Effect of remote ischaemic preconditioning in cardiac dysfunction and end-organ injury following cardiac surgery with cardiopulmonary bypass in children: A translational approach investigating clinical outcome and myocardial molecular biology

Congenital heart disease (CHD) is the most common birth defect, causing an important rate of morbidity and mortality. Treatment of CHD requires surgical correction in a significant percentage of cases which exposes patients to cardiac and end organ injury. Cardiac surgical procedures often require the utilisation of cardiopulmonary bypass (CPB), a system that replaces heart and lungs function by diverting circulation into an external circuit. The use of CPB can initiate potent inflammatory responses, in addition a proportion of procedures require a period of aortic cross clamp during which the heart is rendered ischaemic and is exposed to injury. High O2 concentrations are used during cardiac procedures and when circulation is re-established to the heart which had adjusted metabolically to ischaemia, further injury is caused in a process known as ischaemic reperfusion injury (IRI). Several strategies are in place in order to protect the heart during surgery, however injury is still caused, having detrimental effects in patients at short and long term. Remote ischaemic preconditioning (RIPC) is a technique proposed as a potential cardioprotective measure. It consists of exposing a remote tissue bed to brief episodes of ischaemia prior to surgery in order to activate protective pathways that would act during CPB, ischaemia and reperfusion. This study aimed to assess RIPC in paediatric patients requiring CHD surgical correction with a translational approach, integrating clinical outcome, marker analysis, cardiac function parameters and molecular mechanisms within the cardiac tissue. A prospective, single blinded, randomized, controlled trial was conducted applying a RIPC protocol to randomised patients through episodes of limb ischaemia on the day before surgery which was repeated right before the surgery started, after anaesthesia induction. Blood samples were obtained before surgery and at three post-operative time points from venous lines, additional pre and post-bypass blood samples were obtained from the right atrium. Myocardial tissue was resected during the ischaemic period of surgery. Echocardiographic images were obtained before the surgery started after anaesthetic induction and the day after surgery, images were stored for later off line analysis. PICU surveillance data was collected including ventilation parameters, inotrope use, standard laboratory analysis and six hourly blood gas analysis. Pre and post-operative quantitation of markers in blood specimens included cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP), inflammatory mediators including interleukins IL-6, IL-8, IL-10, tumour necrosis factor (TNF-α), and the adhesion molecules ICAM-1 and VCAM-1; the renal marker Cystatin C and the cardiovascular markers asymmetric dymethylarginine (ADMA) and symmetric dymethylarginine (SDMA). Nitric oxide (NO) metabolites and cyclic guanosine monophosphate (cGMP) were measured before and after bypass. Myocardial tissue was processed at baseline and after incubation at hyperoxic concentration during four hours in order to mimic surgical conditions. Expression of genes involved in IRI and RIPC pathways was analysed including heat shock proteins (HSPs), toll like receptors (TLRs), transcription factors nuclear factor κ-B (NF- κ-B) and hypoxia inducible factor 1 (HIF-1). The participation of hydrogen sulfide enzymatic genes, apelin and its receptor were explored. There was no significant difference according to group allocation in any of the echocardiographic parameters. There was a tendency for higher cTnI values and inotropic score in control patients post-operatively, however this was not statistically significant. BNP presented no significant difference according to group allocation. Inflammatory parameters tended to be higher in the control group, however only TNF- α was significantly higher. There was no difference in levels of Cystatin C, NO metabolites, cGMP, ADMA or SDMA. RIPC patients required shorter PICU stay, all other clinical and laboratory analysis presented no difference related to the intervention. Gene expression analysis revealed interesting patterns before and after incubation. HSP-60 presented a lower expression at baseline in tissue corresponding to RIPC patients, no other differences were found. This study provided with valuable descriptive information on previously known and newly explored parameters in the study population. Demographic characteristics and the presence of cyanosis before surgery influenced patterns of activity in several parameters, numerous indicators were linked to the degree of injury suffered by the myocardium. RIPC did not reduce markers of cardiac injury or improved echocardiographic parameters and it did not have an effect on end organ function; some effects were seen in inflammatory responses and gene expression analysis. Nevertheless, an important clinical outcome indicator, PICU length of stay was reduced suggesting benefit from the intervention. Larger studies with more statistical power could determine if the tendency of lower injury and inflammatory markers linked to RIPC is real. The present results mostly support findings of larger multicentre trials which have reported no cardiac benefit from RIPC in paediatric cardiac surgery.

Diadenosine Polyphosphates in Tears of Sjögren Syndrome Patients

Purpose.: To analyze the levels of diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) in tears of subjects with Sjögren syndrome and to compare them with those in a control group. Methods.: Twelve subjects with a diagnosis of Sjögren syndrome and 20 healthy control subjects were invited to participate in the present study. Schirmer strips were used to measure tear secretion (Schirmer I test) and to collect tears. Ap4A and Ap5A were measured by high-pressure liquid chromatography (HPLC), and a dry eye questionnaire (DEQ) was used to evaluate dry eye symptomatology. Results.: The mean concentrations of Ap4A and Ap5A in the Sjögren syndrome group were 2.54 ± 1.02 and 26.13 ± 6.95 μM, respectively. This group of patients was divided in two subgroups: four patients with normal tear production and eight patients with low tear production. Concentrations of Ap4A, and Ap5A in patients with normal tear production (Schirmer test result, 12.3 ± 1.2 mm) were 0.47 ± 0.20 and 8.03 ± 3.27 μM, respectively. In the patients with low tear production (Schirmer test result, 1.0 ± 0.3 mm), the concentrations were 4.09 ± 1.36 and 39.51 ± 8.46 μM, respectively and in the control group, 0.13 ± 0.03 and 0.04 ± 0.02 μM, respectively. Conclusions.: Patients with Sjögren syndrome have abnormally elevated concentrations of diadenosine polyphosphates, indicating that these compounds could be used in the diagnosis of this disease.

Changes in Diadenosine Polyphosphates during Alignment-Fit and Orthokeratology Rigid Gas Permeable

Purpose.: To evaluate the levels of dinucleotides diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) in tears of patients wearing rigid gas permeable (RGP) contact lenses on a daily wear basis and of patients wearing reverse-geometry RGP lenses overnight for orthokeratology treatment. Methods.: Twenty-two young volunteers (10 females, 12 males; 23.47 ± 4.49 years) were fitted with an alignment-fit RGP lens (paflufocon B) for a month, and after a 15-day washout period they were fitted with reverse-geometry RGP lenses for corneal reshaping (paflufocon D) for another month. During each period, tears were collected at baseline day 1, 7, 15, and 28. Ap4A and Ap5A were measured by high-pressure liquid chromatography (HPLC). Additionally, corneal staining, break-up time (BUT), Schirmer test, and dryness symptoms were evaluated. Results.: Ap4A concentrations increased significantly from baseline during the whole period of daily wear of RGP lenses (P < 0.001); concentration was also significantly higher than in the orthokeratology group, which remained at baseline levels during the study period except at day 1 (P < 0.001) and day 28 (P = 0.041). While BUT and Schirmer remained unchanged in both groups, discomfort and dryness were significantly increased during alignment-fit RGP daily wear but not during the orthokeratology period. Conclusions.: Daily wear of RGP lenses increased the levels of Ap4A due to mechanical stimulation by blinking of the corneal epithelium, and this is associated with discomfort. Also, orthokeratology did not produce symptoms or signs of ocular dryness, which could be a potential advantage over soft contact lenses in terms of contact lens-induced dryness.

Genome-wide detection of CNVs and their association with meat tenderness in Nelore cattle.

Brazil is one of the largest beef producers and exporters in the world with the Nelore breed representing the vast majority of Brazilian cattle (Bos taurus indicus). Despite the great adaptability of the Nelore breed to tropical climate, meat tenderness (MT) remains to be improved. Several factors including genetic composition can influence MT. In this article, we report a genome-wide analysis of copy number variation (CNV) inferred from Illumina1 High Density SNP-chip data for a Nelore population of 723 males. We detected >2,600 CNV regions (CNVRs) representing 6.5% of the genome. Comparing our results with previous studies revealed an overlap in 1400 CNVRs (>50%). A total of 1,155 CNVRs (43.6%) overlapped 2,750 genes. They were enriched for processes involving guanosine triphosphate (GTP), previously reported to influence skeletal muscle physiology and morphology. Nelore CNVRs also overlapped QTLs for MT reported in other breeds (8.9%, 236 CNVRs) and from a previous study with this population (4.1%, 109 CNVRs). Two CNVRs were also proximal to glutathione metabolism genes that were previously associated with MT. Genome-wide association study of CN state with estimated breeding values derived from meat shear force identified 6 regions, including a region on BTA3 that contains genes of the cAMP and cGMP pathway. Ten CNVRs that overlapped regions associated with MT were successfully validated by qPCR. Our results represent the first comprehensive CNV study in Bos taurus indicus cattle and identify regions in which copy number changes are potentially of importance for the MT phenotype.

Innovative photoresponsive materials & supramolecular structures

This thesis focuses on two main topics: photoresponsive azobenzene-based polymers and supramolecular systems generated by the self-assembly of lipophilic guanosines. In the first chapters describe innovative photoresponsive devices and materials capable of performing multiple roles in the field of soft robotics and energy conversion. Chapter 2 describes a device obtained by coupling a photoresponsive liquid-crystalline network and a piezoelectric polymer to convert visible light into electricity. Chapter 3 deals with a material that can assume different shapes when triggered by three different stimuli in different environments. Chapter 4 reports a highly performing artificial muscle that contracts when irradiated. The last two chapters report on supramolecular structures generated from functionalized guanosines dissolved in organic solvents. Chapter 6 illustrates the self-assembly into G-quadruplexes of 8- and 5’-functionalized guanosines in the absence of templating ions. Chapter 7 describes the supramolecular structure generated by the assembly of a lipophilic guanosine in the presence of silver cations. Chapter 6 is reproduced from an already published paper, while the other chapters are going to be submitted to different journals in a couple of months.

Chemiluminescence – based bioanalytical devices for astrobiological, food safety and clinical applications

At the intersection of biology, chemistry, and engineering, biosensors are a multidisciplinary innovation that provide a cost-effective alternative to traditional laboratory techniques. Due to their advantages, biosensors are used in medical diagnostics, environmental monitoring, food safety and many other fields. The first part of the thesis is concerned with learning the state of the art of paper-based immunosensors with bioluminescent (BL) and chemiluminescent (CL) detection. The use of biospecific assays combined with CL detection and paper-based technology offers an optimal approach to creating analytical tools for on-site applications and we have focused on the specific areas that need to be considered more in order to ensure a future practical implementation of these methods in routine analyses. The subsequent part of the thesis addresses the development of an autonomous lab-on-chip platform for performing chemiluminescent-based bioassays in space environment, exploiting a CubeSat platform for astrobiological investigations. An origami-inspired microfluidic paper-based analytical device has been developed with the purpose of assesses its performance in space and to evaluate its functionality and the resilience of the (bio)molecules when exposed to a radiation-rich environment. Subsequently, we designed a paper-based assay to detect traces of ovalbumin in food samples, creating a user-friendly immunosensing platform. To this purpose, we developed an origami device that exploits a competitive immunoassay coupled with chemiluminescence detection and magnetic microbeads used to immobilize ovalbumin on paper. Finally, with the aim of exploring the use of biomimetic materials, an hydrogel-based chemiluminescence biosensor for the detection of H2O2 and glucose was developed. A guanosine hydrogel was prepared and loaded with luminol and hemin, miming a DNAzyme activity. Subsequently, the hydrogel was modified by incorporating glucose oxidase enzyme to enable glucose biosensing. The emitted photons were detected using a portable device equipped with a smartphone's CMOS (complementary metal oxide semiconductor) camera for CL emission detection.