Efeitos do cobre no metabolismo energético do bivalve marinho Mesodesma mactroides

Apesar de ser um micronutriente essencial aos organismos, o cobre (Cu) é tóxico quando presente em elevadas concentrações na água. O mecanismo pelo qual este metal exerce sua toxicidade em invertebrados marinhos ainda não está bem estabelecido. Dentre os diversos efeitos relatados, observa-se uma redução do consumo de oxigênio corporal e tecidual no marisco Mesodesma mactroides exposto (96 h) ao Cu (150 µg L-1 ) em água do mar (salinidade 30). Portanto, o objetivo do presente estudo foi avaliar os efeitos desta exposição ao Cu no metabolismo energético em teciduais do marisco M. mactroides. Os conteúdos de ATP e coenzimas (NAD+ e NADH) nas brânquias, glândula digestiva e músculo pedal não foram alterados pela exposição ao Cu, indicando que estes tecidos mantiveram suas capacidades de produção aeróbica de energia. Porém, foi observada uma redução no conteúdo hemolinfático de ATP. Quanto ao conteúdo de proteínas, houve um aumento na glândula digestiva, que pode estar associado à maior oxidação de proteínas já relatada para esse tecido após exposição ao Cu. Os conteúdos de lipídios, glicogênio e glicose permaneceram inalterados em todos os tecidos analisados, exceto no músculo pedal, onde foi observada uma redução no conteúdo de glicose. Por isso, os conteúdos de piruvato e lactato também foram analisados no músculo pedal e na hemolinfa. Em ambos tecidos, foi observado um aumento do conteúdo de lactato, sem alteração no conteúdo de piruvato. Portanto, os resultados do presente estudo sugerem que os tecidos de M. mactroides utilizam a anaerobiose para obtenção de energia durante a exposição ao Cu, conforme demonstrado no músculo pedal e hemolinfa. Apesar disso, a hemolinfa não é capaz de manter o nível de ATP nas condições experimentais testadas. 

The Outcome of Infantile Onset Pompe Disease in South of Iran

Background: Infantile Onset Pompe Disease (IOPD) is a rare autosomal recessive neuromuscular disorder. It is associated with cardiomegaly, hypotonia, paresis, and death in the first year of life. Since 2006, following the use of Alglucosidase alfa as Enzyme Replacement Therapy (ERT), the patients’ survival is improved to a noticeable extent. Objectives: The purpose of this study is to examine the outcome of IOPD patients in South of Iran and the degree of responsiveness to ERT. Patients and Methods: All patients who were diagnosed with IOPD on the bases of clinical symptoms, and enzyme assay on dried blood spot, were included in the study; and were followed up regarding cardiac function, locomotor activity, and cognition. Results: Six patients with IOPD were identified. All these six patients suffered from Hypertrophic Cardiomyopathy (HCM). Four (67%) of them also had generalized hypotonia. Three patients expired during the first weeks due to severe respiratory infection. One of them also got involved with Acute Cardiopulmonary Failure while receiving the fifth dose of ERT; and expired. However, the remaining two patients had a significant improvement after the maximum of 117 weeks of following up both cardiac and locomotor findings. These two patients were the same patients who showed cardiac symptoms from the beginning but did not have generalized hypotonia. Conclusions: Although ERT has a significant effect on enhancing the survival of IOPD patients, it should be associated with meticulous heart-respiratory cares during the first months of treatment and preventing infection especially nosocomial infections.

Sodium chloride added to transport water and physiological responses of Matrinxã Brycon amazonicus (Teleost: Characidae)

A adição de sal à água tem sido utilizada para a mitigação de estresse e aumento da taxa de sobrevivência em peixes. O presente estudo avaliou o efeito do cloreto de sódio (0,0; 1,0; 3,0 e 6.0 g/l) nas concentrações de cortisol plasmático, glicemia, triglicerídios, proteínas total plasmática, hematócrito, hemoglobina, número de eritrócitos, glicogênio e lipídio hepáticos, e lipídio muscular em matrinxã Brycon amazonicum adultos após quatro horas de transporte e durante período de recuperação de 96 h. Amostras foram coletadas antes e depois do transporte, bem como 24 e 96 h após a chegada. O nível de cortisol plasmático estava mais elevado logo após o transporte quando comparado à condição inicial (pré-transporte), exceto para os peixes transportados com sal nas concentrações 3,0 e 6,0 g/l. Comportamento semelhante foi observado para a glicemia, porém os peixes dos tratamentos 0,0, 1,0 e 3,0 g/l necessitaram de período superior a 24 h para recuperar a condição inicial. Foram registrados níveis mais baixos de glicogênio hepático em peixes do tratamento controle (0,0 g/l). Os parâmetros hemoglobina, número de eritrócitos, proteína plasmática total e lipídio hepático não apresentaram alterações durante o período experimental. Os valores de hematócrito diminuíram logo após o transporte em todos os tratamentos, retornando aos níveis iniciais após 24 h. Todos os tratamentos apresentaram redução nos níveis de lipídio muscular e triglicerídios durante o período de recuperação. Os resultados sugerem que a adição de 6,0 g/l de sal na água de transporte reduz as alterações fisiológicas de estresse e que é necessário período de 96 h após o transporte para a recuperação da condição inicial de matrinxãs transportados sem a adição de sal.

Feeding strategy with alternate fasting and refeeding: effects on farmed pacu production

P>A 36-day trial was conducted to determine the effects of repetitive periods of food restriction and refeeding on growth and energy metabolism in pacu (Piaractus mesopotamicus). A total 264 juvenile fish (36.9 +/- 2.8 g) were fed with the experimental diet for 36 days using three regimes: (i) feeding daily to satiation (FD); (ii) no feed for 3 days, then feeding the same amount offered to the control groups for the next 3 days (NF/R controlled); and (iii) no feed for 3 days, then feeding to apparent satiation for the next 3 days (NF/R at satiation). The treatments were distributed into four tanks each. WG and SGR were higher in FD group. Fish refed showed hyperphagia just up to the second day of refeeding. The worst feed conversion rate and the lowest protein efficiency ratio were found in fish NF/R controlled. The lowest values of visceral fat somatic index were found in both fasted fish groups, particularly in NF/R at satiation. The LL and glycogen concentrations, and the hepatosomatic index were all elevated in both feed restricted fish. Muscle lipid showed a tendency to decrease after the cycle of fasting and refeeding. Plasma free fatty acids and glucose levels were elevated in fish subjected to feeding restrictions while serum triglycerides levels were reduced. Triiodothyronine levels were significantly depressed in fish from the NF/R-controlled group and remained at the same levels as the control fish in fish NF/R at satiation. Results indicated that fish subjected to cyclic periods of 3-day satiation or controlled feeding after 3-days of fasting were unable to achieve the final body weight of fish fed to satiation after 36 days.

Steroid induced neuroprotection of damaged photoreceptor cells

Retinitis Pigmentosa (RP) is the name given to a group of hereditary diseases causing progressive and degenerative blindness. RP affects over 1 in 4000 individuals, making it the most prevalent inherited retinal disease worldwide, yet currently there is no cure. In 2011, our group released a paper detailing the protective effects of the synthetic progestin ‘Norgestrel’. A common component of the female oral contraceptive pill, Norgestrel was shown to protect against retinal cell death in two distinct mouse models of retinal degeneration: in the Balb/c light damage model and the Pde6brd10 (rd10) model. Little was known of the molecular workings of this compound however and thus this study aimed to elucidate the protective manner in which Norgestrel worked. To this aim, the 661W cone photoreceptor-like cell line and ex vivo retinal explanting was utilised. We found that Norgestrel induces a increase in neuroprotective basic fibroblast growth factor (bFGF) with subsequent downstream actions on the inhibition of glycogen synthase kinase 3β. Progesterone receptor expression was subsequently characterised in the C57 and rd10 retinas and in the 661W cell line. Norgestrel caused nuclear trafficking of progesterone receptor membrane complex one (PGRMC1) in 661W cells and thus Norgestrel was hypothesised to work primarily through the actions of PGRMC1. This trafficking was shown to be responsible for the critical upregulation of bFGF and PGRMC1- Norgestrel binding was proven to cause a neuroprotective bFGF-mediated increase in intracellular calcium. The protective properties of Norgestrel were further studied in the rd10 mouse model of retinitis pigmentosa. Using non-invasive diet supplementation (80mg/kg), we showed that Norgestrel gave significant retinal protection out to postnatal day 40 (P40). Overactive microglia have previously been shown to potentiate photoreceptor cell loss in the degenerating rd10 retina and thus we focussed on Norgestrel-mediated changes in photoreceptor-microglial crosstalk. Norgestrel acted to dampen pro-inflammatory microglial cell reactivity, decreasing chemokine (MCP1, MCP3, MIP-1α, MIP-1β) and subsequent damaging cytokine (TNFα, Il-1β) production. Critically, Norgestrel up-regulated photoreceptor-microglial, fractalkine-CX3CR1 signalling 1000-fold in the P20 rd10 mouse. Known to prevent microglial activation, we hypothesise that Norgestrel acts as a vital anti-inflammatory in the diseased retina, driving fractalkine-CX3CR1 signalling to delay retinal degeneration. This study stands to highlight some of the neuroprotective mechanisms utilised by Norgestrel in the prevention of photoreceptor cell death. We identify for the first time, not only a pro-survival pathway activated directly in photoreceptor cells, but also a Norgestreldriven mediation of an otherwise damaging microglial cell response. All taken, these results form the beginning of a case to bring Norgestrel to clinical trials, as a potential therapeutic for the treatment of RP.

Double-trouble for respiratory control in Pompe disease

A commentary on ‘Hypoglossal neuropathology and respiratory activity in Pompe mice’, by Lee, K.-Z., Qiu, K., Sandhu, M. S., Elmullah, M. K., Falk, D. J., Lane, M. A., Reier, P. J., Byrne, B. J., and Fuller, D. D. (2011). Front. Physiol. 2:31. doi: 10.3389/fphys.2011.00031.

Mcardle disease: Update of reported mutations and polymorphisms in the Pygm gene

McArdle disease is an autosomal recessive disorder caused by inherited deficiency of the muscle isoform of glycogen phosphorylase (or ‘myophosphorylase´), which catalyzes the first step of glycogen catabolism, releasing glucose-1-phosphate from glycogen deposits. As a result, muscle metabolism is impaired, leading to different degrees of exercise intolerance. Patients range from asymptomatic to severely affected, including in some cases limitations in activities of daily living. The PYGM gene codifies myophosphoylase and to date 147 pathogenic mutations and 39 polymorphisms have been reported. Exon 1 and 17 are mutational hot-spots in PYGM and 50% of the described mutations are missense.

The role of inflammation in vascular function and pregnancy outcome in the pregnant stroke prone spontaneously hypertensive rat

During pregnancy, the maternal cardiovascular system undergoes major adaptation. One of these changes is a 40-50 % increase in circulating blood volume which requires a systemic remodelling of the vasculature in order to regulate maternal blood pressure and maximise blood supply to the developing placenta and fetus. These changes are broadly conserved between humans and rats making them an appropriate pre-clinical model in which to study the underlying mechanisms of pregnancy-dependent cardiovascular remodelling. Whilst women are normally protected against cardiovascular disease; pregnancy marks a period of time where women are susceptible to cardiovascular complications. Cardiovascular disease is the leading cause of maternal mortality in the United Kingdom; in particular hypertensive conditions are among the most common complications of pregnancy. One of the main underlying pathologies of these pregnancy complications is thought to be a failure of the maternal cardiovascular system to adapt. The remodelling of the uterine arteries, which directly supply the maternal-fetal interface, is paramount to a healthy pregnancy. Failure of the uterine arteries to remodel sufficiently can result in a number of obstetric complications such as preeclampsia, fetal growth restriction and spontaneous pregnancy loss. At present, it is poorly understood whether this deficient vascular response is due to a predisposition from existing maternal cardiovascular risk factors, the physiological changes that occur during pregnancy or a combination of both. Previous work in our group employed the stroke prone spontaneously hypertensive rat (SHRSP) as a model to investigate pregnancy-dependent remodelling of the uterine arteries. The SHRSP develops hypertension from 6 weeks of age and can be contrasted with the control strain, the Wistar Kyoto (WKY) rat. The phenotype of the SHRSP is therefore reflective of the clinical situation of maternal chronic hypertension during pregnancy. We showed that the SHRSP exhibited a deficient uterine artery remodelling response with respect to both structure and function accompanied by a reduction in litter size relative to the WKY at gestational day (GD) 18. A previous intervention study using nifedipine in the SHRSP achieved successful blood pressure reduction from 6 weeks of age and throughout pregnancy; however uterine artery remodelling and litter size at GD18 was not improved. We concluded that the abnormal uterine artery remodelling present in the SHRSP was independent of chronic hypertension. From these findings, we hypothesised that the SHRSP could be a novel model of spontaneously deficient uterine artery remodelling in response to pregnancy which was underpinned by other as yet unidentified cardiovascular risk factors. In Chapter 1 of this thesis, I have characterised the maternal, placental and fetal phenotype in pregnant (GD18) SHRSP and WKY. The pregnant SHRSP exhibit features of left ventricular hypertrophy in response to pregnancy and altered expression of maternal plasma biomarkers which have been previously associated with hypertension in human pregnancy. I developed a protocol for accurate dissection of the rat uteroplacental unit using qPCR probes specific for each layer. This allowed me to make an accurate and specific statement about gene expression in the SHRSP GD18 placenta; where oxidative stress related gene markers were increased in the vascular compartments. The majority of SHRSP placenta presented at GD18 with a blackened ring which encircled the tissue. Further investigation of the placenta using western blot for caspase 3 cleavage determined that this was likely due to increased cell death in the SHRSP placenta. The SHRSP also presented with a loss of one particular placental cell type at GD18: the glycogen cells. These cells could have been the target of cell death in the SHRSP placenta or were utilised early in pregnancy as a source of energy due to the deficient uterine artery blood supply. Blastocyst implantation was not altered but resorption rate was increased between SHRSP and WKY; indicating that the reduction in litter size in the SHRSP was primarily due to late (>GD14) pregnancy loss. Fetal growth was not restricted in SHRSP which led to the conclusion that SHRSP sacrifice part of their litter to deliver a smaller number of healthier pups. Activation of the immune system is a common pathway that has been implicated in the development of both hypertension and adverse pregnancy outcome. In Chapter 2, I proposed that this may be a mechanism of interest in SHRSP pregnancy and measured the pro-inflammatory cytokine, TNFα, as a marker of inflammation in pregnant SHRSP and WKY and in the placentas from these animals. TNFα was up-regulated in maternal plasma and urine from the GD18 SHRSP. In addition, TNFα release was increased from the GD18 SHRSP placenta as was the expression of the pro-inflammatory TNFα receptor 1 (Tnfr1). In order to investigate whether this excess TNFα was detrimental to SHRSP pregnancy, a vehicle-controlled intervention study using etanercept (a monoclonal antibody which works as a TNFα antagonist) was carried out. Etanercept treatment at GD0, 6, 12 and 18 resulted in an improvement in pregnancy outcome in the SHRSP with an increased litter size and reduced resorption rate. Furthermore, there was an improved uterine artery function in GD18 SHRSP treated with etanercept which was associated with an improved uterine artery blood flow over the course of gestation. In Chapter 3, I sought to identify the source of this detrimental excess of TNFα by designing a panel for maternal leukocytes in the blood and placenta at GD18. A population of CD3- CD161+ cells, which are defined as rat natural killer (NK) cells, were increased in number in the SHRSP. Intracellular flow cytometry also identified this cell type as a source of excess TNFα in blood and placenta from pregnant SHRSP. I then went on to evaluate the effects of etanercept treatment on these CD3- CD161+ cells and showed that etanercept reduced the expression of CD161 and the cytotoxic molecule, granzyme B, in the NK cells. Thus, etanercept limits the cytotoxicity and potential damaging effect of these NK cells in the SHRSP placenta. Analysing the urinary peptidome has clinical potential to identify novel pathways involved with disease and/or to develop biomarker panels to aid and stratify diagnosis. In Chapter 4, I utilised the SHRSP as a pre-clinical model to identify novel urinary peptides associated with hypertensive pregnancy. Firstly, a characterisation study was carried out in the kidney of the WKY and SHRSP. Urine samples from WKY and SHRSP taken at pre-pregnancy, mid-pregnancy (GD12) and late pregnancy (GD18) were used in the peptidomic screen. In order to capture peptides which were markers of hypertensive pregnancy from the urinary peptidomic data, I focussed on those that were only changed in a strain dependent manner at GD12 and 18 and not pre-pregnancy. Peptide fragments from the uromodulin protein were identified from this analysis to be increased in pregnant SHRSP relative to pregnant WKY. This increase in uromodulin was validated at the SHRSP kidney level using qPCR. Uromodulin has previously been identified to be a candidate molecule involved in systemic arterial hypertension but not in hypertensive pregnancy thus is a promising target for further study. In summary, we have characterised the SHRSP as the first model of maternal chronic hypertension during pregnancy and identified that inflammation mediated by TNFα and NK cells plays a key role in the pathology. The evidence presented in this thesis establishes the SHRSP as a pre-clinical model for pregnancy research and can be continued into clinical studies in pregnant women with chronic hypertension which remains an area of unmet research need.

Novel dual D3R/GSK-3β modulators: an innovative multitarget strategy for bipolar disorder

Among the psychiatric diseases, bipolar disorder (BD) is the sixth leading cause of disability with a prevalence up to 4 % worldwide. BD is a complex neuropsychiatric condition which alternates episodes of mania with symptoms of depression. Although the neurobiological pathways are not completely clarified, the dopamine (DA) hypothesis, recognized as the leading theory explaining the pathophysiology of the malady, states that the dramatically compromised homeostatic regulation of dopaminergic circuits leads to alternated changes in DA neurotransmission. Modulation of D2 and D3 receptors (D2/3R) through partial agonists represents the first-line therapeutic strategy for psychiatric diseases. Moreover, a deregulation of the enzyme glycogen synthase kinase-3β (GSK-3β) has been reported as peculiar feature of BD. In this scenario, the concomitant modulation of D3R and GSK-3β, by employing multitarget compounds, could offer promises to achieve an effective cure of this illness. In the light of these findings, we rationally envisaged the pharmacophoric model at the basis of the design of several D3R partial agonists, suitable to be exploited for the dual D3R/GSK-3β ligand design. Thus, synthetic efforts were addressed to develop a first set of hybrid molecules able to concurrently modulate the selected targets. For a chemical structure point of view, we employed different spacers to combine a substituted aryl-piperazine moiety, reported in previously discovered D3R modulators, with a pyrazole-based fragment, already identified in GSK-3β inhibitors. A fluorescent and a cellular functional assays were carried out to assess the activity of all synthetized compounds against GSK-3β and on D3R, respectively. Most of the derivatives proved to effectively modulate both GSK-3β and D3R with potencies in the low-µM and low-nM range, respectively. The consistent biological data allowed us to identify some lead candidates worth to be further modified with the aim to optimize their biological profile and to perform a structure-activity relationship (SAR) study.