The role of Hedgehog signaling during zebrafish larvae fin fold regeneration

Several studies have demonstrated that although the structure of the adult and larval zebrafish caudal fin is different, there are similarities at the cellular and molecular level that turn larval zebrafish fin fold a useful model to study the basic principles of regeneration. In this process, while the essential role for Hedgehog (Hh) signaling is well established in the adult zebrafish caudal fin system, its involvement in juvenile tissue regeneration is still unknown. The aim of this Master thesis was therefore to evaluate the contribution of the Hh signaling pathway to the larval zebrafish fin fold regeneration process. Accordingly, we analyzed the expression of several Hh signaling components through in situ hybridization. Here, we showed that several of these genes are effectively expressed in the larval regenerating fin tissue, suggesting a role for Hh signaling also during larval regeneration. However, divergence in the regulation of few Hh signaling components appears to exist between the adult and larval zebrafish fin regeneration processes. Nevertheless, similarly to adult caudal fin regeneration, when Hh signaling was blocked, by using cyclopamine, the larval fin fold regenerative outgrowth is severely impaired. Since larval zebrafish fin fold is ciliated, and primary cilia are closely related to Hh signaling regulation in vertebrate systems, we further addressed the role of primary cilia during larval fin fold regeneration process. To this end, we used the zebrafish iguana mutant, in which primary cilia are not formed, to study the modulation of Hh signaling expression during larval fin fold regeneration in the absence of primary cilia. Here, we found that several genes were expressed with a delay, coincident with the delay in the mutant fin fold regeneration observed in previous work. We show that Hh signaling in the fin fold is crucial to promote cell proliferation. When Hh signaling is blocked using cyclopamine there is a strong blockage of cell proliferation and regeneration is also blocked. Surprisingly, in iguana mutants where Hh signaling is impaired but not totally blocked, cell proliferation is not detected but regeneration still occurs. This raises the question about the requirement of cell proliferation in larvae fin fold regeneration. By blocking the cell cycle using aphidicolin we demonstrate that cell proliferation is not necessary for zebrafish larvae fin fold regeneration.

Long term (five-year) survival following radical surgical treatment plus adjuvant chemotherapy (FAM) in advanced gastric cancer: a controlled study

Several drugs and their associations are being used for adjuvant or complementary chemotherapy with the aim of improving results of gastric cancer treatment. The objective of this study was to verify the impact of these drugs on nutrition and on survival rate after radical treatment of 53 patients with gastric cancer in stage III of the TNM classification. A control group including 28 patients who had only undergone radical resection was compared to a group of 25 patients who underwent the same operative technique followed by adjuvant polychemotherapy with FAM (5-fluorouracil, Adriamycin, and mitomycin C). In this latter group, chemotherapy toxicity in relation to hepatic, renal, cardiologic, neurological, hematologic, gastrointestinal, and dermatological functions was also studied. There was no significant difference on admission between both groups in relation to gender, race, macroscopic tumoral type of tumor according to the Borrmann classification, location of the tumor in the stomach, length of the gastric resection, or response to cutaneous tests on delayed sensitivity. Chemotherapy was started on average, 2.3 months following surgical treatment. Clinical and laboratory follow-up of all patients continued for 5 years. The following conclusions were reached: 1) The nutritional status and incidence of gastrointestinal manifestation were similar in both groups; 2) There was no occurrence of cardiac, renal, neurological, or hepatic toxicity or death due to the chemotherapeutic method per se; 3) Dermatological alterations and hematological toxicity occurred exclusively in patients who underwent polychemotherapy; 4) There was no significant difference between the rate and site of tumoral recurrence, the disease-free interval, or the survival rate of both study groups; 5) Therefore, we concluded, after a 5-year follow-up, chemotherapy with the FAM regimen did not increase the survival rate.

On occurrence, habitat specificity and natural history of adult tiger beetles (Coleoptera: Carabidae: Cicindelinae) near Manaus, Central Amazonia, and key to the larvae of tiger beetle genera

Over a seven year period from 1991 to 1997, 22 species of tiger beetles, representing nine genera, were recorded near Manaus, Brazil. In the Whitewaterfloodplains along the Rio Solimões-Amazonas (Ilha de Marchantaria), three diurnal species inhabit inundation forests and six species (two diurnal, four nocturnal) live in open areas. Data on their natural history and adaptation to living conditions in floodplains are presented. Fifteen species were located on non-flooded uplands (Reserva Florestal A. Ducke). Five diurnal species inhabit the forest floor, two species are canopy dwellers, and eight species (seven diurnal, one nocturnal) live in open areas on whitesand or laterite. Only one species, Pentacomia lacordairei, was found in both floodplain and upland forests. A key to the larvae of tiger beetle genera located near Manaus is presented.

Feeding of the Brycon cephalus, Triportheus elongatus and Semaprochilodus insignis (osteichthyes, characiformes) larvae in Solimões/Amazonas River and floodplain areas.

Information on larval fish feeding is essential for understanding their trophic relations, including the management in conditions totally or partially controlled by humans. An experiment was designed to evaluate the larval diets of three commercially important species. Four varzea-lakes and the adjacent river were sampled with bongo and hand nets from January 1993 to November 1995. Larval diets were evaluated by length-classes and capture sites, and were tested by two factor ANOVA. The larvae were feeding in all habitats, except in the flooded forests. The three species had different diets, which varied with their length and lake. The rotifers were the main initial food item of the three species, replaced by fish larvae in Brycon cephalus, cladocerans in Triportheus elongatus and detritus in Semaprochilodus insignis. The increase of the ingestion limit, as the larvae grew, was higher than the increase in the consumed prey size for the three species.

Preliminary observations on the effect of sudden changes of temperature on survival of young Matrinxã (Brycon cephalus) under laboratory conditions

Matrinxã is a very promising amazonian fish for fish culture in Brazil. This study is aimed at determining the approximate tolerated temperature range in this species. Groups of ten young matrinxã specimens (15.1±0.8 cm average length and 58.3±10.3 g average weight) were subjected to 9 different temperatures for 24 hours without previous acclimation. Fish were transferred from an initial temperature of 27ºC to those ranging from 12 to 39ºC at 3ºC intervals. Both 12ºC and 39ºC temperatures were lethal for this species with 100% mortality rate. Following 2 minutes of exposure to 39ºC fish changed behavior, showing an increase in opercular movements and erratic swimming; mortality reached 100% after 18 minutes. At 12ºC, fish lost equilibrium immediately after exposure and started swimming erratically; after only 4 minutes fish became lethargic and remained immobile on the bottom of the tank. Total mortality was only evident following 24 hours. At 15ºC matrinxã lost equilibrium after 5 to 6 minutes of exposure but mortality was only 20% after 24 hours. Fish tolerated well temperatures ranging from 18 to 36ºC with 100% survival after 24 hours. This preliminary study suggests that temperatures between 18 and 36ºC are the approximate range normally tolerated by this species, although survival at other temperatures may be increased by gradually acclimating fish to the more severe increases or decreases in temperature. In addition, it indicates that matrinxã may be cultivated over a wide geographical area.

Screening of plants found in the State of Amazonas, Brazil for activity against Aedes aegypti larvae

Ethanol, methanol and water extracts representing mostly native plant species found in the Amazon region were prepared, respectively, by maceration, continuous liquid-solid extraction and infusion, followed by evaporation and freeze-drying. The freeze-dried extracts were tested for lethality toward Aedes aegypti larvae at test concentrations of 500 mg / mL. In general, methanol extracts exhibited the greatest larvicidal activity. The following 7 methanol extracts of (the parts of) the indicated plant species were the most active, resulting in 100% mortality in A. aegypti larvae: Tapura amazonica Poepp. (root), Piper aduncum L. (leaf and root), P. tuberculatum Jacq. (leaf, fruit and branch). and Simaba polyphylla (Cavalcante) W.W. Thomas (branch).

Developmental morphology of mouthparts and foregut of the larvae and postlarvae of Lepidophthalmus siriboia Felder & Rodrigues, 1993 (Decapoda: Callianassidae)

In this study, the gross morphology of the mouthparts and foregut of the ghost shrimp Lepidophthalmus siriboia were investigated from larvae and postlarvae reared in the laboratory. The mouthparts (maxillae and maxillipeds) of the zoeae have a reduced number of setae and spines (or is absent in some individuals), and the foregut, under developed, have few minute setae in the cardiac and pyloric chambers. In contrast, after the metamorphosis into megalopa stage, all feeding appendages have many setae and, the foregut shows a well-developed gastric mill with strong lateral teeth. In the juvenile stage occurs an increase of setae and spines in the mouthparts and the foregut becomes more specialized. These observations strongly suggest that a lecithotrophic development occurs during all zoeal stages but the megalopa and juvenile stages are feeding animals. The functional morphology of the feeding structures of L. siriboia and other decapods will be briefly discussed.

The morphology of the foregut of larvae and postlarva of Sesarma curacaoense De Man, 1892: a species with facultative lecithotrophy during larval development

Previous study on the resistance of larvae of Sesarma curacaoense submitted to starvation has revealed a facultative lecithotrophy during zoeal stages, but megalopa and first juvenile stages are exclusively feeding stages. In the present study, the gross morphology and fine structure of the foregut of S. curacaoense were investigated during larval, megalopa and first juvenile stages. The foregut of the zoea I show specific setae and a filter press apparently functional. The foregut undergoes changes in the zoea II (last larval stage) with increment of setae number, mainly on the cardiopyloric valve and complexity of the filter press. After metamorphosis to megalopa stage the foregut become rather complex, with a gastric mill supporting a medial and two lateral teeth well-developed. The foregut of the first juvenile is more specialized compared to the previous stage, showing similar characteristics of the decapod adults. These results provide further evidence of facultative lecithotrophic development in the larvae of S. curacaoense.

Tambaqui growth and survival when exposed to different photoperiods

The use of different photoperiods (light) were investigated during tambaqui (Colossoma macropomum) juvenile growth under captivity. Light intensity tested was: continuous dark (24hrs without light), natural photoperiod simulation (10hrs of light and 14hrs without light) and continuous light (24 with light). No mortality was recorded among treatments. Significant differences was observed after 50 days of experiment among mean fish weight, fish kept under a continuous darkness showed a better specific growth rate (6.02%) when compared to control fish (natural photo period, 3.67%). Fish exposed to continuous light presented the lowest mean specific growth rate (2.04%). It is possible to improve tambaqui juvenile weight gain performance when kept under continuous darkness.

Analysis of toxicity on Bacillus sphaericus from amazonian soils to Anopheles darlingi and Culex quinquefasciatus larvae

Bioassays under laboratory conditions aiming to determine the larvicidal activity of Bacillus sphaericus were carried out on Anopheles darlingi and Culex quinquefasciatus. In order to estimate the toxicity through median lethal concentration (LC50) and the relative potency of the strains to B. sphaericus standard strain 2362, probit analysis was performed utilizing the POLO-PC program. The findings of LC50 pointed out high effectiveness on strains IB15 (0.040 ppm), IB19 and S1116 (0.048 ppm), IB16 (0.052 ppm) and S265 (0.057 ppm). Strain IB15 presented nearly 50% more potency than strain 2362 in bioassays conducted on A. darlingi. It was observed that IB16 and S1116 strains were the most powerful against C. quinquefasciatus, showing to be about 300-400% stronger than 2362 strain. The results show that laboratory conditioned evaluation can be an important way to select promising bacteria with entomopathogenic action on biolarvicides production for use on mosquitoes breeding sites.

Nonparametric estimation of the survival function for ordered multivariate failure time data: a comparative study

In longitudinal studies of disease, patients may experience several events through a follow-up period. In these studies, the sequentially ordered events are often of interest and lead to problems that have received much attention recently. Issues of interest include the estimation of bivariate survival, marginal distributions and the conditional distribution of gap times. In this work we consider the estimation of the survival function conditional to a previous event. Different nonparametric approaches will be considered for estimating these quantities, all based on the Kaplan-Meier estimator of the survival function. We explore the finite sample behavior of the estimators through simulations. The different methods proposed in this article are applied to a data set from a German Breast Cancer Study. The methods are used to obtain predictors for the conditional survival probabilities as well as to study the influence of recurrence in overall survival.

Occurrence of Aelurostrongylus abstrusus (Railliet, 1898) larvae (Nematoda: Metastrongylidae) infecting Achatina (Lissachatina) fulica Bowdich, 1822 (Mollusca: Gastropoda) in the Amazon region

Achatina fulica or "giant African snail" is an exotic species, considered to be one of the world's hundred most invasive species, causing serious environmental damages. In the present study we report, for the first time, the occurrence of Aelurostrongylus abstrusus infecting A.fulica in the Amazon region. This nematode is described parasitizing mainly the pulmonary system of felines, which causes "aelurostrongilose", also known as feline cardio-pulmonary strongyloidosis. New morphometric data of third stage larvae are presented herein. The present study demonstrated that 40% of all the snails were infected by A. abstrusus. Achatina fulica specimens were collected from three different areas in Manaus namely: rural; east and west areas. The east area presents the highest prevalence of 80%. The large number of A.fulica found in inhabited areas increases the chances of emergent zoonoses, which highlights the need of further studies so as to better control this disease.

Seasonality of nematode larvae in Iguanodectes spilurus (Characidae) an ornamental fish from northeastern Para, Brazil

The current study presents a parasitological survey of larval nematodes from freshwater ornamental fish Iguanodectes spilurus caught in the watercourse of the Caete River, in the northeast region of the State of Para, Brazil. A total of 176 specimens, 1.36±0.75 g weight and 5.53±0.98 cm total length, were analyzed. Nematode larvae were identified as Capillaria sp., Procamallanus sp. and Anisakidae, with prevalence of 70.45% and infection intensity ranging from 1.81 to 4.70 larvae. The highest prevalence 57.38% occurred in the liver, but no seasonality was observed, indicating high infection throughout the year. Seasonality was observed in fish parasitized in the stomach, intestine and caecum, with prevalence and mean intensity of 17.61% and 2.32 parasites, 12.5% and 1.81 parasites, 10.79% and 2.21 parasites, respectively. The highest degree of infection was observed in the rainy season, probably due to increased availability of intermediate hosts or food.

Ki-67 expression in CRC lymph node metastasis does not predict survival

Colorectal cancer is one of the most common malignancies and a leading cause of cancer death worldwide. Molecular markers may improve clinicopathologic staging and provide a basis to guide novel therapeutic strategies which target specific tumourassociated molecules according to individual tumour biology; however, so far, no ideal molecular marker has been found to predict disease progression. We tested Ki-67 proliferation marker in primary and lymph node metastasis of CRC. We observed a statistical significant difference between the positive rates of neoplastic cells positively stained byKi-67 in both sites, with remarkable increased number of Ki-67 positive cells in primary tumor cells compared to cancer cells that invaded lymph nodes. We can speculate that the metastatic CRC in lymph node can be more resistant to the drugs that target cellular division.

Joint modelling of longitudinal and survival data on breast cancer

Tese de Doutoramento em Ciências (Especialidade em Matemática)