Automatic diagnosis of liver steatosis by ultrasound using autoregressive tissue characterization

Liver steatosis is mainly a textural abnormality of the hepatic parenchyma due to fat accumulation on the hepatic vesicles. Today, the assessment is subjectively performed by visual inspection. Here a classifier based on features extracted from ultrasound (US) images is described for the automatic diagnostic of this phatology. The proposed algorithm estimates the original ultrasound radio-frequency (RF) envelope signal from which the noiseless anatomic information and the textural information encoded in the speckle noise is extracted. The features characterizing the textural information are the coefficients of the first order autoregressive model that describes the speckle field. A binary Bayesian classifier was implemented and the Bayes factor was calculated. The classification has revealed an overall accuracy of 100%. The Bayes factor could be helpful in the graphical display of the quantitative results for diagnosis purposes.

Helical tomotherapy versus 3D conformal radiotherapy in dosimetric planning for patients with localized prostate cancer

Radiotherapy is one of the therapeutics selected for localized prostate cancer, in cases where the tumour is confined to the prostate, penetrates the prostatic capsule or has reached the seminal vesicles (T1 to T3 stages). The radiation therapy can be administered through various modalities, being historically used the 3D conformal radiotherapy (3DCRT). Other modality of radiation administration is the intensity modulated radiotherapy (IMRT), that allows an increase of the total dose through modulation of the treatment beams, enabling a reduction in toxicity. One way to administer IMRT is through helical tomotherapy (TH). With this study we intent to analyze the advantages of helical tomotherapy when compared with 3DCRT, by evaluating the doses in the organs at risk (OAR) and planning target volumes (PTV).

Dosimetric effect of tissue heterogeneity for 125I prostate implants

Aim - To use Monte Carlo (MC) together with voxel phantoms to analyze the tissue heterogeneity effect in the dose distributions and equivalent uniform dose (EUD) for (125)I prostate implants. Background - Dose distribution calculations in low dose-rate brachytherapy are based on the dose deposition around a single source in a water phantom. This formalism does not take into account tissue heterogeneities, interseed attenuation, or finite patient dimensions effects. Tissue composition is especially important due to the photoelectric effect. Materials and Methods - The computed tomographies (CT) of two patients with prostate cancer were used to create voxel phantoms for the MC simulations. An elemental composition and density were assigned to each structure. Densities of the prostate, vesicles, rectum and bladder were determined through the CT electronic densities of 100 patients. The same simulations were performed considering the same phantom as pure water. Results were compared via dose-volume histograms and EUD for the prostate and rectum. Results - The mean absorbed doses presented deviations of 3.3-4.0% for the prostate and of 2.3-4.9% for the rectum, when comparing calculations in water with calculations in the heterogeneous phantom. In the calculations in water, the prostate D 90 was overestimated by 2.8-3.9% and the rectum D 0.1cc resulted in dose differences of 6-8%. The EUD resulted in an overestimation of 3.5-3.7% for the prostate and of 7.7-8.3% for the rectum. Conclusions - The deposited dose was consistently overestimated for the simulation in water. In order to increase the accuracy in the determination of dose distributions, especially around the rectum, the introduction of the model-based algorithms is recommended.

Influence of constraining and confinement in the molecular mobility of low molecular weight materials

Dissertation presented to obtain a Ph.D. Degree in Chemical Physics

Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

Evaluation of the schistosomicidal efficacy of liposome - Entrapped Oxamniquine

Oxamniquine (OXA) was sucessfully encapsulated in small unilamellar vesicles using a pH gradient method. This procedure led to a high drug encapsulation efficiency (> 85%) at a drug to lipid molar ratio of 1/10. Moreover, these liposomes were found to retain encapsulated OXA efficciently under dialysis conditions at 37º C. Liposome-entrapped OXA (LOXA), OXA, and empty liposomes were tested against Schistosoma mansoni in a murine model. LOXA produced a significant reduction of the worm burden compared to the other preparations, when inoculated by subcutaneous route (s.c.) with 10 mg OXA/kg animal one day before the infection, and 3, 7, and 14 days after. However, LOXA was not effective when given 7 days before, or 35 days after infections. OXA, in the free form, was effective in relation to the untreated group, only when administered 3 days after the infection. Maximum effect of LOXA, with 97% reduction of the parasite number, was observed when the preparation was given s.c.one day before the infection. On the other hand, LOXA inoculated intraperitoneally one day before the infection didn’t show any reduction of the parasite count. It can be concluded that LOXA is more effective than OXA for the treatment of experimental schistosomiasis, particularly when administered subcutaneously at a time close to the infection

Protein glycosylation of exosomes from ovarian carcinoma cells: structures and biological roles

Dissertation presented to obtain the Ph.D. degree in Biology

Optimization of the Tartrate-Resistant Acid Phosphatase Detection by Histochemical Method

According to the new KDIGO (Kidney Disease Improving Global Outcomes) guidelines, the term of renal osteodystrophy, should be used exclusively in reference to the invasive diagnosis of bone abnormalities. Due to the low sensitivity and specificity of biochemical serum markers of bone remodelling,the performance of bone biopsies is highly stimulated in dialysis patients and after kidney transplantation. The tartrate-resistant acid phosphatase (TRACP) is an iso-enzyme of the group of acid phosphatases, which is highly expressed by activated osteoclasts and macrophages. TRACP in osteoclasts is in intracytoplasmic vesicles that transport the products of bone matrix degradation. Being present in activated osteoclasts, the identification of this enzyme by histochemistry in undecalcified bone biopsies is an excellent method to quantify the resorption of bone. Since it is an enzymatic histochemical method for a thermolabile enzyme, the temperature at which it is performed is particularly relevant. This study aimed to determine the optimal temperature for identification of TRACP in activated osteoclasts in undecalcified bone biopsies embedded in methylmethacrylate. We selected 10 cases of undecalcified bone biopsies from hemodialysis patients with the diagnosis of secondary hyperparathyroidism. Sections of 5 μm were stained to identify TRACP at different incubation temperatures (37º, 45º, 60º, 70º and 80ºC) for 30 minutes. Activated osteoclasts stained red and trabecular bone (mineralized bone) was contrasted with toluidine blue. This approach also increased the visibility of the trabecular bone resorption areas (Howship lacunae). Unlike what is suggested in the literature and in several international protocols, we found that the best results were obtained with temperatures between 60ºC and 70ºC. For technical reasons and according to the results of the present study, we recommended that, for an incubation time of 30 minutes, the reaction should be carried out at 60ºC. As active osteoclasts are usually scarce in a bone section, the standardization of the histochemistry method is of great relevance, to optimize the identification of these cells and increase the accuracy of the histomosphometric results. Our results, allowing an increase in osteoclasts contrast, also support the use of semi-automatic histomorphometric measurements.

Neurotoxic activity and ultrastructural changes in muscles caused by the brown widow spider Latrodectus geometricus venom

Brown widow spider (Latrodectus geometricus) venom (BrWSV) produces few local lesions and intense systemic reactions such as cramps, harsh muscle pains, nausea, vomiting and hypertension. Approximately 16 protein bands under reducing conditions and ~ 14 bands under non-reducing conditions on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis were observed. Neurotoxic clinical manifestations were confirmed in vivo, while proteolytic activity was demonstrated on gelatine film. Severe ultrastructural damages in mice skeletal muscles were observed at 3, 6, 12 and 24 h postinjection with at total of 45 µg of venom protein. Infiltration of eosinophils and ruptures of the cellular membranes were observed in the muscles along with swelling of the nuclear cover and interruption of the collagen periodicity. Altered mitochondrias and autophage vacuoles, nuclear indentation and mitochondria without cristae, slight increment of intermyofibrillar and subsarcolemic spaces and myelinic figures formation were also observed. In the capillary, endothelial membrane unfolding into the lumen was noticed; along with myelinic figures compatible with a toxic myopathy. Swollen sarcotubular systems with lysis of membrane, intense mitochondria autophagia and areas without pinocytic vesicles were observed. Swollen mitochondria surrounded by necrotic areas, myofibrillar disorganization and big vacuolas of the sarcotubular system, degenerated mitochondrium with formation of myelinic figure was seen. Glycogenosomes with small particulate, muscle type glycogen was noticed. Autophagic vacuole (autophagolysosomes) and necrotic areas were also noticed. These damages may be due to interactive effects of the multifactorial action of venom components. However, Latrodectus geometricus venom molecules may also be utilized as neuro therapeutic tools, as they affect neuronal activities with high affinity and selectivity. To our knowledge, the present study is the first ultrastructural report in the literature of muscle injuries and neurological and proteolytic activities caused by BrWSV.

Dissecting the molecular interaction between hepatocytes and Plasmodium liver parasites

Dissertation presented to obtain the Ph.D degree in Biology

Hand, Foot, and Mouth Syndrome in an Immunocompetent Adult: a Case Report

BACKGROUND: Hand, foot, and mouth syndrome (HFMS) is a common acute illness. It is characterized by mild clinical symptoms including fever, blisters, and sores in the mouth and on the palms and soles following a 3- to 7-day incubation period. This syndrome is rarely seen in adults. CASE PRESENTATION: A 35-year-old male Caucasian patient had a history of multiple episodes of acute pharyngitis, hypertension, hypercholesterolemia, and occasional abdominal pain. He presented with polyarthralgia in the knees and hands and odynophagia, followed by fever, oral mucosal aphthous lesions, and vesicles on the palms and soles. Three weeks after presentation, he was admitted to the emergency room with acute myocarditis. The in-hospital evaluation revealed positive serology for coxsackie A9 (1:160), positive anti-transglutaminase and anti-gliadin antibodies, normal immunoglobulins, and human immunodeficiency virus negativity. CONCLUSION: We herein describe a case of HFMS that was associated with coxsackie A9 infection complicated by acute myocarditis. Although an association between celiac disease and HFMS has not been described, this patient's immunologic disruption could have favored the development of infection and ultimately HFMS.

Post-Treated Prostate Cancer: Normal Findings and Signs of Local Relapse on Multiparametric Magnetic Resonance Imaging

The use of multiparametric magnetic resonance imaging (mp-MRI) for prostate cancer has increased over recent years, mainly for detection, staging, and active surveillance. However, suspicion of recurrence in the set of biochemical failure is becoming a significant reason for clinicians to request mp-MRI. Radiologists should be able to recognize the normal post-treatment MRI findings. Fibrosis and atrophic remnant seminal vesicles after prostatectomy are often found and must be differentiated from local relapse. Moreover, brachytherapy, external beam radiotherapy, cryosurgery, and hormonal therapy tend to diffusely decrease the signal intensity of the peripheral zone on T2-weighted images (T2WI) due to the loss of water content, consequently mimicking tumor and hemorrhage. The combination of T2WI and functional studies like diffusion-weighted imaging and dynamic contrast-enhanced improves the identification of local relapse. Tumor recurrence tends to restrict on diffusion images and avidly enhances after contrast administration either within or outside the gland. The authors provide a pictorial review of the normal findings and the signs of local tumor relapse after radical prostatectomy, external beam radiotherapy, brachytherapy, cryosurgery, and hormonal therapy.

Interaction of malaria parasites with host late endocytic and autophagic pathways is essential for Plasmodium liver stage development

RESUMO: A Malária é causada por parasitas do género Plasmodium, sendo a doença parasitária mais fatal para o ser humano. Apesar de, durante o século passado, o desenvolvimento económico e a implementação de diversas medidas de controlo, tenham permitido erradicar a doença em muitos países, a Malária continua a ser um problema de saúde grave, em particular nos países em desenvolvimento. A Malária é transmitida através da picada de uma fêmea de mosquito do género Anopheles. Durante a picada, os esporozoítos são injetados na pele do hospedeiro, seguindo-se a fase hepática e obrigatória do ciclo de vida. No fígado, os esporozoítos infetam os hepatócitos onde se replicam, dentro de um vacúolo parasitário (VP) e de uma forma imunitária silenciosa, em centenas de merozoitos. Estas novas formas do parasita são as responsáveis por infetar os eritrócitos, iniciando a fase sanguínea da doença, onde se os primeiros sintomas se manifestam, tais como a característica febre cíclica. A fase hepática da doença é a menos estudada e compreendida. Mais ainda, as interações entre o VP e os organelos da células hospedeira estão ainda pouco caracterizados. Assim, neste estudo, as interações entre os organelos endocíticos e autofágicos da célula hospedeira e o VP foram dissecados, observando-se que os anfisomas, que são organelos resultantes da intersecção do dois processos de tráfego intracelular, interagem com o parasita. Descobrimos que a autofagia tem também uma importante função imunitária durante a fase hepática inicial, ao passo, que durante o desenvolvimento do parasita, já numa fase mais tardia, o parasita depende da interação com os endossomas tardios e anfisomas para crescer. Vesiculas de BSA, EGF e LC3, foram, também, observadas dentro do VP, sugerindo que os parasitas são capazes de internalizar material endocítico e autofágico do hospedeiro. Mais ainda, mostramos que esta interação depende da cinase PIKfyve, responsável pela conversão do fosfoinositidio-3-fosfato no fosfoinositidio-3,5-bifosfato, uma vez que inibindo esta cinase o parasita não é capaz de crescer normalmente. Finalmente, mostramos que a proteína TRPML1, uma proteína efetora do fosfoinositidio-3,5-bifosfato, e envolvida no processo de fusão das membranas dos organelos endocíticos e autofágicos, também é necessária para o crescimento do parasita. Desta forma, o nosso estudo sugere que a membrana do VP funde com vesiculas endocíticas e autofágicas tardias, de uma forma dependente do fositidio-3,5-bifosfato e do seu effetor TRPML1, permitindo a troca de material com a célula hospedeira. Concluindo, os nossos resultados evidenciam que o processo autofágico que ocorre na célula hospedeira tem um papel duplo durante a fase hepática da malaria. Enquanto numa fase inicial os hepatócitos usam o processo autofágico como forma de defesa contra o parasita, já durante a fase de replicação o VP funde com vesiculas autofágicas e endocíticas de forma a obter os nutrientes necessários ao seu desenvolvimento.--------- ABSTRACT: Malaria, which is caused by parasites of the genus Plasmodium, is the most deadly parasitic infection in humans. Although economic development and the implementation of control measures during the last century have erradicated the disease from many areas of the world, it remains a serious human health issue, particularly in developing countries. Malaria is transmitted by female mosquitoes of the genus Anopheles. During the mosquito blood meal, Plasmodium spp. sporozoites are injected into the skin dermis of the vertebrate host, followed by an obligatory liver stage. Upon entering the liver, Plasmodium parasites infect hepatocytes and silently replicate inside a host cell-derived parasitophorous vacuole (PV) into thousands of merozoites. These new parasite forms can infect red blood cells initiating the the blood stage of the disease which shows the characteristic febrile malaria episodes. The liver stage is the least characterized step of the malaria infection. Moreover, the interactions between the Plasmodium spp. PV and the host cell trafficking pathways are poorly understood. We dissected the interaction between Plasmodium parasites and the host cell endocytic and autophagic pathways and we found that both pathways intersect and interconnect in the close vicinity of the parasite PV, where amphisomes are formed and accumulate. Interestingly, we observed a clearance function for autophagy in hepatocytes infected with Plasmodium berghei parasites at early infection times, whereas during late liver stage development late endosomes and amphisomes are required for parasite growth. Moreover, we found the presence of internalized BSA, EGF and LC3 inside parasite vacuoles, suggesting that the parasites uptake endocytic and autophagic cargo. Furthermore, we showed that the interaction between the PV and host traffic pathways is dependent on the kinase PIKfyve, which converts the phosphoinositide PI(3)P into PI(3,5)P2, since PIKfyve inhibition caused a reduction in parasite growth. Finally, we showed that the PI(3,5)P2 effector protein TRPML1, which is involved in late endocytic and autophagic membrane fusion, is also required for parasite development. Thus, our studies suggest that the parasite parasitophorous vacuole membrane (PVM) is able to fuse with late endocytic and autophagic vesicles in a PI(3,5)P2- and TRPML1-dependent manner, allowing the exchange of material between the host cell and the parasites, necessary for the rapid development of the latter that is seen during the liver stage of infection. In conclusion, we present evidence supporting a specific and essential dual role of host autophagy during the course of Plasmodium liver infection. Whereas in the initial hours of infection the host cell uses autophagy as a cell survival mechanism to fight the infection, during the replicative phase the PV fuses with host autophagic and endocytic vesicles to obtain nutrients required for parasite growth.

Storage of water molecules into biomimetic heterostructures: the role of roughness

The development of devices based on heterostructured thin films of biomolecules conveys a huge contribution on biomedical field. However, to achieve high efficiency of these devices, the storage of water molecules into these heterostructures, in order to maintain the biological molecules hydrated, is mandatory. Such hydrated environment may be achieved with lipids molecules which have the ability to rearrange spontaneously into vesicles creating a stable barrier between two aqueous compartments. Yet it is necessary to find conditions that lead to the immobilization of whole vesicles on the heterostructures. In this work, the conditions that govern the deposition of open and closed liposomes of 1.2-dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (sodium Salt) (DPPG) onto polyelectrolytes cushions prepared by the layer-by-layer (LbL) method were analyzed. Electronic transitions of DPPG molecules as well as absorption coefficients were obtained by vacuum ultraviolet spectroscopy, while the elemental composition of the heterostructures was characterized by x-ray photoelectron spectroscopy (XPS). The presence of water molecules in the films was inferred by XPS and infrared spectroscopy. Quartz crystal microbalance (QCM) data analysis allowed to conclude that, in certain cases, the DPPG adsorbed amount is dependent of the bilayers number already adsorbed. Moreover, the adsorption kinetics curves of both adsorbed amount and surface roughness allowed to determine the kinetics parameters that are related with adsorption processes namely, electrostatic forces, liposomes diffusion and lipids re-organization on surface. Scaling exponents attained from atomic force microscopy images statistical analysis demonstrate that DPPG vesicles adsorption mechanism is ruled by the diffusion Villain model confirming that adsorption is governed by electrostatic forces. The power spectral density treatment enabled a thorough description of the accessible surface of the samples as well as of its inner structural properties. These outcomes proved that surface roughness influences the adsorption of DPPG liposomes onto surfaces covered by a polyelectrolyte layer. Thus, low roughness was shown to induce liposome rupture creating a lipid bilayer while high roughness allows the adsorption of whole liposomes. In addition, the fraction of open liposomes calculated from the normalized maximum adsorbed amounts decreases with the cushion roughness increase, allowing us to conclude that the surface roughness is a crucial variable that governs the adsorption of open or whole liposomes. This conclusion is fundamental for the development of well-designed sensors based on functional biomolecules incorporated in liposomes. Indeed, LbL films composed of polyelectrolytes and liposomes with and without melanin encapsulated were successfully applied to sensors of olive oil.

Description of microsporidia in simulids: molecular and morphological characterization of microsporidia in the larvae of Simulium pertinax Kollar (Diptera: Simuliidae)

IntroductionMicrosporidia constitute the most common black fly pathogens, although the species' diversity, seasonal occurrence and transmission mechanisms remain poorly understood. Infections by this agent are often chronic and non-lethal, but they can cause reduced fecundity and decreased longevity. The objective of this study was to identify microsporidia infecting Simulium (Chirostilbia) pertinax (Kollar, 1832) larvae from Caraguatatuba, State of São Paulo, Brazil, by molecular and morphological characterization.MethodsLarvae were collected at a single point in a stream in a rural area of the city and were kept under artificial aeration until analysis. Polydispyrenia spp. infection was characterized by the presence of at least 32 mononuclear spores measuring 6.9 ± 1.0 × 5.0 ± 0.7µm in persistent sporophorous vesicles. Similarly, Amblyospora spp. were characterized by the presence of eight uninucleate spores measuring 4.5 × 3.5µm in sporophorous vesicles.ResultsThe molecular analysis confirmed the presence of microsporidian DNA in the 8 samples (prevalence of 0.51%). Six samples (Brazilian larvae) were related to Polydispyrenia simulii and Caudospora palustris reference sequences but in separate clusters. One sample was clustered with Amblyospora spp. Edhazardia aedis was the positive control taxon.ConclusionsSamples identified as Polydispyrenia spp. and Amblyospora spp. were grouped with P. simulii and Amblyospora spp., respectively, corroborating previous results. However, the 16S gene tree showed a considerable distance between the black fly-infecting Amblyospora spp. and the mosquito-infecting spp. This distance suggests that these two groups are not congeneric. Additional genomic region evaluation is necessary to obtain a coherent phylogeny for this group.