boro, entre outros, não apresentaram resposta.

Avaliação da judicialização da saúde no município de Ribeirão Preto/SP

A Constituição Federal Brasileira institucionalizou o direito a saúde no Brasil, o artigo 196 que diz: A saúde é um direito de todos e um dever do Estado apresenta esse direito. Ao regulamentar a criação do Sistema Único de Saúde a lei 8.080 reafirma a obrigação do Estado com a Saúde da população. Dentro desse contexto a Assistência Farmacêutica (AF) tem importante papel de garantir medicamentos seguros, eficácias, em tempo e quantidade necessária para atender a demanda dos cidadãos, porém apesar das constantes atualizações em prol de promover maior eficiência dos processos da AF, ainda acontecem situações em que o paciente não tem o medicamento requerido, seja por falta nas unidades dispensadoras ou a não presença nas listas de medicamentos padronizados. Essa situação faz com que o cidadão recorra à via judicial na tentativa de garantir o acesso ao medicamento pleiteado, fenômeno conhecido como judicialização da saúde, que traz grandes implicações sobre a gestão da assistência farmacêutica. Diante disso o objetivo do trabalho foi descrever o panorama geral das ações judiciais pleiteando medicamentos e insumos para insulina que foram assumidos pela prefeitura de Ribeirão Preto. Para alcançar esses objetivos, foi realizado um estudo do tipo descritivo. Foram analisados ao todo 1861 processos judiciais sendo 1083 ainda ativos e 778 que já haviam sido encerrados. Na maioria dos processos o juiz dava como prazo máximo 30 dias (99%) para se cumprir a ação, o que é insuficiente para realizar uma licitação pública obrigando a gestão a utilizar via paralela de compra. O Ministério Público foi o principal representante legal (71,7%) utilizado e a maioria das prescrições foram advindas de hospitais e clínicas particulares (50,1%). Os principais diagnósticos referidos nas ações foram diabetes e o transtorno de déficit de atenção e hiperatividade (TDAH). Já os medicamentos mais prevalentes foram as insulinas e o metilfenidato. Dentre os médicos prescritores 3% somam aproximadamente 30% das prescrições. Diante dos resultados expostos, o presente estudo evidenciou o impacto da judicialização da saúde no município de Ribeirão Preto, demandando da gestão pública organização estrutural e financeira para lidar com as demandas judiciais.

Magnetic activity and differential rotation in the young Sun-like stars KIC 7985370 and KIC 7765135

Aims. We present a detailed study of the two Sun-like stars KIC 7985370 and KIC 7765135, to determine their activity level, spot distribution, and differential rotation. Both stars were previously discovered by us to be young stars and were observed by the NASA Kepler mission. Methods. The fundamental stellar parameters (vsini, spectral type, T_eff, log g, and [Fe/H]) were derived from optical spectroscopy by comparison with both standard-star and synthetic spectra. The spectra of the targets allowed us to study the chromospheric activity based on the emission in the core of hydrogen Hα and Ca ii infrared triplet (IRT) lines, which was revealed by the subtraction of inactive templates. The high-precision Kepler photometric data spanning over 229 days were then fitted with a robust spot model. Model selection and parameter estimation were performed in a Bayesian manner, using a Markov chain Monte Carlo method. Results. We find that both stars are Sun-like (of G1.5 V spectral type) and have an age of about 100–200 Myr, based on their lithium content and kinematics. Their youth is confirmed by their high level of chromospheric activity, which is comparable to that displayed by the early G-type stars in the Pleiades cluster. The Balmer decrement and flux ratio of their Ca ii-IRT lines suggest that the formation of the core of these lines occurs mainly in optically thick regions that are analogous to solar plages. The spot model applied to the Kepler photometry requires at least seven persistent spots in the case of KIC 7985370 and nine spots in the case of KIC 7765135 to provide a satisfactory fit to the data. The assumption of the longevity of the star spots, whose area is allowed to evolve with time, is at the heart of our spot-modelling approach. On both stars, the surface differential rotation is Sun-like, with the high-latitude spots rotating slower than the low-latitude ones. We found, for both stars, a rather high value of the equator-to-pole differential rotation (dΩ ≈ 0.18 rad d^-1), which disagrees with the predictions of some mean-field models of differential rotation for rapidly rotating stars. Our results agree instead with previous works on solar-type stars and other models that predict a higher latitudinal shear, increasing with equatorial angular velocity, that can vary during the magnetic cycle.

Cold DUst around NEarby Stars (DUNES). First results. A resolved exo-Kuiper belt around the solar-like star ζ2 Ret

We present the first far-IR observations of the solar-type stars δ Pav, HR 8501, 51 Peg and ζ^2 Ret, taken within the context of the DUNES Herschel open time key programme (OTKP). This project uses the PACS and SPIRE instruments with the objective of studying infrared excesses due to exo-Kuiper belts around nearby solar-type stars. The observed 100 μm fluxes from δ Pav, HR 8501, and 51 Peg agree with the predicted photospheric fluxes, excluding debris disks brighter than L_dust/L_* ~ 5 x 10^-7 (1σ level) around those stars. A flattened, disk-like structure with a semi-major axis of ~100 AU in size is detected around ζ2 Ret. The resolved structure suggests the presence of an eccentric dust ring, which we interpret as an exo-Kuiper belt with L_dust/L_* ≈ 10^-5.

Towards Closed-loop Deep Brain Stimulation: Behavior Recognition from Human STN

Deep brain stimulation (DBS) provides significant therapeutic benefit for movement disorders such as Parkinson’s disease (PD). Current DBS devices lack real-time feedback (thus are open loop) and stimulation parameters are adjusted during scheduled visits with a clinician. A closed-loop DBS system may reduce power consumption and side effects by adjusting stimulation parameters based on patient’s behavior. Thus behavior detection is a major step in designing such systems. Various physiological signals can be used to recognize the behaviors. Subthalamic Nucleus (STN) Local field Potential (LFP) is a great candidate signal for the neural feedback, because it can be recorded from the stimulation lead and does not require additional sensors. This thesis proposes novel detection and classification techniques for behavior recognition based on deep brain LFP. Behavior detection from such signals is the vital step in developing the next generation of closed-loop DBS devices. LFP recordings from 13 subjects are utilized in this study to design and evaluate our method. Recordings were performed during the surgery and the subjects were asked to perform various behavioral tasks. Various techniques are used understand how the behaviors modulate the STN. One method studies the time-frequency patterns in the STN LFP during the tasks. Another method measures the temporal inter-hemispheric connectivity of the STN as well as the connectivity between STN and Pre-frontal Cortex (PFC). Experimental results demonstrate that different behaviors create different m odulation patterns in STN and it’s connectivity. We use these patterns as features to classify behaviors. A method for single trial recognition of the patient’s current task is proposed. This method uses wavelet coefficients as features and support vector machine (SVM) as the classifier for recognition of a selection of behaviors: speech, motor, and random. The proposed method is 82.4% accurate for the binary classification and 73.2% for classifying three tasks. As the next step, a practical behavior detection method which asynchronously detects behaviors is proposed. This method does not use any priori knowledge of behavior onsets and is capable of asynchronously detect the finger movements of PD patients. Our study indicates that there is a motor-modulated inter-hemispheric connectivity between LFP signals recorded bilaterally from STN. We utilize a non-linear regression method to measure this inter-hemispheric connectivity and to detect the finger movements. Our experimental results using STN LFP recorded from eight patients with PD demonstrate this is a promising approach for behavior detection and developing novel closed-loop DBS systems.