Recovery of linear and nonlinear heart rate dynamics after coronary artery bypass grafting surgery

Background: Conventional coronary artery bypass grafting (C-CABG) and off-pump CABG (OPCAB) surgery may produce different patients' outcomes, including the extent of cardiac autonomic (CA) imbalance. the beneficial effects of an exercise-based inpatient programme on heart rate variability (HRV) for C-CABG patients have already been demonstrated by our group. However, there are no studies about the impact of a cardiac rehabilitation (CR) on HRV behaviour after OPCAB. the aim of this study is to compare the influence of both operative techniques on HRV pattern following CR in the postoperative (PO) period.Methods: Cardiac autonomic function was evaluated by HRV indices pre- and post-CR in patients undergoing C-CABG (n = 15) and OPCAB (n = 13). All patients participated in a short-term(approximately 5 days) supervised CR programme of early mobilization, consisting of progressive exercises, from active-assistive movements at PO day 1 to climbing flights of stairs at PO day 5.Results: Both groups demonstrated a reduction in HRV following surgery. the CR programme promoted improvements in HRV indices at discharge for both groups. the OPCAB group presented with higher HRV values at discharge, compared to the C-CABG group, indicating a better recovery of CA function.Conclusion: Our data suggest that patients submitted to OPCAB and an inpatient CR programme present with greater improvement in CA function compared to C-CABG.

Neonatal exposure to estradiol in female rats influences neuroactive steroid concentrations and behaviour in the adult rat

The gonadal steroids, in particular estradiol, exert an important action during perinatal period in the regulation of sexual dimorphism and neuronal plasticity, and in the growth and development of nervous system. Exposure of the developing female to estrogens during perinatal period may have long-lasting effects that are now regarded as “programming” the female neuroendocrine axis to malfunction in adulthood. The purpose of this study was to describe the effect of a single administration of a low dose (10 μg) of β-estradiol 3-benzoate (EB) to female rats on the day of birth on brain and plasma concentrations of the neuroactive steroid allopregnanolone, general behaviours and behavioral sensitivity to benzodiazepines. Neonatal administration of EB induces a dramatic reduction in the cerebrocortical and plasma levels of allopregnanolone and progesterone that were apparent in both juvenile (21 days) and adult (60 days). In contrast, this treatment did not affect 17β-estradiol levels. Female rats treated with β-estradiol 3-benzoate showed a delay in vaginal opening, aciclicity characterized by prolonged estrus, and ovarian failure. Given that allopregnanolone elicits anxiolytic, antidepressive, anticonvulsant, sedative-hypnotic effects and facilitates social behaviour, we assessed whether this treatment might modify different emotional, cognitive and social behaviours. This treatment did not affect locomotor activity, anxiety- and mood-related behaviours, seizures sensitivity and spatial memory. In contrast, neonatal β-estradiol 3-benzoate-treated rats showed a dominant, but not aggressive, behaviour and an increase in body investigation, especially anogenital investigation, characteristic of male appetitive behaviour. On the contrary, neonatal administration of β-estradiol 3-benzoate to female rats increases sensitivity to the anxiolytic, sedative, and amnesic effects of diazepam in adulthood. These results indicate that the marked and persistent reduction in the cerebrocortical and peripheral concentration of the neuroactive steroid allopregnanolone induced by neonatal treatment with β-estradiol 3-benzoate does not change baseline behaviours in adult rats. On the contrary, the low levels of allopregnanolone seems to be associated to changes in the behavioural sensitivity to the positive allosteric modulator of the GABAA receptor, diazepam. These effects of estradiol suggest that it plays a major role in pharmacological regulation both of GABAergic transmission and of the abundance of endogenous modulators of such transmission during development of the central nervous system.

Progettazione e sintesi di nuovi antagonisti CB1 a struttura pirazolica come potenziali radiofarmaci per la PET imaging

Cannabinoid receptors are members of the large family of G-protein coupled receptors. Two types of cannabinoid receptor have been discovered: CB1 and CB2. CB1 receptors are localised predominantly in the brain whereas CB2 receptors are more abundant in peripheral nervous system cells. CB1 receptors have been related with a number of disorders, including depression, anxiety, stress, schizophrenia, chronic pain and obesity. For this reason, several cannabinoid ligands were developed as drug candidates. Among these ligands, a prominent position is occupied by SR141716 (Rimonabant), which is a pyrazole derivative with inverse agonist activity discovered by Sanofi-Synthelabo in 1994. This compound was marketed in Europe as an anti-obesity drug, but subsequently withdrawn due to its side-effects. Since the relationship between the CB1 receptors’ functional modification, density and distribution, and the beginning of a pathological state is still not well understood, the development of radio-ligands suitable for in vivo PET (Positron Emission Tomography) functional imaging of CB1 receptors remains an important area of research in medicine and drug development. To date, a few radiotracers have been synthesised and tested in vivo, but most of them afforded unsatisfactory brain imaging results. A handful of radiolabelled CB1 PET ligands have also been submitted to clinical trials in humans. In this PhD Thesis the design, synthesis and characterization of three new classes of potential high-affinity CB1 ligands as candidate PET tracers is described.

Fisiopatologia da Dor

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

As neurotoxinas de clostridium sp.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Lupus Eritematoso Sistémico

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Novos sistemas terapêuticos para administração nasal de fármacos

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Occupational lead neurotoxicity: A behavioural and electrophysiological evaluation. study design and year one results.

To evaluate the effects of chronic lead exposure on the nervous system in adults, a set of neurobehavioural and electrophysiological tests was administered to 99 lead exposed foundry employees and 61 unexposed workers. Current and past blood lead concentrations were used to estimate the degree of lead absorption; all previous blood lead concentrations had been less than or equal to 90 micrograms/100 ml. Characteristic signs (such as wrist extensor weakness) or symptoms (such as colic) of lead poisoning were not seen. Sensory conduction in the sural nerve was not affected. By contrast, various neurobehavioural functions deteriorated with increasing lead burden. Workers with blood lead concentrations between 40 and 60 micrograms/100 ml showed impaired performance on tests of verbal concept formation, visual/motor performance, memory, and mood. Thus impairment in central nervous system function in lead exposed adults occurred in the absence of peripheral nervous system derangement and increased in severity with increasing lead dose.

A Neural Network Model for the Spatial and Temporal Response of Retinal Ganglion Cells

This article introduces a quantitative model of early visual system function. The model is formulated to unify analyses of spatial and temporal information processing by the nervous system. Functional constraints of the model suggest mechanisms analogous to photoreceptors, bipolar cells, and retinal ganglion cells, which can be formally represented with first order differential equations. Preliminary numerical simulations and analytical results show that the same formal mechanisms can explain the behavior of both X (linear) and Y (nonlinear) retinal ganglion cell classes by simple changes in the relative width of the receptive field (RF) center and surround mechanisms. Specifically, an increase in the width of the RF center results in a change from X-like to Y-like response, in agreement with anatomical data on the relationship between α- and

A Unified Model of Spatiotemporal Processing in the Retina

A computational model of visual processing in the vertebrate retina provides a unified explanation of a range of data previously treated by disparate models. Three results are reported here: the model proposes a functional explanation for the primary feed-forward retinal circuit found in vertebrate retinae, it shows how this retinal circuit combines nonlinear adaptation with the desirable properties of linear processing, and it accounts for the origin of parallel transient (nonlinear) and sustained (linear) visual processing streams as simple variants of the same retinal circuit. The retina, owing to its accessibility and to its fundamental role in the initial transduction of light into neural signals, is among the most extensively studied neural structures in the nervous system. Since the pioneering anatomical work by Ramón y Cajal at the turn of the last century[1], technological advances have abetted detailed descriptions of the physiological, pharmacological, and functional properties of many types of retinal cells. However, the relationship between structure and function in the retina is still poorly understood. This article outlines a computational model developed to address fundamental constraints of biological visual systems. Neurons that process nonnegative input signals-such as retinal illuminance-are subject to an inescapable tradeoff between accurate processing in the spatial and temporal domains. Accurate processing in both domains can be achieved with a model that combines nonlinear mechanisms for temporal and spatial adaptation within three layers of feed-forward processing. The resulting architecture is structurally similar to the feed-forward retinal circuit connecting photoreceptors to retinal ganglion cells through bipolar cells. This similarity suggests that the three-layer structure observed in all vertebrate retinae[2] is a required minimal anatomy for accurate spatiotemporal visual processing. This hypothesis is supported through computer simulations showing that the model's output layer accounts for many properties of retinal ganglion cells[3],[4],[5],[6]. Moreover, the model shows how the retina can extend its dynamic range through nonlinear adaptation while exhibiting seemingly linear behavior in response to a variety of spatiotemporal input stimuli. This property is the basis for the prediction that the same retinal circuit can account for both sustained (X) and transient (Y) cat ganglion cells[7] by simple morphological changes. The ability to generate distinct functional behaviors by simple changes in cell morphology suggests that different functional pathways originating in the retina may have evolved from a unified anatomy designed to cope with the constraints of low-level biological vision.

Characterisation of the non-muscle α-actinins

Actinins are cytoskeleton proteins that cross-link actin filaments. Evolution of the actinin family resulted in the formation of Ca++-insensitive muscle isoforms (actinin-2 and- 3) and Ca++-sensitive non-muscle isoforms (actinin-1 and -4) with regard to their actin-binding function. Despite high sequence similarity, unique properties have been ascribed to actinin-4 compared with actinin-1. Actinin-4 is the predominant isoform reported to be associated with the cancer phenotype. Actinin-4, but not actinin-1, is essential for normal glomerular function in the kidney and and is able to translocate to the nucleus to regulate transcription. To understand the molecular basis for such isoform-specific functions I have comprehensively compared these proteins in terms of localisation, migration, alternative splicing, actin-binding properties, heterodimer formation and molecular interactions for the first time. This work characterises a number of commercially available actinin antibodies and in doing so, identifies actinin-1, -2 and -4 isoform-specific antibodies that enabled studies of actinin expression and localisation. This work identifies the actinin rod domain as the predominant domain that influences actinin localisation however localisation is likely to be effected by the entire actinin protein. si-RNA- mediated knockdown of actinin-1 and -4 did not affect migration in a number of cell lines highlighting that migration may only require a fraction of total non-muscle actinin levels. This work finds that the Ca++-insensitive variant of actinin-4 is expressed only in the nervous system and thus cannot be regarded as a smooth muscle isoform, as is the case for the Ca++-insensitive variant of actinin-1. This work also identifies a previously unreported exon 19a+19b expressing variant of actinin-4 in human skeletal muscle. This work finds that alternative splice variants of actinin-1 and -4 are co-expressed in a number of tissues, in particular the brain. In contrast to healthy brain, glioblastoma cells express Ca++-sensitive variants of both actinin-1 and -4. Actin-binding properties of actinin-1 and -4 are similar and are unlikely to explain isoform-specific functions. Surprisingly, this work reveals that actinin-1/-4 heterodimers, rather than homodimers, are the most abundant form of actinin in many cancer cell lines. Taken together this data suggests that actinin-1 and -4 cannot be viewed as distinct entities from each other but rather as proteins that can exist in both homodimeric and heterodimeric forms. Finally, this work employs yeast two-hybrid and proteomic approaches to identify actinin-interacting proteins. In doing so, this work identifies a number of putative actinin-4 specific interacting partners that may help to explain some of the unique functions attributed the actinin-4. The observation of alternative splice variants of actinin-1 and -4 combined with the observed potential of these proteins to form homodimers and heterodimers suggests that homodimers and heterodimers with novel actin-binding properties and interaction networks may exist. The ability to behave in this manner may have functional implications. This may be of importance considering that these proteins are central to such processes as cell migration and adhesion. This significantly alters our view of the non-muscle actinins.

Investigation of micro-devices for neurobiological applications

The aim of this project is to integrate neuronal cell culture with commercial or in-house built micro-electrode arrays and MEMS devices. The resulting device is intended to support neuronal cell culture on its surface, expose specific portions of a neuronal population to different environments using microfluidic gradients and stimulate/record neuronal electrical activity using micro-electrode arrays. Additionally, through integration of chemical surface patterning, such device can be used to build neuronal cell networks of specific size, conformation and composition. The design of this device takes inspiration from the nervous system because its development and regeneration are heavily influenced by surface chemistry and fluidic gradients. Hence, this device is intended to be a step forward in neuroscience research because it utilizes similar concepts to those found in nature. The large part of this research revolved around solving technical issues associated with integration of biology, surface chemistry, electrophysiology and microfluidics. Commercially available microelectrode arrays (MEAs) are mechanically and chemically brittle making them unsuitable for certain surface modification and micro-fluidic integration techniques described in the literature. In order to successfully integrate all the aspects into one device, some techniques were heavily modified to ensure that their effects on MEA were minimal. In terms of experimental work, this thesis consists of 3 parts. The first part dealt with characterization and optimization of surface patterning and micro-fluidic perfusion. Through extensive image analysis, the optimal conditions required for micro-contact printing and micro-fluidic perfusion were determined. The second part used a number of optimized techniques and successfully applied these to culturing patterned neural cells on a range of substrates including: Pyrex, cyclo-olefin and SiN coated Pyrex. The second part also described culturing neurons on MEAs and recording electrophysiological activity. The third part of the thesis described integration of MEAs with patterned neuronal culture and microfluidic devices. Although integration of all methodologies proved difficult, a large amount of data relating to biocompatibility, neuronal patterning, electrophysiology and integration was collected. Original solutions were successfully applied to solve a number of issues relating to consistency of micro printing and microfluidic integration leading to successful integration of techniques and device components.

The role of glucocorticoid-induced tumour necrosis factor receptor in developing mouse sympathetic neurons

Hereditary sensory autonomic neuropathy IV (HSAN IV) is an autosomal recessive disorder characterised by inability to feel pain and anhidrosis and is a consequence of defective NGF/TrkA signalling and growth of sensory and sympathetic neurons. Glucocortiocoid-induced tumour necrosis factors receptor (GITR), a transmembrane protein, activated by its specific ligand, GITRL, is well known for its role in the regulation of innate and acquired immune system responses. Recently, GITR was found to be required for NGF-dependant and extracellular signal-related kinase 1/2 (ERK1/2)-induced neurite growth and target innervation in the developing sympathetic nervous system (SNS). Given this novel role of GITR, it is possible that strategies targeting GITR have potential therapeutic benefit in promoting neurite growth in autonomic neuropathies such as HSAN IV. Using P1 mouse SCG neurons as a model, in addition to various SCG cell treatments, knock down models and transfection methods, we investigated whether GITR increases the sensitivity of sympathetic neurons to NGF; the region of GITR required for the enhancement of NGF-promoted growth, the signalling pathways downstream of GITR and how extensively GITR is involved in regulating peripheral innervation of the SNS. Results indicate that the region responsible for the growth promoting effects of GITR lies in its juxtamembrane intracellular region (here termed the growth promoting domain (GPD)) of GITR. The GPD of GITR activates ERK1/2 and inhibits nuclear factor kappa B (NF-κB) in an inverse fashion to provide an optimal cellular growth environment for P1 SCG neurons. While deleting the GPD of GITR had no effect on TrkA expression, constitutive phosphorylation of specific sites in the GPD reduced TrkA expression indicating a possible role for GITR in increasing the sensitivity of SCG neurons to NGF by the regulation of these sites, TrkA expression and subsequent NGF/TrkA binding. GITR appears to be heterogeneously required for NGF-promoted target innervation of SCG neurons in some organs, implying additional factors are involved in extensive NGF-target innervation of the SNS. In conclusion, this study answers basic biological questions regarding the molecular mechanism behind the role of GITR in the development of the SNS, and provides a basis for future research if GITR modulation is to be developed as a strategy for promoting axonal growth.

Renal failure and the neural control of the kidney

Renal failure (RF) is associated with an over activation of the sympathetic nervous system. The aim of this thesis was to investigate the hypothesis that as the kidney progresses into RF there is an inappropriate and sustained activation of renal afferent nerves which results in a dysregulation of basal RSNA and reflexly controlled RSNA by the high and low pressure baroreceptors. Baroreflex gain curves for both RSNA and HR were generated in control and RF rats. This study clearly showed a blunted high-pressure baroreflex in RF rats, an impairment which was almost completely corrected by bilateral renal denervation. The integrity of the low-pressure cardiopulmonary receptors to inhibit RSNA was investigated using acute saline volume. Again, a blunted reflex sympatho-inhibition of RSNA was observed, which was corrected by renal denervation. Finally a functional study to examine how the renal excretory response to volume expansion differed in RF was carried out. This study revealed an impairment of the low-pressure baroreflex control of the sympathetic outflow. The result of these studies suggest that cisplatin induced RF initiates a neural signal from within the kidney, which over rides the normal reflex regulation of RSNA by the high and low – pressure baroreceptors and that this impairment in function can be normalised by renal denervation. This raises further questions as to the mechanisms involved in the afferent over activation arising from the diseased kidneys.

Modified cyclodextrins as novel non-viral vectors for neuronal siRNA delivery: focus on Huntington’s disease

Huntington’s Disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a mutant Huntingtin (muHTT) protein. Therefore, preventing the expression of muHTT by harnessing the specificity of the RNA interference (RNAi) pathway is a key research avenue for developing novel therapies for HD. However, the biggest caveat in the RNAi approach is the delivery of short interfering RNA (siRNAs) to neurons, which are notoriously difficult to transfect. Indeed, despite the great advances in the field of nanotechnology, there remains a great need to develop more effective and less toxic carriers for siRNA delivery to the Central Nervous System (CNS). Thus, the aim of this thesis was to investigate the utility of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as non-viral vectors for siRNA delivery for HD. Modified CDs were able to bind and complex siRNAs forming nanoparticles capable of delivering siRNAs to ST14A-HTT120Q cells and to human HD fibroblasts, and reducing the expression of the HTT gene in these in vitro models of HD. Moreover, direct administration of CD.siRNA nanoparticles into the R6/2 mouse brain resulted in significant HTT gene expression knockdown and selective alleviation of rotarod motor deficits in this mouse model of HD. In contrast to widely used transfection reagents, CD.siRNA nanoparticles only induced limited cytotoxic and neuroinflammatory responses in multiple brain-derived cell-lines, and also in vivo after single direct injections into the mouse brain. Alternatively, we have also described a PEGylation-based formulation approach to further stabilise CD.siRNA nanoparticles and progress towards a systemic delivery nanosystem. Resulting PEGylated CD.siRNA nanoparticles showed increased stability in physiological saltconditions and, to some extent, reduced protein-induced aggregation. Taken together, the work outlined in this thesis identifies modified CDs as effective, safe and versatile siRNA delivery systems that hold great potential for the treatment of CNS disorders, such as HD.