Padronização e validação de marcadores moleculares para o diagnóstico da Leishmaniose Tegumentar

O diagnóstico da leishmaniose tegumentar (LT) baseia-se em critérios clínicos e epidemiológicos podendo ser confirmado por exames laboratoriais de rotina como a pesquisa direta do parasito por microscopia e a intradermorreação de Montenegro. Atualmente, os métodos moleculares, principalmente a reação da cadeia da polimerase (PCR), têm sido considerados para aplicação em amostras clínicas, devido a sua alta sensibilidade e especificidade. Este trabalho teve como objetivo a padronização e validação das técnicas de PCR-RFLP com diferentes iniciadores (kDNA, its1, hsp70 e prp1), visando o diagnóstico e a identificação das espécies de Leishmania presentes em amostras de DNA provenientes de lesões de pele ou mucosa de 140 pacientes com suspeita de leishmaniose tegumentar. Para tal, realizamos ensaios de: 1) sensibilidade das PCRs com os diferentes iniciadores, 2) especificidade dos ensaios utilizando DNAs de diferentes espécies de referência de Leishmania, de tripanossomatídeos inferiores e de fungos, 3) validação das técnicas de PCR-RFLP com os iniciadores estudados em amostras de DNA de lesões de pele ou mucosas de pacientes com LT. Os resultados dos ensaios de limiar de detecção das PCR (sensibilidade) mostraram que os quatro iniciadores do estudo foram capazes de detectar o DNA do parasito, porém em quantidades distintas: até 500 fg com os iniciadores para kDNA e its1, até 400 fg com hsp70 e até 5 ng com prp1. Quanto à especificidade dos iniciadores, não houve amplificação dos DNAs fúngicos. Por outro lado, nos ensaios com os iniciadores para kDNA e hsp70, verificamos amplificação do fragmento esperado em amostras de DNA de tripanossomatídeos. Nos ensaios com its1 e prp1, o padrão de amplificação com os DNAs de tripanossomatídeos foi diferente do apresentado pelas espécies de Leishmania. Verificou-se nos ensaios de validação que o PCR-kDNA detectou o parasito em todas as 140 amostras de DNA de pacientes e, assim, foi utilizado como critério de inclusão das amostras. A PCR-its1 apresentou menor sensibilidade, mesmo após a reamplificação com o mesmo iniciador (85,7% ou 120/140 amostras). Para os ensaios da PCR-hsp70, as amostras de DNA foram amplificadas com Repli G, para obter uma sensibilidade de 68,4% (89/140 amostras). A PCR-prp1 não detectou o parasito em amostras de DNA dos pacientes. Quanto aos ensaios para a identificação da espécie presente na lesão, a PCR-kDNA-RFLP-HaeIII e a PCR-its1- RFLP-HaeIII permitem a distinção de L. (L.) amazonensis das outras espécies pertencentes ao subgênero Viannia. A PCR-hsp70-RFLP-HaeIII-BstUI, apesar de potencialmente ser capaz de identificar as seis espécies de Leishmania analisadas, quando utilizada na avaliação das amostras humanas permitiu apenas a identificação de L. (V.) braziliensis. No entanto, é uma técnica com várias etapas e de difícil execução, o que pode inviabilizar o seu uso rotineiro em centros de referência em diagnósticos. Assim, recomendamos o uso dessa metodologia apenas em locais onde várias espécies de Leishmania sejam endêmicas. Finalmente, os resultados indicam que o kDNA-PCR devido à alta sensibilidade apresentada e facilidade de execução pode ser empregada como exame de rotina nos centros de referência, permitindo a confirmação ou exclusão da LT.

Rapid Scan Electron Paramagnetic Resonance (EPR) and Digital EPR Development

Rapid scan electron paramagnetic resonance (EPR) was developed in the Eaton laboratory at the University of Denver. Applications of rapid scan to wider spectra, such as for immobilized nitroxides, spin-labeled proteins, irradiated tooth and fingernail samples were demonstrated in this dissertation. The scan width has been increased from 55 G to 160 G. The signal to noise (S/N) improvement for slowly tumbling spin-labeled protein samples that is provided by rapid scan EPR will be highly advantageous for biophysical studies. With substantial improvement in S/N by rapid scan, the dose estimation for irradiated tooth enamels became more reliable than the traditional continuous wave (CW) EPR. An alternate approach of rapid scan, called field-stepped direct detection EPR, was developed to reconstruct wider EPR signals. A Mn2+ containing crystal was measured by field-stepped direct detection EPR, which had a spectrum more than 6000 G wide. Since the field-stepped direct detection extends the advantages of rapid scan to much wider scan ranges, this methodology has a great potential to replace the traditional CW EPR. With recent advances in digital electronics, a digital rapid scan spectrometer was built based on an arbitrary waveform generator (AWG), which can excite spins and detect EPR signals with a fully digital system. A near-baseband detection method was used to acquire the in-phase and quadrature signals in one physical channel. The signal was analyzed digitally to generate ideally orthogonal quadrature signals. A multiharmonic algorithm was developed that employed harmonics of the modulation frequencies acquired in the spectrometer transient mode. It was applied for signals with complicated lineshapes, and can simplify the selection of modulation amplitude. A digital saturation recovery system based on an AWG was built at X-band (9.6 GHz). To demonstrate performance of the system, the spin-lattice relaxation time of a fused quartz rod was measured at room temperature with fully digital excitation and detection.

Analysis and optimisation of the performance of nonlinear optical communication systems

We investigate the feasibility of simultaneous suppressing of the amplification noise and nonlinearity, representing the most fundamental limiting factors in modern optical communication. To accomplish this task we developed a general design optimisation technique, based on concepts of noise and nonlinearity management. We demonstrate the immense efficiency of the novel approach by applying it to a design optimisation of transmission lines with periodic dispersion compensation using Raman and hybrid Raman-EDFA amplification. Moreover, we showed, using nonlinearity management considerations, that the optimal performance in high bit-rate dispersion managed fibre systems with hybrid amplification is achieved for a certain amplifier spacing – which is different from commonly known optimal noise performance corresponding to fully distributed amplification. Required for an accurate estimation of the bit error rate, the complete knowledge of signal statistics is crucial for modern transmission links with strong inherent nonlinearity. Therefore, we implemented the advanced multicanonical Monte Carlo (MMC) method, acknowledged for its efficiency in estimating distribution tails. We have accurately computed acknowledged for its efficiency in estimating distribution tails. We have accurately computed marginal probability density functions for soliton parameters, by numerical modelling of Fokker-Plank equation applying the MMC simulation technique. Moreover, applying a powerful MMC method we have studied the BER penalty caused by deviations from the optimal decision level in systems employing in-line 2R optical regeneration. We have demonstrated that in such systems the analytical linear approximation that makes a better fit in the central part of the regenerator nonlinear transfer function produces more accurate approximation of the BER and BER penalty. We present a statistical analysis of RZ-DPSK optical signal at direct detection receiver with Mach-Zehnder interferometer demodulation

Time resolved bit error rate analysis of a fast switching tunable laser for use in optically switched networks

We investigate the use of different direct detection modulation formats in a wavelength switched optical network. We find the minimum time it takes a tunable sampled grating distributed Bragg reflector laser to recover after switching from one wavelength channel to another for different modulation formats. The recovery time is investigated utilizing a field programmable gate array which operates as a time resolved bit error rate detector. The detector offers 93 ps resolution operating at 10.7 Gb/s and allows for all the data received to contribute to the measurement, allowing low bit error rates to be measured at high speed. The recovery times for 10.7 Gb/s non-return-to-zero on–off keyed modulation, 10.7 Gb/s differentially phase shift keyed signal and 21.4 Gb/s differentially quadrature phase shift keyed formats can be as low as 4 ns, 7 ns and 40 ns, respectively. The time resolved phase noise associated with laser settling is simultaneously measured for 21.4 Gb/s differentially quadrature phase shift keyed data and it shows that the phase noise coupled with frequency error is the primary limitation on transmitting immediately after a laser switching event.

Chromatic dispersion compensation using full-field maximum-likelihood sequence estimation

We investigate full-field detection-based maximum-likelihood sequence estimation (MLSE) for chromatic dispersion compensation in 10 Gbit/s OOK optical communication systems. Important design criteria are identified to optimize the system performance. It is confirmed that approximately 50% improvement in transmission reach can be achieved compared to conventional direct-detection MLSE at both 4 and 16 states. It is also shown that full-field MLSE is more robust to the noise and the associated noise amplifications in full-field reconstruction, and consequently exhibits better tolerance to nonoptimized system parameters than full-field feedforward equalizer. Experiments over 124 km spans of field-installed single-mode fiber without optical dispersion compensation using full-field MLSE verify the theoretically predicted performance benefits.

Unrepeatered DPSK transmission over 360 km SMF-28 fibre using URFL based amplification

Unrepeatered 42.7 Gb/s per channel RZ-DPSK transmission over standard SMF-28 fibre with novel URFL based amplification using fibre Bragg gratings is investigated. OSNR and gain performance are studied experimentally and through simulations. Error free transmission for 16 channels across the full C-band with direct detection was experimentally demonstrated for 280 km span length, as well as 6-channel transmission at 340 km and single-channel transmission up to 360 km (75 dB) without employing ROPA or specialty fibres.©2013 Optical Society of America.

Gain switched multi-carrier transmitter and pilot tone based receiver for long reach access networks

A novel and cost effective long reach PON downlink scenario is proposed employing a multi-carrier transmitter and pilot tone aided direct detection at the receiver. Error free performance with QPSK and 50km transmission is presented. © 2012 OSA.

Optimum bias point in broadband subcarrier multiplexing with optical IQ modulators

This paper develops a theoretical analysis of the tradeoff between carrier suppression and nonlinearities induced by optical IQ modulators in direct-detection subcarrier multiplexing systems. The tradeoff is obtained by examining the influence of the bias conditions of the modulator on the transmitted single side band signal. The frequency components in the electric field and the associated photocurrent at the output of the IQ modulator are derived mathematically. For any frequency plan, the optimum bias point can be identified by calculating the sensitivity gain for every subchannel. A setup composed of subcarriers located at multiples of the data rate ensures that the effects of intermodulation distortion are studied in the most suitable conditions. Experimental tests with up to five QPSK electrical subchannels are performed to verify the mathematical model and validate the predicted gains in sensitivity.

Time resolved bit error rate analysis of a fast switching tunable laser for use in optically switched networks

We investigate the use of different direct detection modulation formats in a wavelength switched optical network. We find the minimum time it takes a tunable sampled grating distributed Bragg reflector laser to recover after switching from one wavelength channel to another for different modulation formats. The recovery time is investigated utilizing a field programmable gate array which operates as a time resolved bit error rate detector. The detector offers 93 ps resolution operating at 10.7 Gb/s and allows for all the data received to contribute to the measurement, allowing low bit error rates to be measured at high speed. The recovery times for 10.7 Gb/s non-return-to-zero on–off keyed modulation, 10.7 Gb/s differentially phase shift keyed signal and 21.4 Gb/s differentially quadrature phase shift keyed formats can be as low as 4 ns, 7 ns and 40 ns, respectively. The time resolved phase noise associated with laser settling is simultaneously measured for 21.4 Gb/s differentially quadrature phase shift keyed data and it shows that the phase noise coupled with frequency error is the primary limitation on transmitting immediately after a laser switching event.

Unrepeatered 8 x 40Gb/s transmission over 320km SMF-28 using ultra-long raman fibre laser based amplification

Unrepeatered transmission over SMF-28 fibre is investigated using ultra-long Raman fibre laser based amplification. Experiments and simulations demonstrate 8 x 42.7Gb/s transmission up to 320km (67dB) span length using DPSK and ASK modulation with direct detection. © 2012 OSA.

Reductions of peak-to-average power ratio and optical beat interference in cost-effective OFDMA-PONs

The peak-to-average power ratio (PAPR) and optical beat interference (OBI) effects are examined thoroughly in orthogonal frequency-division multiplexing access (OFDMA)-passive optical networks (PONs) at a signal bit rate up to ∼ 20 Gb/s per channel using cost-effective intensity-modulation and direct-detection (IM/DD). Single-channel OOFDM and upstream multichannel OFDM-PONs are investigated for up to six users. A number of techniques for mitigating the PAPR and OBI effects are presented and evaluated including adaptive-loading algorithms such as bit/power-loading, clipping for PAPR reduction, and thermal detuning (TD) for the OBI suppression. It is shown that the bit-loading algorithm is a very efficient PAPR reduction technique by reducing it at about 1.2 dB over 100 Km of transmission. It is also revealed that the optimum method for suppressing the OBI is the TD + bit-loading. For a targeted BER of 1 × 10-3, the minimum allowed channel spacing is 11 GHz when employing six users. © 2013 Springer Science+Business Media New York.

Development of micro immunosensors to study genomic and proteomic biomarkers related to cancer and Alzheimer's disease

A report from the National Institutes of Health defines a disease biomarker as a “characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.” Early diagnosis is a crucial factor for incurable disease such as cancer and Alzheimer’s disease (AD). During the last decade researchers have discovered that biochemical changes caused by a disease can be detected considerably earlier as compared to physical manifestations/symptoms. In this dissertation electrochemical detection was utilized as the detection strategy as it offers high sensitivity/specificity, ease of operation, and capability of miniaturization and multiplexed detection. Electrochemical detection of biological analytes is an established field, and has matured at a rapid pace during the last 50 years and adapted itself to advances in micro/nanofabrication procedures. Carbon fiber microelectrodes were utilized as the platform sensor due to their high signal to noise ratio, ease and low-cost of fabrication, biocompatibility, and active carbon surface which allows conjugation with biorecognition moieties. This dissertation specifically focuses on the detection of 3 extensively validated biomarkers for cancer and AD. Firstly, vascular endothelial growth factor (VEGF) a cancer biomarker was detected using a one-step, reagentless immunosensing strategy. The immunosensing strategy allowed a rapid and sensitive means of VEGF detection with a detection limit of about 38 pg/mL with a linear dynamic range of 0–100 pg/mL. Direct detection of AD-related biomarker amyloid beta (Aβ) was achieved by exploiting its inherent electroactivity. The quantification of the ratio of Aβ1-40/42 (or Aβ ratio) has been established as a reliable test to diagnose AD through human clinical trials. Triple barrel carbon fiber microelectrodes were used to simultaneously detect Aβ1-40 and Aβ1-42 in cerebrospinal fluid from rats within a detection range of 100nM to 1.2μM and 400nM to 1μM respectively. In addition, the release of DNA damage/repair biomarker 8-hydroxydeoxyguanine (8-OHdG) under the influence of reactive oxidative stress from single lung endothelial cell was monitored using an activated carbon fiber microelectrode. The sensor was used to test the influence of nicotine, which is one of the most biologically active chemicals present in cigarette smoke and smokeless tobacco.

Scaling multimode fibre IM/DD transmission capacity through spatial-spectral multiplexing

Data centre connections can greatly benefit from parallel transmission channels on one multimode fibre (MMF). Shortwave wavelength division multiplexing (SWDM) achieves parallel transmission through spectral multiplexing. Furthermore, MMFs offer a spatial dimension that should be exploited to increase parallel transmission, albeit in a cost-effective way. In this paper, it is shown that SWDM and spatial multiplexing can be combined in intensity modulation and direct detection MMF transmission systems that use selective offset excitation and mode-selective spatial filtering.

Gas, water and volume characteristics during controlled degassing experiments on ODP Leg 164 PCS cores

A pressurized core with CH4 hydrate or dissolved CH4 should evolve gas volumes in a predictable manner as pressure is released over time at isothermal conditions. Incremental gas volumes were collected as pressure was released over time from 29 pressure core sampler (PCS) cores from Sites 994, 995, 996, and 997 on the Blake Ridge. Most of these cores were kept at or near 0ºC with an ice bath, and many of these cores yielded substantial quantities of CH4. Volume-pressure plots were constructed for 20 of these cores. Only five plots conform to expected volume and pressure changes for sediment cores with CH4 hydrate under initial pressure and temperature conditions. However, other evidence suggests that sediment in these five and at least five other PCS cores contained CH4 hydrate before core recovery and gas release. Detection of CH4 hydrate in a pressurized sediment core through volume-pressure relationships is complicated by two factors. First, significant quantities of CH4-poor borehole water fill the PCS and come into contact with the core. This leads to dilution of CH4 concentration in interstitial water and, in many cases, decomposition of CH4 hydrate before a degassing experiment begins. Second, degassing experiments were conducted after the PCS had equilibrated in an ice-water bath (0ºC). This temperature is significantly lower than in situ values in the sediment formation before core recovery. Our results and interpretations for PCS cores collected on Leg 164 imply that pressurized containers formerly used by the Deep Sea Drilling Project (DSDP) and currently used by ODP are not appropriately designed for direct detection of gas hydrate in sediment at in situ conditions through volume-pressure relationships.

Ionization of Atoms by Slow Heavy Particles, Including Dark Matter

Atoms and molecules can become ionized during the scattering of a slow, heavy particle off a bound electron. Such an interaction involving leptophilic weakly interacting massive particles (WIMPs) is a promising possible explanation for the anomalous 9σ annual modulation in the DAMA dark matter direct detection experiment [R. Bernabei et al., Eur. Phys. J. C 73, 2648 (2013)]. We demonstrate the applicability of the Born approximation for such an interaction by showing its equivalence to the semiclassical adiabatic treatment of atomic ionization by slow-moving WIMPs. Conventional wisdom has it that the ionization probability for such a process should be exponentially small. We show, however, that due to nonanalytic, cusplike behavior of Coulomb functions close to the nucleus this suppression is removed, leading to an effective atomic structure enhancement. We also show that electron relativistic effects actually give the dominant contribution to such a process, enhancing the differential cross section by up to 1000 times.