959 resultados para imaging of connective tissues


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We present whole-rock and zircon rare earth element (REE) data from two early Archaean gneisses (3.81 Ga and 3.64 Ga) from the Itsaq gneiss complex, south-west Greenland. Both gneisses represent extremely rare examples of unaltered, fresh and relatively undeformed igneous rocks of such antiquity. Cathodoluminescence imaging of their zircons indicates a single crystallisation episode with no evidence for either later metamorphic and/or anatectic reworking or inheritance of earlier grains. Uniform, single-population U/Pb age data confirm the structural simplicity of these zircons. One sample, a 3.64 Ga granodioritic gneiss from the Gothabsfjord, yields a chondrite-normalised REE pattern with a positive slope from La to Lu as well as substantial positive Ce and slight negative Eu anomalies, features generally considered to be typical of igneous zircon. In contrast, the second sample, a 3.81 Ga tonalite from south of the Isua Greenstone Belt, has variable but generally much higher light REE abundances, with similar middle to heavy REE. Calculation of zircon/melt distribution coefficients (D-REE(zircon/melt)) from each sample yields markedly different values for the trivalent REE (i.e. Ce and Eu omitted) and simple application of one set of D-REE(zircon/melt) to model the melt composition for the other sample yields concentrations that are in error by up to two orders of magnitude for the light REE (La-Nd). The observed light REE overabundance in the 3.81 Ga tonalite is a commonly observed feature in terrestrial zircons for which a number of explanations ranging from lattice strain to disequilibrium crystallisation have been proposed and are further investigated herein. Regardless of the cause of light REE overabundance, our study shows that simple application of zircon/melt distribution coefficients is not an unambiguous method for ascertaining original melt composition. In this context, recent studies that use REE data to claim that > 4.3 Ga Hadean detrital zircons originally crystallised from an evolved magma, in turn suggesting the operation of geological processes in the early Earth analogous to those of the present day (e.g. subduction and melting of hydrated oceanic crust), must be regarded with caution. Indeed, comparison of terrestrial Hadean and > 3.9 Ga lunar highland zircons shows remarkable similarities in the light REE, even though subduction processes that have been used to explain the terrestrial zircons have never operated on the Moon. (C) 2002 Elsevier Science B.V. All rights reserved.

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Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl- secretion and inhibit amiloride-sensitive Na+ transport. CFTR has been suggested to conduct adenosine 5'-triphosphate (ATP) or to control ATP release to the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na+ channels (ENaC) could be by release of ATP or uridine 5'-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after coexpression of P2Y(2) receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl- channel blocker 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl- transport or inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N-2,2'-O-dibutyrylguanosine 3',5-cyclic monophosphate (cGMP) and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl-. (C) 2002 Elsevier Science B.V. All rights reserved.

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Although the majority of dental abscesses in children originate from dental caries or trauma, a few are associated with unusual conditions which challenge diagnosis and management. Recent research findings have shed light on these unusual entities and greatly improved understanding of their clinical implications. These conditions include developmental abnormalities such as dens invaginatus in which there is an invagination of dental tissues into the pulp chamber and dens evaginatus in which a tubercle containing pulp is found on the external surface of a tooth crown. In addition, inherited conditions which show abnormal dentine such as dentine dysplasia, dentinogenesis imperfecta, and osteogenesis imperfecta predispose the dentition to abscess formation. Furthermore, 'spontaneous' dental abscesses are frequently encountered in familial hypophosphataemia, also known as vitamin D-resistant rickets, in which there is hypomineralization of dentine and enlargement of the pulp. In addition to developmental conditions, there are also acquired conditions which may cause unusual dental abscesses,. These include pre-eruptive intracoronal resorption which was previously known as 'pre-eruptive caries' or the 'fluoride bomb'. In addition, some undiagnosed infections associated with developing teeth are now thought to be the mandibular infected buccal cysts which originate from infection of the developing dental follicles. In the present paper, these relatively unknown entities Which cause unusual abscesses in children are reviewed with the aim of updating the general practitioner in their diagnosis and management.

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Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyltransferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARgamma/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.

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This paper investigates the possible link between non-workplace cadmium (Cd) exposure, cytochrome P450 expression and hypertension. We present results of our investigation into the relationships between liver and kidney Cd burdens and the abundance of the CYP isoform 4A11. Our data show associations between non-workplace Cd exposure and changes in the abundance of hepatic and renal cortical CYP4A11. In liver the levels of immunochemically detectable CYP4A11 were positively correlated with tissue Cd content while in contrast CYP4A11 abundance was inversely correlated with kidney Cd burden. These differences are most likely related to the different Cd burden of the tissues. These observations suggest the potential for involvement of Cd as a mediator of CYP4A11 expression in kidney cortex and indicate that elevations in kidney Cd content may be involved in hypertension via alteration of the expression of this particular isoform. Potential mechanisms by which Cd may alter CYP4A11 expression are discussed briefly. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

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The rapid growth in genetics and molecular biology combined with the development of techniques for genetically engineering small animals has led to increased interest in in vivo small animal imaging. Small animal imaging has been applied frequently to the imaging of small animals (mice and rats), which are ubiquitous in modeling human diseases and testing treatments. The use of PET in small animals allows the use of subjects as their own control, reducing the interanimal variability. This allows performing longitudinal studies on the same animal and improves the accuracy of biological models. However, small animal PET still suffers from several limitations. The amounts of radiotracers needed, limited scanner sensitivity, image resolution and image quantification issues, all could clearly benefit from additional research. Because nuclear medicine imaging deals with radioactive decay, the emission of radiation energy through photons and particles alongside with the detection of these quanta and particles in different materials make Monte Carlo method an important simulation tool in both nuclear medicine research and clinical practice. In order to optimize the quantitative use of PET in clinical practice, data- and image-processing methods are also a field of intense interest and development. The evaluation of such methods often relies on the use of simulated data and images since these offer control of the ground truth. Monte Carlo simulations are widely used for PET simulation since they take into account all the random processes involved in PET imaging, from the emission of the positron to the detection of the photons by the detectors. Simulation techniques have become an importance and indispensable complement to a wide range of problems that could not be addressed by experimental or analytical approaches.

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Male mice (NMRI strain) of 3 and 5 g were inoculated i. p. with 8 x 10(6) and 9 x 10(4) metatrypomastigotes/g harvested from a 12-day-old LIT culture of Trypanosoma rangeli of the "Dog-82" strain. At regular intervals after inoculation, the animals were sacrificed and portions of heart, liver, spleen, lung, thigh, kidney, stomach, intestine, brain, sternum, and vertebral column were embedded in paraffin, sectioned, and stained with haematoxylin-eosin and Giemsa colophonium. Pathology was encountered in the first five tissues cited above. The subcutaneous, periosteal, interstitial, and peribronchial connective tissues, and later the muscle cells of the heart, were heavily parasitized by amastigotes and trypomastigotes. The possible reasons for the decrease in tissue parasitosis at the same time that the parasitemia is reaching its peak, and for the low level of inflammation in the parasitized tissues, are discussed. The observations of other workers, as well as the results described here, indicate that certain strains of T. rangeli under certain conditions may well cause pathological alterations in mammals.

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Whipple's disease (WD) is a rare systemic disease of infectious etiology which involves the small intestine but can virtually affect any organ. We present here five cases (four males and one female) ranging in age from 20 to 59 years. All patients had intestinal involvement associated or not with clinical manifestations linked to this organ. Vegetation in the tricuspid valve was observed in one patient, suggesting endocarditis caused by Tropheryma whippelii, with disappearance of the echocardiographic alterations after treatment. In one of the male patients the initial clinical manifestation was serologically negative spondylitis, with no diarrhea occurring at any time during follow-up. Ocular involvement associated with intestinal malabsorption and significant weight loss were observed in one case. In the other two cases, diarrhea was the major clinical manifestation. All patients were diagnosed by histological examination of the jejunal mucosa and, when indicated, of extraintestinal tissues by light and electron microscopy. After antibiotic treatment, full remission of symptoms occurred in all cases. A control examination of the intestinal mucosa performed after twelve months of treatment with sulfamethoxazole-trimethoprim revealed the disappearance of T. whippelii in four patients. The remaining patient was lost to follow-up.

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INTRODUCTION: Synovial sarcoma is a high-grade, soft-tissue sarcoma that most frequently is located in the vicinity of joints, tendons or bursae, although it can also be found in extra-articular locations. Most patients with synovial sarcoma of the hand are young and have a poor prognosis, as these tumors are locally aggressive and are associated with a relatively high metastasis rate. According to the literature, local recurrence and/or metastatic disease is found in nearly 80% of patients. Current therapy comprises surgery, systemic and limb perfusion chemotherapy, and radiotherapy. However, the 5-year survival rate is estimated to be only around 27% to 55%. Moreover, most authors agree that synovial sarcoma is one of the most commonly misdiagnosed malignancies of soft tissues because of their slow growing pattern, benign radiographic appearance, ability to change size, and the fact that they may elicit pain similar to that caused by common trauma. CASE PRESENTATION: We describe an unusual case of a large synovial sarcoma of the hand in a 63-year-old Caucasian woman followed for 12 years by a multidisciplinary team. In addition, a literature review of the most pertinent aspects of the epidemiology, diagnosis, treatment and prognosis of these patients is presented. CONCLUSION: Awareness of this rare tumor by anyone dealing with hand pathology can hasten diagnosis, and this, in turn, can potentially increase survival. Therefore, a high index of suspicion for this disease should be kept in mind, particularly when evaluating young people, as they are the most commonly affected group.

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This study investigated the sero-conversion period in which dogs from endemic areas test positive for visceral leishmaniasis (VL) as well as the early post-infection period in which renal alterations are observed. Dogs that were initially negative for Canine Visceral Leishmaniasis (CVL) were clinically evaluated every three months by serological, parasitological and biochemical tests until sero-conversion was confirmed, and six months later a subsequent evaluation was performed. Samples of kidney tissues were processed and stained with Hematoxylin and Eosin (H&E), Periodic Acid Schiff (PAS) and Masson’s trichrome stain and lesions were classified based on the WHO criteria. Of the 40 dogs that initially tested negative for VL, 25 (62.5%) exhibited positive serological tests during the study period. Of these 25 dogs, 15 (60%) tested positive within three months, five (20%) tested positive within six months and five (20%) tested positive within nine months. The dogs exhibited antibody titers between 1:40 and 1:80 and 72% of the dogs exhibited clinical symptoms. The Leishmania antigen was present in the kidneys of recently infected dogs. We found higher levels of total protein and globulin as well as lower levels of albumin in the infected dogs when compared to the control dogs. Additionally, infected dogs presented levels of urea and creatinine that were higher than those of the uninfected dogs. Glomerulonephritis was detected in some of the dogs examined in this study. These data suggest that in Teresina, the sero-conversion for VL occurs quickly and showed that the infected dogs presented abnormal serum proteins, as well as structural and functional alterations in the kidneys during the early post-infection period.

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Breast cancer is the most common cancer among women, being a major public health problem. Worldwide, X-ray mammography is the current gold-standard for medical imaging of breast cancer. However, it has associated some well-known limitations. The false-negative rates, up to 66% in symptomatic women, and the false-positive rates, up to 60%, are a continued source of concern and debate. These drawbacks prompt the development of other imaging techniques for breast cancer detection, in which Digital Breast Tomosynthesis (DBT) is included. DBT is a 3D radiographic technique that reduces the obscuring effect of tissue overlap and appears to address both issues of false-negative and false-positive rates. The 3D images in DBT are only achieved through image reconstruction methods. These methods play an important role in a clinical setting since there is a need to implement a reconstruction process that is both accurate and fast. This dissertation deals with the optimization of iterative algorithms, with parallel computing through an implementation on Graphics Processing Units (GPUs) to make the 3D reconstruction faster using Compute Unified Device Architecture (CUDA). Iterative algorithms have shown to produce the highest quality DBT images, but since they are computationally intensive, their clinical use is currently rejected. These algorithms have the potential to reduce patient dose in DBT scans. A method of integrating CUDA in Interactive Data Language (IDL) is proposed in order to accelerate the DBT image reconstructions. This method has never been attempted before for DBT. In this work the system matrix calculation, the most computationally expensive part of iterative algorithms, is accelerated. A speedup of 1.6 is achieved proving the fact that GPUs can accelerate the IDL implementation.

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RESUMO:O glicosilfosfatidilinositol (GPI) é um complexo glicolipídico utlizado por dezenas de proteínas, o qual medeia a sua ancoragem à superfície da célula. Proteínas de superfície celular ancoradas a GPI apresentam várias funções essenciais para a manutenção celular. A deficiência na síntese de GPI é o que caracteriza principalmente a deficiência hereditária em GPI, um grupo de doenças autossómicas raras que resultam de mutações nos genes PIGA, PIGL, PIGM, PIGV, PIGN, PIGO e PIGT, os quais sao indispensáveis para a biossíntese do GPI. Uma mutação pontual no motivo rico em GC -270 no promotor de PIGM impede a ligação do factor de transcrição (FT) Sp1 à sua sequência de reconhecimento, impondo a compactação da cromatina, associada à hipoacetilação de histonas, e consequentemente, impedindo a transcrição de PIGM. Desta forma, a adição da primeira manose ao GPI é comprometida, a síntese de GPI diminui assim como as proteínas ligadas a GPI à superficie das células. Pacientes com Deficiência Hereditária em GPI-associada a PIGM apresentam trombose e epilesia, e ausência de hemólise intravascular e anemia, sendo que estas duas últimas características definem a Hemoglobinúria Paroxística Nocturna (HPN), uma doença rara causada por mutações no gene PIGA. Embora a mutação que causa IGD seja constitutiva e esteja presente em todos os tecidos, o grau de deficiência em GPI varia entre células do mesmo tecido e entre células de tecidos diferentes. Por exemplo nos granulócitos e linfócitos B a deficiência em GPI é muito acentuada mas nos linfócitos T, fibroblastos, plaquetas e eritrócitos é aproximadamente normal, daí a ausência de hemólise intravascular. Os eventos transcricionais que estão na base da expressão diferencial da âncora GPI nas células hematopoiéticas são desconhecidos e constituem o objectivo geral desta tese. Em primeiro lugar, os resultados demonstraram que os níveis de PIGM mRNA variam entre células primárias hematopoiéticas normais. Adicionalmente, a configuração dos nucleossomas no promotor de PIGM é mais compacta em células B do que em células eritróides e tal está correlacionado com os níveis de expressão de PIGM, isto é, inferior nas células B. A presença de vários motivos de ligação para o FT específico da linhagem megacariocítica-eritróide GATA-1 no promotor de PIGM sugeriu que GATA-1 desempenha um papel regulador na sua transcrição. Os resultados mostraram que muito possivelmente GATA-1 desempenha um papel repressor em vez de activador da expressão de PIGM. Resultados preliminares sugerem que KLF1, um factor de transcrição restritamente eritróide, regula a transcrição de PIGM independentemente do motivo -270GC. Em segundo lugar, a investigação do papel dos FTs Sp demonstrou que Sp1 medeia directamente a transcrição de PIGM em ambas as células B e eritróide. Curiosamente, ao contrário do que acontece nas células B, em que a transcrição de PIGM requer a ligação do FT geral Sp1 ao motivo -270GC, nas células eritróides Sp1 regula a transcrição de PIGM ao ligar-se a montante e não ao motivo -270GC. Para além disso, demonstrou-se que Sp2 não é um regulador directo da transcrição de PIGM quer nas células B quer nas células eritróides. Estes resultados explicam a ausência de hemólise intravascular nos doentes com IGD associada a PIGM, uma das principais características que define a HPN. Por último, resultados preliminares mostraram que a repressão da transcrição de PIGM devida à mutação patogénica -270C>G está associada com a diminuição da frequência de interacções genómicas em cis entre PIGM e os seus genes “vizinhos”, sugerindo adicionalmente que a regulação de PIGM e desses genes é partilhada. No seu conjunto, os resultados apresentados nesta tese contribuem para o conhecimento do controlo transcricional de um gene housekeeping, específico-detecido, por meio de FTs genéricos e específicos de linhagem.-------------ABSTRACTC: Glycosylphosphatidylinositol (GPI) is a complex glycolipid used by dozens of proteins for cell surface anchoring. GPI-anchored proteins have various functions that are essential for the cellular maintenance. Defective GPI biosynthesis is the hallmark of inherited GPI deficiency (IGD), a group of rare autosomal diseases caused by mutations in PIGA, PIGL, PIGM, PIGV, PIGN, PIGO and PIGT, all genes indispensable for GPI biosynthesis. A point mutation in the -270GC-rich box in the core promoter of PIGM disrupts binding of the transcription factor (TF) Sp1 to it, imposing nucleosome compaction associated with histone hypoacetylation, thus abrogating transcription of PIGM. As a consequence of PIGM transcriptional repression, addition of the first mannose residue onto the GPI core and thus GPI production are impaired; and expression of GPI-anchored proteins on the surface of cells is severely impaired. Patients with PIGM-associated IGD suffer from life-threatening thrombosis and epilepsy but not intravascular haemolysis and anaemia, two defining features of paroxysmal nocturnal haemoglobinuria (PNH), a rare disease caused by somatic mutations in PIGA. Although the disease-causing mutation in IGD is constitutional and present in all tissues, the degree of GPI deficiency is variable and differs between cells of the same and of different tissues. Accordingly, GPI deficiency is severe in granulocytes and B cells but mild in T cells, fibroblasts, platelets and erythrocytes, hence the lack of intravascular haemolysis.The transcriptional events underlying differential expression of GPI in the haematopoietic cells of PIG-M-associated IGD are not known and constitute the general aim of this thesis. Firstly, I found that PIGM mRNA levels are variable amongst normal primary haematopoietic cells. In addition, the nucleosome configuration in the promoter of PIGM is more compacted in B cells than in erythroid cells and this correlated with the levels of PIGM mRNA expression, i.e., lower in B cells. The presence of several binding sites for GATA-1, a mega-erythroid lineage-specific transcription factor (TF), at the PIGM promoter suggested that GATA-1 has a role on PIGM transcription. My results showed that GATA-1 in erythroid cells is most likely a repressor rather than an activator of PIGM expression. Preliminary data suggested that KLF1, an erythroid-specific TF, regulates PIGM transcription but independently of the -270GC motif. Secondly, investigation of the role of the Sp TFs showed that Sp1 directly mediates PIGM transcriptional regulation in both B and erythroid cells. However, unlike in B cells in which active PIGM transcription requires binding of the generic TF Sp1 to the -270GC-rich box, in erythroid cells, Sp1 regulates PIGM transcription by binding upstream of but not to the -270GC-rich motif. Additionally, I showed that Sp2 is not a direct regulator of PIGM transcription in B and erythroid cells. These findings explain lack of intravascular haemolysis in PIGM-associated IGD, a defining feature of PNH. Lastly, preliminary work shows that transcriptional repression of PIG-M by the pathogenic -270C>G mutation is associated with reduced frequency of in cis genomic interactions between PIGM and its neighbouring genes, suggesting a shared regulatory link between these genes and PIGM. Altogether, the results presented in this thesis provide novel insights into tissuespecific transcriptional control of a housekeeping gene by lineage-specific and generic TFs.

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RESUMO: Enthesitis is the hallmark of spondyloarthritis (SpA), and is observed in all subtypes. Wide information on SpA abnormalities, including synovitis, tendinitis and enthesitis, can be efficiently perceived by Doppler ultrasound. Furthermore, several studies on imaging of enthesis showed that imaging techniques are better than clinical examination to detect enthesis alterations; and vascularized enthesitis detected by Doppler ultrasound appears to be a valuable diagnostic tool to confirm SpA diagnosis. However, data published until now concerning entheseal elementary alterations that characterize SpA enthesitis (enthesis inflammatory activity) or enthesopathy (permanent structural changes) reflect rather the authors’ empiric opinion than a methodological validation process. In this sense it seems crucial to identify elementary entheseal lesions associated with activity or damage, in order to improve monitoring and treatment response in SpA patients. The development of better assessment tools is today a challenge and a need in SpA. The first study of this thesis focused on the analysis of the reliability of inter-lector and inter-ultrasonography equipment of Madrid sonography enthesitis index (MASEI). Fundamental data for the remaining unrolling project validity. In the second and third studies we concerned about two entheseal elemental lesions: erosions and bursa. In literature erosions represent a permanent structural damage, being useful for monitoring joint injury, disease activity and therapeutic response in many rheumatic diseases; and to date, this concept has been mostly applied in rheumatoid arthritis (RA). Unquestionably, erosion is a tissue-related damage and a structural change. However, the hypothesis that we decided to test was if erosions represent a permanent structural change that can only grow and worsen over time, as occurs in RA, or a transitory alteration. A longitudinal study of early SpA patients was undertaken, and the Achilles enthesis was used as a model. Our results strongly suggested that previously detected erosions could disappear during the course of the disease, being consistent with the dynamic behavior of erosion over time. Based on these striking results it seems reasonable to suggest that the new-bone formation process in SpA could be associated with the resolution of cortical entheseal erosion over time. These results could also be in agreement with the apparent failure of anti-tumor necrosis factor (TNF) therapies to control bone proliferation in SpA; and with the relation of TNF-α, Dickkopf-related protein 1 (Dkk-1) and the regulatory molecule of the Wnt signaling pathway in the bone proliferation in SpA. In the same model, we then proceeded to study the enthesis bursa. Interestingly, the Outcome Measures in Rheumatology Clinical Trials (OMERACT) enthesopathy definition does not include bursa as an elementary entheseal lesion. Nonetheless, bursa was included in 46% of the enthesis studies in a recently systematic literature review, being in agreement with the concept of “synovio-entheseal complex” that includes the link between enthesitis and osteitis in SpA. It has been clarified in recent data that there is not only a close functional integration of the enthesis with the neighboring bone, but also a connection between enthesitis and synovitis. Therefore, we tried to assess the prevalence and relevance of the bursa-synovial lesion in SpA. Our findings showed a significant increase of Achilles bursa presence and thickness in SpA patients compared to controls (healthy/mechanical controls and RA controls). These results raise awareness to the need to improve the enthesopathy ultrasonographic definition. In the final work of this thesis, we have explored new perspectives, not previously reported, about construct validity of enthesis ultrasound as a possible activity outcome in SpA. We performed a longitudinal Achilles enthesis ultrasound study in patients with early SpA. Achilles ultrasound examinations were performed at baseline, six- and twelve-month time periods and compared with clinical outcome measures collected at basal visit. Our results showed that basal erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are higher in patients with Doppler signal in enthesis, and even that higher basal ESR, CRP and Ankylosing Spondylitis Disease Activity Score (ASDAS) predicted a higher Doppler signal (an ultrasound alteration accepted as representative of inflammation) six months later. Patients with very high disease activity assessed by ASDAS (>3.5) at baseline had significantly higher Achilles total ultrasound score verified at the same time; and ASDAS <1.3 predicted no Doppler signal at six and twelve months. This seems to represent a connection between classical biomarkers and clinical outcomes associated with SpA activity and Doppler signal, not only at the same time, but also for the following months. Remarkably, patients with inactive disease (ASDAS < 1.3) at baseline had no Doppler signal at six and twelve months. These findings reinforce the potential use of ultrasound related techniques for disease progression assessment and prognosis purposes. Intriguingly, Ankylosing Spondylitis Disease Activity Index (BASDAI) didn’t show significant differences between different cut-offs concerning ultrasound lesions or Doppler signal, while verified with ASDAS. These results seem to indicate that ASDAS reflects better than BASDAI what happens in the enthesis. The work herein discussed clearly shows the potential utility of ultrasound in enthesis assessment in SpA patients, and can be important for the development of ultrasound activity and structural damage scores for diagnosis and monitoring purposes. Therefore, local promotion of this technique constitutes a medical intervention that is worth being tested in SpA patients for diagnosis, monitoring and prognosis purposes.

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The Stiles-Crawford effect (SCE) is the well-known phenomenon in which the brightness of light perceived by the human eye depends upon its entrance point in the pupil. This physiological characteristic is due to the directional sensitivity of the cone photoreceptors in the retina and it displays an approximately Gaussian dependency which is altered in a number of pathologies. Retinal imaging, a widely spread clinical practice, may be used to evaluate the SCE and thus serve as diagnostic tool. Nonetheless, its use for such a purpose is still underdeveloped and far from the clinical reality. In this project a fundus camera was built and used to assess the cone photoreceptor directionality by reflective imaging of the retina in healthy individuals. The physical and physiological implications of its development are addressed in detail in the text: the optical properties of the human eye, illumination issues, acquiring a retinal image formed by the eye, among others. A full description of the developmental process that led to the final measuring method and results is also given. The developed setup was successfully used to obtain high quality images of the eye fundus and in particular the parafoveal cone photoreceptors. The SCE was successfully observed and characterized. Even though considerable improvements could be done to the measurement method, the project showed the feasibility of using retinal imaging to evaluate the SCE thus motivating its usage in a clinical environment.

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RESUMO: Mutações em genes envolvidos na formação do coração e anomalias em qualquer etapa deste processo causam frequentemente malformações cardíacas, que representam o tipo mais comum de defeitos em neonatais, afetando cerca de 1% dos nascimentos por ano. Assim, estima-se que 20 milhões de pessoas sejam portadoras de um defeito cardíaco congénito. O coração da Drosophila melanogaster (mosca-da-fruta), denominado vaso dorsal, é um órgão relativamente simples que actua como uma bomba muscular, contraindo automaticamente para permitir a circulação da hemolinfa através do corpo. A formação do vaso dorsal na mosca é muito semelhante ao desenvolvimento do coração em vertebrados, representando por isso, um poderoso modelo para estudar a rede de genes e os padrões regulatórios relacionados com o desenvolvimento deste órgão. Anteriormente, nós identificámos um gene em Drosophila, darhgef10, fortemente expresso no coração em desenvolvimento e cuja deleção induz anormalidades cardíacas subtis mas prevalentes. Os mutantes para darhgef10 são viáveis e férteis no ambiente controlado de laboratório. Este trabalho teve como objectivos caracterizar fenotipicamente os mutantes nulos para darhgef10, determinar a localização subcelular da proteína dArhgef10 e investigar a base celular subjacente ao defeito no alinhamento dos cardioblastos observado nos mutantes. Os nossos resultados revelaram que a deleção de darhgef10 provoca uma severa redução da viabilidade, sem no entanto comprometer o tempo de desenvolvimento e a longevidade. Por outro lado, o aumento da expressão de darhgef10 em músculos, glândulas salivares e no disco imaginal do olho afeta drasticamente a integridade destes tecidos. A expressão ectópica de darhgef10 in vitro e in vivo revelou que a proteína está localiza no citoplasma com enriquecimento junto à membrana celular, com associação à actina F. Live imaging de embriões mutantes para darhgef10 revelou que os defeitos observados no coração podem estar associados a um defeito na adesão dos músculos alary e/ou das células pericardiais ao vaso dorsal. O homólogo humano de darhgef10, ARHGEF10, também é expresso no coração e está associação a uma maior susceptibilidade para a ocorrência de acidentes vasculares cerebrais aterotrombóticos, sugerindo que o que aprendemos sobre darhgef10 em Drosophila pode ter implicações do ponto de vista clínico para a saúde humana. ----------------------------- ABSTRACT: Mutations in genes controlling heart development and abnormalities in any of its steps frequently cause cardiac malformations, the most common type of birth defects in humans, affecting nearly 1% of births per year. Hence around 20 million adults are expected to live with a congenital heart defect. The Drosophila melanogaster heart, called dorsal vessel, is a relatively simple organ that acts as a muscular pump contracting automatically to allow the circulation of hemolymph. Drosophila heart formation shares many similarities with heart development in vertebrates providing a powerful system to study gene networks and regulatory pathways involved in heart development. We have previously identified a Drosophila gene, darhgef10, which is strongly expressed in the developing heart and when deleted, leads to flies with highly prevalent yet subtle heart abnormalities, compatible with unchallenged life in the laboratory. Our aims were to phenotypically characterize homozygous null darhgef10 mutants, characterize the subcellular localization of dArhgef10 and to study the cellular basis of the misaligned cardioblasts defect. We found that about half of darhgef10 mutants die during development. However, the survivors surprisingly have a nearly normal developmental time, adult locomotor behavior and total lifespan. Detection of transgene-derived dArhgef10 protein in vitro and in vivo using custom antibodies revealed a cytosolic protein slightly enriched in the cellular membranes and associated with F-actin. Tissue-specific darhgef10 expression disrupts the normal morphology of developing muscles, salivary glands and the eye. Live imaging of darhgef10 mutant embryos revealed that heart defect could be caused by a reduced capacity of attachment of pericardial cells and/or alary muscle to dorsal vessel. The human homolog of darhgef10 is also expressed in the heart and is a susceptibility gene for atherothrombotic stroke, suggesting that what we learn about the function of this gene and its phenotypes in Drosophila could have implications to human health.