Translational regulation of nuclear gene COX4 expression by mitochondrial content of phosphatidylglycerol and cardiolipin in Saccharomyces cerevisiae.

Previous results indicated that translation of four mitochondrion-encoded genes and one nucleus-encoded gene (COX4) is repressed in mutants (pgs1Delta) of Saccharomyces cerevisiae lacking phosphatidylglycerol and cardiolipin. COX4 translation was studied here using a mitochondrially targeted green fluorescence protein (mtGFP) fused to the COX4 promoter and its 5' and 3' untranslated regions (UTRs). Lack of mtGFP expression independent of carbon source and strain background was established to be at the translational level. The translational defect was not due to deficiency of mitochondrial respiratory function but was rather caused directly by the lack of phosphatidylglycerol and cardiolipin in mitochondrial membranes. Reintroduction of a functional PGS1 gene under control of the ADH1 promoter restored phosphatidylglycerol synthesis and expression of mtGFP. Deletion analysis of the 5' UTR(COX4) revealed the presence of a 50-nucleotide fragment with two stem-loops as a cis-element inhibiting COX4 translation. Binding of a protein factor(s) specifically to this sequence was observed with cytoplasm from pgs1Delta but not PGS1 cells. Using HIS3 and lacZ as reporters, extragenic spontaneous recessive mutations that allowed expression of His3p and beta-galactosidase were isolated, which appeared to be loss-of-function mutations, suggesting that the genes mutated may encode the trans factors that bind to the cis element in pgs1Delta cells.

Subacute neural stem cell therapy for traumatic brain injury.

INTRODUCTION: Traumatic brain injury (TBI) frequently results in devastating and prolonged morbidity. Cellular therapy is a burgeoning field of experimental treatment that has shown promise in the management of many diseases, including TBI. Previous work suggests that certain stem and progenitor cell populations migrate to sites of inflammation and improve functional outcome in rodents after neural injury. Unfortunately, recent study has revealed potential limitations of acute and intravenous stem cell therapy. We studied subacute, direct intracerebral neural stem and progenitor cell (NSC) therapy for TBI. MATERIALS AND METHODS: The NSCs were characterized by flow cytometry and placed (400,000 cells in 50 muL 1x phosphate-buffered saline) into and around the direct injury area, using stereotactic guidance, of female Sprague Dawley rats 1 wk after undergoing a controlled cortical impact injury. Immunohistochemistry was used to identify cells located in the brain at 48 h and 2 wk after administration. Motor function was assessed using the neurological severity score, foot fault, rotarod, and beam balance. Cognitive function was assessed using the Morris water maze learning paradigm. Repeated measures analysis of variance with post-hoc analysis were used to determine significance at P < 0.05. RESULTS: Immunohistochemistry analysis revealed that 1.4-1.9% of infused cells remained in the neural tissue at 48 h and 2 wk post placement. Nearly all cells were located along injection tracks at 48 h. At 2 wk some cell dispersion was apparent. Rotarod motor testing revealed significant increases in maximal speed among NSC-treated rats compared with saline controls at d 4 (36.4 versus 27.1 rpm, P < 0.05) and 5 (35.8 versus 28.9 rpm, P < 0.05). All other motor and cognitive evaluations were not significantly different compared to controls. CONCLUSIONS: Placement of NSCs led to the cells incorporating and remaining in the tissues 2 wk after placement. Motor function tests revealed improvements in the ability to run on a rotating rod; however, other motor and cognitive functions were not significantly improved by NSC therapy. Further examination of a dose response and optimization of placement strategy may improve long-term cell survival and maximize functional recovery.

The effectiveness of Alexander Technique on music performance and musicians’ health and well-being – a systematic review

Introduction Musicians often suffer injuries related to their music playing. Therefore, some use Alexander Technique (AT), a mental-physical method that facilitates to release unnecessary muscle tension and to re-educate non-beneficial movement patterns through enhanced kinaesthetic awareness. According to a recent review AT may be effective for chronic back pain [1]. This review aimed to evaluate the evidence for the effectiveness of AT lessons on music performance and musicians’ health and well-being. Methods The following electronic databases were searched up to July 2012 for relevant literature: PUBMED, Google Scholar, CINAHL and EMBASE. The search criteria were "Alexander technique" AND "music*" [all fields]. References were searched, and experts and societies of AT or musicians' medicine contacted for further publications. Results 100 studies were identified. 24 studies were included for further analysis, 5 of which were randomised controlled trials (RCTs), 5 controlled but not randomised (CTs), 5 without control group, 2 mixed methods (RCT and case studies), and 7 surveys. 13 to 72 musicians participated per RCT. In 5 RCTs AT groups received between 12 and 20 one-to-one lessons. In 4 RCTs control groups received no interventions. Primary outcomes were performance anxiety, music performance, "use" as well as respiratory function and pain. Performance anxiety decreased by AT in 3 of 4 RCTs and in 3 of 3 CTs. Music performance was improved by AT in 1 RCT, yet in 2 RCTs comparing neurofeedback (NF) to AT, only NF showed improvements. Discussion and Conclusion To investigate the effectiveness of AT in musicians a variety of study designs and outcome measures have been used. Evidence from RCTs suggests that AT may improve performance anxiety in musicians. Effects on music performance, body use and respiratory function yet remain inconsistent. Future trials with scientifically sound study designs are warranted to further and more reliably explore the potential of AT as a relatively low cost and low risk method in the interest of musicians. References [1] Woodman JP, Moore NR. Evidence for the effectiveness of Alexander Technique lessons in medical and health-related conditions: a systematic review. Int J Clin Pract 2012;66(1):98-112.

The Effectiveness of Alexander Technique on Music Performance and Musicians' Health and Well-being – a Systematic Review

Purpose Musicians often suffer injuries related to their music playing. Therefore, some use the Alexander Technique (AT), a psycho-physical method that helps to release unnecessary muscle tension and re-educates non-beneficial movement patterns through enhanced kinaesthetic awareness. According to a recent review AT may be effective for chronic back pain. This review aimed to evaluate the evidence for the effectiveness of AT lessons on music performance and musicians’ health and well-being. Methods The following electronic databases were searched up to July 2012 for relevant literature: PUBMED, Google Scholar, CINAHL and EMBASE. The search criteria were "Alexander technique" AND "music*" [all fields]. References were searched, and experts and societies of AT or musicians' medicine contacted for further publications. Results 100 studies were identified. 35 studies were included for further analysis, 5 of which were randomised controlled trials (RCTs), 5 controlled but not randomised, 5 not controlled, 5 qualitative case studies, 2 mixed-models (RCT and case studies), 7 surveys, 4 qualitative case reports and 2 unpublished pilot studies. 13 to 72 musicians participated per RCT. In 5 RCTs AT groups received between 12 and 20 one-to-one lessons. In 4 RCTs control groups received no interventions. Primary outcomes were performance anxiety, performance, "use" as well as respiratory function and pain. Performance anxiety decreased by AT in 3 of 4 RCTs. Music performance was improved by AT in 1 RCT, yet in 2 RCTs comparing neurofeedback (NF) to AT, only NF showed improvements. Conclusions To investigate the effectiveness of AT in musicians a variety of study designs and outcome measures have been used. Evidence from RCTs suggests that AT may improve performance anxiety in musicians. Effects on music performance, body use and respiratory function yet remain inconsistent. Future trials with well-established study designs are warranted to further and more reliably explore the potential of AT as a relatively low cost and low risk method in the interest of musicians.

The Alexander Technique and musicians: a systematic review of controlled trials

BACKGROUND: Musculoskeletal disorders, stress and performance anxiety are common in musicians. Therefore, some use the Alexander Technique (AT), a psycho-physical method that helps to release unnecessary muscle tension and re-educates non-beneficial movement patterns through intentional inhibition of unwanted habitual behaviours. According to a recent review AT sessions may be effective for chronic back pain. This review aimed to evaluate the evidence for the effectiveness of AT sessions on musicians' performance, anxiety, respiratory function and posture. METHODS: The following electronic databases were searched up to February 2014 for relevant publications: PUBMED, Google Scholar, CINAHL, EMBASE, AMED, PsycINFO and RILM. The search criteria were "Alexander Technique" AND "music*". References were searched, and experts and societies of AT or musicians' medicine contacted for further publications. RESULTS: 237 citations were assessed. 12 studies were included for further analysis, 5 of which were randomised controlled trials (RCTs), 5 controlled but not randomised (CTs), and 2 mixed methods studies. Main outcome measures in RCTs and CTs were music performance, respiratory function, performance anxiety, body use and posture. Music performance was judged by external experts and found to be improved by AT in 1 of 3 RCTs; in 1 RCT comparing neurofeedback (NF) to AT, only NF caused improvements. Respiratory function was investigated in 2 RCTs, but not improved by AT training. Performance anxiety was mostly assessed by questionnaires and decreased by AT in 2 of 2 RCTs and in 2 of 2 CTs. CONCLUSIONS: A variety of outcome measures have been used to investigate the effectiveness of AT sessions in musicians. Evidence from RCTs and CTs suggests that AT sessions may improve performance anxiety in musicians. Effects on music performance, respiratory function and posture yet remain inconclusive. Future trials with well-established study designs are warranted to further and more reliably explore the potential of AT in the interest of musicians.

Cessation and resuming of alglucosidase alfa in Pompe disease: a retrospective analysis.

Enzyme replacement therapy (ERT) with recombinant human alglucosidase alfa (rhGAA) in late-onset Pompe disease is moderately effective. Little is known about the clinical course after treatment termination and the resumption of ERT. In Switzerland, rhGAA therapy for Pompe disease was temporarily withdrawn after the federal court judged that the treatment costs were greatly out of proportion compared to the benefits. Re-treatment was initiated after the therapy was finally licensed. We retrospectively analysed seven Pompe patients, who underwent cessation and resumption of ERT (median age 43 years). The delay from first symptoms to final diagnosis ranged from 4 to 20 years. The demographics, clinical characteristics, assessments with the 6-min walking test (6-MWT), the predicted forced vital capacity (FVC) and muscle strength were analysed. Before initiation of ERT, all patients suffered from proximal muscle weakness of the lower limbs; one was wheelchair-bound and two patients received night-time non-invasive ventilation. Initial treatment stabilised respiratory function in most patients and improved their walking performance. After treatment cessation, upright FVC declined in most and the 6-MWT declined in all patients. Two patients needed additional non-invasive ventilatory support. Twelve months after resuming ERT, the respiratory and walking capacity improved again in most patients. However, aside for one patient, none of the patients reached the same level of respiratory function or distance walked in 6 min, as at the time of ERT withdrawal. We conclude that cessation of ERT in Pompe disease causes a decline in clinical function and should be avoided. Resuming treatment only partially recovers respiratory function and walking capacity.

Cross-sectional study of 168 patients with hepatorenal tyrosinaemia and implications for clinical practice

BackgroundHepatorenal tyrosinaemia (Tyr 1) is a rare inborn error of tyrosine metabolism. Without treatment, patients are at high risk of developing acute liver failure, renal dysfunction and in the long run hepatocellular carcinoma. The aim of our study was to collect cross-sectional data.MethodsVia questionnaires we collected retrospective data of 168 patients with Tyr 1 from 21 centres (Europe, Turkey and Israel) about diagnosis, treatment, monitoring and outcome. In a subsequent consensus workshop, we discussed data and clinical implications.ResultsEarly treatment by NTBC accompanied by diet is essential to prevent serious complications such as liver failure, hepatocellular carcinoma and renal disease. As patients may remain initially asymptomatic or develop uncharacteristic clinical symptoms in the first months of life newborn mass screening using succinylacetone (SA) as a screening parameter in dried blood is mandatory for early diagnosis. NTBC-treatment has to be combined with natural protein restriction supplemented with essential amino acids. NTBC dosage should be reduced to the minimal dose allowing metabolic control, once daily dosing may be an option in older children and adults in order to increase compliance. Metabolic control is judged by SA (below detection limit) in dried blood or urine, plasma tyrosine (<400 ¿M) and NTBC-levels in the therapeutic range (20¿40 ¿M). Side effects of NTBC are mild and often transient.Indications for liver transplantation are hepatocellular carcinoma or failure to respond to NTBC. Follow-up procedures should include liver and kidney function tests, tumor markers and imaging, ophthalmological examination, blood count, psychomotor and intelligence testing as well as therapeutic monitoring (SA, tyrosine, NTBC in blood).ConclusionBased on the data from 21 centres treating 168 patients we were able to characterize current practice and clinical experience in Tyr 1. This information could form the basis for clinical practice recommendations, however further prospective data are required to underpin some of the recommendations.

A numerical model for inert gas transport in the lung based on fractal airway morphology

Patients suffering from cystic fibrosis (CF) show thick secretions, mucus plugging and bronchiectasis in bronchial and alveolar ducts. This results in substantial structural changes of the airway morphology and heterogeneous ventilation. Disease progression and treatment effects are monitored by so-called gas washout tests, where the change in concentration of an inert gas is measured over a single or multiple breaths. The result of the tests based on the profile of the measured concentration is a marker for the severity of the ventilation inhomogeneity strongly affected by the airway morphology. However, it is hard to localize underlying obstructions to specific parts of the airways, especially if occurring in the lung periphery. In order to support the analysis of lung function tests (e.g. multi-breath washout), we developed a numerical model of the entire airway tree, coupling a lumped parameter model for the lung ventilation with a 4th-order accurate finite difference model of a 1D advection-diffusion equation for the transport of an inert gas. The boundary conditions for the flow problem comprise the pressure and flow profile at the mouth, which is typically known from clinical washout tests. The natural asymmetry of the lung morphology is approximated by a generic, fractal, asymmetric branching scheme which we applied for the conducting airways. A conducting airway ends when its dimension falls below a predefined limit. A model acinus is then connected to each terminal airway. The morphology of an acinus unit comprises a network of expandable cells. A regional, linear constitutive law describes the pressure-volume relation between the pleural gap and the acinus. The cyclic expansion (breathing) of each acinus unit depends on the resistance of the feeding airway and on the flow resistance and stiffness of the cells themselves. Special care was taken in the development of a conservative numerical scheme for the gas transport across bifurcations, handling spatially and temporally varying advective and diffusive fluxes over a wide range of scales. Implicit time integration was applied to account for the numerical stiffness resulting from the discretized transport equation. Local or regional modification of the airway dimension, resistance or tissue stiffness are introduced to mimic pathological airway restrictions typical for CF. This leads to a more heterogeneous ventilation of the model lung. As a result the concentration in some distal parts of the lung model remains increased for a longer duration. The inert gas concentration at the mouth towards the end of the expirations is composed of gas from regions with very different washout efficiency. This results in a steeper slope of the corresponding part of the washout profile.

Potential Role of Adenosine Signaling in Acetic Acid Activation of Murine Sensory Neurons

Chronic respiratory illnesses are a significant cause of morbidity and mortality, and acute changes in respiratory function often lead to hospitalization. Air pollution is known to exacerbate asthma, but the molecular mechanisms of this are poorly understood. The current studies were aimed at clarifying the roles of nerve subtypes and purinergic receptors in respiratory reflex responses following exposure to irritants. In C57Bl/6J female mice, inspired adenosine produced sensory irritation, shown to be mediated mostly by A-delta fibers. Secondly, the response to inhaled acetic acid was discovered to be dually influenced by C and A-delta fibers, as indicated by the observed effects of capsaicin pretreatment, which selectively destroys TRPV1-expressing fibers (mostly C fibers) and pretreatment with theophylline, a nonselective adenosine receptor antagonist. The responses to both adenosine and acetic acid were enhanced in the ovalbumin-allergic airway disease model, although the particular pathway altered is still unknown.

Mitochondrial dysfunction leads to NOX activation: A novel mechanism to maintain high glycolysis in cancer cells

Increased glycolysis and oxidative stress are common features of cancer cells. These metabolic alterations are associated with mitochondrial dysfunction and can be caused by mitochondrial DNA (mtDNA) mutations, oncogenic signals, loss of tumor suppressor, and tumor tissue hypoxia. It is well established that mitochondria play central roles in energy metabolism, maintenance of redox balance, and regulation of apoptosis. However, the biochemical and molecular mechanisms that maintain high glycolysis in cancer cells (the Warburg effect) with mitochondrial dysfunction and oxidative stress remain to be determined. The major goals of this study were to establish a unique experimental system in which the mitochondrial respiratory function can be regulated as desired, and to use this system to investigate the mechanistic link between mitochondrial dysfunction and the Warburg effect along with oxidative stress in cancer cells. To achieve these goals, I have established a tetracycline-inducible system in which a dominant negative form of mitochondrial DNA polymerase y (POLGdn) expression could be regulated by tetracycline; thus controlling mitochondrial respiratory function. Using this cell system, I demonstrated that POLGdn expression resulted in mitochondrial dysfunction through decreasing mtDNA content, depletion of mtDNA encoded mRNA and protein expression. This process was mediated by TFAM proteasome degradation. Mitochondrial dysfunction mediated by POLGdn expression led to a significant increase in cellular glycolysis and oxidative stress. Surprisingly, mitochondrial dysfunction also resulted in increased NAD(P)H oxidase (NOX) enzyme activity, which was shown to be essential for maintaining high glycolysis. Chemical Inhibition of NOX activity by diphenyliodonium (DPI) preferentially impacted the survival of mitochondrial defective cells. The colon cancer HCT116-/- cells that have lost transcriptional regulation of the mitochondrial assembling enzyme SCO2, leading to compromised mitochondrial respiratory function, were found to have increased NOX activity and were highly sensitive to DPI treatment. Ovarian epithelial cells with Ras transformation also exhibited an increase in NOX gene expression and NOX enzyme activity, rendering the cells sensitive to DPI inhibition especially under hypoxic condition. These data together suggest that NOX plays a novel role in maintaining high glycolysis in cancer cells with mitochondrial defects, and that NOX may be a potential target for cancer therapy. ^

Expanding the functional human mitochondrial DNA database by the establishment of primate xenomitochondrial cybrids

The nuclear and mitochondrial genomes coevolve to optimize approximately 100 different interactions necessary for an efficient ATP-generating system. This coevolution led to a species-specific compatibility between these genomes. We introduced mitochondrial DNA (mtDNA) from different primates into mtDNA-less human cells and selected for growth of cells with a functional oxidative phosphorylation system. mtDNA from common chimpanzee, pigmy chimpanzee, and gorilla were able to restore oxidative phosphorylation in the context of a human nuclear background, whereas mtDNA from orangutan, and species representative of Old-World monkeys, New-World monkeys, and lemurs were not. Oxygen consumption, a sensitive index of respiratory function, showed that mtDNA from chimpanzee, pigmy chimpanzee, and gorilla replaced the human mtDNA and restored respiration to essentially normal levels. Mitochondrial protein synthesis was also unaltered in successful “xenomitochondrial cybrids.” The abrupt failure of mtDNA from primate species that diverged from humans as recently as 8–18 million years ago to functionally replace human mtDNA suggests the presence of one or a few mutations affecting critical nuclear–mitochondrial genome interactions between these species. These cellular systems provide a demonstration of intergenus mtDNA transfer, expand more than 20-fold the number of mtDNA polymorphisms that can be analyzed in a human nuclear background, and provide a novel model for the study of nuclear–mitochondrial interactions.

Escoliose no contexto de distrofia muscular

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Colestase intra-hepática da gravidez

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Avaliação dos internamentos em endocrinologia

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Análise descritiva da função respiratória de adultos com fibrose quística

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014