Peptide analogs to pathogenic epitopes of the human acetylcholine receptor alpha subunit as potential modulators of myasthenia gravis.

Myasthenia gravis is an autoimmune disease in which T cells specific to epitopes of the autoantigen, the human acetylcholine receptor, play a role. We identified two peptides, p195-212 and p259-271, from the alpha subunit of the receptor, which bound to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) from peripheral blood lymphocytes of myasthenia gravis patients and stimulated lymphocytes of >80% of the patients. We have prepared analogs of these myasthenogenic peptides and tested their ability to bind to MHC class II determinants and to interfere specifically with T-cell stimulation. We first determined relative binding efficiency of the myasthenogenic peptides and their analogs to APCs of patients. We found that single substituted analogs of p195-212 (Ala-207) and p259-271 (Lys-262) could bind to human MHC molecules on APCs as efficiently as the original peptides. Moreover, dual analogs containing the two single substituted analogs in one stretch (either sequentially, Ala-207/Lys-262, or reciprocally, Lys-262/Ala-207) could also bind to APCs of patients, including those that failed to bind one of the single substituted analogs. The single substituted analogs significantly inhibited T-cell stimulation induced by their respective myasthenogenic peptides in >95% of the patients. The dual analogs were capable of inhibiting stimulation induced by either of the peptides: They inhibited the response to p195-212 and p259-271 in >95% and >90% of the patients, respectively. Thus, the dual analogs are good candidates for inhibition of T-cell responses of myasthenia gravis patients and might have therapeutic potential.

Activity-Regulated microRNAs: Modulators of Synaptic Growth at the Drosophila Neuromuscular Junction

It is well established that long-term changes in synaptic structure and function are mediated by rapid activity-dependent gene transcription and new protein synthesis. A growing body of evidence supports the involvement of the microRNA (miRNA) pathway in these processes. We have used the Drosophila neuromuscular junction (NMJ) as a model synapse to characterize activity-regulated miRNAs and their important mRNA targets. Here, we have identified five neuronal miRNAs (miRs-1, -8, -289, -314, and -958) that are significantly downregulated in response to neuronal activity. Furthermore we have discovered that neuronal misexpression of three of these miRNAs (miR-8, -289, and -958) is capable of suppressing new synaptic growth in response to activity suggesting that these miRNAs control the translation of biologically relevant target mRNAs. Putative targets of the activity-regulated miRNAs-8 and -289 are significantly enriched in clusters mapping to functional processes including axon development, pathfinding, and axon growth. We demonstrate that activity-regulated miR-8 regulates the 3'UTR of wingless, a presynaptic regulatory protein involved in the process of activity-dependent axon terminal growth. Additionally, we show that the 3'UTR of the protein tyrosine phosophatase leukocyte antengen related (lar), a protein required for axon guidance and synaptic growth, is regulated by activity-regulated miRNAs-8, -289, and -958 in vitro. Both wg and lar were identified as relevant putative targets for co-regulation based through our functional cluster analysis. One putative target of miR-289 is the Ca2+/calmodulin-dependent protein kinase II (CamKII). While CamKII is not predicted as a target for co-regulation by multiple activity-regulated miRNAs we identified it as an especially pertinent target for analysis in our system for two reasons. First, CamKII has an extremely well characterized role in postsynaptic plasticity, but its presynaptic role is less well characterized and bears further analysis. Second, local translation of CamKII mRNA is regulated in part by the miRNA pathway in an activity-dependent manner in dendrites. We find that the CamKII 3'UTR is regulated by miR-289 in-vitro and this regulation is alleviated by mutating the `seed region' of the miR-289 binding site within the CamKII 3'UTR. Furthermore, we demonstrate a requirement for local translation of CamKII in motoneurons in the process of activity-regulated axon terminal growth.

Impaired renal endothelial nitric oxide synthase and reticulocyte production as modulators of hypertension induced by recombinant human erythropoietin in the rat

INTRODUCTION AND AIMS: Hypertension is a common side effect of recombinant human erythropoietin (rHuEPO) therapy; however, the exact pathways remain to be elucidated. The discovery of non-hematopoietic actions of rHuEPO increased the number of patients that could putatively benefit from this therapy; however, to achieve those effects higher doses are usually needed, which increase the risk and incidence of adverse events. Our aim was to study the effect of a broad range of rHuEPO doses on hematological and biochemical parameters, blood pressure and renal function and damage in the rat, focusing on endothelial nitric oxide synthase (eNOS) and hypoxia-inducible factors (HIFs). METHODS: Male Wistar rats were divided in 5 groups receiving different doses of rHuEPO (100, 200, 400 and 600 IU/kg body weight (BW)/week) and saline solution (control), during 3 weeks. Blood and 24h urine were collected to perform hematological and biochemical analysis. Blood pressure (BP) was measured by the tail-cuff method. The kidney tissue was collected to mRNA and protein expression assays and to characterize renal lesions. RESULTS: A dose-dependent increase in red blood cells count, hematocrit and hemoglobin levels was found with rHuEPO therapy, in rHuEPO200, rHuEPO400 and rHuEPO600 groups. Increased reticulocyte count was found in the rHuEPO400 and rHuEPO600 groups. BP raised in all groups receiving rHuEPO. The rHuEPO200 and rHuEPO600 groups presented increased kidney protein levels of HIF2α and a reduction in kidney protein levels of eNOS, along with the highest grade of vascular and tubular renal lesions. CONCLUSIONS: Our study showed that rHuEPO-induced hypertension might involve indirect (hematological) and direct (renal) effects which varies according to the dose used. Thus, rHuEPO therapy should be performed rationally and under adequate surveillance, as hypertension develops even with lower doses. Especial caution with higher doses should be taken, as rHuEPO-induced hypertension leads to early renal damage without alterations in traditional markers of renal function, thus masking the serious adverse effects and risks.

Hemorheological alterations in sickle cell anemia and their clinical consequences: the role of genetic modulators

Sickle cell anemia (SCA) is an autosomal recessive chronic hemolytic anemia, caused by homozygosity for the HBB:c.20A>T mutation. The disease presents with high clinical heterogeneity, stroke being the most devastating manifestation. This study aimed to identify genetic modulators of severe hemolysis and stroke risk in children with SCA, as well as understand their consequences at the hemorheological level. Sixty-six children with SCA were categorised according to their degree of cerebral vasculopathy (Stroke/Risk/Control). Relevant data were collected from patients’ medical records. Several polymorphic regions in genes related to vascular cell adhesion and tonus were characterized by molecular methodologies. Data analyses were performed using R software. Several in silico tools (e.g. TFBind, MatInspector) were applied to investigate the main variant consequences. Some genetic variants in vascular adhesion molecule-1 gene promoter and endothelial nitric oxide synthase gene were associated with higher levels of hemolysis and stroke events. They modify important transcription factor binding sites or disturb the corresponding protein structure/function. Our findings emphasize the relevance of the genetic variants in modulating the degree of hemolysis and development of cerebral vasculopathy due to their effect on gene expression, modification of protein biological activities related with erythrocyte/endothelial interactions and consequent hemorheological abnormalities in SCA.

Novel electro-optical light modulators.

Vita.

Ciguatoxins: Cyclic polyether modulators of voltage-gated Iion channel function

Ciguatoxins are cyclic polyether toxins, derived from marine dinoflagellates, which are responsible for the symptoms of ciguatera poisoning. Ingestion of tropical and subtropical fin fish contaminated by ciguatoxins results in an illness characterised by neurological, cardiovascular and gastrointestinal disorders. The pharmacology of ciguatoxins is characterised by their ability to cause persistent activation of voltage-gated sodium channels, to increase neuronal excitability and neurotransmitter release, to impair synaptic vesicle recycling, and to cause cell swelling. It is these effects, in combination with an action to block voltage-gated potassium channels at high doses, which are believed to underlie the complex of symptoms associated with ciguatera. This review examines the sources, structures and pharmacology of ciguatoxins. In particular, attention is placed on their cellular modes of actions to modulate voltage-gated ion channels and other Na+-dependent mechanisms in numerous cell types and to current approaches for detection and treatment of ciguatera.

All optical networking beyond 10 Gbits/s OTDM networks based on electro-optic modulators and fibre ring lasers

This thesis examines options for high capacity all optical networks. Specifically optical time division multiplexed (OTDM) networks based on electro-optic modulators are investigated experimentally, whilst comparisons with alternative approaches are carried out. It is intended that the thesis will form the basis of comparison between optical time division multiplexed networks and the more mature approach of wavelength division multiplexed networks. Following an introduction to optical networking concepts, the required component technologies are discussed. In particular various optical pulse sources are described with the demanding restrictions of optical multiplexing in mind. This is followed by a discussion of the construction of multiplexers and demultiplexers, including favoured techniques for high speed clock recovery. Theoretical treatments of the performance of Mach Zehnder and electroabsorption modulators support the design criteria that are established for the construction of simple optical time division multiplexed systems. Having established appropriate end terminals for an optical network, the thesis examines transmission issues associated with high speed RZ data signals. Propagation of RZ signals over both installed (standard fibre) and newly commissioned fibre routes are considered in turn. In the case of standard fibre systems, the use of dispersion compensation is summarised, and the application of mid span spectral inversion experimentally investigated. For green field sites, soliton like propagation of high speed data signals is demonstrated. In this case the particular restrictions of high speed soliton systems are discussed and experimentally investigated, namely the increasing impact of timing jitter and the downward pressure on repeater spacings due to the constraint of the average soliton model. These issues are each addressed through investigations of active soliton control for OTDM systems and through investigations of novel fibre types respectively. Finally the particularly remarkable networking potential of optical time division multiplexed systems is established, and infinite node cascadability using soliton control is demonstrated. A final comparison of the various technologies for optical multiplexing is presented in the conclusions, where the relative merits of the technologies for optical networking emerges as the key differentiator between technologies.

40-Gbaud 16-QAM transmitter using tandem IQ modulators with binary driving electronic signals

We propose a novel 16-quadrature amplitude modulation (QAM) transmitter based on two cascaded IQ modulators driven by four separate binary electrical signals. The proposed 16-QAM transmitter features scalable configuration and stable performance with simple bias-control. Generation of 16-QAM signals at 40 Gbaud is experimentally demonstrated for the first time and visualized with a high speed constellation analyzer. The proposed modulator is also compared to two other schemes. We investigate the modulator bandwidth requirements and tolerance to accumulated chromatic dispersion through numerical simulations, and the minimum theoretical insertion attenuation is calculated analytically.

Multi-wavelength source using low drive-voltage amplitude modulators for optical communications

A simple and cost-effective technique for generating a flat, square-shaped multi-wavelength optical comb with 42.6 GHz line spacing and over 0.5 THz of total bandwidth is presented. A detailed theoretical analysis is presented, showing that using two concatenated modulators driven with voltages of 3.5 Vp are necessary to generate 11 comb lines with a flatness below 2dB. This performance is experimentally demonstrated using two cascaded Versawave 40 Gbit/s low drive voltage electro-optic polarisation modulators, where an 11 channel optical comb with a flatness of 1.9 dB and a side-mode-suppression ratio (SMSR) of 12.6 dB was obtained.

Optimisation of optical regeneration at 40 Gbit/s in dispersion-managed transmission lines with modified synchronous modulators

Numerical optimisation of a 40 Gbit/s dispersion-managed soliton transmission system with in-line synchronous intensity modulation is performed. Using a time-saving numerical approach, superiority of the modified synchronous modulation over conventional synchronous modulation is demonstrated and an optimal operational regime is found.

Optical regeneration at 40 Gb/s in dispersion-managed transmission lines with in-line synchronous modulators

Numerical optimization is performed of the 40-Gb/s dispersion-managed (DM) soliton transmission system with in-line synchronous intensity modulation. Stability of DM soliton transmission results from a combined action of dispersion, nonlinearity, in-line filtering, and modulation through effective periodic bandwidth management of carrier pulses. Therefore, analysis of the multiparametric problem is typically required. A two-stage time-saving numerical optimization procedure is applied. At the first step, the regions of the stable carrier propagation are determined using theoretical models available for DM solitons, and system parameters are optimized. At the second stage, full numerical simulations are undertaken in order to verify the tolerance of optimal transmission regimes. An approach developed demonstrates feasibility of error-free transmission over 20 000 km in a transmission line composed of standard fiber and dispersion compensation fiber at 40 Gb/s.

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.

Multi-wavelength source using low drive-voltage amplitude modulators for optical communications

A simple and cost-effective technique for generating a flat, square-shaped multi-wavelength optical comb with 42.6 GHz line spacing and over 0.5 THz of total bandwidth is presented. A detailed theoretical analysis is presented, showing that using two concatenated modulators driven with voltages of 3.5 Vp are necessary to generate 11 comb lines with a flatness below 2dB. This performance is experimentally demonstrated using two cascaded Versawave 40 Gbit/s low drive voltage electro-optic polarisation modulators, where an 11 channel optical comb with a flatness of 1.9 dB and a side-mode-suppression ratio (SMSR) of 12.6 dB was obtained.

Two cascaded SOAs used as intensity modulators for adaptively modulated optical OFDM signals in optical access networks

Detailed theoretical and numerical investigations of the transmission performance of adaptively modulated optical orthogonal frequency division multiplexed (AMOOFDM) signals are undertaken, for the first time, in optical amplification and chromatic dispersion (CD) compensation free single mode fiber (SMF) intensity-modulated and directdetection (IMDD) systems using two cascaded semiconductor optical amplifiers in a counterpropagating configuration as an intensity modulator (TC-SOA-CC-IM). A theoretical model describing the characteristics of this configuration is developed. Extensive performance comparisons are also made between the TC-SOA-CC and the single SOA intensity modulators. It is shown that, the TC-SOA-CC reaches its strongly saturated region using a lower input optical power much faster than the single SOA resulting in significantly reduced effective carrier lifetime and thus wide TC-SOA-CC bandwidths. It is shown that at low input optical power, we can increase the signal line rate almost 115% which will be more than twice the transmission performance offered by single SOA. In addition, the TCSOA-CC-IM is capable of supporting signal line rates higher than corresponding to the SOA-IM by using 10dB lower input optical powers. For long transmission distance, the TC-SOA-CC-IM has much stronger CD compensation capability compared to the SOA-IM. In addition the use of TC-SOA-CC-IM is more effective regarding the capability to benefit from the CD compensation for shorter distances starting at 60km SMF, whilst for the SOA-IM starting at 90km. © 2014 Optical Society of America.